Download MK-886 and DITPA Attenuate Global Myocardial Ischemia

Heider SH. Qassam
MSc. PH. & TH.
Heart transplantation
It is a widely accepted therapy for most
patients under 65 years of age with
advanced heart failure who remain
symptomatic with the expectation of high
intermediate term mortality, despite
optimal heart failure medications.
Heterotopic Heart Transplantation
 The phrase `heterotopic' describes placing the
heart in an ectopic position without removing
the native heart. The heterotopic technique has
been used in experimental transplantation.
 It can be categorized into `working' and
`nonworking' models.
 It has several advantages , including technical
simplicity, better accessibility for biopsies, and
survival of the recipient even in case of graft
rejection.
Transplantation–Induced (I/R) Injury
 Organ transplantation is a unique situation
where grafts are successively subjected to global
cold ischemia, warm ischemia, and blood
reperfusion.
 These events are believed to impair graft
function.
 The pathophysiology of (I/R) injury shows several
characteristics of inflammatory responses
including activation of complement, endothelial
cells, infiltration of neutrophils, the release of
oxygen–derived free radicals, and cytokines.
Pathogenesis of Myocardial
(I/R) Injury
Ion accumulation
 Intracellular calcium overload.
 Increased intracellular sodium.
 Decrease in pH with rapid normalization upon
reperfusion.
Dissipation of mitochondrial
membrane potential
MPTP
Free radical formation/ROS
Generation from xanthine oxidase.
Release of reactive mitochondrial
intermediates.
Neutrophil infiltration.
NO metabolism
 Loss of NO-vasodilation.
 Accumulation of reactive peroxynitrite.
Apoptosis
Endothelial dysfunction
 Cytokine and chemokine signaling.
 Expression of cellular adhesion markers.
 Impaired vasodilation.
Immune activation
Innate immunity (e.g., complement
activation, expression of Toll-like
receptors)
Neutrophil accumulation
Cell-mediated damage (macrophage
and T cell)
Proinflammatory Mediators in
Myocardial (I/R) Injury
Tumour necrosis factor alpha
 It is a pleiotropic cytokine that has many
proinflammatory actions with negative inotropic
effects.
 It is produced by a host in response to
inflammation, tissue injury and has recently been
recognized as a myocardial depressant substance.
 TNF-α is released from macrophages, monocytes and
mast cells within minutes in response to myocardial
I/R injury.
 Interleukine-1 Beta
 IL-1β is a proinflammatory cytokine produced by
activated macrophages and monocytes. It functions
in the generation of systemic and local responses to
infection, injury, and is the primary cause of
inflammation.
 It has functions similar to TNF-α such as induction
of
chemokine production and expression of
adhesion molecules by endothelial cells.
 IL-1β plays a crucial role in initiating inflammatory
responses and in recruitment and activation
neutrophils to inflammation sites.
Intercellular Adhesion Molecule
(ICAM-1)
 It is a member of the immunoglobulin superfamily
and is critical for the firm arrest and
transmigration of leukocytes out of blood vessels
and into tissues.
 ICAM-1 plays an important role in the early
stages of cardiac damage following ischemia–
reperfusion injury. In this process, ICAM-1mediated leukocyte adhesion and subsequent
infiltration into the infarct area could be
responsible for myocyte damage via released free
radicals.
Cardiac Troponin I
Troponin I is a contractile protein " Part of
the thin filament regulatory complex that
confers calcium sensitivity to the ATP
activity of the striated muscle actin-myosin
complex".
 cTnI was found exclusively in cardiac
muscle and thus making it a specific
marker for myocardial injury.
MK-886
 It is a highly potent inhibitor of leukotriene
formation.
 This compound inhibits leukotriene biosynthesis
indirectly by a mechanism through the binding of
a membrane bound (FLAP), thereby inhibiting the
translocation and activation of 5-lipoxygenase
which is catalyses the conversion of arachidonic
acid to (LTA4).
It was found to be a potent and specific
inhibitor of both LTB4 and LTC4 synthesis in
human phagocytes and moderately potent
PPARα antagonist.
It was also found to prevents postischemic
leukotriene accumulation after I/R .
DITPA
 It is a TH analog with positive inotropic activity
but minimal effect on heart rate or metabolic rate
compared with thyroid hormone itself.
 DITPA can promote angiogenesis by interacting with
membrane-bound integrin αVβ3 and activating the
MAPK cascade.
 DITPA attenuates the acute inflammatory
response and reduces myocardial infarct size
following myocardial ischemia .
 DITPA enhances endothelial NO-mediated
vasorelaxation.
 DITPA prevent abnormal sarcoplasmic reticulum
calcium transport and abnormal contractile
function associated with MI.
Aim of the Study
 this study was undertaken to assess the
possible protective effect of MK-886 and
DITPA against global myocardial I/R injury
after heart transplantation via interfering
with inflammatory pathways.
Animals and Study Design
The rats were randomized into six groups as
follow
 Sham group: rats underwent the same
anesthetic and surgical procedures but with no
HHT .
 Control group: rats underwent 30 min of
global myocardial ischemia followed by 60
min of reperfusion via HHT.
 Control vehicle (1) group: Donors rats received
vehicle of MK-886 30 min before HHT, and the
same dose was repeated for recipients upon
reperfusion.
 Control vehicle (2) group: Donors and recipients rats
pretreated with DITPA vehicle for 7days before HHT.
 MK-886 treated group: Donors rats received MK886 (0.6 mg/kg) i.p. 30 min before HHT , and the
same dose was repeated for recipients upon
reperfusion.
 DITPA treated group: Donors and recipients rats
pretreated with DITPA(3.75 mg/kg) s.c. for 7days
before HHT.
Methods Overview
Isolate donor heart
 Bilateral
thoractomy
was made, and interior
chest wall was opend.
 All the vessels, except
the aorta and
pulmonary artery, were
ligated
 The heart removed &
stored in cold lactated
ringer solution.
Prepare recipient
animals
 The right CA was
exposed and then
occluded with clamp.
 Distal portion of CA
was ligated
 CA was then incised
and passed through
cuff.
 REJV was prepared the
in same way.
Heart
transplantation
 The donor heart was
placed in the right neck
of the recipient.
 The arterial cuff was
inserted into donor aorta
and fixed with a ligature.
 IV cuff was inserted into
the donor PA and fixed
with ligature.
Isolated donor
heart
Operation
Prepare recipient
rats
Heart
transplantation
Preparation of Samples
Blood Sampling for measurement of
plasma cTnI
 At the end of experiment, about 2 ml of blood
was collected from the heart and placed in a tube
containing Na2EDTA and used for determination
plasma cTnI.
 Tissue Sampling for Histopathology
 The apical part of the heart was fixed in 10%
formalin to be used for histopathological
evaluation
Tissue preparation for TNF-α, IL-1β and ICAM1 measurement
 10% homogenates of heart tissue were prepared
in PBS that contained 1% Triton X-100 and a
protease inhibitor cocktail.
 The homogenate was centrifuged at 2,500 g for
20 min at 4°C. The supernatant was collected for
determination of TNF-α, IL-1β and ICAM-1.
Histopathological Evaluation of
Rat Heart
 The scoring system for histological findings
was divided into 4 scores:
 Score 0, no damage.
 Score 1, interstitial edema and focal necrosis.
 Score 2, diffuse myocardial cell swelling and necrosis.
 Score 3, necrosis with the presence of contraction
bands, neutrophil infiltration and the capillaries were
compressed.
 Score 4, widespread necrosis with the presence of
contraction bands, neutrophil infiltration, capillaries
compressing and hemorrhage.
Statistical Analysis




ANOVA test was used for the multiple comparison
among all groups followed by post-hoc tests using
LSD method.
Mann-Whitney and Krusakl-Wallis tests were used to
assess
the
statistical
significance
of
histopathological parameters.
Pearson correlation coefficient was used to assess
the associations between two quantitative variable.
Spearman correlation coefficient was used to assess
the associations between two quantitative variable
when one of them was non-normally distributed.
This result is consistent with
Gurevitch et al. (1996) were the
first to demonstrate a significant
release of TNF-α in the rat
coronary effluent at 1 min after
reperfusion.
*
Ѱ,*
†,*
Figure (6): The mean of cardiac TNF-α level (pg/ml) in the six
experimental groups at the end of the experiment.
* vs. sham group, ѱ vs. control vehicle (1) group, †vs. control vehicle (2) group.
The data expressed as mean ±SEM (p<0.05)
This result is consistent with
Herskowitz et al . (1995) demonstrated
induced myocardial gene expression of
IL-1β after permanent LAD occlusion
and
temporary
LAD
occlusion
followed by reperfusion.
*
Ѱ,*
†,*
Figure (7): The mean of cardiac IL-1β level (pg/ml) in the six
experimental groups at the end of the experiment.
* vs. sham group, ѱ vs. control vehicle (1) group, †vs. control vehicle (2) group.
The data expressed as mean ±SEM (p<0.05)
This result is in agreement with
Kukielka et al. (1993) ) were reported
that
ICAM-1
gene
expression
increased in reperfused myocardium
after 1 hr coronary occlusion and 1 hr
reperfusion
*
Ѱ,*
†,*
Figure (8): The mean of cardiac ICAM-1 level (pg/ml) in the six
experimental groups at the end of the experiment
* vs. sham group, ѱ vs. control vehicle (1) group, †vs. control vehicle (2) group.
The data expressed as mean ±SEM (p<0.05)
This result is consistent with
Bertinchant et al. (1999) ) showed
that cTnI released in 1 min during
60 min of reperfusion after 20 min,
30 min, 40 min or 60 min of global
ischemia by using Langendorffperfused rat hearts model.
*
Ѱ,*
†,*
Figure (9): The mean of plasma (cTnI) level (ng/ml) in the six
experimental groups at the end of the experiment
* vs. sham group, ѱ vs. control vehicle (1) group, †vs. control vehicle (2) group.
The data expressed as mean ±SEM (p<0.05)
This result is cosistent with
zingarelli et al. (2002) showed that
a marked disruption of the
myocardial structure in
I/R
injury was characterized by
appearance of extensive necrosis
and contraction bands.
*
Ѱ,*
†,*
Figure (11): Error bar chart shows the difference in mean ±SEM values of
total severity scores in the six experimental groups.
* vs. sham group, ѱ vs. control vehicle (1) group, †vs. control vehicle (2) group.
The data expressed as mean ±SEM (p<0.05)
Figure (12 ): Photomicrograph of cardiac section
of normal rats shows the normal architecture.
The section stained with Haematoxylin and Eosin
(X 40).
Figure (13): Photomicrograph of cardiac section showed
interstitial edema and focal necrosis. The section stained
with Haematoxylin and Eosin (X 40).
Figure (14): Photomicrograph of cardiac section
showed neutrophil infiltration (arrows). The section
stained with Haematoxylin and Eosin (X 40).
Figure (15): Photomicrograph of cardiac section showed
capillary compressing. The section stained with
Haematoxylin and Eosin (X 40).
Figure (16 ): Photomicrograph of cardiac section showed
contraction bands. The section stained with Haematoxylin
and Eosin (X 40).
Figure (17): Photomicrograph of cardiac section
showed hemorrhage. The section stained with
Haematoxylin and Eosin (X 40).
Figure (18 ): Photomicrograph of cardiac
section in MK-886 treated group. The
section stained with Haematoxylin and
Eosin (X 40).
Figure (19): Photomicrograph of cardiac
section in DITPA treated group. The
section stained with Haematoxylin and
Eosin (X 40).
Conclusions
 The findings of this study suggest the followings:



Both MK-886 and DITPA ameliorate myocardial injury
associated with HT by reducing inflammatory mediators and
adhesion molecule. This may give an evidence for their
protective effect.
Both MK-886 and DITPA reduce cTnI associated with MI/R
injury induced by HT which is a specific marker for cardiac
injury.
This study supports the hypothesis that inflammatory
pathways are involved in global MI/R injury induced by HT.
Recommendations
we recommend the following:
 These findings have put MK-886 and DITPA as a future
therapeutic agents in situations including global MI/R injury
following heart transplantation through their interfering with
inflammatory pathways.
 Measuring of cardiac myeloperoxidase activity to estimate
tissue PMNs accumulation in inflamed hearts.
 Measuring of infarct size to evaluate the cytoprotection of
drugs used in this study and this is done by (TTC) staining.
 Measuring of LTB4 and LTC4 in the heart to further support
their role in the pathogenesis of MI/R injury.