Download Up-regulation of HO-1 Attenuates Left Ventricular Remodeling Post Myocardial by

Up-regulation of HO-1 Attenuates Left Ventricular Remodeling Post Myocardial
Infarction in Rats
by
Rebecca E. Tee
A thesis submitted to the Department of Physiology
in conformity with the requirements for
the degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
September, 2007
Copyright © Rebecca Elizabeth Tee, 2007
Abstract
Background/Objective: Reperfusion injury is a serious consequence of blood flow
reestablishment after myocardial infarction (MI) mediated by reactive oxygen species and
neutrophilic cellular damage.
Following MI, the left ventricle (LV) undergoes
remodeling characterized by progressive wall thinning and cavity dilatation. HemeOxygenase-1 (HO-1) dependent decrease in oxidative stress may attenuate injury in part
by inhibiting transcription factor NFκB-mediated inflammation.
Hypothesis: I
hypothesized that upregulation of HO-1 by hemin administration confers acute and
chronic cardioprotection against I/R injury in rats and attenuates LV remodeling post-MI.
I proposed the HO-1-dependent decrease in oxidative stress attenuates post-ischemic
myocardial injury in part by inhibiting NFκB-mediated inflammation. Methods: Six
week old male Wistar rats were randomly assigned to sham, vehicle, or hemin-treated
groups.
Vehicle and hemin were administered intraperitoneally once daily for 3
consecutive days prior to left anterior descending (LAD) coronary artery occlusion.
Administration resumed 48 hours post-operatively and continued once every 3 days.
Infarct size was determined by H&E histological analysis and fibrosis was quantified by
Masson’s Trichrome staining. Transthoracic echocardiography was used to assess LV
parameters and wall motion. Results: Hemin increased HO-1 expression, decreased
infarct size and fibrosis, and attenuated LV remodeling in the short-term (4 days postinfarction). The decrease in infarct size and area of fibrosis in the hemin group was
accompanied by a decrease in NFκB activity. No significant difference in infarct size
and area of fibrosis between hemin and vehicle-treated groups was observed at 3 months.
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LV diameter and cardiac function did not differ significantly between the two groups at 3
months despite an attenuation of anterior wall thinning in the hemin group. Conclusion:
HO-1 upregulation by hemin administration conferred acute cardioprotection and
attenuated LV remodeling, possibly by inhibiting NFκB-mediated inflammation.
However, chronic treatment with hemin did not prevent long-term post-infarction LV
remodeling. It is possible that cardioprotection afforded by HO-1 upregulation is strong
enough to curtail inflammation post-reperfusion and prevent LV remodeling acutely, but
is not robust enough to protect the myocardium to the same degree in the long-term.
Future research should focus on optimal HO-1 upregulation to attenuate long-term LV
remodeling due to reperfusion injury.
iii
Statement of Co-Authorship
The ideas and hypotheses presented in this thesis are the result of a collaboration
between Dr. Luis G. Melo, Dr. Kofoworola Ogunyankin, and Rebecca Tee. All in vivo
and in vitro experiments were performed by Rebecca Tee. All echocardiography studies
and analysis were performed by Rebecca Tee. All protocols used in this thesis were
developed by Rebecca Tee with the help of Keith Brunt and Dr. Xiaoli Liu under the
guidance of Dr. Melo and Dr. Ogunyankin.
iv
Acknowledgements
First and foremost, I would like to thank my parents and sister who provided me
with meaningful advice and endless support. My father taught me at an early age that
nothing worth doing was easy and, if something was easy, everyone would be doing it.
This piece of wisdom kept me motivated during many long nights in the lab alone. I
would like to thank Dr. Luis Melo for his vision and guidance throughout this project. I
am very grateful to Dr. Kofoworola Ogunyankin for his expertise in echocardiography
and his patience when teaching me proper echocardiography techniques. I have learned
more during my graduate studies than I could have imagined. Thank you for being
patient with my steep learning curve.
Many individual people have helped me along the way. I would like to thank Dr.
Steven Pang and the Department of Anatomy and Cell Biology for their willingness to
help me and provide me with equipment whenever necessary. I would also like to thank
the Echocardiography Lab at Kingston General Hospital for being instrumental to my
echocardiography studies. Many, many thanks also go to Dr. Xiaoli Liu for teaching me
small animal surgical skills and histology techniques. I would like to make special
mention of Keith Brunt who answered innumerable questions of mine and took much
time to instruct me and help me troubleshoot. A great deal of what I have learned has
come from you. Finally, thank you to all my friends who made my experience at Queens
memorable.
v
Table of Contents
Abstract………...……………………………………………………………………….....ii
Statement of Co-Authorship……………………………………………………………...iv
Acknowledgements………………………………………………………………………..v
Table of Contents………………………………………………………………………....vi
List of Tables …………………………………………………………………………….ix
List of Figures.………………………………………………………………………….....x
List of Abbreviations…………………………………………………………………….xii
Chapter 1:
Introduction…………………………………………………………………………..........1
1.1 Preamble………………………………………………………………………1
1.2 Functional Consequences of Metabolism……………………………………..2
1.2.1 Metabolism and Mechanical Function…………………………….2
1.2.2 Organelle Remodeling and Ion Handling…………………………3
1.2.3 Ischemia-induced Cytokine Production…………………………...4
1.3 Myocardial Reperfusion.………………………………………………………7
1.3.1 Reperfusion Injury………………………………………………...7
1.3.2 Metabolism and Mechanical Function Following Reperfusion…...7
1.3.3 Ion Handling and Ca2+ Overload During Reperfusion……………9
1.3.4 Contracture During Reperfusion………………………………….9
1.4 Reperfusion-induced myocardial inflammation……………………………..10
1.4.1 Neutrophil Infiltration and Injury………………………………..11
1.4.2 Mechanisms of Neutrophil-induced Injury………………………13
1.4.3 Reactive Oxygen Species………………………………………...13
1.4.4 Consequences of ROS Activity………………………………….17
1.4.5 Other Mechanisms of Neutrophil-mediated Injury………………18
1.4.5.1 Neutrophil-derived Proteolytic Enzymes………………...18
1.4.5.2 Arachidonic Acid Metabolites…………………………...18
1.5 Inflammatory Cytokines and Chemokines…………………………………...19
1.5.1 Inflammatory Cytokines…………………………………………19
1.5.2 Chemokines………………………………………………………20
1.5.3 Regulation of Cytokine Gene Expression During I/R Injury…….21
1.6 Wound Healing………………………………………………………………23
1.6.1 Scar Formation…………………………………………………...23
1.7 Ventricular Remodeling……………………………………………………...25
1.7.1 Infarct Expansion………………………………………………...25
1.7.2 Compensatory Hypertrophy and Ventricular Dilatation…………27
1.8 Echocardiography……………………………………………………………30
1.9 Heme-oxygenase System…………………………………………………….34
1.9.1 Cardioprotective Effects of Hemin and HO-1…………………...37
1.10 Rationale and Hypotheses………………………………………………..37
1.10.1 Rationale…………………………………………………………37
1.10.2 Hypotheses……………………………………………………….40
Chapter 2: Materials and Methods……………………………………………………….41
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2.1 Animals………………………………………………………………………41
2.2 Hemin Administration……………………………………………………….41
2.3 Left Anterior Descending (LAD) Coronary Artery Ligation………………..41
2.4 Transthoracic Echocardiography…………………………………………….42
2.5 Tissue Harvest……………………………………………………………….44
2.6 Isolation of Cytoplasmic and Nuclear Proteins……………………………...45
2.7 Western Blot Analysis……………………………………………………….45
2.8 NFκB Binding Activity Assay……………………………………………….46
2.9 Preparation of Histological Specimens………………………………………47
2.10 Morphometric Analysis…………………………………………………….48
2.11 Statistical Analysis………………………………………………………….48
Chapter 3: Results………………………………………………………………………..50
3.1 The Effect of Hemin Treatment in the Setting of Myocardial I/R Injury……50
3.1.1 Effect of Hemin Administration on HO-1 Expression…………….50
3.1.2 Effect of Ischemia on HO-1 Expression in Hemin and Vehicletreated Animals ………………………………………………………….50
3.1.3 Effect of Hemin Treatment on Ischemic and Reperfused
Myocardium……………………………………………………………...53
3.2 Quantification of Myocardial Infarct Size…………………………………...53
3.2.1 Myocardial Area at Risk for Infarction…………………………….53
3.2.2 Myocardial Infarct Size…………………………………………….56
3.2.3 Quantification of Myocardial Fibrosis……………………………..61
3.3 Quantification of LV Structure and Function by Echocardiography………...65
3.3.1 LV Wall and Chamber Dimensions………………………………..65
3.3.2 Echocardiography Assessment of Cardiac Function………………72
3.3.2a Mid Level Ejection Fraction, Fractional Shortening and LV
Mass……………………………………………………………...72
3.3.2b Apex Level Ejection Fraction, Fractional Shortening and
LV Mass………………………………………………………….75
3.4 Quantification of NFκB Binding Post-reperfusion…………………………..81
3.5 Effect of Hemin Treatment on Hematocrit…………………………………..81
Chapter 4: Discussion……………………………………………………………………84
4.1 Major Findings……………………………………………………………….84
4.2 Histological Findings………………………………………………………...85
4.2.1 Myocardial Infarctions……………………………………………..85
4.2.2 Myocardial Fibrosis………………………………………………..87
4.3 Echo Findings………………………………………………………………..88
4.4 HO-1 Upregulation and Preservation of Coronary Vascular Homeostasis….92
4.5 Effect of HO-1 Upregulation of Post-infarction Left Ventricular Function…92
4.6 Comparison of Short and Long Term Studies……………………………….94
4.7 Changes in Cardiac Mass…………………………………………………….95
4.8 Role of NFκB in I/R Injury…………………………………………………..96
4.9 Hematocrit and Cardiac Function Post-I/R Injury…………………………...98
4.10 Potential Therapeutic Application of HO-1 Upregulation in Myocardial
Infarction and Reperfusion………………………………………………………99
4.11 Study Consideration.………………………………………………………101
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4.12 Study Limitations………………………………………………………….102
4.12.1 Sample Size……………………………………………………...102
4.12.2 Use of Volatile Anaesthetic……………………………………..103
4.12.3 Echocardiography Technique…………………………………...103
4.13 Conclusions and Future Directions………………………………………..104
References………………………………………………………………………………106
viii
List of Tables
Table 1A. Left ventricle wall and chamber dimensions in sham-operated groups….….66
Table 1B. Left ventricle wall and chamber dimensions in hemin-treated groups…...….67
Table 1C. Left ventricle wall and chamber dimensions in buffer-treated groups…...….68
Table 2. Percent change in left ventricle dimensions from baseline echo………………70
Table 3. Percent change in functional parameters from baseline echo………………….79
Table 4. Percent change in left ventricle mass…………………………………………..80
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List of Figures
Figure 1. Consequences of ischemia and reperfusion…………………………………….8
Figure 2. Neutrophil infiltration…………………………………………………………12
Figure 3. Reactive oxygen species………………………………………………………16
Figure 4. NFκB pathway………………………………………………………………...22
Figure 5. Left ventricle remodeling post-infarction……………………………………..29
Figure 6. Sections of the left ventricle…………………………………………………..32
Figure 7. M-mode echocardiograms…………………………………………………….33
Figure 8. The heme oxygenase-1 pathway. …………………………………………….36
Figure 9. Left anterior descending coronary artery occlusion…………………………..39
Figure 10. HO-1 western blot…………………………………………………………...51
Figure 11. HO-1 expression in ischemic and non-ischemic myocardium………………52
Figure 12. HO-1 expression in reperfused and non-reperfused myocardium…………...54
Figure 13. Area at risk for infarction……………………………………………………55
Figure 14. Myocardial infarction after I/R in vehicle and hemin-treated rats…………..57
Figure 15. Hematoxylin & eosin stained left ventricle myocardium post-I/R injury…...59
Figure 16. Infarct size relative to area at risk……………………………………………60
Figure 17. Myocardial fibrosis…………………………………………………………..62
Figure 18. Area of fibrosis relative to the area at risk…………………………………...64
Figure 19. Mid level functional echo parameters…………………………….................73
Figure 20. Mid LV mass to body weight………………………………………………..74
Figure 21. Apex level functional echo parameters……………………………………...76
x
Figure 22. Apex LV mass to body weight………………………………………………77
Figure 23. NFκB-DNA binding activity after I/R…………………………………….....82
Figure 24. Hematocrit at cardiac harvest………………………………………………..83
xi
List of Abbreviations
ANOVA = analysis of variance
ASE = American Society of Echocardiography
BR = bilirubin
BV = biliverdin
BVr = biliverdin reductase
CAD = coronary artery disease
cGMP = cyclic guanylyl monophosphate
C5a = complement factor 5a
CHF = congestive heart failure
CO = carbon monoxide
CO = cardiac output
EPO = erythropoietin
H 2 O 2 = hydrogen peroxide
HO = heme oxygenase
HO-1 = heme oxygenase-1
HSP-32 = heat shock protein-32
ICAM = intercellular adhesion molecule
IκB = inhibitory protein of NFκB
IL = interleukin
I/R = ischemia reperfusion
IVS = interventricular septum
IVSdA = interventricular septum during diastole, apex level
xii
IVSdB = interventricular septum during diastole, base level
IVSdM = interventricular septum during diastole, mid level
IVSsA = interventricular septum during systole, apex level
IVSsB = interventricular septum during systole, base level
IVSsM = interventricular septum during systole, mid level
LAD = left anterior descending (coronary artery)
LV = left ventricle
LVD = left ventricle diameter
LVDdA = left ventricle diameter during diastole, apex level
LVDdB = left ventricle diameter during diastole, base level
LVDdM = left ventricle diameter during diastole, mid level
LVDsA = left ventricle diameter during systole, apex level
LVDsB = left ventricle diameter during systole, base level
LVDsM = left ventricle diameter during systole, mid level
MCP-1 = monocyte chemoattractant protein 1
MI = myocardial infarction
MMP = matrix metalloproteinase
NAD = nicotinamide adenine dinucleotide
NADPH = nicotinamide adenine dinucleotide phosphate, reduced form
NFκB = nuclear factor kappa B
NO = nitric oxide
OS = oxidative stress
PAF = platelet activating factor
xiii
PECAM = platelet/endothelial cellular adhesion molecule
PSLX = parasternal long axis
PSSX = parasternal short axis
PW = posterior wall
PWdA = posterior wall during diastole, apex level
PWdB = posterior wall during diastole, base level
PWdM = posterior wall during diastole, mid level
PWsA = posterior wall during systole, apex level
PWsB = posterior wall during systole, base level
PWsM = posterior wall during systole, mid level
ROS = reactive oxygen species
RT = room temperature
SEM = standard error of the mean
SOD = superoxide dismutase
TGF-β = transforming growth factor-beta
TIMP = tissue inhibitor of matrix metalloproteinase
TNFα = tumour necrosis factor alpha
VCAM = vascular cellular adhesion molecule
VEGF = vascular endothelial growth factor
xiv
Chapter 1: Introduction
1.1 Preamble
Myocardial infarction and reperfusion injury is a major cause of death in
developed countries (Liu et al., 1997). An estimated 7.9 million Americans suffered
myocardial infarctions in 2004 (Heart Disease and Stroke Statistics 2007 Update,
American Heart Association). Coronary artery disease (CAD) frequently progresses to
an occlusion of a major coronary artery, thereby restricting blood flow to the myocardium
and compromising metabolism. Early reperfusion is essential to prevent cell death after
coronary artery occlusion (Di Napoli et al., 2002). Paradoxically, sudden reoxygenation
of ischemic myocardium may cause further tissue injury due to excess production of
reactive oxygen species (ROS). The oxygen paradox during reperfusion describes the
contradictory need to deliver oxygen and the resultant molecular reduction of oxygen to
form free radicals that are involved in macromolecule oxidation, membrane dysfunction,
apoptosis, and damaged calcium-sequestering ability (reviewed by Hoffman et al., 2004).
These cell-damaging events are amplified by the hyper-activation of neutrophils, which
promote the formation of pro-inflammatory mediators and oxygen radicals (Hoffman et
al., 2004) as well as endothelial dysfunction. Myocardial necrosis and apoptosis result in
increased mechanical loading of the surviving myocardium, causing changes in
ventricular architecture involving the infarct and peri-infarct zones as well as the remote
non-infarcted tissue. The resultant increase in load triggers a cascade of biochemical
intracellular signaling processes that initiates and subsequently modulates reparative
changes, which include dilatation, hypertrophy and the formation of a discrete collagen
1
scar (St John Sutton and Sharpe, 2000). It is clear that substantial alterations in gene
expression occur in order to confer such profound changes within the cells of the
remodeling myocardium (Stanton et al., 2000). Coordinated changes in expression of
more than 400 genes related to cellular and extracellular architecture are consistent with
the remodeling process in the post-ischemic heart (Stanton et al., 2000). Myocardial
ischemia/reperfusion (I/R) injury is a cascade of events that requires an understanding of
cellular and molecular events to explain the pathogenesis and subsequent functional
consequences of ventricular remodeling.
1.2 Functional Consequences of Myocardial Ischemia
1.2.1 Metabolism and Mechanical Function
Myocardial infarction is most commonly caused by a ruptured atherosclerotic
lesion within a major coronary artery, wherein the vessel is occluded and blood flow to
the myocardium is severely restricted. Within the first 10-15 seconds after the occlusion,
the tissue partial oxygen pressure decreases, mitochondrial respiration is inhibited, and
the myocytes begin to use anaerobic glycolysis as the principal source of new highenergy phosphate (Reimer et al., 1983). The delivery of glucose is markedly reduced and
myocardial glycogen stores must provide the substrate for anaerobic glycolysis.
In
prolonged ischemia, lactate accumulates in the tissue because it cannot be further
metabolized in the absence of oxygen nor can it be cleared by the systemic circulation.
Eventually, glycolysis is inhibited by the accumulation of its own products, leading to
further reduction of glucose metabolism in the absence of adequate ATP production
2
(Vanoverschelde et al., 1994). Protons, Na+, and Ca2+ continue to accumulate, leading to
an acidotic microenvironment and transmembrane ionic imbalances.
Meanwhile, the reactions that continue to require high energy phosphate (ATP)
during ischemia include the myosin ATPase of the myofibrils, Na+/K+ ATPase, the Ca2+
ATPases of the sarcolemma and sarcoplasmic reticulum, adenylyl cyclase, fatty acidCoA synthetase, and other ATPases of the myocytes (Jennings and Reimer, 1991). The
demand for ATP rapidly exceeds the supply. Electrical conduction continues, however,
resulting in unproductive and/or sustained myocyte contractions (excitation contraction
uncoupling), further requiring ATP for Ca2+ and Na+/K+ ATPases. Effective contractions
terminate shortly thereafter. The metabolic changes occurring with ischemia thus include
cessation of aerobic metabolism, onset of anaerobic glycolysis, and accumulation of
glycolytic products (Jennings and Reimer, 1991). The degree of metabolic disturbance is
proportional to the severity of ischemia. The metabolic recovery of the myocardium is
dependent on rapid reestablishment of blood flow. In the absence of timely reperfusion,
myocytes undergo irreversible damage, leading to cell death.
1.2.2 Organelle Remodeling and Ion Handling
Prolonged (>30 minutes) ischemia causes biologically irreversible injury
(Jennings and Reimer, 1991).
Ultrastructural changes include diffuse mitochondrial
swelling, the exhaustion of glycogen stores, marked peripheral aggregation of nuclear
chromatin, and the appearance of discontinuities in the plasmalemma of the sarcolemma
3
(Jennings and Reimer, 1991). One of the hallmarks of myocyte death is sarcolemmal
disruption (Jennings et al., 1983). Once breaks in the sarcolemma develop, myocytes
become osmotically fragile, lose low molecular weight molecules, and accumulate
extracellular electrolytes such as Na+ and Ca2+ (Jennings et al., 1990). Ca2+-induced
activation of proteases causes alterations in contractile proteins, decreased sensitivity to
Ca2+, and sustained impairment of contractility despite the elevated cytosolic Ca2+
(Steenbergen et al., 1987; Buja, 2005). A decreased Ca2+ sensitivity via Ca2+ overload
and ischemia-induced conformational and functional changes in the channels cause
contractile deficiencies. Furthermore, the increased cytosolic Ca2+ load may trigger
mitochondrial release of cytochrome c and subsequently activate apoptotic caspases
leading to cell death (Xu et al., 2001).
Mitochondrial function is impaired during ischemia. Ischemia-induced depletion
of ATP and subsequent cellular Ca2+ overload can lead to the opening of large
permeability transition pores (MTP) in the mitochondria (Halestrap et al., 1998). The
mitochondria not only lose small ions via these nonspecific pores, and thereby the ability
to separate charges and to maintain membrane polarization, but also lose essential
substrates like NAD/NADH (Vetterlein et al., 2003).
1.2.3 Ischemia-induced Cytokine Production
Cytokine production and inflammation play crucial roles in determining the
outcome of post-myocardial infarction. While it is widely accepted that reperfusion of
4
the ischemic tissue enables the inflammatory reaction, it is important to consider that the
inflammatory reactions begin during the ischemic period (Neumann et al., 1995).
Cytokines and chemokines are important pleiotropic mediators of inflammation
(Neumann et al., 1995; Deten et al., 2002; Sharma and Das, 1997; Frangogiannis and
Entman, 2005; Nian et al., 2004).
As effectors of the immune system and the
inflammatory response, cytokines modulate gene expression and transcription factor
activity, and promote chemotaxis of inflammatory cells (Sun et al., 2004; Ono et al.,
1998; Mercurio et al., 1997; Chandrasekar et al., 2001; Nian et al., 2004). Hypoxia and
membrane disruption are likely the initial triggers for the upregulation of cytokines like
TNF-α, IL-1β, and IL-6 through multiple cell signaling pathways (Deten et al., 2002).
Cytokines such as TNF-α and IL-6 are rapidly released in the central ischemic zone but
are also found in high concentration in the border zone (Gwechenberger et al., 1999).
Ischemic stress is a powerful trigger for cytokine production, but direct myocardial
mechanical stretch, which is maximal in the infarct and peri-infarct zone, is also a
powerful stimulus for cytokine production (Nian et al., 2004). If the infarct size is small,
the cytokine upregulation may promptly return to baseline. Deten and colleagues (2002)
found that IL-1β and IL-6 mRNA were strongly induced in the myocardium after
permanent coronary artery occlusion in rats. This induction persisted over the first 12
hours after infarction and rapidly declined to control levels thereafter (Deten et al., 2002).
In contrast, if the infarction size is large, robust cytokine expression may be sustained
chronically or may exhibit a second wave of upregulation, in keeping with the process of
ventricular remodeling (Nian et al., 2004).
5
Several independent groups including Frangogiannis et al. (1998) have confirmed
the presence of mast cells in the infarcted myocardium of different species. Mast cells
are regulated by their local micro-environment and, in the heart, are involved in
complement activation and the release of proinflammatory and vasoactive mediators
(Patella et al., 1995). Initial studies by Frangogiannis et al. (1998) established the
presence of preformed TNF-α in cardiac mast cells in normal myocardium in both
control hearts and areas of ischemic and reperfused dog heart. Complement factor C5a is
known to induce degranulation in cardiac mast cells and is present exclusively in the area
surrounding the myocardial injury before initiation of reperfusion (Frangogiannis et al.,
1998; Patella et al., 1995; Rossen et al., 1994).
TNF-α binds the cell surface receptors
and triggers intracellular signaling cascades that can result in activation of transcription
factors such as NF-κB or death domain effectors such as caspases (Sun et al., 2004). It is
possible that the degranulation of cardiac mast cells and the release of preformed TNF-α
represent the stimulus for the initiation of a cytokine cascade before the reestablishment
of coronary blood flow.
Cytokines have the unique ability to self-amplify their biological effects through a
positive feedback loop targeting the transcription factor NF-κB (Irwin et al., 1999). A
strong amplification of the cytokine response, however, generally requires the
recruitment of inflammatory cells- including leukocytes, macrophages and mast cells- to
the site of injury, requiring perfusion. This concept is a cornerstone of reperfusion injury
and will be discussed further in the setting of reperfusion.
6
1.3 Myocardial Reperfusion
1.3.1 Reperfusion Injury
Several investigators believe that reperfusion of ischemic tissue simply
accelerates the injury caused during the ischemic period and that “reperfusion injury”
does not truly exist (Zweier et al., 1987; Zahger et al., 1995; Vetterlein et al., 2003).
However, morphologic observations show that reperfusion of severely ischemic
myocardium causes structural d.erangements exceeding the amount of damage seen with
ischemia alone, indicating that reperfusion is a truly pathologic phenomenon (Vetterlein
et al., 2003). Moreover, it is well established that reperfusion of ischemic myocardium
with oxygenated blood generates a “respiratory burst” (Figure 1), characterized by high
consumption of oxygen in association with metabolic disturbances, biochemical changes,
and clinical manifestations triggered largely by neutrophil-mediated inflammation and
the production of oxygen free radicals (Duilio et al., 2001; Goldhaber and Weiss 1992).
1.3.2 Metabolism and Mechanical Function Following Reperfusion
The functional recovery following reperfusion is mainly determined by the extent
of accumulation of metabolic end products during ischemia and the substrate availability
at reperfusion (Depre et al., 1999). When glycolysis is stimulated in the reperfused
myocardium, the cytosolic accumulation of Ca2+ decreases (Jeremy et al., 1992).
However, elevated circulating levels of free fatty acids, due to lipolysis caused by fasting
and stress hormone release, inhibit glucose utilization during ischemia and reperfusion
(Hendrickson et al., 1997). As a result, high glycolytic rates during reperfusion and low
7
O2 ,
High energy phosphates
ATP
H+
Lactate
pH
Sarcolemma disruption, mitochondrial injury, cytoskeletal damage
K+
Na2+
Ischemic contracture,
Cl-
Ca2+
Cell death
osmolar load, cell swelling
Reperfusion
Respiratory burst
Free radical
generation
•Oxidative stress
•Protein denaturation
•Lipid peroxidation
Pro-inflammatory
mediator release
Neutrophil
Accumulation
Cytokine
Elaboration
Protease
Release
•Chemotaxis
•Amplification
•ECM degradation
•Myocyte slippage
“No re-flow”
Figure 1. Consequences of ischemia and reperfusion. Inadequate oxygen supply
resulting in anaerobic glycolysis produces an accumulation of waste products and
intracellular acidosis that weaken cytoskeletal structure and damage organelles. Altered
ion distribution and dysfunctional organelles increase the osmolar load and promote cell
swelling as well as encourage ischemic contracture, all of which can lead to necrotic cell
death or programmed cell death by apoptosis. Reperfusion induces a respiratory burst
with a multi-component deleterious cascade, at which point cells still viable after the
period of ischemia are subjected to conditions which could potentially induce necrosis or
apoptosis.
8
oxidation rates can result in substantial uncoupling of glycolysis from glucose oxidation
(Lopaschuk and Stanley, 1995). This uncoupling contributes to the production of protons
and to ischemic reperfusion injury and a decrease in cardiac efficiency (Liu et al., 1996).
1.3.3 Ion Handling and Ca2+ Overload during Reperfusion
One of the primary causes of functional impairment in post-ischemic myocardium
is cellular Ca2+ overload (Depre et al., 1999). Substantial evidence exists to support the
idea that the Na+/Ca2+ exchanger serves as the major physiological route for Ca2+ entry
during reperfusion (Bagchi et al., 1997). During ischemia, increased cellular acidosis
favours the exchange of intracellular H+ for Na+. Sudden normalization of tissue pH
upon reperfusion favours hypercontracture and contributes to further Ca2+ overload
(Bond et al., 1991; Schafer et al., 2000). Grinwald and Brosnahan (1987) demonstrated
that the Na+ pump is inhibited during hypoxia and sustained during reperfusion, and this
system could account for the Na+ load and massive Ca2+ influx via Na+/Ca2+ exchange.
Rapid H+ extrusion and Na+/H+ exchange activation cause a net influx of Na+ and,
depending on its ability to remove the excess load of Na+, it may in turn activate the
Na+/Ca2+ exchange mechanism in the “reverse mode”, enhancing the pre-existing Ca2+
overload (Piper et al., 1998).
1.3.4 Contracture during Reperfusion
Contracture refers to a sustained shortening and stiffening of the myocardium that
develops during ischemia as a result of insufficient ATP to dissociate myosin crossbridges from actin filaments (Kingsley et al., 1991; Piper et al., 2003). Hypercontracture
9
occurs during the first few minutes of reperfusion because of sudden ATP availability
(“re-energization”) in the presence of abnormally high intracellular Ca2+; myocytes
respond to re-energization by developing an extreme distortion of their architecture
resulting from development of excessive contractile force (Ruiz-Meana et al., 1999;
Garcia-Dorado et al., 1997; Ladilov et al., 1997; Ganote 1983). Hypercontracture is
associated with sarcolemmal disruption and enzyme release which cause dramatic
changes in cytosolic composition, in turn propagated to adjacent cells through gap
junctions (Ruiz-Meana et al., 1999).
Moreover, increased mural tension due to
contracture further constricts small vessels within the area of ischemia, making any
residual oxygen and glucose delivery impossible. This is sometimes called a loss of
“perfusability”.
1.4 Reperfusion-induced Myocardial Inflammation
Acute
myocardial
ischemia-reperfusion
is
associated
with
an
intense
inflammatory reaction that plays a part in both acute myocardial injury as well as repair
of the myocardium (Nossuli et al., 2000). Reactive oxygen species and neutrophils are
key players in reperfusion injury, interacting with each other at multiple sites, as well as
with other elements such as the complement system, endothelial cells, lymphocytes,
macrophages and myocytes (Litt et al., 1989; Liu et al., 1997; Amsterdam et al., 1995;
Kaminski et al., 2002). Principal components of the myocardial inflammation response
include cell-mediated inflammation, humoral inflammatory response, the role of
10
inflammatory cytokines and transcription factor NFκB, endothelial and vascular
dysfunction, and the initiation of wound healing.
1.4.1 Neutrophil Infiltration and Injury
One of the earliest events of reperfusion involves neutrophil trapping in the
microvasculature, whereby neutrophils are entrapped in the coronary microcirculation
prior to their involvement in inflammation (Frangogiannis et al., 2002).
This is
commonly referred to as the “no-reflow” phenomenon and can result in a delayed,
progressive decrease in blood flow. Cellular adhesion molecules promote neutrophil
adhesion to endothelial cells, lining the capillaries. These adhesion molecules include
selectins, β2-integrins and the immunoglobulin family, including ICAM-1, VCAM-1 and
platelet-endothelial cell adhesion molecule-1 (PECAM-1) (Jordan et al., 1999). For
neutrophils, firm adhesion requires activation of the β2-integrins and results in binding to
one of the intercellular adhesion molecules on the surfaces of endothelial cells
(Frangogiannis et al., 2002). After the inflammatory stimulus is initiated, leukocytes roll
along the post-capillary venules (but not arterioles or small arteries) at a slower velocity
than that of flowing blood (Frangogiannis et al., 2002). This occurs as a multi-step
process (Figure 2). The slower movement enables a secure contact, mediated by ICAM1, between the vessel endothelium and the leukocyte.
Dreyer and colleagues (1989) reported peak chemotactic activity released into the
cardiac lymph within the first hour of reperfusion. This sharp rise in chemotaxis could
explain the accelerated neutrophil accumulation, especially as the reduction in neutrophil
11
Basement membrane
Endothelium
Blood Flow
Vessel Lumen
Interstitium
Neutrophil rolling, firm adhesion and transmigration
P-selectin glycoprotein 1
P-selectin
Leukocyte β2 integrin
ICAM-1
Figure 2. Neutrophil infiltration. The cell-mediated inflammatory response post-MI is
dependent upon neutrophil-endothelial cell (EC) interaction to allow extravasation from
the bloodstream into the tissue. Neutrophils roll along the endothelium at a reduced
velocity and are tethered to the EC mediated by selectins, leading to neutrophil integrin
activation. Firm adhesion of the neutrophil via neutrophil integrins binding to members
of the immunoglobulin superfamily expressed on EC is necessary for transmigration into
the extravascular tissue (adapted from Frangogiannis et al., 2002)
12
accumulation shares a close temporal relationship with a decrease in chemotactic activity
over the same period (Dreyer et al., 1989; Dreyer et al., 1991). Resident mast cells in the
reperfused area release prepackaged proinflammatory mediators and growth factors.
Stem cell factor, secreted by a subset of macrophages in the reperfused area, may also be
an important factor for mast cell mobilization (Nian et al., 2004). These inflammatory
cells accumulate by three days post-reperfusion and are capable of further attracting
mononuclear cells to the site of injury.
1.4.2 Mechanisms of Neutrophil-Induced Injury
Myocardial injury is greatly enhanced if neutrophils emigrate to the extravascular
tissue or adhere directly to myocytes.
As with neutrophil-endothelial interactions,
adherence is low without activation of either cell type; however, when both the
neutrophils and myocytes are stimulated by C5a and/or cytokines, adhesion is markedly
increased (Smith et al., 1991; Entman et al., 1991; Amsterdam et al., 1995).
The
mechanisms involved in tissue injury mediated by neutrophils include the generation of
reactive oxygen species, neutrophil-derived proteases and arachidonic acid metabolites,
and the elaboration of the cytokine cascade.
1.4.3 Reactive Oxygen Species
Reactive oxygen species (ROS) are atoms or compounds of oxygen with
hydrogen and nitrogen with unpaired electrons in their outer orbit (Frangogiannis et al.,
2002). These byproducts of oxygen metabolism, which include the superoxide anion
(•O2-), hydrogen peroxide (H2O2), and the hydroxyl radical (•OH), are highly reactive
13
molecules capable of damaging tissue during I/R injury (Myers et al., 1985; Chambers et
al., 1985). The cellular sources of ROS within the heart include myocytes, endothelial
cells and neutrophils (Myers et al., 1985; Zweier et al., 1988, Becker et al., 1999).
Within myocytes, ROS are produced by mitochondrial electron transport, NADPH
oxidase, and xanthine dehydrogenase/xanthine oxidase (Myers et al., 1985; Becker et al.,
1999; Chambers et al., 1985; Zorov et al., 2000). Xanthine oxidase and nitric oxide
synthase (NOS) are sources of ROS production in the endothelial cell (Liu et al., 1997;
Chambers et al., 1985).
Neutrophils are the primary source of ROS, especially
superoxide anions, generated by activity of NADPH oxidase after prolonged ischemia
(Duilio et al., 2001). The production of ROS by neutrophils stimulated by
proinflammatory mediators exemplifies a respiratory burst, characterized by high
metabolic activity and increased consumption of oxygen.
Superoxide is formed by the transfer of an electron to molecular oxygen.
Superoxide dismutase (SOD) catalyzes the formation of H2O2 from •O2- (Chambers et al.,
1985). Contrary to •O2-, H2O2 is a moderately strong oxidant and a highly stable small
molecule that crosses membranes freely as its diffusion properties are similar to water
(Antunes and Cadenas, 2000; Hool, 2006). This makes H2O2 a significant pro-oxidant
species and signaling molecule. H2O2 can participate in one-electron reactions with
metal ions (Fenton reaction, see Figure 3a) and can react with •O2- to generate the very
toxic hydroxyl radical (•OH-) through the Haber-Weiss reaction (Chambers et al., 1985;
Hool, 2006; see Figure 3b). Other reactive species are derived from nitric oxide: NO
14
reacts with many molecules including oxygen, thiol groups and metals or with superoxide
to produce peroxynitrite (ONOO-) (Liu et al., 1997; Lalu et al., 2002; see Figure 3c).
15
A.
Fenton Reaction
Mn+ + H2O2
B.
Mn+1 + OH•
Haber-Weiss Reaction
O2•- + H2O2
C.
+ OH-
O2 +
OH•
+ OH-
Peroxynitrite Formation
NO• + O2•-
ONOO-
Figure 3. Reactive oxygen species. Superoxide is generated in abundance during
reperfusion. SOD converts •O2- to H2O2. H2O2 can then participate in one-electron
reactions with metal ions, iron in particular, to produce a hydroxyl radical through the
Fenton reaction (Panel A). H2O2 can also react with •O2- to generate a through the HaberWeiss reaction (Panel B). At the same time that •O2- is generated, the myocardium
produces a burst of NO by inducible NOS. NO and •O2- react rapidly to form ONOO-.
16
1.4.4 Consequences of ROS Activity
Direct consequences of ROS production include DNA nicking, lipid peroxidation,
degradation of interstitial matrix molecules, as well as oxidation of amino acids and
sulfhydryl groups within proteins, denaturing ion channels, receptors, enzymes and
transport proteins (Fantone and Ward, 1982; Xu et al., 1997; Ambrosio et al., 1991).
Proteins modified by ROS are resistant to proteolytic degradation by proteosome and this
leads to a cytosolic accumulation of modified proteins (Bulteau et al., 2001). Lipid
peroxidation renders the sarcolemmal and intracellular membranes weak, compromising
cellular integrity. ROS alter nucleic acid and gene function, especially mitochondrial
DNA (mtDNA), as it is in direct proximity to the site of myocyte ROS formation and has
limited repair activity (Ide et al., 2001; Zorov et al., 2000). In addition, endothelial
damage by ROS and neutrophil adhesion attenuates the release of endothelium-derived
factors with anti-neutrophil properties, such as basal release of NO, and attenuates
endothelium-dependent vascular relaxation, which increases endothelial permeability and
neutrophil transmigration (Zhao et al., 2000). Endothelial dysfunction may also lead to
increased expression of adhesion molecules and production of inflammatory cytokines
(Kharbanda et al., 2001).
Under physiological conditions, the toxic effects of ROS can be prevented by
endogenous antioxidant enzymes SOD, catalase, glutathione peroxidase and the nonenzymatic antioxidants including vitamin E, ubiquinol, ascorbate, and reduced
glutathione (Haramaki et al., 1998; Tsutsui, 2006; Hool, 2006). Myocardial recovery
17
from reperfusion injury depends partly upon its endogenous anti-oxidant reserve prior to
ischemia as well as the extent of the oxidative stress.
1.4.5 Other Mechanisms of Neutrophil-mediated Injury
1.4.5.1 Neutrophil-derived Proteolytic Enzymes
Activated neutrophils also release other cytotoxic metabolites including serine
proteases (Murohara et al., 1995). The serine proteases that have been implicated in
cardiac tissue injury are human leukocyte elastase, cathepsin G, and PR3 as they are
stored in an active form within leukocyte granules (Murohara et al., 1995; Pruefer et al.,
2002; Weiss, 1989; Owen and Campbell, 1999). These proteases share a broad and
overlapping spectrum of activity: extracellular matrix proteolysis of elastin, fibronectin,
laminin and collagen; vascular endothelial and basement membrane injury; activation of
TNFα and Il-1β pro-forms; degradation of plasma proteins (complement components and
immunoglobulins); enhance neutrophil release of the chemotactic platelet activating
factor (PAF) and leukotriene B4 (Buerke et al.,1994; Zimmerman and Granger, 1990;
Camussi et al., 1988; Camussi et al., 1989; Owen and Campbell, 1999).
1.4.5.2 Arachidonic Acid Metabolites
Stimulation of phospholipase A2 after neutrophil activation mobilizes membrane
lipids and results in generation of 5-lipoxygenase products (i.e., leukotriene B4) and the
phospholipid PAF (Hansen, 1995). The elevation of intracellular Ca2+ during ischemia
may also activate phospholipase C to stimulate arachidonic acid metabolism, which
generates ROS as by-products (Kuehl et al., 1980). Leukotriene B4 and PAF are potent
18
stimulants of neutrophil chemotaxis, adhesion to endothelial cells, oxidative metabolism
and degranulation and may serve to amplify neutrophil-mediated tissue injury and
vascular permeability (Hansen, 1995).
1.5 Inflammatory Cytokines and Chemokines
1.5.1 Inflammatory Cytokines
Interleukin 1β, acting as the “first line” cytokine stimulates chemotaxis of
neutrophils but may also induce apoptosis by acting in concert with free radicals
(Rampart and Williams, 1988; Arnstall et al., 1999; Kaminski et al., 2002). IL-1β is a
powerful stimulus for increased IL-6 production (VanDamme et al., 1987). It has been
reported that IL-6 is rapidly induced after reperfusion and induces myocyte ICAM-1
expression which may cause lethal myocyte injury due to neutrophil adhesion and
transmigration (Kukielka et al., 1995). Cardiac release of IL-6, TNF-α and IL-8 during
the first few minutes of reperfusion has been confirmed by various investigators (Nossuli
et al., 2000; Neumann et al., 1995; Kukielka et al., 1995). The release of these cytokines
is likely induced and sustained by paracrine release of TNF-α from resident leukocytes
and macrophages (Neumann et al., 1995; Frangogiannis et al., 1998). Macrophages
resident in cardiac tissue are considered to be a major source of IL-1β and TNF-α early
during reperfusion (Herskowitz et al., 1995; Chensue et al., 1988).
Cytokine
amplification takes place in part through direct recruitment of more inflammatory cells to
the site of injury (Nian et al., 2004). In addition, free radicals activate redox-sensitive
transcription factors, including nuclear factor-κB (NFκB), and trigger the expression of
19
IL-1ß and TNF-α, that are themselves potent inducers of NF-κB activation
(Chandrasekar et al., 2001).
While IL-1ß, TNF-α, and IL-6 are predominantly pro-inflammatory in the
reperfusion model, IL-10 serves to limit the burst of inflammatory cytokines (Yang et al.,
2000).
Based on the ability of exogenously administered IL-10 to suppress the
expression of proinflammatory cytokines and adhesion molecules and the effect of
exogenous IL-10 in limiting the neutrophil infiltration, Yang and colleagues (2000)
hypothesized that the primary actions of endogenous IL-10 are related to the modulation
of the neutrophil activation and their tissue infiltration. Transforming growth factor-β1
(TGF-β1) has also demonstrated a cardioprotective effect in the setting of I/R by
decreasing circulating TNF-α levels (Lefer et al., 1990).
1.5.2 Chemokines
Chemokines are a family of low molecular weight proteins capable of stimulating
neutrophil chemotaxis as well as modulating leukocyte adhesion, activation and gene
expression, mitogenesis, and apoptosis (Frangogiannis and Entman, 2005). In the canine
model of myocardial infarction, IL-8 upregulation markedly increased adhesion of
neutrophils to isolated canine cardiac myocytes suggesting a potential role in neutrophilmediated injury (Kukielka et al., 1995; Frangogiannis and Entman, 2005). Monocyte
chemoattractant protein-1 (MCP-1) is capable of modulating fibroblast phenotype and
action by altering collagen expression and regulating matrix metalloproteinase synthesis
as well as attracting neutrophils (Gharaee-Kermani et al., 1996). Furthermore, MCP-1
20
has been implicated in the pathogenesis of ischemic cardiomyopathy whereby prolonged
chemokine-driven inflammatory response is triggered in the absence of cellular necrosis
and may play a significant role in the progression of fibrosis (Frangogiannis and Entman,
2005; Dewald et al., 2005).
1.5.3 Regulation of Cytokine Gene Expression during I/R injury
Free radicals activate redox-sensitive transcription factors, including nuclear
factor-κB, and trigger the expression of IL-1β and TNF-α and other inflammatory
mediators (Chandrasekar et al., 2001; Fan et al., 2002). Increased NFκB/DNA binding
activity has been confirmed in the I/R rat model by multiple investigators (Chandrasekar
et al., 2001; Li et al., 1999). The transcription factor is normally present in the cytosol in
its inactive form, bound by inhibitory proteins IκB. The phosphorylation and degradation
of IκB proteins are key steps in NFκB activation, translocation to the nucleus, and
stimulation of gene expression (Li et al., 2001; McDonald et al., 1997; see Figure 4).
The cytokines involved in ischemia and reperfusion injury, including IL-1β, TNFα, IL-6 and IL-8, feature κB or κB-like binding motifs in their 5’ regulatory region
(McDonald et al., 1997). Reperfusion after myocardial ischemia in a rat model initiates a
biphasic increase in both NFκB protein levels and DNA-binding activity possibly
corresponding to a primary activation by ROS and a secondary activation by proinflammatory cytokines produced by the first activation (Chandrasekar and Freeman,
1997).
21
UV radiation
Hypoxia/anoxia
Cytokines (IL-1, TNF-α)
Reactive Oxygen Species
Virus
Bacterial Antigens
IκB kinases
P
IκB proteins
NFκB
Iκκ complex
Ubiquitination, degradation
Translocation to nucleus
NFkB responsive gene
Regulation of gene expression
-cytokines
-cell adhesion molecules
-matrix metalloproteinases
-inducible NOS
Figure 4. NFκB pathway. Inhibitory proteins (IκB) retain the inactive transcription
factor in the cytoplasm until a stimulus triggers the phosphorylation and degradation of
IκB. NFκB then translocates to the nucleus and regulates the expression of many
inflammatory-related genes. NFκB activation is bi-phasic, peaking after 15 min and 3h
reperfusion, possibly corresponding to a primary activation by ROS and a secondary
activation by pro-inflammatory cytokines produced by the first activation.
22
1.6 Wound Healing and Scar Formation
After reperfusion and the induction of the acute inflammatory phase, removal of
injured cells by leukocytes and macrophages is followed by proliferation, migration, and
activation of mesenchymal cells, and deposition of extracellular matrix proteins
(Frangogiannis et al., 2005).
Successful cardiac recovery depends upon controlling
inflammation and establishing a reparative scar in the infarct zone to maintain ventricle
wall integrity and cardiac function. Infarct healing can be divided into 3 overlapping
phases: the inflammatory phase, the proliferative phase, and the maturation phase
(Frangogiannis et al., 2005).
1.6.1 Scar Formation
The inflammatory phase is largely dominated by the infiltration of neutrophils and
mononuclear cells that phagocytose dead cells and matrix debris and produce growth
factors, inducing fibroblast migration, proliferation, and activation (Dobaczewski et al.,
2006). In the early stages of wound healing, extravasation of plasma proteins results in
the formation of a complex fibrillar provisional matrix composed primarily of fibrin and
fibronectin (Dobaczewski et al., 2006).
myofibroblast
progenitors
and
During the proliferative phase, circulating
normal
interstitial
fibroblasts
transform
into
myofibroblasts that are hypersynthetic, less migratory and possess contractile properties
(Serini and Gabbiani, 1999; Powell et al., 1999; Freed et al., 2005). Platelet-derived
growth factor (PDGF), stem cell factor (SCF) and TGF-β in particular are the primary
stimuli for the transformation of myofibroblasts (Desmouliere et al., 1993).
23
Peak
proliferation of myofibroblasts occurs at 4 days post-MI, predominantly in the border
zone (Virag and Murry, 2003).
As repair proceeds, the myofibroblasts deposit a network of collagen, specifically
types I and III, and the provisional matrix is resorbed (Virag and Murry, 2003; Sun et al.,
2000). The peak of type III collagen production (as early as 3 days post-infarct) is
followed by a lower and slower developing peak of type I collagen, generating tensile
strength and stiffness to the healing wound (Cleutjens et al., 1999; Blankesteijn et al.,
2001; Dobaczewski et al., 2006). During the maturation phase, myofibroblasts form
highly organized arrays in the infarct area, parallel to the epi- and endocardium, and an
organized assembly of fibers in the form of scar tissue becomes evident by 2-3 weeks
post-MI (Blankesteijn et al., 2001). Contraction of scar tissue during wound healing is
well documented and is due, in part, to the contractile features of myofibroblasts
(Tomasek et al., 2002).
Formation of new blood vessels is critical for supplying the healing infarcted
myocardium with oxygen and nutrients necessary to sustain metabolism (Lu et al., 2004).
VEGF is critical to the development of collateral vessels and its production is
significantly increased during ischemia (Nordlie et al., 2006).
Immediately after
reperfusion, mast cells release TNF-α which upregulates interferon-inducible protein 10
(IP-10), a known angiostatic chemokine, inhibiting neovascularization (Frangogiannis et
al., 2001). After 24 hours, however, TGF-β-mediated IP-10 downregulation and VEGF
upregulation shift the balance toward angiogenesis (Nian et al., 2004).
24
1.7 Ventricular Remodeling
Ventricular remodeling after myocardial infarction describes alterations in the
ventricle structure that include gross morphologic, histological and molecular changes in
both the infarcted and non-infarcted myocardium. The process of remodeling involves
thinning of the myocardial wall, enlargement of the ventricular cavity and hypertrophy of
myocytes in the remote region. Animal experiments have defined myocardial infarct size
as a major determinant of the severity of left ventricular dilatation (Gaudron et al., 1993),
and left ventricular dilatation and dysfunction are large predictors of post-MI prognosis.
Post-infarction remodeling has been arbitrarily divided into an early phase (within 72
hours) and a late phase (beyond 72 hours) (St. John Sutton and Sharpe, 2000). Early
remodeling includes extracellular matrix remodeling and infarct expansion leading to
wall thinning. Late remodeling includes ventricular dilatation, compensatory myocyte
hypertrophy, and altered myocardial geometry.
1.7.1 Infarct Expansion
Infarct expansion was originally defined using post-mortem examinations of
patients dying of acute phase infarction whose left ventricle exhibited regional thinning
and elongation within the scar (Hutchins and Bulkley, 1978). Infarct expansion has been
defined as acute dilatation and thinning of the area of infarction not explained by
additional myocardial necrosis (Hutchins and Bulkley, 1978).
However, infarct
expansion does not occur within all infarcts; it occurs within large, transmural anterior
and antero-lateral infarcts. Most importantly, it is associated with increased mortality and
25
may be important in the development of aneurysms (McKay et al., 1986). Prevention of
infarct expansion attenuates a decrease in ejection fraction and preserves ventricular
function (Kelley et al.,1999).
Before and during the period of resorption of necrotic tissue but before there is
extensive deposition of collagen and an increase in the tensile strength, the infarcted
region can thin and elongate (Pfeffer and Braunwald, 1990). This involves degradation
of the extracellular scaffolding, allowing side-by-side slippage of muscle bundles to
occur, resulting in a reduction of the number of myocytes across the infarcted region
(Weisman et al., 1988). Central to expansion is the degradation of the inter-myocyte
collagen struts by serine proteases and the activation of matrix metalloproteinases
(MMPs) released from neutrophils (Cleutjens et al., 1995; Deten et al., 2002).
Tissue
inhibitors of MMPs (TIMPs) confine collagenolytic activity to the region of injury and
control matrix degradation. Synthesis of MMPs and TIMPs is regulated by locally
generated growth factors including TGF-β1 (Sun et al., 2000). Infarct expansion reflects
disequilibrium between MMPs and TIMPs (Cleutjens et al., 1995).
Infarct expansion leads to functional alterations due to changes in wall stress.
Infarctions involving the apex are particularly vulnerable to remodeling. Given the
normal ellipsoid shape of the ventricle, the apex is the most highly curved as well as the
thinnest region and has the lowest wall tension (Pfeffer, 1995). Any expansion or wall
thinning at the apex is exposed to greater distortion forces. Wherever the wall thinning
occurs, it is exposed to the same tension per unit muscle thickness as the rest of the
26
myocardium but increased mechanical stress. This phenomenon can further perpetuate
wall thinning.
Transthoracic short-axis two-dimensional echocardiography at the
papillary muscle level is used to determine anterior and posterior segment lengths to
confirm the presence of expansion (Pfeffer and Braunwald, 1990).
1.7.2 Compensatory Hypertrophy and Ventricular Dilatation
In addition to changes that occur in the infarct zone, progressive changes in the
non-infarcted regions of the left ventricle contribute to the overall process of chamber
enlargement (Pfeffer and Braunwald, 1990). Myocyte hypertrophy of remaining viable
myocardium is initiated by neurohormonal activation, myocardial stretch, the activation
of the local tissue renin-angiotensin-system (RAS), and paracrine/autocrine factors
(Anversa et al., 1985; St John Sutton and Sharpe, 2000). Increased myocyte shortening
and increased heart rate from sympathetic stimulation result in hyperkinesis of the noninfarcted myocardium and temporary circulatory compensation (St John Sutton and
Sharpe, 2000). However, the cellular hypertrophy exhibited in the remote area is often
insufficient to counteract the loss of myocyte contractility within the infarct territory.
The end result is slow developing ventricular dilatation.
The enlargement of the LV cavity can lead to restoration of stroke volume
without concomitant improvement in ejection fraction (Pfeffer, 1995). Stroke volume
can thus be maintained initially by the dilated ventricle cavity (with or without
compensatory hypertrophy) with less energy expenditure and less muscle exertion. The
disadvantage of this initial compensation is that the architectural changes that create
27
greater loading conditions on the viable myocardium promote further enlargement as well
as hypertrophy and dysfunction (Pfeffer, 1995). Considering Laplace’s Law, whereby
tension is a function of pressure and radius, dilatation increases both diastolic and systolic
wall stress. Although early infarct expansion is associated with immediate ventricle
enlargement, cavity dilatation continues for months thereafter, depending on the severity
of the infarct.
Increased cavity volume, together with insufficient compensatory
hypertrophy, results in loading conditions promoting further enlargement and dysfunction
(Pfeffer and Braunwald, 1990; Figure 5).
28
Figure 5. Left ventricle remodeling post-infarction. Infarct expansion results from the
degradation of the extracellular structure, allowing side-by-side slippage of muscle
bundles to occur. Infarct expansion and wall thinning promote ventricle distortion forces
initially compensated by chamber dilatation. These architectural changes create greater
loading conditions on the viable myocardium thereby promoting further dilatation as well
as hypertrophy and dysfunction.
29
1.8 Echocardiography
Ultrasound imaging encompasses a wide range of imaging modes and techniques
that utilize the interaction of sound waves with living tissues to produce an image of the
tissues (Coatney, 2001). The development of high definition echocardiographic imaging
systems with high frequency transducers has permitted imaging of the heart in small
animal models of myocardial infarction to evaluate systolic and diastolic function, infarct
size and left ventricular remodeling (Nozawa et al., 2006). The advantages of using
transthoracic echocardiography compared with other image acquisition systems include
its versatility, safety, and non-invasiveness.
Two-dimensional mode imaging involves a gray scale cross-sectional image of
the tissue while M-mode displays the image over time, indicating the tissue depth on the
Y-axis and time on the X-axis. Of particular importance is the high temporal resolution
of M-mode imaging. Information that can be obtained from an M-mode echocardiogram
includes LV wall thickness and chamber dimensions at various time points throughout
the cardiac cycle, but commonly at end systole and diastole (Coatney, 2001).
End
diastolic and end systolic volumes as well as LV mass can be measured from direct LV
chamber dimensions. Electrocardiogram (ECG) leads are attached to the animal to
monitor electrical activity. With heart rate monitored, quantification of stroke volume,
cardiac output and percent ejection fraction are possible. Serial echo studies permit the
investigator to assess and follow cardiac function and phenotype longitudinally as left
ventricular remodeling develops over time.
30
Standardization of measurements in echocardiography had been inconsistent and
less successful compared with other imaging techniques and, consequently,
echocardiographic measurements were sometimes perceived as less reliable (Lang et al.,
2005). The American Society of Echocardiography (ASE), working together with the
European Association of Echocardiography, has critically reviewed literature and updated
recommendations for quantifying cardiac chamber dimensions (Lang et al., 2005). The
ASE established the “leading edge convention” wherein dimensions are measured from
the leading edge of echocardiographic borders, resulting in the inclusion of endocardial
echo from the interventricular septum and posterior wall and the exclusion of endocardial
echo from the left ventricle internal diameter (Deague et al., 1999). Meticulous attention
is required to obtain correct orientation of imaging planes with regard to internal
landmarks (Schiller et al., 1989). In general, a standardized long axis view should
maximize the size of the left ventricular cavity, whereas a standardized short-axis view
should minimize it within the guidelines defining each particular view (Schiller et al.,
1989). A precise endocardial definition is central to accurate quantification (Schiller et
al., 1989).
Two-dimensionally oriented M-mode images, perpendicular from the
longitudinal axis slightly above the papillary muscle level (see Figure 6), are widely
employed in clinical practice and epidemiological studies (Foppa et al., 2005; see Figure
7). As a research tool, noninvasive ultrasound imaging represents not only a refinement
in technique, but also a significant advancement in the ability to evaluate and quantify
target organ structure and function in rodent species (Coatney, 2001).
31
Base
Mid
Papillary Muscle
Apex
Figure 6. Sections of the left ventricle. The left ventricle is divided into base, mid, and
apex sections to facilitate echocardiography studies. Mid sections, at the level of the
papillary muscle, are frequently used in short- and long-axis echo studies because they
are easier to image with clear endocardial borders.
32
A
B
Figure 7. M-mode echocardiograms. M-mode echo studies are often guided by 2D
images in order to ensure correct placement of the echo cursor. This visual aid enables
the investigator to distinguish base, mid, or apical sections of the left ventricle. By
modifying the placement and angle of the echo probe on the subject’s chest, the
investigator can obtain a parasternal long-axis (PSLX; Panel A) image or a parasternal
short-axis (PSSX; Panel B) image.
33
1.9 Heme-Oxygenase System
The heme-heme oxygenase system has recently been recognized to possess
important
regulatory
properties
tightly
involved
in
both
physiological
and
pathophysiological processes including cytoprotection, apoptosis, and inflammation
(Wagener et al., 2003; Maines, 1997; Wagener et al., 2003).
The heme molecule
promotes mostly biological oxidation processes and is vital to cellular and whole body
homeostasis (Ryter and Tyrrell, 2000). Heme is involved as a carrier in oxygen transport
and storage (myoglobin and hemoglobin), mitochondrial respiration (cytochromes within
the electron transport chain), drug metabolism (cytochrome P450 family), endogenous
anti-oxidants (catalases and peroxidases), and many others (Maines, 1992; Wagener et
al., 2003). Besides its function as a prosthetic moiety in heme proteins, heme itself may
influence the expression of many genes including down-regulating its own production
and upregulating heme metabolism (Wagener et al., 2003b). Not only does heme affect
the expression of globin, cytochromes, myeloperoxidase, and heme oxygenase-1, it also
modulates cellular differentiation and proliferation. However, the pleiotropic functions
of hemin are not exclusively pro-survival. It may promote deleterious iron-dependent
reactions leading to ROS generation, membrane lipid peroxidation, and organelle and
cellular membrane disruption (Vercellotti et al., 1994; Jeney et al., 2002; Ryter and
Tyrrell, 2000).
An increased cellular “free heme pool” by exogenous heme
administration, increased heme biosynthesis, or increased hemoprotein degradation
results in oxidative stress and can potentially cause cellular injury if endogenous antioxidant activity is insufficient. In summary, heme exerts a dual role: in small amounts it
34
acts by itself or as the functional group of heme proteins and provides diverse and
indispensable cellular functions, whereas in excessive amounts, free heme can cause
severe tissue damage (Wagener et al., 2001; Ryter and Tyrrell, 2000).
The heme molecule can be degraded by both enzymatic and chemical
mechanisms, both utilizing molecular oxygen and requiring a reducing agent (Maines,
1997). Heme oxygenase is the rate-limiting enzyme in the catabolism of heme, which
yields equimolar amounts of biliverdin, free iron (Fe2+), and carbon monoxide (Maulik et
al., 1996; Csonka et al., 1999; Hangaishi et al., 2000; Figure 8).
subsequently converted to bilirubin by biliverdin reductase.
Biliverdin is
Three isoforms of the
enzyme have been identified to date: inducible HO-1, constitutively-expressed HO-2, and
recently discovered HO-3. HO-1 has garnered a lot of attention because accumulating
experimental evidence has shown that the byproducts of heme degradation exert
cytoprotective properties in numerous in vitro and in vivo cellular and tissue models of
injury (Figure 8).
Although heme is the major inducer of HO-1, other stimuli also upregulate HO-1
activity including oxidative stress, hypoxia, hyperoxia, cytokines, heat shock, hydrogen
peroxide, ultraviolet radiation, shear stress, peroxynitrite and others (Clark et al., 2000;
Maines, 1997) (Figure 8). HO-1 induction involves many signal cascades including
protein kinase C, cGMP-dependent protein kinase and cAMP-dependent protein kinase
(Terry et al., 1999; Immenschuh et al., 1998; Maines, 1997). Moreover, the HO-1 gene
35
Heme
Stimuli
-Hypoxia, hyperoxia
-Ischemia
-Oxidative stress
-Cytokines
-Shear stress
-NO
-Prostaglandins, endotoxins
Source
-Endogenous biosynthesis
-Exogenous administration
Heme Oxygenase
O2
NADPH
H2 O
NADP+
CO
Biliverdin
Biliverdin
Reductase
Bilirubin
Fe2+
Oxidant
Species
Ferritin
•Vasodilator
•Anti-inflammatory
•Anti-apoptotic
•Anti-thrombotic
•Anti-oxidant
•Anti-inflammatory
•Anti-oxidant
•Iron homeostasis
Figure 8. The heme oxygenase-1 pathway. HO-1 catalyzes the degradation of heme to
yield equimolar CO, biliverdin and Fe2+. Biliverdin is subsequently converted to
bilirubin by biliverdin reductase. The production of Fe2+ induces an upregulation of
ferritin which sequesters the free iron.
36
has regulatory elements in the promoter region including DNA-binding sites for redoxsensitive transcription factors AP-2 and NFκB (Lavrovsky et al., 1994).
1.9.1 Cardioprotective Effects of Hemin and HO-1
The exogenous administration of hemin and subsequent upregulation of HO-1
confer potent resistance to stress and cellular injury by mediating oxidative stress and
acute inflammation (Clark et al., 2000; Weber et al., 2001). There is considerable
interest in the cardioprotective potential of HO-1 activity in the setting of myocardial
infarction and ischemia/reperfusion injury.
Hangaishi et al. (2000) observed the
upregulation of cardiac HO-1 expression induced by hemin injection decreased infarct
size 2 hours after reperfusion. Melo et al. (2002) reported a pre-emptive gene therapy
strategy for myocardial protection from ischemia/reperfusion injury using adenoassociated virus to deliver HO-1 to the heart. In an allograft transplantation model,
transgenic mice overexpressing HO-1 systemically sustained improved allograft
outcomes with inhibition of the rejection response (Araujo et al., 2003), implicating antiinflammatory, anti-oxidant and anti-apoptotic properties.
Most recently, Liu et al.
(2006) showed complete recovery of function and normalization of ventricular
dimensions in intramyocardial HO-1-delivered rats 1 year after MI.
1.10 Rationale and Hypotheses
1.10.1 Rationale
The introduction of genetic manipulation into molecular medicine represents an
advancement in the treatment of cardiovascular diseases. Controlling post-infarction
37
tissue inflammation through regulation of gene expression is an attractive means of
limiting organ damage. Evaluating viability and contractile function in cultured cardiac
myocytes is a good model for distinguishing between the simulated ischemia and
reperfusion phases. However, in vitro studies frequently do not simulate the severity of
reperfusion injury observed in vivo due to the absence of endothelial and inflammatory
cells, the absence of inflammatory mediators, the lack of sufficient muscle for
hypercontracture damage, and fewer sources of ROS (Vanden Hoek et al., 1996).
Surgically induced myocardial infarct in rats is a widely accepted in vivo model of
myocardial injury (Moises et al., 2000). The major advantage of this model is the
possibility of keeping infarcted animals alive for extended periods of time, thereby
allowing investigation of healing and recovery of the infarcted myocardium (Moises et
al., 2000).
Furthermore, left anterior descending coronary artery occlusions are
especially useful to study because the left ventricle territory it supplies is large,
compromising the anterior lateral wall (see Figure 9). Serial echocardiographic studies,
including baseline and post-op studies for each animal, permit the investigation of left
ventricular structure and function and abnormalities therein.
38
Figure 9. Left anterior descending coronary artery occlusion. The LAD coronary
artery supplies most of the left ventricle territory and occlusions therein create large
antero-lateral apical to mid-level infarcts making it very useful to study.
39
1.10.2 Hypotheses
The purpose of this study was to investigate the effects of HO-1 upregulation in
vivo in myocardial tissue recovering from I/R injury. I hypothesized that exogenous
upregulation of HO-1 by hemin administration confers acute and chronic cardioprotection
against I/R injury in rats and attenuates post-infarction left ventricular remodeling. I
proposed the HO-1-dependent decrease in oxidative stress attenuates post-ischemic
myocardial injury in part by inhibiting NFκB-mediated inflammation. Using histological
methods, I quantified infarct size and left ventricle fibrosis in hemin- and buffer-treated
animals subjected to ligation and release of the LAD coronary artery. Echocardiography
was used to examine left ventricle wall and chamber dimensions as well as cardiac
function.
Transcription factor NFκB binding activity was measured.
This study
confirmed the hypothesis that HO-1 upregulation reduces acute injury after myocardial
infarction at 24 hours and 4 days post-op. However, treatment with hemin did not reduce
reperfusion injury nor prevent long term LV remodeling 3 months after infarction.
40
Chapter 2: Materials and Methods
2.1 Animals
All animal handling and procedures were carried out according to the Canadian
Council of Animal Care (CCAC) guidelines and approved by the University Animal Care
Committee at Queen’s University. Six week old male Wistar rats (180-200 g) from
Charles River, Canada were randomly assigned to sham, buffer, or hemin-treated groups.
The animals were kept on a 12:12 hour light: dark cycle at an ambient temperature of 2123°C and 60% humidity. Food and water were provided ad libitum.
2.2 Hemin Administration
Hemin (Sigma Aldrich, Oakville, ON) was dissolved in buffer consisting of 0.1N
NaOH and 0.01M KPO4 monobasic to a concentration of 75 μM and administered at a
concentration of 50 μMol/kg body weight.
Buffer and hemin were administered
intraperitoneally once daily for 3 consecutive days prior to left anterior descending
(LAD) coronary artery occlusion. Intraperitoneal administration was resumed 48 hours
post-operatively and continued once every 3 days. Sham animals were injected with
vehicle solution.
2.3 Left Anterior Descending (LAD) Coronary Artery Ligation
Animals were anaesthetized using 60 mg/kg sodium pentobarbital given
intraperitoneally and additional doses were given as needed to maintain surgical plane.
Animals were intubated and ventilated using a rodent ventilator (model 683, Harvard
Apparatus; Montreal, QC) with 100% oxygen throughout the procedure. Rats were
41
ventilated at a tidal volume of 1.25 ml/100 g body weight (to a maximum tidal volume of
5 ml) and rate of 72 breaths per minute. Left horizontal thoracotomy was performed in
the second intercostal space. The pectoral muscles were separated by blunt dissection
and intercostal muscles were cut to expose the heart in the thoracic cavity. The second
and third ribs were retracted and the pericardium was torn to expose the left ventricle
wall. Polypropylene suture thread (6.0 Ethicon; Johnson and Johnson, Markham, ON)
was pulled through the left ventricular myocardium, beneath the left auricle. A small
(1mm diameter) piece of cotton was placed within the ligature knot to prevent crushing of
the vessel. The ligature was tied tightly for 30 minutes and subsequently loosened,
allowing reperfusion of the left anterior descending coronary artery. Sham animals were
subjected to thoracotomy and ligature placement without vessel occlusion. The ligature
was left in place, the cotton removed from the knot, and the thoracotomy was closed
using 3.0 Ethicon suture thread.
Phosphate buffered saline (PBS) (Sigma) was
administered intraperitoneally at the onset of ischemia and during recovery to maintain
hydration and restore any potential blood loss. Once extubated, buprenorphine (0.3
mg/ml) was administered subcutaneously at 0.05 mg/kg post-op and again 8 hours later
for pain control.
2.4 Transthoracic echocardiography
Echocardiography was performed on all treatment groups 1 day pre-operatively
and 4 days post-operatively using a Phillips HDI-5000 Ultrasound System with a P 12.5MHz transducer at 2 cm depth.
For some groups, an additional echo study was
performed at 3 months. Measurements of LV chamber and wall dimensions were made
42
according to the leading edge guidelines set by the American Society of
Echocardiography. For image acquisition, the animals were shaved and sedated with
halothane (Sigma) by nose cone inhalation. Animals were placed on a ramp with a fixed
heating pad and linen cloth to maintain body temperature and were positioned such that a
comparable left lateral decubitus position was achieved. Three electrocardiograph lead
wires were inserted sub-cutaneously on the left arm, left leg, and right arm in order to
monitor heart rate. Two-dimensional and M-mode images from the parasternal long and
short axes were taken from the basal (at the tip of the mitral valve leaflets), mid (at the
papillary muscle), and apical (distal to the papillary muscle) levels and recorded on an
optical disk for later review.
Image analysis and measurements were performed blind to the treatment group.
Echocardiography studies were reviewed and the best frames within an animal study
were selected. Three consecutive contraction cycles were measured within a single frame
at each level of the heart, in both parasternal long and short axes. Endocardial borders
were traced to calculate the interventricular septum, cavity diameter, and posterior wall
thickness during diastole and systole. Values were recorded in Microsoft Excel and
imported to Microsoft Access for data organization and queries, and formula derivation
to calculate left ventricle mass, fractional shortening, and ejection fraction. LV mass was
calculated using the ASE formula (1.04 x [LVd + PWd + AWd)3- LVd3] where 1.04 is
the specific gravity of myocardial tissue. LVd is LV diameter during diastole and PWd
and AWd are end diastolic posterior wall and anterior wall (or interventricular septum)
thicknesses respectively. Fractional shortening (FS) was calculated by (LVD-LVs)/LVd
43
x 100, where LVs is end-systolic diameter. Ejection fraction (EF) is calculated by (LV
diastolic volume- LV systolic volume)/ LV diastolic volume.
2.5 Tissue harvest
Hearts were harvested at 24 hours, 4 days and 3 months post-op. The animals
were anaesthetized with sodium pentobarbital (60 mg/kg) by intraperitoneal injection and
intubated. The sternum was cut mid-sagitally and the thoracic cage was retracted. The
right carotid artery was bluntly dissected and cannulated with 1 mm plastic tubing with
suture thread to secure the cannula in place. The thymus was removed to expose the
aortic arch and heart. Suture thread was placed around the aortic arch between the right
brachiocephalic and the left common carotid artery. A second suture was placed around
the right subclavian branch. Arterial blood was collected in a 3 ml syringe and then
centrifuged at 3200 rcf for 10 minutes at 4°C. Serum was collected and stored at -80°C.
Capillary tubes were filled with arterial blood and spun for 6 minutes in a micro
hematocrit centrifuge (International Equipment Company; Fisher Scientific, Ottawa, ON)
to determine the blood hematocrit by micro-capillary reader (IEC). Next, the heart was
perfused through the carotid cannula with cold PBS + KCl (0.01M) to arrest the heart in
diastole. The LAD suture thread was tied tightly and Evan’s blue dye (1 mg/ml PBS;
Sigma) was infused via the carotid cannula. Evan’s blue dye perfused and stained the
area of the LV not at risk for infarction while the myocardium distal to the LAD ligature
was not perfused, clearly marking the ventricular area at risk. Hearts were quickly
excised and washed with cold PBS.
44
2.6 Isolation of Cytoplasmic and Nuclear Proteins
Infarcted and non-infarcted tissues were quickly dissected from the harvested
heart and immediately immersed in liquid nitrogen. Tissues were pulverized in liquid N2
using a mortar and pestle and stored at -80°C until use. Cytosolic and nuclear fractions
were obtained from each sample.
Samples were first homogenized with ice-cold
hypotonic buffer (10mM HEPES, 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM
DTT, protease inhibitor, phosphatase inhibitor; 500 μl/100 mg dry weight) using a
Polytron PT-2100, carried out on ice, to lyse the cell membrane. Homogenate was
centrifuged at 2000 rpm for 1 minute (4°C) and the supernatant was collected and
incubated on ice for 20 minutes.
10% Igepal was added (50 μl/100 mg) to the
supernatant, vortexed for 30 seconds, and centrifuged at 10,000 rpm (1min, 4°C). The
cytosolic fraction was collected and stored at -80°C. The pellet was rinsed once in
hypotonic buffer, repelleted by centrifugation and the supernatant was discarded. The
pellet was then resuspended in ice-cold hypertonic buffer (20mM HEPES, 0.4M NaCl,
1mM EDTA, 1mM EGTA, 1mM DTT, protease inhibitor, phosphatase inhibitor; 100
μl/100 mg original tissue weight), homogenized using a Dounce homogenizer and
incubated for 40 minutes on ice with frequent mixing. The nuclear homogenate was then
centrifuged for 15 minutes at 14,000 rpm. This supernatant was collected as nuclear
extract and stored at -80°C.
2.7 Western Blot Analysis
Total protein concentration was determined for each sample by using the
Bradford assay with a spectrophotometer (DU® 530; Beckman Coulter, Mississauga, ON,
45
Canada) at 530 ηm. Protein samples (25 μg/μl) were mixed with 4x Laemmli buffer with
β-mercaptoethanol and were electrophoresed on a 12% poly-acrylamide gel using a Bio
Rad system (Bio Rad Laboratories, Mississauga, ON, Canada). Transfer onto PVDF
membrane (0.45 μM; Immobilon-P, Millipore, Billierica, MA, USA) was carried out
using a Bio Rad semi-dry apparatus. Membranes were blocked with 4% non-fat dry milk
and probed for human HO-1 primary antibody (dilution 1:5000; SPA 895, Stressgen,
Victoria, BC). After the membranes were washed 3x 15 minutes with TBST, they were
incubated in 1:5000 horseradish peroxidase-conjugated anti-rabbit secondary antibody
(#7074, Cell Signaling; Danvers, Massachusetts, USA).
Following 3 x 15 minutes
washes, membranes were exposed using enhanced chemiluminescence according to the
manufacturer (Pierce; Fisher Scientific, Ottawa, ON) and an AlphaEaseTM gel
documentation system (Fluorchem 8900; Alpha Innotech Corp., San Leandro, CA, USA)
for visualization of the protein. Blot images were analyzed for spot densitometry using
Image J software. Membranes were stripped using a 0.2M glycine, pH 2.6 stripping
buffer solution for 1 hour with rocking and were re-probed for β-actin (1:5000) for
normalization of in vivo western blots.
2.8 NFκB Binding Activity Assay
Transcription factor activity was quantified using the NoShiftTM II NFκB
Transcription Factor Assay kit (Novagen; VWR CANLAB; Mississauga, ON). Briefly,
binding solution and nuclear samples were distributed to sample wells in a 96-well plate
and were incubated for 20 minutes at RT. Binding reagent was added and the plate was
incubated for another 20 minutes at RT. Digestion reaction buffer was added and the
46
plate was incubated at 37°C for 20 minutes. Hybridization reaction buffer was added to
corresponding wells of the NoShift II Capture Microplate. Samples were then transferred
to the microplate and incubated for 45 minutes at RT. Samples were washed four times
with wash buffer and aspirated. Detection reagent was added to each well and the
microplate was incubated for 30 minutes at RT.
The detection reagent was then
aspirated, and the wells were washed four times. Chemiluminescent substrate was added
and the microplate was incubated for 30 minutes at 37°C. Chemiluminescence was
measured with luminometer using a plate reader (Fluostar Optima, BMG Biotech) and
results were expressed as relative light units (RLU) per μg of protein.
2.9 Preparation of Histological Specimens
Warmed agarose gel (2%; Sigma) was poured over the excised heart to
temporarily fix it in place. Left ventricles were cut into 3-4 sections, approximately 3-4
mm thick, and were photographed with a digital camera fixed to a dissecting microscope
for morphometric analysis of the risk and non-risk areas. Sections were washed in cold
PBS. Samples were immersed in 2,3,5-triphenyltetrazolium chloride (TTC) solution (1
mg/ml PBS; Sigma) and incubated for 12 minutes at 37°C. Samples were photographed
for morphometric analysis of the area of infarction.
Sections were placed in 10%
formalin (Accustain, Sigma) and stored at 4°C overnight and then stored in 70% ethanol
at 4°C until paraffin embedding.
47
2.10 Morphometric Analysis
Ventricular sections from the apex, mid, and basal segments of the heart were
embedded in paraffin wax (TissuePrep, Fisher Scientific), cut into 5 μm thick with a
“820” Spencer Microtome (American Optical Corporation; Colorado, USA), and placed
on 3-(aminopropyl)triethoxysilane-coated histology glass slides. Sections were stained
with hematoxylin and eosin (H&E) and Masson’s Trichrome (Accustain, Sigma). To
image the entire heart section, slides were scanned using a high resolution Epson scanner.
Adobe Photoshop 5.5 was used to quantify the area of left ventricular infarction using
multiple (3-4) coronal sections of each animal specimen. The area of infarction, ventricle
cavity and the entire left ventricle were measured using the lasso tool and were expressed
in relative pixels. This lasso method was also used to quantify the area at risk of
infarction, delineated by the Evan’s Blue infusion, relative to the entire ventricle once
photographed with the dissecting microscope. The area of infarction was then expressed
relative to the area at risk in order to normalize any variation in infarct size. The same
method was used to measure the area of fibrosis relative to the area at risk of infarction.
2.11 Statistical Analysis
Statistical analysis was performed by unpaired t-test or 1-way ANOVA with P
value set at <0.05 and post-hoc Bonferroni multiple comparison tests using GraphPad
Prism 4.0 (GraphPad Software, Inc.; San Diego, California). To determine the statistical
significance in area at risk, infarct size and collagen fraction between hemin- and buffertreated animals, unpaired t-test was performed between the two groups at each time point
(24 hours, 4 days, 3 months). To determine the statistical significance in NFκB-DNA
48
binding activity, unpaired t-test was also performed between the two groups at each time
point. Echocardiographic parameters were compared between sham, hemin- and buffertreated groups using 1-way ANOVA followed by Bonferroni multiple comparison test.
Comparisons were made between the groups at each of the time points (pre-op, 4 days, 3
months).
49
Chapter 3: Results
3.1 The Effect of Hemin Treatment in the Setting of Myocardial I/R Injury
3.1.1 Effect of Hemin Administration on HO-1 Expression
The effect of hemin administration on myocardial HO-1 protein expression is
shown in Figure 10. HO-1 protein expression was increased 24 hours and 4 days post I/R
in the hemin-treated animals compared to vehicle after daily intraperitoneal injection of
hemin for 3 days prior to and 2 days after I/R. These results confirm that hemin
administration upregulates myocardial HO-1 expression in the setting of I/R. By day 4,
the HO-1 protein levels were approximately two-fold higher in the hemin-treated animals
compared to the vehicle-treated animals.
3.1.2 Effect of Ischemia on HO-1 Expression in Hemin and Vehicle-treated Animals
Since ischemia per se may induce HO-1 expression, I compared expression of
HO-1 protein in the ischemic and non-ischemic region of the hemin and vehicle-treated
animals. Twenty-four hours after hemin or vehicle treatments, HO-1 protein levels were
comparable in the ischemic and non-ischemic myocardium of the vehicle-treated animals
(Figure 11) indicating that I/R had no direct effect on endogenous HO-1 expression. In
contrast, HO-1 protein expression was markedly increased in the ischemic and nonischemic myocardium in the hemin-treated animals (Figure 11).
50
A
Spot Density
(arbitrary units)
300
200
100
4d
H
4d
B
H
B
24
h
24
h
0
B4d
H4d
B
C
HO-1 +ve
control
B24h
H24h
β-actin reprobe
Figure 10. HO-1 western blot. A) Densitometric analysis of HO-1 expression in
control, buffer- and hemin-treated left ventricle myocardial tissue at 24 hours and 4 days.
HO-1 expression in hemin-treated animals was greater at 24 hour and 4 days after I/R
(n=1). B) Representative HO-1 western blot showing HO-1 protein expression at 24
hours and 4 days in both hemin- and buffer-treated animals. C) β-actin reprobing of the
same blot reveals equal loading.
51
A
Spot Density
(arbitrary units)
500
400
300
200
100
B24h
. I.
N
Is
ch
.
N
Is
ch
.
. I.
0
H24h
B
HO-1 +ve
control
C
I
NI
B24h
I
NI
H24h
β-actin reprobe
Figure 11. HO-1 expression in ischemic and non-ischemic myocardium. A)
Densitometric analysis of HO-1 protein expression in ischemic and non-ischemic tissue
in both buffer- and hemin-treated animals at 24 hours. No difference was noted between
ischemic and non-ischemic tissue in the buffer treated sample. In contrast, HO-1
expression was markedly increased in both the ischemic and non-ischemic myocardium
of hemin-treated animals. B) Representative western blot showing HO-1 expression in
the ischemic and non-ischemic regions of the myocardium in vehicle and hemin-treated
animals 24 hours after I/R and C) β-actin reprobing of the same blot as in (B).
52
3.1.3 Effect of Hemin Treatment on Ischemic and Reperfused Myocardium
Since reperfusion by itself generates ROS and may induce HO-1 protein
expression, the isolated effects of myocardial reperfusion and ischemia on HO-1
expression were determined. The results are shown in Figure 12. Although, hemin was
shown to induce HO-1 expression in ischemic and non-ischemic myocardium (Figure
11), reperfusion had an additive effect on HO-1 expression in both ischemic and nonischemic regions of the myocardium after reperfusion. This indicates that reperfusion by
itself compounds the induction of HO-1 by hemin administration.
3.2 Quantification of Myocardial Infarct Size
3.2.1 Myocardial Area at Risk for Infarction
A prerequisite for the assessment of myocardial injury is that the area at risk of
I/R injury is comparable between the study groups. Thus, area at risk for infarction was
calculated to assess infarct size accuracy and to normalize for infarct size discrepancies
between animals. The area of risk for the various hemin and vehicle-treated groups was
calculated by measuring the area of the LV unstained by Evan’s blue dye, expressing it as
a percent of the entire LV. The percent of the LV area at risk for infarction was not
significantly different between the hemin and buffer-treated animals. However, the mean
area at risk for the hemin-treated animals tended to be greater than the vehicle group at
every time point. Interestingly, there was a time-dependent trend towards smaller areas
of risk in both groups at later time points, possibly because of thinning of the infarcted
wall and concurrent hypertrophy of the remote non-infarcted myocardium.
53
A
Spot Density
(arbitrary units)
400
300
200
100
ch
ic
on
-Is
ch
em
Reperfused
N
Is
N
Is
on
-Is
ch
em
ic
ch
0
Non-reperfused
B
HO-1 +ve
control
I
NI
Reperfused heart
I
NI
Non-reperfused
C
β-actin reprobe
Figure 12. HO-1 expression in reperfused and non-reperfused myocardium. A)
Densitometric analysis of HO-1 protein expression in hemin-treated animals. Ischemic
reperfused myocardium expressed greater HO-1 than non-ischemic myocardium and
ischemic non-reperfused myocardium (n=1). B) Representative western blot and C) βactin reprobe to confirm equal protein loading.
54
% Left Ventricle Area
80
60
40
20
B
uf
fe
r
3m
3m
in
em
H
B
uf
fe
r
4d
4d
in
em
H
r2
4h
uf
fe
B
H
em
in
24
h
0
Figure 13. Area at risk for infarction. Percentage of left ventricle area at risk for
infarction after LAD occlusion in hemin- and buffer-treated animals at 24 hours, 4 days,
and 3 months. Though no significant differences were observed among the groups,
hemin-treated animals showed a general trend of greater mean areas at risk compared
with buffer-treated animals at corresponding time points. (24 hours, hemin, n=7, buffer,
n=10; 4 days, hemin, n=6, buffer, n=6; 3 months, hemin, n=8, buffer, n=4; P<0.05)
55
3.2.2 Myocardial Infarct Size
Figure 14 depicts representative thick ventricular sections from the infarcted or
sham-operated LV from each treatment group.
Evan’s blue staining represents
myocardial tissue not at risk for infarction, while red or pink staining signifies viable
tissue and white staining represents dead myocardium. The buffer 24 hour representative
image reveals a very large, transmural infarct with minimal viable myocardium within
the ventricle’s infarct zone while the representative buffer 4 day image reveals a massive
circumferential infarct. The buffer 3 month sample shows considerable myocardial injury
and wall thinning with no visible myocardial salvage within the infarct zone. It was
observed upon organ harvest that the lateral ventricle wall was fixed to the chest well
with scar tissue and was difficult to dissect; it was technically challenging to maintain the
shape of the ventricle once sliced and the true nature of the paper-thin infarct zone is
somewhat lost. The hemin samples, on the other hand, display discrete bands of injury
within the left ventricle wall and confirm the presence of viable tissue in the
subendocardium and subepicardium. Differences in the shades of viable red tissue are
due to lighting differences from the dissecting microscope upon taking the photographs.
56
Sham
Hemin
Buffer
24 hours
4 days
3 months
Figure 14. Myocardial infarction after I/R in vehicle and hemin-treated rats. Thick
ventricular sections from the apical-mid region of infarcted hemin and vehicle-treated
animals were stained with TTC and compared to sections from sham-operated animals.
White area indicates infarction, whereas red or pink tissue indicates viable tissue. Blue
staining from the Evan’s blue infusion indicates the area not at risk for infarction. The
area excluding Evan’s blue is the area at risk for infarction. Circumferential and nearcircumferential transmural infarctions were present in the buffer-treated animals at 24
hours and 4 days compared to the smaller infarcts in the hemin-treated animals.
Extensive wall thinning was seen in the buffer-treated animal 3 months after infarction.
57
H&E histologically stained samples of LV myocardium are shown in Figure 15.
Panels A, D, and G show sham-operated H&E stained myocardium at 24 hours, 4 days,
and 3 months post-op, respectively, and the maintenance of myocardial integrity is clear.
Panel B illustrates a hemin-treated animal 24 hours after reperfusion and the beginning of
the injury development, manifested by tissue edema and disorganization, is clear.
However, the injury shown in the buffer-treated 24 hour animal (Panel C) appears far
greater in area and injury intensity. Panels E and F compare a hemin-treated 4 day
animal with a buffer-treated 4 day animal, respectively, and the difference in the area of
injury is evident. Panels H and I show hemin 3 month and buffer 3 month images side by
each.
Wall thinning is apparent in both images, however, myocardial salvage is
noticeable in the hemin 3 month animal (Panel H) and the integrity of the remaining
myocardium is comparable to the sham-operated 3 month animal (Panel G). In contrast,
extreme wall thinning and transmural scar formation are evident in the buffer 3 month
animal.
Infarct size was normalized to the area at risk within each treatment group. Once
this was taken into account, significant differences in infarct size (expressed as a fraction
of the LV) were observed between the hemin- and buffer-treated groups at 24 hours
(0.211 ± 0.001 vs. 0.435 ± 0.003, respectively) and 4 days (0.344 ± 0.004 vs. 0.682 ±
0.004, respectively) post-MI (Figure 16). Surprisingly, no significant difference was seen
between the treatment groups at 3 months, although the mean infarct size of the buffertreated animals showed a trend towards larger infarcts (0.507 ± 0.091) compared to the
hemin-treated animals (0.459 ± 0.063).
58
A
B
C
D
E
F
G
H
I
Figure 15. Hematoxylin & eosin stained left ventricle myocardium post-I/R injury.
Panels A, D, and G show sham-operated H&E stained myocardium at 24 hours, 4 days,
and 3 months post-op, respectively. Hematoxylin stains nuclear material dark purpleblue and eosin stains cytoplasmic material (including proteins, connective tissue and
collagen) pink-red. Panels B, E, and H demonstrate hemin-treated animals 24 hours, 4
days, and 3 months post-reperfusion, respectively, while panels C, F, and I reveal buffertreated animals 24 hours, 4 days, and 3 months post-reperfusion, respectively.
(Magnification 10x; inserts 20x)
59
0.7
0.6
*
0.5
0.4
0.3
0.2
0.1
3m
B
uf
fe
r
3m
in
4d
H
em
B
uf
fe
r
4d
H
em
in
B
uf
fe
r
H
em
in
24
h
0.0
24
h
Infarct Size: Area at Risk
Ratio
*
0.8
Figure 16. Infarct size relative to area at risk. Infarct size was normalized to area at
risk to account for differences in area at risk between animals. Infarct size was
significantly greater in the buffer-treated animals compared to the hemin-treated animals
at 24 hours and 4 days after I/R (P<0.05). No significant difference was seen between
buffer and hemin-treated groups at 3 months, although the mean ratio of the buffertreated animals tended to be greater (0.507 ± 0.091) than that of the hemin-treated
animals (0.459 ± 0.063). (24 hours, hemin, n=7, buffer, n=10; 4 days, hemin, n=6,
buffer, n=6; 3 months, hemin, n=7, buffer, n=4)
60
3.2.3 Quantification of Myocardial Fibrosis
Myocardial fibrosis in the paraffin-embedded sections stained with Masson’s
Trichrome is shown in Figure 17. Masson’s Trichrome staining represents the presence
of collagen by blue staining, while white staining reveals dead tissue and red staining
reveals viable tissue. The hemin 4 day representative image reveals a typical hemin
infarct whereby there is a longitudinal band of injury and a limited development of
fibrosis beyond the peri-vascular ring of collagen. The buffer 4 day image reveals
considerable peri-vascular fibrosis and the beginning of a transmural collagen network
wherein fibrosis present at 4 days is evident as widespread, diffuse blue specks within the
infarct zone. The 3 month hemin representative image displays a mature scar wherein
collagen has completely replaced the infarcted myocardium.
However, the
subendocardium has been salvaged (nearly half the width of the ventricle’s free wall) and
its architecture has been maintained. The 3 month buffer image displays a transmural
infarct that has been replaced entirely with collagen and no viable myocardium remains.
The difference in staining intensity between the 4 day and the 3 month samples likely
resulted simply from different experimental conditions or variation in the length of
staining.
61
A
B
C
D
Figure 17. Myocardial fibrosis. Panel A displays a representative hemin 4 day image
with minimal fibrosis and maintenance of the subepicardium and endocardium. The
development of fibrosis is more noticeable in Panel B, a buffer-treated animal at 4 days,
particularly peri-vascularly. Note the large size of the infarct with very little viable
myocardium. Panel C, a hemin 3 month image, shows significant fibrosis despite
considerable myocardial salvage. Panel D shows a 3 month buffer-treated animal’s nearcompletely fibrotic left ventricular free wall, including diffuse fibrosis of the papillary
muscle.
62
The quantification of fibrosis requires some clarification. Figure 18, which shows
the fibrotic left ventricular area normalized for the area at risk at 4 days and 3 months,
should be interpreted as follows: at 4 days, the results represent the area of the LV at risk
that has shown evidence of the development of fibrosis, not complete replacement of the
myocardial architecture (myocytes, endothelial cells) by collagen.
The process of
collagen deposition requires the homing and trans-differentiation of fibroblasts into
myofibroblasts, proliferation of myofibroblasts (which peaks at 4 days) and
synthesis/elaboration of collagen (Virag and Murray, 2003). By this definition, fibrosis
observed at 4 days has only just commenced. It would be incorrect to interpret the data at
4 days in Figure 18 as the area of the LV that has been replaced with collagen entirely as
it biologically impossible to have a mature scar of collagen at 4 days post-I/R injury. The
results at 3 months, however, can be interpreted as the ratio of the LV which has been
replaced by collagen normalized to the area at risk. Figure 17 clearly showed the
differences in the qualitative nature of acute and long-term fibrosis remodeling.
The area of myocardium displaying evidence of collagen deposition was
significantly greater in the buffer group at 4 days compared to the hemin group (0.382 ±
0.005 vs. 0.802 ± 0.007, P< 0.05; Figure 18). However, no significant difference in
fibrosis was seen between the two groups at 3 months, although the hemin-treated group
exhibited a trend towards greater mean area of fibrosis (0.869 ± 0.121) than the buffertreated group (0.784 ± 0.081). This may be due to the small sample size and the
variability between samples.
63
Fibrosis: Area at Risk
1.2
1.0
*
0.8
0.6
0.4
0.2
m
B
uf
fe
r3
3m
H
em
in
fe
r4
uf
B
H
em
in
4d
d
0.0
Figure 18. Area of fibrosis relative to the area at risk. Post-MI fibrosis in the left
ventricle between hemin and vehicle-treated groups at 4 days and 3 months. A
significant difference (P<0.05) was observed between hemin- and buffer-treated animals
at 4 days. No significant difference in fibrosis between the two groups was seen at 3
months. (4 days, hemin, n=10, buffer n=8; 3 months, hemin, n=7, buffer, n=4).
64
3.3 Quantification of LV Structure and Function by Echocardiography
3.3.1 LV Wall and Chamber Dimensions
The time course of echocardiographic measurements is summarized in Table 1.
Because of the anatomy of the LAD and the position of the ligature, the infarcts are
predominantly in the mid-papillary and apical region of the LV; the most striking
differences between hemin and vehicle animals are seen in those regions. At 4 days, wall
thickness was significantly reduced in the buffer-treated group compared to the hemintreated group (Table 1). Significant changes in posterior wall thickness were observed at
the apex only in the buffer group. This is likely due to chamber remodeling resulting
from the expansion and thinning of the infarct.
At 3 months after I/R, hemin and buffer-treated animals showed interventricular
septal wall thinning and chamber dilatation at the apical, mid, and base levels. Posterior
wall thinning at 3 months was seen in the apex in the buffer group and the apex and mid
sections in the hemin group. Anterior wall thinning at the base level in both treatment
groups may result from distorted forces exerted upon the interventricular myocardial wall
resulting from the infarct expansion in the apical and mid levels and subsequent
dysfunctional wall motion rather than infarction at the base level. This wall thinning at
the base also may explain the observed chamber dilatation at the base level.
It is important to highlight the fact that some LV parameters from the buffertreated groups displayed a significant difference from the hemin-treated group as well as
65
PARAMETER
SHAM
Wall Dimensions
Pre-op
4 Days Post-op
3 Months Post-op
IVSdA
IVSsA
IVSdM
IVSsM
IVSdB
IVSsB
0.127 ± 0.002
0.169 ± 0.002
0.138 ± 0.003
0.172 ± 0.006
0.121 ± 0.001
0.161 ± 0.006
0.122 ± 0.006
0.168 ± 0.001
0.141 ± 0.001
0.179 ± 0.003
0.146 ± 0.004
0.168 ± 0.006
0.155 ± 0.003
0.198 ± 0.007
0.158 ± 0.003
0.216 ± 0.003
0.161 ± 0.001
0.204 ± 0.003
PWdA
PWsA
PWdM
PWsM
PWdB
PWsB
0.146 ± 0.009
0.197 ± 0.004
0.139 ± 0.004
0.208 ± 0.008
0.134 ± 0.006
0.189 ± 0.014
0.146 ± 0.005
0.199 ± 0.008
0.148 ± 0.006
0.204 ± 0.010
0.147 ± 0.008
0.195 ± 0.012
0.180 ± 0.011
0.230 ± 0.005
0.168 ± 0.005
0.247 ± 0.001
0.174 ± 0.004
0.228 ± 0.007
0.596 ± 0.060
0.364 ± 0.059
0.631 ± 0.059
0.401 ± 0.052
0.664 ± 0.020
0.436 ± 0.022
0.682 ± 0.034
0.404 ± 0.024
0.816 ± 0.022
0.514 ± 0.044
0.791 ± 0.029
0.497 ± 0.014
Chamber Dimensions
LVDdA
LVDsA
LVDdM
LVDsM
LVDdB
LVDsB
0.558 ± 0.055
0.357 ± 0.061
0.603 ± 0.010
0.338 ± 0.015
0.649 ± 0.018
0.418 ± 0.015
Table 1A. Left ventricle wall and chamber dimensions in sham-operated groups.
Pre-op, 4 day, and 3 month echo measurements for sham groups (measured in cm ±
SEM). (Sham, pre-op, n=3; 4 days, n=3; 3 months, n=3)
66
PARAMETER
HEMIN
Wall Dimensions
Pre-op
4 Days Post-op
3 Months Post-op
IVSdA
IVSsA
IVSdM
IVSsM
IVSdB
IVSsB
0.128 ± 0.003
0.165 ± 0.003
0.132 ± 0.003
0.167 ± 0.003
0.140 ± 0.006
0.172 ± 0.006
0.108 ± 0.004
0.131 ± 0.006 *
0.124 ± 0.003
0.149 ± 0.006
0.127 ± 0.003 *
0.152 ± 0.005
0.106 ± 0.007 *
0.141 ± 0.008 *
0.128 ± 0.008
0.161 ± 0.009 *
0.147 ± 0.003
0.182 ± 0.004 *
PWdA
PWsA
PWdM
PWsM
PWdB
PWsB
0.140 ± 0.003
0.192 ± 0.005
0.149 ± 0.004
0.204 ± 0.005
0.154 ± 0.005
0.209 ± 0.006
0.147 ± 0.004
0.190 ± 0.006
0.144 ± 0.003
0.198 ± 0.006
0.144 ± 0.004
0.200 ± 0.006
0.148 ± 0.004 *
0.178 ± 0.005 *
0.155 ± 0.009
0.206 ± 0.010 *
0.167 ± 0.002
0.217 ± 0.004
0.590 ± 0.019
0.416 ± 0.020
0.648 ± 0.017
0.445 ± 0.018
0.669 ± 0.017
0.480 ± 0.017
0.849 ± 0.036 *
0.658 ± 0.047 *
0.956 ± 0.034 *
0.745 ± 0.038 *
0.933 ± 0.039
0.696 ± 0.037 *
Chamber Dimensions
LVDdA
LVDsA
LVDdM
LVDsM
LVDdB
LVDsB
0.529 ± 0.018
0.303 ± 0.016
0.589 ± 0.017
0.365 ± 0.015
0.599 ± 0.017
0.360 ± 0.003
Table 1B. Left ventricle wall and chamber dimensions in hemin-treated groups.
Pre-op, 4 day, and 3 month echo measurements for hemin groups (measured in cm ±
SEM). Significant difference (P<0.05) from sham is denoted by (*). (Hemin, pre-op,
n=15; 4 days, n=15; 3 months, n=7)
67
PARAMETER
BUFFER
Wall Dimensions
Pre-op
4 Days Post-op
3 Months Post-op
IVSdA
IVSsA
IVSdM
IVSsM
IVSdB
IVSsB
0.132 ± 0.003
0.174 ± 0.005
0.138 ± 0.004
0.172 ± 0.005
0.135 ± 0.004
0.172 ± 0.003
0.096 ± 0.004 *
0.114 ± 0.005 *
0.105 ± 0.004 *^
0.122 ± 0.006 *^
0.115 ± 0.004 *^
0.146 ± 0.005
0.095 ± 0.005 *
0.105 ± 0.006 *^
0.096 ± 0.005 *^
0.117 ± 0.010 *^
0.142 ± 0.006 *
0.180 ± 0.001 *
PWdA
PWsA
PWdM
PWsM
PWdB
PWsB
0.147 ± 0.004
0.204 ± 0.006
0.152 ± 0.004
0.206 ± 0.008
0.154 ± 0.004
0.200 ± 0.009
0.129 ± 0.005^
0.163 ± 0.010
0.139 ± 0.005
0.182 ± 0.007
0.133 ± 0.004
0.196 ± 0.006
0.131 ± 0.008 *
0.164 ± 0.004 *
0.151 ± 0.010
0.205 ± 0.011
0.169 ± 0.003
0.224 ± 0.003
0.661 ± 0.022
0.494 ± 0.026
0.725 ± 0.015^
0.551 ± 0.021 *^
0.715 ± 0.023
0.518 ± 0.029
0.864 ± 0.026 *
0.680 ± 0.032 *
1.010 ± 0.041 *
0.791 ± 0.044 *
0.944 ± 0.034
0.711 ± 0.047 *
Chamber Dimensions
LVDdA
LVDsA
LVDdM
LVDsM
LVDdB
LVDsB
0.552 ± 0.018
0.324 ± 0.014
0.615 ± 0.012
0.377 ± 0.020
0.637 ± 0.010
0.408 ± 0.016
Table 1C. Left ventricle wall and chamber dimensions in buffer-treated groups.
Pre-op, 4 day, and 3 month echo measurements for buffer groups (measured in cm ±
SEM). Significant difference (P<0.05) from sham is denoted by (*) while significant
difference (P<0.05) between hemin and buffer groups is denoted by (^). (Buffer, pre-op,
n=11; 4 days, n=11; 3 months, buffer, n=4).
68
from the sham-operated group. At 4 days after I/R, significantly reduced interventricular
septal wall thickness, more pronounced at the mid level, was seen in the buffer group
compared to hemin. The upregulation of HO-1 by hemin likely decreased ROS and
prevented expansion to some degree at the mid-level, resulting in a significant difference
from sham in the hemin group but also protecting the infarct zone from further expansion
as seen in the buffer group. This also explains the significant difference in LV chamber
dilatation seen at 4 days between the two treatment groups at the mid level, as infarct
expansion and wall thinning beget chamber dilatation. Similarly, at 3 months after
infarction, a significant difference in interventricular septal wall thinning was seen
between hemin and buffer groups, where the IVS wall thinning was attenuated at the
apex and mid level LV in the hemin group compared with the buffer group. This is likely
due to the prevention of infarct expansion to some degree in the hemin group relative to
the buffer group.
The percent change of each echocardiographic parameter relative to its baseline is
shown in Table 2. At 4 days post-I/R, the hemin-treated group showed considerably less
interventricular septal wall thinning than the buffer-treated animals, at all levels of the
LV, although this was more pronounced in the apex and mid sections.
Similarly,
chamber dilatation was more than doubled in the buffer-treated group at both the apex
and mid sections, with comparable changes occurring at the base level. The increased
posterior wall thickness observed at the apex section of the hemin-treated group may be
due to regional compensatory hypertrophy of the remote myocardium. The posterior wall
69
PARAMETER
% Change at 4 days
% Change at 3 months
Wall Dimensions Sham
Hemin
Buffer
Sham
Hemin
Buffer
IVSdA
IVSsA
IVSdM
IVSsM
IVSdB
IVSsB
-3.58 ± 3.9
-0.95 ± 1.5
2.09 ± 2.2
4.41± 5.3
20.2 ± 2.2
9.01 ± 1.8
-15.6 ± 3.1
-20.1 ± 3.6*
-5.19 ± 2.9
-10.5 ± 3.9
-7.4 ± 3.2*
-11.0 ± 3.0
-27.2 ± 3.0*^
-36.6 ± 2.4*^
-23.2 ± 4.3*^
-31.5 ± 3.2*^
-13.3 ± 3.7*
-25.5 ± 5.2*^
24.5 ± 0.8
16.7 ± 4.8
14.1 ± 1.7
23.4 ± 6.1
32.6 ± 0.8
27.0 ± 6.7
-7.41 ± 6.9*
-12.2 ± 6.1*
-0.97 ± 7.6*
0.06 ± 6.3
9.7 ± 2.9*
9.31 ± 3.3
-28.3 ± 6.4*
-41.7 ± 5.8*^
-30.3 ± 2.2*
-32.8 ± 6.6*^
9.04 ± 5.3*
3.93 ± 4.7*
PWdA
PWsA
PWdM
PWsM
PWdB
PWsB
0.58 ± 9.5
0.91 ± 4.3
6.17 ± 5.7
-1.02 ± 7.4
9.93 ± 9.7
3.52 ± 3.1
5.63 ± 3.2
-0.73 ± 3.2
-5.7 ± 3.0
-2.3 ± 2.7
-4.3 ± 3.2
-5.9 ± 4.8
-13.8 ± 4.3^
-26.7 ± 3.8*^
-9.7 ± 3.7
-11.6 ± 7.5
-14.5 ± 3.9
-3.6 ± 5.7
23.2 ± 5.7
16.9 ± 4.7
20.4 ± 1.2
19.5 ± 4.5
29.6 ± 6.0
21.2 ± 5.0
5.32 ± 3.8
-8.2 ± 5.1*
6.4 ± 7.4
2.39 ± 7.9
12.5 ± 3.9
7.4 ± 4.4
-10.3 ± 4.3*
-22.5 ± 4.8*
0.56 ± 5.1
-14.5 ± 3.3
12.1 ± 6.1
6.5 ± 8.7
12.3 ± 3.4
36.5 ± 6.7
11.0 ± 3.9
25.1 ± 8.8
14.4 ± 2.9
35.7 ± 8.3
22.7 ± 8.6
56.4 ± 9.3*
20.4 ± 3.1
60.3 ± 12.5
14.6 ± 3.8
34.9 ± 9.1
23.3 ± 6.3
18.2 ± 14.6
35.3 ± 4.6
52.2 ± 10.7
21.8 ± 8.8
17.8 ± 2.3
58.7 ± 13.4
96.5 ± 17.7*
61.0 ± 13.3
106 ± 28.0
52.9 ± 8.8
83.8 ± 17.7
57.2 ± 5.3
137 ± 14.3*
62.4 ± 3.4
148 ± 24.8
48.9 ± 6.7
83.3 ± 9.4
Chamber Dimensions
LVDdA
LVDsA
LVDdM
LVDsM
LVDdB
LVDsB
6.89 ± 4.9
3.5 ± 8.1
4.42 ± 8.4
18.6 ± 13.1
2.7 ± 5.9
4.69 ± 6.9
Table 2. Percent change in left ventricle dimensions from baseline echo. Results are
expressed as percent change from mean pre-op echo dimension to mean 4 day and 3
month echo dimension within each treatment group. Significant difference from
comparable sham time point is denoted by (*) (P<0.05), while significant difference
between hemin and buffer groups within the same time point is denoted by (^) (P<0.05).
(Percent change at 4 days, sham, n=3, hemin, n=15, buffer, n=11; 3 months, sham n=3,
hemin, n=7, buffer, n=4)
70
experienced less percent change from baseline in both groups as it is the more remote
area of the LV.
Attenuation of wall thinning at 3 months in the hemin-treated group is observed at
the apical and mid levels only, as there is little difference in percent change between the
groups at the base level. At 3 months, chamber dilation during diastole and systole is
relatively comparable between the hemin and buffer groups. The percent change in PW
observed at the base level of both hemin- and buffer-treated groups is small but likely
reflects the animals’ growth from 6 weeks of age to 4.5 months of age. Similar to the
acute time-point, the small percent change observed at the apex of the PW in the hemintreated group at 3 months is difficult to reconcile and may be due to the intricacy of
imaging the apical PW endocardial borders.
Overall, at 4 days, less change was observed at every level of the anterior and
posterior ventricle wall in the hemin group compared to the buffer group (except for a
small increase in change at the base level of the posterior wall). Similarly, a stark trend
of reduced change- nearly by half- in chamber dimension in the hemin group (save for
the base level) was observed. At 3 months, changes in anterior and posterior wall
thickness were abrogated at the apex and mid levels and comparable at the base in the
hemin group compared to buffer. Changes in LV diameter in the hemin group were
comparable to buffer in the apex and mid levels during diastole and were considerably
reduced during systole, while changes in LV diameter at the base level were similar in
both groups.
71
3.3.2 Echocardiography Assessment of Cardiac Function
Cardiac function was assessed by calculating ejection fraction (EF), fractional
shortening (FS) and LV mass using standard echocardiographic formulas.
3.3.2a Mid Level Ejection Fraction, Fractional Shortening and LV Mass
The mid ventricle region of the LV showed the greatest degree of post-I/R change
in EF and FS (Figure 19) in both the hemin and buffer-treated groups. Percent change
relative to baseline results is shown in Table 2 for each group. Only in the 4-day buffertreated animals was EF significantly decreased relative to its pre-op value and relative to
the 4 day hemin animals. At 4 days after I/R, EF was preserved in the hemin-treated
animals (Figure 19A). At 3 months, EF was nearly identical between the buffer and
hemin groups (0.523 ± 0.026 and 0.526 ± 0.029, respectively).
Mid level FS (Figure 19B) showed a trend similar to EF. FS at 4 days in the
buffer-treated animals was significantly decreased relative to the sham and hemin groups
at the same time point. The difference in FS between the hemin and buffer groups at 3
months was almost indistinguishable (21.90 ± 1.57% and 22.26 ± 1.58%, respectively).
No significant differences in LV mass: body weight (Figure 20) were observed
among the three study groups at the mid level of the left ventricle at 3 months.
72
A
1.00
0.75
EF
*^
*
*
0.50
0.25
eo
P p
re
-o
p
S
4d
H
4d
B
4d
S
3m
H
3m
B
3m
B
Pr
H
S
Pr
eop
0.00
B
50
FS (%)
40
*^
30
*
*
20
10
S
3m
B
3m
H
3m
S
4d
B
H
4d
4d
S
Pr
e
H -o p
Pr
e
B - op
P
re
-o
p
0
Figure 19. Mid level functional echo parameters. Comparison of A) ejection fraction and B) fractional
shortening at mid level ventricle among all treatment groups, pre-op and 4 days and 3 months post-MI.
Significant difference from sham is denoted by (*, P<0.05), while significant difference between hemin and
buffer groups is denoted by (^, P<0.05). (Pre-op, sham, n=3, hemin, n=15, buffer, n=11; 4 days, sham,
n=3, hemin, n=15, buffer, n=11; 3 months, sham n=3, hemin, n=7, buffer, n=4)
73
0.004
0.003
0.002
0.001
3m
B
3m
H
3m
S
B
Pr
eop
Pr
H
Pr
S
eop
0.000
eop
LV Mass to Weight Ratio
0.005
Figure 20. Mid LV mass to body weight. Left ventricle mass normalized for body
weight at mid level (measured along the parasternal long axis) among all treatment
groups, pre-op and 3 months post-MI. No significant difference was observed. (Pre-op,
sham, n=3, hemin, n=7, buffer, n=4; 3 months, sham n=3, hemin, n=7, buffer, n=4)
74
3.3.2b Apex Level Ejection Fraction, Fractional Shortening and LV Mass
Significant difference in apical EF from sham was observed in both hemin and
buffer groups at 3 months and the values are similar (0.5386 ± 0.04 and 0.5100 ± 0.03,
respectively; Figure 21A). Though no significant difference was observed at the 4 day
time-point among all treatment groups, the mean EF in the hemin group is greater than
the mean EF in the buffer group (0.6387 ± 0.032 and 0.5608 ± 0.057, respectively). The
trend observed in EF is also seen in the FS results (Figure 21B). Significant difference
from sham was observed in both hemin and buffer groups at 3 months and the values are
similar (23.07 ± 2.25% and 21.40 ± 1.66%, respectively).
Though no significant
difference was noted among treatment groups at 4 days (likely due to considerable
standard error of the mean), the mean FS of the hemin group is greater than the mean FS
in the buffer group (29.70 ± 2.14 and 25.38 ± 2.92, respectively).
No significant differences in LV mass: body weight (Figure 22) were observed
among the three study groups at the apex level of the left ventricle at 3 months.
75
A
1.00
EF
0.75
*
*
0.50
0.25
S
Pr
e
H -o p
Pr
e
B - op
P
re
-o
p
S
4d
H
4d
B
4d
S
3m
H
3m
B
3m
0.00
B
50
FS (%)
40
*
30
*
*
20
10
S
3m
B
3m
H
3m
S
4d
B
H
4d
4d
S
Pr
e
H -o p
Pr
e
B - op
P
re
-o
p
0
Figure 21. Apex level functional echo parameters. Comparison of A) ejection fraction and B)
fractional shortening at the level of the apex (measured along the parasternal long axis) among all
treatment groups, pre-op and 4 days and 3 months post-MI. Significant difference from sham is
denoted by (*, P<0.05), while significant difference between hemin and buffer groups is denoted
by (#, P<0.05). (Pre-op, sham, n=3, hemin, n=15, buffer, n=11; 4 days, sham, n=3, hemin, n=15,
buffer, n=11; 3 months, sham n=3, hemin, n=7, buffer, n=4)
76
0.004
0.003
0.002
0.001
3m
B
3m
H
3m
S
H
Pr
e
S
Pr
eop
B
Pr
eop
0.000
-o
p
LV Mass to Weight Ratio
0.005
Figure 22. Apex LV mass to body weight. Left ventricle mass normalized for body
weight at the level of the apex (measured along the parasternal long axis) among all
treatment groups, pre-op and 3 months post-MI. No significant difference was observed.
(Pre-op, sham, n=3, hemin, n=7, buffer, n=4; 3 months, sham n=3, hemin, n=7, buffer,
n=4)
77
The percent change of each derived parameter from its baseline measurement is
shown in Table 3. The decrease in EF observed at the apex and mid levels in the buffer
group at 4 days is attenuated in the 4 day hemin group at the apex and mid levels but not
at the level of the base. A similar trend is observed in FS at 4 days. While there was no
observed statistically significant difference in EF and FS between the hemin and buffer 3
month groups, there is a trend whereby the decline in function in the hemin group is
attenuated by approximately 5-15% compared to the change observed in the buffer 3
month group. The percent change in EF and FS within the sham group is marginal at the
apex and base, but there was an unexpected yet modest decrease in EF and FS at the mid
level. This could be due to the anaesthesia used during the echo procedure.
LV mass at 3 months decreased in a similar fashion among the sham, hemin, and
buffer groups (Table 4). There was no significant difference among the groups and the
decrease is representative of the animals’ growth from 6 weeks of age to 4.5 months,
thereby decreasing the ration of LV mass to body weight.
78
% Change at 4 days
LV Function
SHAM
HEMIN
BUFFER
% Change at 3 months
SHAM
HEMIN
BUFFER
EF Apex
5.1 ± 3.8
-20.9 ± 4.3
-32.4 ± 7.4* 8.9 ± 8.0
-30.9 ± 6.5* -39.8 ± 3.6*
EF Mid
-9.4 ± 3.1
-10.7 ± 3.7
-29.9 ± 4.7^ -9.5 ± 3.9
-28.7 ± 5.3
-38.4 ± 3.6*
EF Base
-1.5 ± 3.7
-15.4 ± 4.7
-12.2 ± 5.7
-20.2 ± 6.2
-25.3 ± 3.4*
FS Apex
8.2 ± 6.4
-30.1 ± 5.7* -42.2 ± 6.98* 15.0 ± 14.6
-41.6 ± 7.4* -54.7 ± 3.9*
FS Mid
-16.3 ± 4.9
-15.5 ± 5.2
-39.4 ± 6.1^ -12.9 ± 3.4
-38.2 ± 6.5
-53.3 ± 4.6*
FS Base
-2.5 ± 5.9
-22.4 ± 6.6
-24.2 ± 6.6
-28.7 ± 9.0
-36.1 ± 3.2*
3.9 ± 2.9
6.8 ± 5.0
Table 3. Percent change in functional parameters from baseline echo. Results are
expressed as percent change from mean pre-op echo values to mean 4 day and 3 month
echo values within each treatment group. (Percent change at 4 days, sham, n=3, hemin,
n=15, buffer, n=11; Percent change at 3 months, sham, n=3, hemin, n=7, buffer, n=4;
P<0.05)
79
% Change at 3 months
Ventricle Mass
SHAM
HEMIN
BUFFER
LV Mass:Wt Apex
-49.1 ± 4.0
-39.8 ± 2.9
-46.6 ± 4.3
LV Mass:Wt Mid
-45.4 ± 3.9
-34.0 ± 3.7
-39.5 ± 3.6
LV Mass:Wt Base
-42.9 ± 5.9
-25.2 ± 4.0
-26.4 ± 2.9
Table 4. Percent change in left ventricle mass. Results are expressed as a ratio of LV
mass normalized to body weight. No significance was observed at 3 months. (Percent
change at 3 months, sham n=3, hemin, n=7, buffer, n=4)
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3.4 Quantification of NFκB-DNA Binding Post-reperfusion
NFκB-DNA binding activity was significantly attenuated in the 4 day hemin
group compared to buffer (Figure 23). While not statistically significant, the NFκB
activity (represented by relative light units) in the buffer 24 hour group is notably greater
than the NFκB activity observed in the hemin 24 hour group. Together, this suggests that
HO-1 upregulation abrogates the transcription factor’s translocation to the nucleus and/or
activity despite redox stimulation from ROS due to reperfusion.
3.5 Effect of Hemin Treatment on Hematocrit
Hematocrit was measured at cardiac harvest at all three time points within all
three treatment groups, though no significant differences were observed (Figure 24). In
fact, very little variation in the mean hematocrit was seen between sham, hemin, and
buffer groups within each time group, though the mean hematocrit results are slightly
lower at the 24 hour time point.
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Relative Light Units
(RLU/μ g)
1.5
*
1.0
0.5
4d
B
4d
H
24
h
B
H
24
h
0.0
Figure 23. NFκB-DNA binding activity after I/R. NFκB activity post-reperfusion was
significantly elevated in the buffer-treated animals at 4 days post-MI (P<0.05). While not
statistically different, the mean NFκB activity observed in the buffer 24 hour group is
greater than the mean observed in the hemin 24 hour group. (24 hours, hemin, n=3,
buffer, n=3; 4 days, hemin, n=4, buffer, n=4)
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Hematocrit (%)
60
40
20
S2
4h
H
24
h
B
24
h
S4
d
H
4d
B
4d
S3
m
H
3m
B
3m
0
Figure 24. Hematocrit at cardiac harvest. No significant difference was observed
among the groups at each time point. However, the mean hematocrit value for each
treatment group is slightly lower at 24 hours compared with the other time points. (24
hours, sham, n=4, hemin, n=3, buffer, n=5; 4 days, sham, n=3, hemin, n=6, buffer, n=5; 3
months, sham, n=3, hemin, n=6, buffer, n=5)
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Chapter 4: Discussion
4.1 Major Findings
This thesis tested the hypothesis that HO-1 upregulation via hemin administration
attenuates acute and chronic I/R-induced myocardial injury and subsequent postmyocardial infarction left ventricular remodeling. The results showed that treatment with
hemin prior to I/R significantly reduced acute myocardial injury, leading to reduced
infarction size and improved post-MI recovery at 4 days. However, chronic hemin
treatment did not prevent long term left ventricular remodeling, despite its acute
cardioprotective effects. The reasons for this discrepancy have not been elucidated in the
present study, but could possibly be attributed, at least in part, to the reduced sample size
of the 3 month study as well as biological differences in infarct size. Alternatively, HO-1
upregulation may not be maintained by chronic hemin administration such that it
continues to exert cardioprotective properties. The acute protective effect of hemin
treatment was associated with reduced myocardial fibrosis and was accompanied by a
decrease in DNA binding activity of the redox-sensitive transcription factor NFκB,
suggesting that inhibiting this pro-inflammatory transcription factor may underlie the
mechanism by which hemin reduces acute myocardial injury. However, the intermediate
signalling pathways leading to inhibition of NFκB by hemin are not known.
The
injection of hemin was associated with induction of HO-1 protein expression and with
reduced myocardial injury, suggesting that the enzyme confers cardioprotection via the
byproducts of heme catabolism, CO, biliverdin, and iron. However, the isolated effects
of the byproducts on myocardial protection were not assessed in this thesis.
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4.2 Histological Findings
4.2.1 Myocardial Infarctions
Because of inter-animal variability in risk area after ligation of the LAD, infarct
size was calculated as a fraction of the area at risk for all animals. The predominant acute
histopathological finding is decreased infarct size in the hemin-treated animals at 24
hours and 4 days after I/R compared with buffer-treated animals. H&E staining in the
hemin-treated animals revealed reduced area of infarction and the maintenance of
myocardial subendocardium within the area at risk compared to buffer-treated animals.
Generally, infarction in hemin-treated animals looked like a discrete longitudinal band of
injury within the cross-section of the left ventricle with a distinct infarct border.
Furthermore, histology revealed non-infarcted, viable subendocardium with architecture
similar to sham-operated animals in nearly all 24 hour and 4 day hemin-treated animals.
Notwithstanding biological differences in coronary artery distribution and architecture,
this may be a genuine pattern of injury among HO-1-upregulated animals, although
proving a direct association between HO-1 levels and this pattern of myocardial injury is
beyond the scope of this thesis.
Buffer-treated animals at the same time points
experienced infarcts that appeared more diffuse and involved all layers of the
myocardium, including the subendocardium and the subepicardium, with borders that
extend beyond the anterolateral wall of the left ventricle. Circumferential and nearcircumferential infarcts were observed only in buffer-treated animals.
The reason for the observed reduced infarct size within the 24 hour and 4 day
hemin-treated animals is likely multifactorial.
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The HO-1 byproducts decrease the
inflammatory reaction upon reperfusion by decreasing endothelial dysfunction and by
enhancing the anti-oxidant reserve, leading to increased tolerability of the myocytes to
the pro-inflammatory and pro-oxidant conditions of the infarct environment. It is likely
these protective functions of HO-1 together are responsible for decreased neutrophil
infiltration and decreased ROS generation, curtailing the cytokine/chemokine elaboration
and decreasing the release of proteolytic enzymes. This in turn limits the cellular injury
and decreases myocyte and endothelial cell apoptosis, as evidenced by a decreased area
of infarction and the maintenance of myocardial integrity within the subendocardium.
Residual myocardial viability after infarction is important not only for contraction and
cardiac performance but the thickness of the surviving myocardium is also inversely
associated with infarct expansion within the infarct zone (Pirolo et al., 1986). While a
certain degree of necrosis is unavoidable after prolonged ischemia, Clark et al. (2000)
reported that rat hearts displaying high levels of HO-1 protein expression showed
considerably reduced infarct size and preserved mitochondrial integrity after ischemia
reperfusion. Vulapalli et al. (2002) reported an antiapoptotic activity of specific HO-1
over-expression in protecting cardiomyocytes from I/R-induced apoptosis in vivo, and
Tang et al. (2004) found that Bcl-2, an anti-apoptotic protein, is upregulated in
myocardium treated with a hypoxia-sensitive hHO-1 plasmid compared with myocardium
treated with saline. The precise mechanism responsible for the observed reduction in
infarct size among the hemin-treated animals in unknown but is likely a combination of
the enzyme’s pleiotropic activities.
The 3 month hemin and buffer groups, on the other hand, had comparable mean
areas of infarction. There was greater variability in the appearance of the infarcts within
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the hemin group, unlike the acute time points. This may be attributable, at least in part,
to technical variability as well as biological variation between the animals. In addition, it
is also possible the animals did not adapt to the initial I/R insult in the same manner
which may have been exaggerated over 3 months. Hemodynamic changes including
blood volume, arterial blood pressure, systemic vascular resistance and pulmonary
pressure may have changed secondary to left ventricular remodeling, which would impair
basal LV function and hemodynamics and would be more prevalent at 3 months after
reperfusion.
4.2.2 Myocardial Fibrosis
The difference in the development of fibrosis at 4 days post-MI between heminand buffer-treated animals was significant. The mean infarct size in the buffer 4 day
group was significantly greater and is therefore expected to be followed by extensive
reparative fibrosis to preserve structural integrity of the infarcted tissue (Sun et al., 2000).
In contrast, there was no statistically significant difference between the two groups at 3
months.
Fibrosis developed beyond the infarct zone and in remote regions in both
groups with web-like spindles of collagen deposited throughout the ventricle unlike the
concentrated areas of fibrosis in the acute study.
Both 3 month treatment groups
experienced significant infarcts and left ventricular remodeling (including chamber
dilatation) as well as reduced cardiac performance, suggesting the animals were
progressing to heart failure. It is now recognized that accumulation of collagen occurs in
the interstitial space of the hypertrophied and failing heart, a process termed “reactive
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interstitial fibrosis” (Weber and Brilla, 1991; Sabbah et al., 1995). The exact mechanism
that promotes the accumulation of fibrosis in the remote areas of the myocardium is not
fully understood, but has been attributed, in part, to enhanced activity of the reninangiotensin system (Weber and Brilla, 1991). Cleutjens et al. (1995) demonstrated that
myofibroblasts are responsible for type I and III collagen synthesis in the repairing
myocardium; however, myofibroblasts are not found within interstitial fibrosis at
noninfarcted remote sites after MI (Sun et al., 2000). This suggests that some other
mechanism, dissimilar to reparative fibrosis, is responsible for the collagen accumulation
in remote myocardium, perhaps increased TGF-β1 production by interstitial macrophages
(Sun et al., 2000). The development of fibrosis beyond the infarct zone may be a
compensatory attempt to strengthen the myocardial wall post-I/R injury or may simply be
a response to chronically elevated TGF-β1 levels. This possibility may explain why
some of the values of fibrosis relative to the area at risk were greater than 1.0. It is also
noteworthy that this reactive interstitial fibrosis is maladaptive in that the expansion of
the interstitial space by the deposition of collagen results in decreased capillary distance
and increased oxygen diffusion distance, which could lead to chronic hypoxia and
adversely influence the function and viability of the collagen-encircled myocyte (Sabbah
et al., 1995).
4.3 Echo Findings
In agreement with the histological findings, the echocardiographic analysis in LV
wall and chamber dimensions revealed reduced wall thinning and decreased enlargement
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of the LV cavity in the hemin group relative to the buffer group at 4 days post-MI.
Changes in wall thickness were localized to the anterior LV wall in the hemin group,
whereas the buffer group showed significant thinning of the posterior wall compared to
the sham and hemin groups. The anterior wall thinning observed in the 4 day hemin
group at the base level is unexpected, particularly in light of the absence of significant
thinning at the mid level. There is a possibility this is a technical error in which the
placement of the 2D echo image cursor along the theoretical border between the base and
mid levels of the left ventricle was too close to the mid section but mistaken as base level
instead. This is likely the reason since there is no other remodeling of the LV at the base
level in the hemin 4 day group. Although it may seem surprising to see significant LV
remodeling at all 3 levels of the ventricle in the buffer group just 4 days post-MI, similar
findings have been reported by others in animals with large infarcts. For example,
Weisman and colleagues (1985) showed significant wall thinning and increased regional
radius of curvature in adjacent and remote regions of the heart within just 3 days after
infarction.
At 3 months, the degree of anterior wall thinning was still attenuated in the
hemin-treated animals compared to the buffer-treated animals despite comparable infarct
sizes. However, in spite of the attenuated wall thinning in the hemin-treated animals, LV
cavity enlargement did not differ significantly from the buffer-treated groups. Even
though significant attenuation in anterior wall thinning at the mid and apical levels was
observed in the hemin group, the mean infarct size was still large (> 40%), suggesting
that the presence of cavity enlargement may be an indication of blood volume congestion
in an attempt to maintain adequate CO in the face of reduced EF (from 75% pre-op to
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50% at 3 months). If the injury to the myocardium is substantial enough to affect pump
function, then it is plausible there is a compensatory increase in renin production in an
attempt to normalize blood volume and cardiac output. This increased volume exerts an
outward pressure on the ventricle walls in all directions, thereby explaining the cavity
dilatation despite attenuated anterior wall thinning. Thus, global myocardial remodeling
after infarction leads to a condition of overload on the surviving myocardium that
consists primarily of a volume component though a pressure component may be operative
as well (Olivetti et al., 1990). Congestion within the circulatory system significant
enough to effect cavity dilatation would occur over the long term and would indeed be
seen at 3 months post-MI in rats.
Interestingly, wall thinning and chamber dilatation were seen at all levels of the
LV at 3 months in both groups. It is surprising to observe anterior wall thinning at the
base of the ventricle (occurring in both treatment groups) since the area at risk for
infarction is primarily the mid and apical sections. However, both groups experienced
infarcts large enough to induce significant thinning at the anterior and posterior apical
wall as well as the anterior mid region.
When disproportionate wall thinning and
chamber dilatation occur in the infarct region, they are accompanied by a distortion in
shape of the entire heart including the normal remote myocardium (Weisman et al.,
1985). In both 3 month treatment groups, the mean chamber diameter changed very little
from the level of the apex to the level of the base, indicating both groups had adopted a
sac-like cardiac phenotype. The degree of ventricular remodeling was great enough that
the normal ellipsoid shape of the ventricle was lost. Upon volume overload, increased
end-diastolic pressure causes an increase in diastolic wall stress. Chamber enlargement
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accommodates the volume overload and returns diastolic pressure toward normal at first,
but chamber enlargement also results in increased systolic wall stress (McKay et al.,
1986). The anterior wall could have been subjected to forces and/or mechanical stretch
from blood volume congestion and aneurysm formation at the apex and mid level,
culminating in an apparent reduction in wall thickness at the base, despite a lack of
infarction at that level. Furthermore, wall thinning and architectural distortion at the base
could potentially lead to the cavity dilatation observed at the base level of both treatment
groups.
The significantly reduced posterior wall thickness at the mid level of the 3 month
hemin group is surprising, especially because it is nearly identical to the mean observed
in the buffer group (0.206 ± 0.10 cm vs 0.205 ± 0.011 cm, respectively). In both 3 month
treatment groups, the mid level posterior wall measurements are nearly identical to the
measurements at the base. This could indicate simply that the mid posterior wall did not
grow over 3 months in the groups that underwent LAD ligature compared with shamoperated group. It is unlikely that either treatment group experienced infarctions so large
as to induce wall thinning at the mid level posterior wall; however this possibility can not
be totally discounted. It is more probable, though, that the absence of increased posterior
wall thickness is due instead to considerable architectural changes at the apex and
anterior mid section, generating distorting forces and potentially subjecting the mid
posterior wall to mechanical stretch mistakenly interpreted as wall thinning due to
infarction.
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4.4 HO-1 Upregulation and Preservation of Coronary Vascular Homeostasis
A principal component in preventing left ventricular remodeling is myocardial
salvage and the prevention of wall thinning and infarct expansion.
As previously
mentioned, HO-1 upregulation reduces infarct size through multiple mechanisms, one of
which is likely a decrease in myocyte apoptosis which could explain the preservation of
the subendocardium revealed by H&E as well as the attenuated wall thinning observed in
the 4 day hemin group. One of the primary goals of reperfusion is to achieve infarctrelated artery patency which also influences the progression of ventricular remodeling. It
is critical that the remaining viable myocardium has unobstructed blood flow in order to
restore cellular ATP, re-establish cellular homeostasis and reverse the injury induced by
reperfusion. It is possible that some of the protection afforded by HO-1 upregulation
occurs through decreased neutrophil adhesion to the coronary microcirculation and
decreased occurrence of “neutrophil plugging”, thereby allowing prompt reperfusion to
the ischemic area. The production of CO through heme metabolism may ameliorate I/Rinduced endothelial dysfunction and reduce coronary vascular tone and possibly prevent
the no-reflow phenomenon.
4.5 Effect of HO-1 Upregulation of Post-Infarction Left Ventricular Function
In agreement with the hypothesis that HO-1 upregulated animals exhibit
attenuated ventricular remodeling, post-infarction ejection fraction and fractional
shortening were maintained in the 4 day hemin-treated animals while the buffer-treated
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group revealed significantly reduced EF and FS compared to sham and hemin groups.
The preservation of left ventricle structural integrity in the 4 day hemin-treated group
likely enables the ventricles to maintain proper pump function.
Although LV wall thinning was attenuated in the 3 month hemin-treated group
compared to the buffer group, the mid level ejection fraction and fractional shortening at
this time point were nearly identical in both groups. This may be an indication that the
degree to which the wall thinning was attenuated was not sufficient to prevent volume
congestion and subsequent pump dysfunction, evidenced by the significantly reduced EF
and FS in the hemin group at 3 months compared to sham.
Acute compensatory
responses serve to maintain pump function, including distension of the viable
myocardium and the operation of the Frank-Starling mechanism, and augmentation of
chronotropic and inotropic receptor stimulation (Pfeffer and Braunwald, 1990). It is
possible that the hemin-treated animals involved in the long-term study had sufficient
acute compensatory mechanisms to maintain normal cardiac function at 4 days but not
once they reached the 3 month time point. Moreover, it is important to consider that
post-infarction left ventricular remodeling in rats takes several months to complete.
Apical level EF and FS followed the same pattern observed at mid-level with the
exception that apical level EF was not significantly reduced at 4 days in the buffer-treated
group compared to sham. The mean EF was, however, less than both the sham and
hemin-treated groups. This may simply reflect varying degrees of infarction of the apical
region in this group, which could be due to differences in the architecture of the coronary
microvasculature in this region.
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4.6 Comparison of Short and Long Term Studies
While it was surprising at first to observe the unmistakeable prevention of acute
LV remodeling in the hemin group compared with the significant remodeling that took
place by 3 months, it should not be understated that the long term remodeling in the
hemin group was attenuated compared to the buffer group and may be biologically
significant. The percent change from baseline measurements in the hemin 3 month group
was noticeably decreased at the apex and mid level anterior and posterior walls.
Moreover, the expansion of the LV in the hemin group at the apex and mid levels was
reduced by 30% during systole compared to buffer. Coupled with the attenuation of wall
thinning at the apex and mid levels compared to buffer, this could indicate some
preservation of systolic function compared to buffer.
The 4 day time point is quite early within the remodeling process when it is not
yet complete. It is possible that HO-1 upregulation prevents remodeling in the short
term, before the insidious onset of architectural changes, or minimizes and/or delays the
process. It is also feasible that long term hemin administration does not upregulate HO-1
as expected. It is possible that long term hemin administration actually causes some
degree of toxicity rather than protecting tissues by repeated exposure of oxidative stress;
this could likely be the problem since hemin administration post-MI induces additional
oxidative stress independent of the oxidative stress due to the generation and activity of
ROS upon reperfusion. Alternatively, it is possible that continued hemin administration
does upregulate HO-1, but the window of opportunity for cardioprotection expires
beyond 4 days.
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Failing heart muscle generally exhibits distinct changes in intracellular Ca2+
handling including impaired removal of cytosolic Ca2+; reduced Ca2+ loading of the
cardiac sarcoplasmic reticulum (SR) with downregulation of SR Ca2+-ATPase 2
(SERCA2); and defects in SR Ca2+ release accompanied by impairment of cardiac
relaxation and systolic function (Marx et al., 2000; Morgan et al., 1990; Nordlie et al.,
2006). It is possible that increased HO-1 expression and a codependent activation of
soluble guanylyl-cyclase could lead to an increase in cGMP levels and to a decrease in
calcium overload (Maines, 1997), all of which involves components of the calcium
handling system including SERCA2a and phospholamban.
Indeed, SERCA2a gene
transfer has proven to normalize LV volumes and improve cardiac function in rats with
induced heart failure, while ablated phospholamban resulted in complete prevention of
the heart failure phenotype in a mouse model of dilated cardiomyopathy, along with
improved cardiac SR Ca2+ storage and release (del Monte et al., 2001; Minamisawa et al.,
1999). Considering the evidence provided above, one might have anticipated that the
hemin 3 month group would have exhibited preserved cardiac performance, at least to
some degree. The fact that it didn’t could strengthen the hypothesis that the upregulation
of HO-1 was not as robust in the 3 month hemin group as it was observed in the 4 day
hemin group.
4.7 Changes in Cardiac Mass
There were no significant differences in LV mass post-infarction between the
hemin and buffer-treated animals compared to sham at any level of the ventricle at 3
95
months. LV mass (expressed as a ratio to body weight) decreased markedly in all 3
experimental groups because the animals’ body weight increased far more over 3 months
than did their heart mass. There were also no significant differences among the groups in
the percent change from baseline within each group. If ventricular volume had been
measured instead, there likely would have been significant increases in both hemin and
buffer 3 month groups considering the LV chamber dilatation observed in both groups.
Pfeffer et al. (1982) examined cardiac mass and volume at 2, 21, and 100 days after MI
and noted a progressive decrease in the ratio of mass to volume, indicating that LV
volume was increasing out of proportion to mass. This suggests that volume-overload
hypertrophy, marked by sarcomere replication in series, fiber elongation and chamber
enlargement, occurs perhaps in response to increased end-diastolic wall stress (McKay et
al., 1986; Pfeffer et al., 1982). Considering there was significant cavity enlargement but
no significantly increased cardiac mass in both 3 month groups in the current study, it is
possible that both hemin and buffer 3 month groups experienced volume-overload as
proposed by McKay et al. (1986).
4.8 Role of NFkB in I/R injury
A significantly increased level of NFκB binding activity was observed in the 4
day buffer group compared with the hemin group at the same time.
Although no
statistical significance between the hemin and buffer groups was found at 24 hours postMI, the buffer group displayed a trend towards higher NFκB-DNA binding activity.
Much of the reperfusion-induced inflammatory response is due to modified gene
96
expression involving NFκB activity (Brown et al., 2005; Mercurio and Manning, 1999).
It is possible the smaller infarct size within the hemin-treated groups at 24 hours and 4
days is partly due to decreased apoptosis and reduced inflammatory response due to
lower NFκB activation than seen in the buffer-treated group. NFκB has been implicated
in the regulation of a myriad of pro-inflammatory and pro-apoptotic genes that participate
in I/R injury such as intracellular adhesion and chemoattractant molecules as well as
inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) (Brown et al., 2005). Brown et
al. (2005) reported >70% reduction in infarct size in NFκB activation-blocked transgenic
mice, clearly demonstrating the overall activity of NFκB in the heart after I/R leads to
injury.
The significant difference between the two groups at 4 days represents the second
wave of NFκB activation in keeping with the second wave of cytokine expression within
the remodeling phase. While the NFκB DNA binding activity at 24 hours was not
statistically significant (this could be due in part to the small sample size), there is a trend
towards greater NFκB activation among the buffer-treated animals. An argument could
be made that the decreased NFκB activation in the hemin 24 hours group is biologically
significant considering how central NFκB activation is to the propagation of
inflammation in reperfusion injury.
Since stimulation of NFκB by oxidative stress triggers the release of NFκB from
IκB proteins and results in its translocation to the nucleus to bind to DNA, one of the
mechanisms by which increased HO-1 expression attenuates NFκB binding activity may
occur by increasing antioxidant capacity even prior to the I/R and subsequently reducing
97
activation of NFκB.
NFκB activation is dependent on the phosphorylation and
degradation of IκB-α (Henkel et al., 1993); it is possible that increased HO-1 activity via
its by-products interferes with one or several essential components of the signal
transduction cascade involved in activation of NFκB (Rossi et al., 1997). Moreover,
Anderson et al. (1994) reported that tyrosine kinase activity is a common requirement for
NFκB activation and that tyrosine kinase activity is increased by oxidants (ROS upon
reperfusion, for example) and decreased by antioxidants (bilirubin, for example).
4.9 Hematocrit and Cardiac Function Post-I/R Injury
Erythropoetin (EPO), a cytokine produced by the adult kidney, is a well known
hematopoietic factor and increased HO-1 expression has recently been linked to EPO
treatment in hemodialysis patients (Moon et al., 2003; Calo et al., 2006). A benefit of
increasing the oxygen-carrying capacity of blood by increasing the red blood cell mass
(hematocrit) by blood transfusion has been reported in elderly patients with acute MI
(Wu et al., 2001).
The advantage of increasing oxygen-carrying capacity in a state of
oxidative stress is obvious. However, in this study, acute and chronic upregulation of
HO-1 by hemin did not result in significant differences in hematocrit at cardiac harvest
among any of the groups. It is possible the upregulation of HO-1 by hemin was not
robust enough to mediate changes in EPO expression. Conversely, EPO expression may
not be amplified or influenced by increased HO-1 activity in the setting of I/R injury.
Although EPO’s antiapoptotic effects strongly point to a central role for HO-1 as a
98
mediator, the literature reveals a very limited understanding of the interaction between
HO-1 and EPO and the pathways they influence (Calo et al., 2006).
4.10 Potential Therapeutic Application of HO-1 Up-regulation in Myocardial
Infarction
This project demonstrated a beneficial effect of hemin pre-treatment on
myocardial reperfusion injury via HO-1 upregulation in the short term and a mild
attenuation of injury in the long term. The trend of current and future research continues
to focus on methods of translating the protective properties of the endogenous HO-1
stress response into therapeutic strategies for the treatment of human disease (Ryter et al.,
2006). For example, experimentation with natural antioxidants and pharmaceuticals has
raised the possibility that HO-1 induction in tissue could be stimulated by agents selected
for their capacity to invoke the response without eliciting tissue damage (Ryter et al.,
2006). Some investigators believe hemin in particular is contra-indicated during a period
of tissue vulnerability. Balla et al. (1991) showed that hemin rapidly intercalates into
cultured endothelial cells resulting in their marked hypersusceptibility to subsequent
oxidant-mediated cytolysis by H2O2 or adherent polymorphonuclear leukocytes. These
authors further described the dichotomous effects of heme exposure on endothelial cells:
heme amplifies oxidant-mediated cytotoxicity during brief (2 h) exposure, yet markedly
protects oxidant-exposed target cells if provided for longer durations (Balla et al., 1992).
Because free iron is a potent pro-oxidant, increasing its tissue concentration via HO-1
upregulation during a reperfusion-induced respiratory burst is not clinically feasible.
99
Furthermore, it takes time for HO-1-upregulation to result in subsequent ferritin
upregulation and iron sequestration.
For this reason, the administration of CO and
biliverdin and/or bilirubin, rather than the induction of HO-1, may prove to be a safer and
more practical means of conferring cytoprotection in a clinical setting (Kirkby and Adin,
2006). Patients stratified at high risk for MI could be considered for pre-emptive therapy
involving the upregulation of HO-1 directly or administration of HO-1 byproducts. As
previously mentioned, carbon monoxide releasing molecules (CORMs) provide a safer,
alternative method of CO delivery than does direct CO inhalation which could induce
tissue hypoxia. Indeed, the administration of CORMs just before reperfusion has proven
to cause a reduction in infarct size similar to the effect afforded by early or late
preconditioning (Guo et al., 2004). The administration of CORMs and albumin-bound
bilirubin at the time of reperfusion in clinical practice might be an attractive method of
limiting reperfusion damage and enhancing cellular antioxidant defense.
Another
promising application of HO-1 upregulation is the development of hypoxia-regulated
vectors for delivery of HO-1. Because myocardial ischemia oftens occurs silently with
multiple episodes in the clinical setting, prompt treatment is required in spite of the
absence of symptoms. Pachori et al. (2006) provided the first evidence of the therapeutic
efficacy of pre-emptive adeno-associated virus-HO-1 delivery for the prevention against
recurrent ischemic injury. Furthermore, our laboratory has already shown the feasibility
of AAV-based HO-1 gene delivery for prevention of LV remodeling after single and
recurrent episodes of I/R (Liu et al., 2006; Melo et al., 2002).
It should be recognized, however, that the systemic effects of chronic HO-1
upregulation have not yet been fully elucidated. In fact, constitutive overexpression of
100
HO-1 may lead to cellular toxicity that may in turn lead to kernicterus and tissue hypoxia
caused by the catalytic byproducts of heme degradation (Platt and Nath, 1998). Tang et
al. (2004) developed a hypoxia-sensitive vigilant human HO-1 plasmid in mice that
provided cardioprotection through decreased apoptosis and fibrosis in the setting of
myocardial ischemia. The next step in the evolution of this endogenous cellular defense
system fit for short term protection may be to construct an adeno-associated viral plasmid
to make a viral vigilant long-term cardioprotective system (Tang et al., 2004).
4.11 Study Consideration
It is important to appreciate that coronary artery ligation in rats is not akin to the
complex development of coronary artery disease leading to myocardial infarction
requiring interventional reperfusion in the clinical setting. However, investigating postI/R histological and LV functional changes in animals provides insight to the changes
that occur from the onset of ischemia to reperfusion that lead to infarct expansion, left
ventricle chamber dilatation, ventricle function depression, and decreased cardiac output,
all of which may be likened to dilated cardiomyopathy and CHF. Pfeffer et al. (1979)
observed a wide range of physiological outcomes in the rat infarction model: normal
baseline ventricular filling pressures and cardiac output were observed in rat infarctions
constituting less than 47% of the left ventricle while more extensive infarcts were
associated with overt depression of cardiac performance manifested by elevated left
ventricle filling pressures and reduced mean arterial pressure and cardiac output. They
concluded that the rat model of coronary artery ligation provides an entire range of postinfarction function from an absence of detectable impairment to gross failure (Pfeffer et
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al., 1979). It is therefore important to recognize that recovery from ischemia reperfusion
injury after a large infarction (i.e. >40% of the left ventricle) in humans may not be as
successful as is often noted among rats and likely involves more complicated,
multivariate pathologies. In fact, ischemic heart disease is the most common cause of
CHF among human patients and often begins after acute myocardial infarction;
moreover, it is associated with a twofold increase in in-hospital morbidity and a fourfold
increase in in-hospital one year mortality (O’Connor et al., 1997). Although conclusions
drawn from animal models in basic science research may not necessarily be directly
applied to the clinical setting, the endeavour to reduce reperfusion injury and salvage
viable myocardium post-MI through experimentation is warranted and should remain a
priority.
4.12 Study Limitations
There are several technical and design limitations to consider within the current
study.
4.12.1 Sample size
Sample sizes upwards of twenty to thirty for each treatment group would have
been impossible to achieve due to the volume of experiments and time required for each
animal. This project focused on whole animal experiments to organ function studies and
molecular studies, all of which incurred a huge potential for biologic variation at any
point from the LAD ligation surgery to organ harvest. Hence, one must be careful about
102
drawing robust conclusions from the modest sample size in the long-term (3 month)
study. Surprisingly, three of the seven animals within the buffer-treated group did not in
fact experience a myocardial infarct in the LAD coronary artery (confirmed by histology)
and were excluded. An intrinsic limitation to this study was the inability to confirm with
certainty the production of a myocardial infarct while keeping the animal alive.
4.12.2 Use of volatile anaesthetic
Halothane was used to sedate the animals for the echo procedures. This volatile
anaesthetic was selected for its rapid induction of sedation and relative ease to administer.
However, volatile anaesthetics are extremely fast-acting and no gauge was available to
monitor the amount given to each animal. Two animals died not from coronary artery
ligation nor reperfusion injury but from halothane overdose due to unpredictable
responses to the drug. Furthermore, the effects of halothane on cardiac function were not
addressed. It would have been more prudent to use a ventilator equipped to deliver
measurable concentrations of anaesthesia known not to depress cardiac function.
4.12.3 Echocardiography technique
The animals’ chests were shaved using rodent hair clippers and commercially
available disposable razors.
In order to obtain clear 2-D and M-mode images, the
animals needed to be shaved very closely and required applying pressure to the chest wall
with each stroke of the razor. Because this was done while the animals were sedated, it
was challenging to accomplish good skin preparation without imposing pressure on the
103
heart. It may prove advantageous instead to use dilapitory cream for hair removal in
preparation for any future echocardiography studies in the lab.
Imaging of the rat’s apex is technically challenging. This is in part due to the size
of the rat heart’s apex and due to its position in the chest.
During human
echocardiography studies, a sonographer can ask patients to reposition or modify their
decubital position or take deep breaths to better image the apex during inspiration. It is
not possible to do this in immobilized animals. For this reason, mid-papillary level
images are often used for experimental studies.
4.13 Conclusions and Future Directions
The results of this project provide support to the general hypothesis that HO-1
upregulation via intraperitoneal administration of hemin in rats confers a cardioprotective
effect after I/R injury in the short term, as evidenced through histological and
echocardiographic examination of hemin-treated animals compared with buffer-treated
and sham-operated animals.
The cardioprotective effect was accompanied by the
induction of HO-1 and decreased NFκB activation; however this study provides no direct
evidence that the by-products of heme catabolism are responsible for the acute
cardioprotective effects of hemin. In the long term, chronic treatment with hemin did not
prevent post-infarction LV remodeling, though it was mildly attenuated. These findings
are limited by the small sample size. Future research should focus on optimizing HO-1
upregulation and its effect on left ventricular remodeling months after I/R injury once
104
remodeling is complete. The development of an ideal strategy for long term HO-1
upregulation (i.e. long term hemin dose and schedule or direct cardiac injection with a
hypoxia-sensitive HO-1 plasmid) will help establish whether or not HO-1 manipulation
yields therapeutic value in the setting of reperfusion injury, LV remodeling, and
congestive heart failure.
105
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