Download Vagal Stimulation, Through its Nicotinic Action, Limits Infarct Size

ORIGINAL ARTICLE
Vagal Stimulation, Through its Nicotinic Action, Limits
Infarct Size and the Inflammatory Response to Myocardial
Ischemia and Reperfusion
Laura Calvillo, PhD,* Emilio Vanoli, MD,†‡ Elisa Andreoli, PhD,‡ Alessandra Besana, PhD,*
Elisabetta Omodeo, PhD,§ Massimiliano Gnecchi, MD, PhD,†¶ Pietro Zerbi, MD,§
Gianluca Vago, MD,§ Giuseppe Busca,k and Peter J. Schwartz, MD†¶**††
Abstract: Vagal activity has protective effects in ischemic heart
disease. We tested whether vagal stimulation (VS) could modulate the
inflammatory reaction, a major determinant of cardiac injury after
ischemia/reperfusion. Four groups of male rats underwent myocardial
ischemia (30 minutes) and reperfusion (24 hours). One group underwent VS (40 minutes), 1 VS plus atrial pacing (VS + Pacing), and
1 VS plus nicotinic inhibition by mecamylamine (VS + MEC). After
24 hours, the area at risk, infarct size, inflammation parameters, and
apoptosis were quantified. Infarct size was reduced in all VS-treated
rats (controls, 53 6 18%; VS, 6.5 6 3%; VS + Pacing, 23 6 6%;
VS + MEC, 33 6 9%; P , 0.005 vs. controls). The infarct size in the
VS + MEC group was larger than that in VS-treated animals, despite
similar heart rate, suggesting partial loss of protection. The number of
macrophages, neutrophils, and apoptotic cells in the area at risk and the
plasma cytokines levels were significantly reduced in all VS-treated
animals. In conclusion, VS decreases infarct size and inflammatory
markers during ischemia/reperfusion independent of the heart rate. The
anti-inflammatory and antiapoptotic properties of the nicotinic pathway
are the primary underlying mechanism. The vagally mediated modulation of inflammatory responses may prove valuable in the clinical
management of acute coronary syndromes and of heart failure.
Key Words: vagal activity, ischemia/reperfusion, myocardial infarction, autonomic nervous system, infarct size, inflammatory
markers
(J Cardiovasc Pharmacol TM 2011;58:500–507)
Received for publication May 12, 2011; accepted June 28, 2011.
From the *Laboratory of Cardiovascular Genetics, IRCCS Istituto Auxologico
Italiano, Milan, Italy; †Section of Cardiology, Department of Lung, Blood
and Heart, University of Pavia, Pavia, Italy; ‡Department of Cardiology,
Policlinico di Monza, Monza, Italy; §Pathology Unit, Institute of
Biomedical Sciences, L. Sacco Hospital, The University of Milan, Italy;
{Department of Cardiology, Fondazione IRCCS Policlinico S. Matteo,
Pavia, Italy; kDepartment of Cardiovascular Medicine, The University of
Milan, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico,
Milan, Italy; **Cardiovascular Genetics Laboratory, Hatter Institute for
Cardiovascular Research in Africa, Department of Medicine, University of
Cape Town, South Africa; and ††Department of Family and Community
Medicine, College of Medicine, King Saud University, Riyadh, Saudi
Arabia.
The authors report no conflict of interest.
Reprints: Peter J. Schwartz, MD, Department of Cardiology, Fondazione
IRCCS Policlinico S. Matteo, Viale Golgi 19, 27100 Pavia, Italy (e-mail:
[email protected]).
Copyright 2011 by Lippincott Williams & Wilkins
500
| www.jcvp.org
INTRODUCTION
Recent clinical evidence suggests that chronic vagal
stimulation (VS) is feasible in man and that it might be
beneficial in the setting of chronic ischemic cardiomyopathy
and heart failure.1,2 We are now advancing the hypothesis that
VS might have protective effects also at a much earlier stage,
when the initial ischemic insult occurs.
Ischemia and reperfusion have complex and deleterious
effects on the myocardium. Inflammatory mediators are
essential in the healing process of injured tissue, but when,
after reperfusion, their response is excessive, they may worsen
myocardial injury.3
Reperfusion is followed by rapid cellular infiltration of
neutrophils and monocytes and by the release of several
proinflammatory cytokines which orchestrate inflammation
and tissue repair processes.3,4 Among them, lipopolysaccharide-inducible CXC chemokine (LIX) and monocyte chemoattractant protein-1 (MCP-1) play a pivotal role.5,6 There is
a fine balance between the inflammatory reaction, which
follows reperfusion with the potential of extending myocardial
damage, and the mechanisms, which can positively affect
tissue repair. A sound approach to an effective management of
this important clinical problem has been hampered by what seems
to be a physiological catch 227: on one hand, inflammatory
mediators are essential in the healing of injured tissue; on the
other hand, an excessively intense inflammatory response after
reperfusion may actually worsen myocardial injury.
A rationale solution might be provided by identifying
means to physiologically modulate this inflammatory reaction.
One of the areas, largely unexplored and of potential relevance
in the modulation of the responses to ischemia and
reperfusion, is the neural control of the heart. We consider
the possibility that vagal activity might represent 1 such
physiological modulator. We focused on the vagus nerve
because there is evidence suggesting its significant role and
because over the years, our experimental and clinical studies
on the neural control of the heart8 have provided data relevant
to the present study. We demonstrated the protective effect of
VS in the prevention of ischemia-induced ventricular
fibrillation,9 the association between depressed vagal reflexes,
and increased cardiac mortality after a myocardial infarction,10,11 and more recently that chronic VS is not only feasible
and safe in man but that it can effectively contribute to the
management of patients with advanced heart failure.1,2
J Cardiovasc Pharmacol Volume 58, Number 5, November 2011
J Cardiovasc Pharmacol Volume 58, Number 5, November 2011
A protective role of 24-hour VS in experimental
myocardial ischemia was suggested because of its association
with decreased infarct size, cytokine expression, and neutrophil
infiltration in reperfusion injury12,13 and represents an area of
active research.14 To better understand the mechanisms underlying this apparent protection, we discriminated between the
nicotinic and muscarinic actions of vagal activity and we also
tested whether a more clinically realistic 40-minute stimulation
would retain its efficacy.
MATERIALS AND METHODS
Our investigation conforms with the Guide for the Care
and Use of Laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85-23, revised
1996), it was approved by the Ethics Review Board of the
Italian Ministry of Health, and all procedures were performed
in accordance with the animal care guidelines of Federation
of Laboratory Animal Science Associations.15 Ischemic injury,
with or without VS, was made in male Sprague-Dawley rats
(250–295 g).
Experimental Protocol
Sprague-Dawley rats were randomized into 5 groups,
and results are reported for the 44 animals that completed the
protocol. All treatment animals received a bolus of 5 mg/kg
carprofen SC 30 minutes before surgery as analgesic. The
protocol of each group is as follows:
Group 1 (n = 13): controls; ischemia (30 minutes) and
reperfusion (24 hours). The right vagus was exteriorized but
not stimulated.
Group 2 (n = 6): ischemia and reperfusion with VS (the right
cervical vagus was stimulated from 5 minutes before
ischemia to 5 minutes after reperfusion).
Group 3 (n = 9): same as group 2 but with the addition of left
atrial pacing to maintain heart rate at same level present in
group 1.
Group 4 (n = 9): same as group 2 but with the addition of
mecamylamine (MEC) injection in the heart to inhibit
nicotinic receptors.16
Group 5 (n = 7): sham-operated rats, no ischemia and
reperfusion.
Ischemia and Reperfusion
All rats were anesthetized with isoflurane and ventilated
through an endotracheal cannula. Anesthetized animals were
continuous monitored for body temperature and electrocardiogram (ECG).
The left coronary artery was ligated with 4-0 silk suture
(Ethicon), and a plain knot was tied over 2 pieces of suture that
were pulled after 30 minutes to reperfuse the heart.17 The chest
was closed under negative pressure. Ischemia was always
confirmed by the appearance of discoloration of the cardiac
surface and ST elevation on ECG, whereas reperfusion was
verified by reddening of previously discolored area and by the
presence of arrhythmias on ECG. Sham-operated rats underwent the same surgical procedures, without ligation of the
left coronary artery.
2011 Lippincott Williams & Wilkins
VS Protects From Ischemia/Reperfusion
Nicotinic Inhibition
MEC, the inhibitor of nicotinic receptors, was injected in
the coronary arteries of the animals assigned to group 4. After
clamping the ascending tract of the aortic arch, a bolus of
MEC (2.1 mg/kg) was injected in the left ventricle. The clamp
was removed after the injection; overall, the procedure lasted
less than 30 seconds.
VS and Pacing
Electrical stimulation was performed by a digital
stimulator Grass S88. The right vagus nerve was exteriorized
and isolated at the cervical level through a single midline
incision, and an electrode was placed around it. Stimulus
parameters were 2.5 V, 0.5-ms pulse duration, and 8–10 Hz
frequency.9 Atrial pacing was performed by means of a
29-gauge needle electrode placed in the left atrium, whereas
the reference electrode was placed on the chest. In the VS +
Pacing group, heart rate was maintained at 330 beats per
minute (bpm), (the average value observed in the control rats).
Sample Collection and Determination of Area
at Risk and Infarct Size
After 24 hours of reperfusion, the chest was reopened
under anesthesia and ventilation. One milliliter of blood was
withdrawn in heparin and centrifuged for 14 minutes at 3500
revolutions per minute at 4C, and plasma was stored at
220C. Then the ligature around the coronary artery was
tightened again and 2 mL of 5% Evans Blue in saline solution
was injected into the right ventricular chamber to distinguish
the area at risk from the perfused area of the left ventricle. The
heart was then excised and cut transversally into 3 main slices.
In the first slice, the septum and the area at risk were excised
and quickly frozen in liquid nitrogen. The second slice was
fixed in formalin for histology and immunohistochemistry.
The third slice was frozen at 220C in optimal cutting
temperature compound for tissue inclusion and cut transversally in 1-mm serial sections with the McIlwain tissue
chopper and incubated for 20 minutes at 37C in triphenyltetrazolium chloride. Photographs of the sections were taken, and
the areas of perfused tissue (blue), viable ischemic tissue (red),
and necrotic ischemic tissue (white) were measured with an
image analyzer (Image Tool UTHSCSA for Windows) and was
used to quantify the perfused left ventricle, the area at risk, and
the necrotic area (Fig. 1). The necrotic area was expressed as the
percentage of the area at risk.
Determination of Macrophage and Neutrophil
Infiltration, a-7 Subunit of the Nicotinic
Acetylcholine Receptor Expression on Cardiac
Macrophages, and Apoptosis
One coronal slice of the heart was formalin-fixed and
paraffin-embedded; then, 4 serial sections were cut from each
block and the following immunostaining reactions were
performed.
Macrophages
After deparaffinization in xylene and endogenous
peroxidase blockade with 3% hydrogen peroxide for 20 minutes,
antigen retrieval was carried out by microwave treatment in 0.01
www.jcvp.org |
501
Calvillo et al
J Cardiovasc Pharmacol Volume 58, Number 5, November 2011
FIGURE 1. Determination of area at
risk and infarcted zone. Evans Blue in
saline solution was injected into the
right ventricle to distinguish the area
at risk from the perfused area of the
left ventricle. Photographs of the
sections were taken, and the areas of
perfused tissue (blue), viable ischemic tissue (red), and necrotic tissue
(white) were measured with an
image analyzer and used to quantify
the perfused left ventricle, the area
at risk, and the necrotic area. Twenty-four hours after surgery, the infarcted area was significantly reduced in VS and in VS + Pacing
(VS + PAC)–treated animals with respect to controls.
M EDTA buffer. Slides were incubated for 2 hours at room
temperature with a mouse antirat CD68 monoclonal antibody
(1:100; AbD Serotec, Oxford, United Kingdom). After washing
with Tris buffer solution, slides were incubated with a ‘‘mouse
on rat’’ HRP-polymer kit (Biocare Medical, Concord, CA) and
Super Sensitive IHC detection system (BioGenex, San Ramon,
CA), respectively. Peroxidase activity was visualized with
diaminobenzidine. Slides were counterstained with hematoxylin.
Double Immunofluorescence Staining for Macrophages and a-7nAChR
After incubation with the primary antibodies (mouse
antirat CD68 at 1:50 and a rabbit antirat polyclonal antibody
a-7nAChR 1:10 from Abcam, Cambridge, United Kingdom),
slides were incubated with secondary antibodies (Alexa Fluor
488 antirabbit and IgG and Alexa Fluor 594 antimouse IgG;
Invitrogen, Carlsbad, CA) both at 1:500 dilutions. Nuclei were
counterstained with Hoechst (1:500).
Polymorphonuclear Leukocytes
Naphthol AS-D chloroacetate esterase kit (Sigma-Aldrich,
Inc, St Louis, MO) was used to detect polymorphonuclear
leukocytes (PMN).
Apoptosis
In situ detection of apoptosis was performed using
CardioTacs kit (Trevigen, Inc, Gaithersburg, MD) according to
the manufacturer’s instructions. Optimized conditions in our
laboratory were permeabilization with proteinase K at 37C
for 15 minutes and labeling procedure with TdT enzyme for
90 minutes.
Plasma Cytokines Expression
Cytokine Array
To detect LIX expression, we used a Rat Cytokine Array
Panel A kit (R&D Systems) following the kit instructions.
Briefly, the membranes were blocked with a blocking buffer
and then plasma was incubated for 1 hour at room temperature.
The blocking buffer was then removed from the membranes,
and the sample–antibody mix was added for the overnight
incubation at 4C. After washing, the membranes were
incubated at room temperature for 30 minutes with 1.5 mL of
horseradish peroxidase–conjugated streptavidin and then
502
| www.jcvp.org
thoroughly washed. The membranes were then developed by
using enhanced chemiluminescence technique (GE Healthcare, formerly Amersham Biosciences), exposed to x-ray film,
and processed by autoradiography.
To detect MCP-1 expression, we used a Rat Cytokine
Antibody Array kit (RayBiotech) following the kit instructions. Briefly, the membranes were blocked with a blocking
buffer and then incubated with the plasma samples for 2 hours
at room temperature. The membranes were washed and then
incubated with 1 mL of primary biotin-conjugated antibody at
4C overnight. After washing, the membranes were incubated
at room temperature for 2 hours with 2 mL of horseradish
peroxidase–conjugated streptavidin and then thoroughly
washed. The membranes were then developed by using
enhanced chemiluminescence technique, exposed to X-ray
film and processed by autoradiography.18 For data analysis,
autoradiographs of the arrays were scanned to determine the
density of the protein array positions and pixel intensity was
measured with the image analyzer NIH ImageJ 1.41 software
for Windows. The values from scans were adjusted based on
the intensity of control spots on the filter corners.
Statistical Analysis
Continuous variables are presented as mean 6 SD. Their
comparisons between groups were performed by 1-way analysis
of variance. All variables, with the exception of heart rate and area
at risk, for which the analysis of variance showed significant
differences were further analyzed by post hoc comparisons using
the Bonferroni method. Whenever the assumption of homogeneity of variance, evaluated by the Levene test, was questionable,
the Games–Howell test for multiple comparisons was used. P ,
0.05 was considered significant.
RESULTS
Effect of VS on Infarct Size and Heart Rate
At baseline, heart rate was similar in all groups (Table 1).
Similarly, the area at risk, calculated as percentage of the left
ventricle, was not significantly different (61 6 16% in the control
group, 52 6 8% in the VS group, 60 6 10% in the VS + Pacing
group, and 48 6 8% in the VS + MEC group; P = NS). VS
markedly decreased infarct size, compared with control, from
53 6 18% to 6.5 6 3% (P , 0.001). Keeping heart rate
controlled by atrial pacing blunted, but did not eliminate, the
2011 Lippincott Williams Wilkins
J Cardiovasc Pharmacol Volume 58, Number 5, November 2011
VS Protects From Ischemia/Reperfusion
TABLE 1. Effect of VS and Nicotinic Receptor Inhibition on Heart Rate, Area at Risk, Infarct Size, Apoptosis, Macrophages, and
PMN After Ischemia and Reperfusion
Groups
Control
VS
VS + Pacing
VS + MEC
Sham
Basal
HR (bpm)
332
303
301
327
344
6
6
6
6
6
43
38
41
25
54
HR After
Ischemia or
Sham (bpm)
334
234
330
251
303
6
6
6
6
6
39
15†
0{
17†
49
AAR/LV
(%)
61
52
60
48
6 16
68
6 10
68
NA
IS/AAR
(%)
53
6.5
23
33
6 18
6 3†
6 6**f
6 9*{
NA
No.
Macrophages
in AAR
1406
491
600
962
33
6
6
6
6
6
343
240†§
215†¤
381¤
28†
No. PMN
in AAR
174
48
60
142
42
6
6
6
6
6
59
39**
41**
85§
27†
No. Apoptotic
Cell in
AAR
190
49
59
153
45
6
6
6
6
6
88
27*
10*
67§§
9*
*P , 0.05, **P , 0.01, †P , 0.001 vs. control.
§P , 0.05, fP , 0.01, {P , 0.001 VS + Pacing or VS + MEC vs. VS.
§P , 0.05 vs. sham; ¤P , 0.01 vs. sham.
AAR, area at risk; HR, heart rate; IS, infarct size; LV, left ventricle; NA, not applicable.
protective effect of VS, as infarct size was still significantly
reduced to 23 6 6% in the VS + Pacing group (P , 0.01; Fig. 1).
Inhibition of cardiac nicotinic receptors by MEC reduced the
protective effect of VS because the infarct size in group 4 (VS +
MEC) was significantly larger (33 6 9% vs. 6.5 6 3%, P ,
0.001) despite a similar reduction in heart rate during ischemia
(234 6 12 bpm in the VS group, 251 6 17 bpm in the VS +
MEC group; P = NS).
VS and Inflammatory Response
Effects on Infiltrating Cells
Compared to control animals, VS significantly reduced
the number of infiltrated macrophages in all treatment groups
with the exception of VS + MEC (Figs. 2A, B and Table 1,
P , 0.001 for all comparisons). Pacing did not affect the result
of VS, as the number of infiltrating macrophages in the area at
risk remained essentially the same in these 2 groups (491 6
240 and 600 6 215 in the VS and the VS + Pacing groups,
respectively). Cardiac nicotinic receptor inhibition associated
with VS produced a nonsignificantly greater number of
macrophages compared with VS (962 6 381 vs. 491 6 240,
NS). In the sham-operated animals, the number of infiltrated
macrophages in the left ventricle free wall was extremely low
(33 6 28) and significantly lower compared with all the other
groups (Table 1). The macrophages infiltrating the infarcted
and reperfused myocardial tissue expressed immunoreactivity
for the a-7nAChR (Fig. 3).
VS had a similar effect in reducing the number of PMN
in myocardial tissue 24 hours after ischemia and reperfusion.
FIGURE 2. Macrophage and PMN
infiltration in the area at risk. Immunohistochemical staining shows macrophage and PMN infiltration in
control animals (A and C, respectively) and in animals treated with VS
(B and D) (magnification, 320).
2011 Lippincott Williams & Wilkins
www.jcvp.org |
503
Calvillo et al
J Cardiovasc Pharmacol Volume 58, Number 5, November 2011
FIGURE 3. Immunostaining for the
a-7nAChR and CD68. A, Staining for
a-7nAChR in green. B, CD68 staining in red; the microphotograph in
(C) represents the merge of (A) and
(B). D, Another representative double staining for a-7nAChR and
CD64, clearly demonstrating that
the infiltrating macrophages coexpress a-7nAChR receptor.
Compared with control animals, the number of PMN was
reduced significantly (P , 0.01) in both VS and VS + Pacing
groups (Figs. 2C, D and Table 1). In contrast, the number of
PMN in the VS + MEC group was not significantly different
from the control group and from the VS and VS + Pacing
groups.
In the sham-operated animals, the number of PMN was
42 6 27, markedly lower versus controls (P , 0.001), lower
versus VS + MEC group (P , 0.05), but similar to that of VS
and VS + Pacing groups.
Effects on Cytokines
A rat cytokine-array approach, to detect the presence of
proteins in the plasma, was used to identify specific cytokines
released after myocardial ischemia and reperfusion injury.
Data from a representative array are shown in Figure 4. The
signal intensity for the 2 cytokines involved in the recruitment
of neutrophils (LIX) and macrophages (MCP-1) was 2-fold
higher in the heart of control rats when compared with that in
sham-operated rats. VS, even in presence of controlled HR,
decreased the signal intensity by almost 2-fold, thus reversing
FIGURE 4. Rat protein array analysis was used to
determine the differences in the level of rat plasma
cytokines during ischemia and reperfusion injury
(control). Cytokines levels were decreased by VS +
Pacing (VS + PAC) treatment. Autoradiographs of the
arrays were scanned to determine the density of the
protein array positions. The values from scans were
adjusted based on the intensity of control spots on
the filter corners, and the level decreases for specific
cytokines are shown.
504
| www.jcvp.org
2011 Lippincott Williams Wilkins
J Cardiovasc Pharmacol Volume 58, Number 5, November 2011
the consequences of ischemia and reperfusion on cytokine
plasma levels. Local inhibition of nicotinic receptors in VS +
MEC–treated rats did not affect the plasma levels of LIX and
MCP-1 with respect to VS-treated animals.
VS and Apoptosis
Compared with control myocardial tissue, which contained 190 6 88 apoptotic cells per area at risk, the myocardium
of rats undergoing VS showed (Fig. 5) a marked and significant
reduction in their number (49 6 27), which was not modified by
pacing (59 6 10) (P , 0.05 for both comparisons). Cardiac
nicotinic receptor inhibition resulted in an increased number of
apoptotic cells with respect to VS group (153 6 67 in VS +
MEC group vs. 49 6 27 in VS-treated rats, P # 0.05). In the
sham-operated animals, this number was 45 6 9, almost
identical to that observed with VS and similar to that of VS +
Pacing (NS); In contrast, it was significantly (P , 0.05) lower
compared with controls and also with VS + MEC, thus showing
that nicotinic receptor inhibition had largely interfered with the
protective effect of VS.
DISCUSSION
The main finding of the present study is that a brief
period of VS, independent of its muscarinic action, drastically
limits infarct size and favorably attenuates the physiological
response to acute myocardial ischemia and reperfusion by its
nicotinic pathway. This protective effect is independent from
heart rate changes, is demonstrated by a significant reduction
in the number of macrophages, PMN, and apoptotic cells, and
is paralleled by decreased levels of circulating proinflammatory cytokines.
These results provide new insights on how the neural
control of the heart may modulate cardiac responses to lifethreatening events and limit, or worsen, the damage produced
by ischemia and reperfusion. Also, they may offer a clue to
understand, at least in part, some recent results obtained by
chronic VS in patients with heart failure, which are not entirely
explained by heart rate changes.1,2
Cardiac Responses During
Ischemia–Reperfusion and the Role of VS
At the time of reperfusion, blood containing leukocytes
enters an area rich in chemotactic factors and inflammatory
mediators.19,20 VS inhibits not only 2 of the key players in this
VS Protects From Ischemia/Reperfusion
process, LIX (IL-8 analogue in the rat) and MCP-1, but also
the infiltration by PMN and macrophages, which is essential in
the entire innate immune process after myocardial reperfusion
injury.21
LIX is a murine chemokine with almost the same function
as IL-8, responsible for 80% of PMN infiltration into an ischemic
myocardium.5 MCP-1 (CCL2) is a small cytokine with profibrotic
properties that recruits monocytes, memory T cells, and dendritic
cells to sites of tissue injury and seems to regulate fibrous tissue
deposition in the injured heart, critically regulating also mononuclear cell recruitment and activation in healing myocardial
infarcts.6,22 All these cytokines have proinflammatory properties
that seem to be clinically relevant. LIX attracts PMN in the site of
injury, thus allowing the intense reaction triggered by myeloperoxidase (MPO). MPO is a peroxidase present in PMN that
damages cardiomyocytes during reperfusion. MPO serum levels
in patients with acute coronary syndrome, monitored during a 6month follow-up, correlate significantly with increased risk for
subsequent cardiovascular events.23
MCP-1, responsible for the recruitment of monocytes to
sites of inflammation, seems to play a critical role in
atherosclerosis and in remodeling after myocardial infarction.
In a large cohort of patients with acute coronary syndromes, an
elevated baseline level of MCP-1 was associated both with
traditional risk factors for atherosclerosis and with an
increased risk for death or myocardial infarction.24 Moreover,
in patients with congestive heart failure, MCP-1 levels were
significantly inversely correlated with left ventricular ejection
fraction and were particularly high in those with heart failure
of ischemic origin.25
VS inhibits also the apoptotic process in the ischemic
and reperfused myocardium, a possible explanation being
a PI3K/Akt/HIF-1alpha pathway activation inside the target
cells. This would be supported by the evidence that
acetylcholine had protective effects on rat cardiomyocytes
subjected to normoxia/hypoxia by increasing Akt phosphorylation and preventing hypoxia-induced apoptosis and
mitochondrial membrane potential collapse.26 Activation of
the same pathway by VS could explain our results.
When taken together, these data strongly suggest that the
immune reactions occurring during acute myocardial ischemia
can worsen prognosis and that the molecules analyzed in the
present study and modified by VS are likely to play a major
role in the harmful side effects of innate immune responses.
FIGURE 5. Apoptotic cells in the left
ventricle. Myocardial tissue from
control animals (A) had significantly
higher number of apoptotic cells/
slice compared with vagal stimulated rats (B) (magnification, 320).
2011 Lippincott Williams & Wilkins
www.jcvp.org |
505
Calvillo et al
J Cardiovasc Pharmacol Volume 58, Number 5, November 2011
VS and Nicotinic Inhibition
The specific functional role of the nicotinic pathway in
our results was assessed by inhibiting nicotinic receptors with
MEC during VS. The myocardial nicotinic receptors were
inhibited by a local injection in the heart without affecting the
systemic cholinergic preganglionic neurons. Despite a similar
heart rate–lowering effect, cardiac nicotinic receptor inhibition
by MEC largely prevented the reduction of both the infarct size
and the number of macrophages, PMN and apoptotic cells
produced by VS alone. Furthermore, local inhibition of the
nicotinic receptors did not affect the plasma content of LIX
and MCP-1 with respect to VS-treated animals, suggesting
that the nicotinic pathway importantly contributed to the
vagally mediated cardioprotection.
It is true that 77% of the infarct size reduction produced
by VS was lost after inhibition of the nicotinic receptors, but
this also means that 23% is contributed by the muscarinic
component. We have elected not to block muscarinic receptors
because the ensuing large increase in heart rate would have
introduced a significant confounder.
The Cholinergic Anti-inflammatory Pathway
We explored whether it might be reasonable to suggest
an involvement of the cholinergic anti-inflammatory pathway
in myocardial ischemia and reperfusion injury. This pathway
is a bidirectional communication between the brain and
the immune system that seems to play a critical role in the
control of inflammation as cholinergic neurons inhibit acute
inflammatory response and, conversely, inflammation in
peripheral tissue alters neural signaling in hypothalamus.27,28
It seems that one of the main cell types involved in this
process is the macrophage, through the a-7nAChR on its
surface.28–30
We tested whether the macrophages infiltrated in the
heart would express a-7nAChR on their surface after ischemia
and reperfusion, which would suggest a possible involvement
of the cholinergic anti-inflammatory pathway in myocardial
reperfusion injury. By performing a double staining with
immunofluorescence on macrophages infiltrated in the myocardial injured tissue, we did indeed observe that they exhibited
immunoreactivity for the subunit a7 on their surface. The
macrophages expressing a-7nAChR were detected in several
zones of the area at risk.
The lack of a commercial antibody able to neutralize
bioactivity in vivo hampered our possibility of inhibiting
a-7nAChR receptors on infiltrated macrophages or in other
cells in the rat. As a consequence, we regard the a-7nAChR
staining on macrophages surface just as a further step
suggesting the involvement of the cholinergic anti-inflammatory pathway in myocardial reperfusion injury. However,
despite the evidence of an anti-inflammatory and cardioprotective property of the nicotinic component of VS, we could
not discriminate between the different subunits of the nicotinic
receptor. It is reasonable to posit that nicotinic protection could
actually be mediated by the a-7 receptor subunit, but the
evidence for the real contribution of the cholinergic antiinflammatory pathway to the myocardial response to reperfusion injury must await further proof.
506
| www.jcvp.org
Relationship With Heart Failure of Ischemic
Origin and Clinical Implications
The present data are clinically relevant, given that the
strong evidence, experimental and clinical,13–21 linking depressed vagal activity to poor outcome in patients with ischemic
heart disease and heart failure, has already prompted a first-inman assessment of the feasibility and safety of chronic VS with
encouraging results.1,2 Our data show that even a brief VS,
lasting only 40 minutes during ischemia and reperfusion can
protect the myocardium from inflammatory and ischemic injury.
Uemura et al13 observed cardioprotection after 24 to 72 hours of
continuous VS after ischemia and reperfusion. A shorter (,1
hour) VS would be clinically implementable and might be
considered in conjunction with angioplasty or other clinical
conditions with a potential for reperfusion injury, such as bypass
grafting or cardiac transplant.
VS shows a remarkable ability to modulate innate
immune responses, cellular networks, and organ homeostasis.
A major difference between a simple pharmacological
inhibition of cytokines or leukocytes and VS is that the latter
represents an already existing physiological anti-inflammatory
mechanism, which just needs reinforcement. The possibility of
fine-tuning, physiologically a complex process involving
cytokines, PMN, and neurohormones, is especially attractive
in light of the current failure of pharmacological interventions
aimed to modulate the inflammatory responses to ischemic
heart disease and heart failure.31,32 The fact that all these
responses can be modulated by VS points to unexpected cross
talk between pathways present in the myocardium and raises
the possibility of novel therapeutic interactions.33
ACKNOWLEDGMENT
The authors are grateful to Dr Lidia Cova and Dr Renato
Pataccini for their very helpful technical advices and for their
professional support, to Dr. Carla Spazzolini for expert
statistical assistance, and to Pinuccia De Tomasi for expert
editorial support.
REFERENCES
1. Schwartz PJ, De Ferrari GM, Sanzo A, et al. Long term vagal stimulation
in patients with advanced heart failure. First experience in man. Eur J
Heart Fail. 2008;10:884–891.
2. De Ferrari GM, Crijns HJ, Borggrefe M, et al; for the CardioFit
Multicenter Trial Investigators. Chronic vagus nerve stimulation: a new
and promising therapeutic approach for chronic heart failure. Eur Heart J.
2011;32:847–855.
3. Entman ML, Smith CW. Postreperfusion inflammation: a model for
reaction to injury in cardiovascular disease. Cardiovasc Res. 1994;28:
1301–1311.
4. Hawkins HK, Entman ML, Zhu JY, et al. Acute inflammatory reaction
after myocardial ischemic injury and reperfusion. Development and use of
a neutrophil-specific antibody. Am J Pathol. 1996;148:1957–1969.
5. Chandrasekar B, Smith JB, Freeman GL. Ischemia-reperfusion of rat
myocardium activates nuclear factor-kb and induces neutrophil infiltration
via lipopolysaccharide-induced CXC chemokine. Circulation. 2001;103:
2296–2302.
6. Frangogiannis NG, Dewald O, Xia Y, et al. Critical role of monocyte
chemoattractant protein-1/CC chemokine ligand 2 in the pathogenesis of
ischemic cardiomyopathy. Circulation. 2004;115:584–592.
7. Heller J. Catch 22. Great Britain, United Kingdom: Jonathan Cape Ltd;
1962.
2011 Lippincott Williams Wilkins
J Cardiovasc Pharmacol Volume 58, Number 5, November 2011
8. Levy MN, Schwartz PJ. Vagal Control of the Heart: Experimental
Basis and Clinical Implications. Armonk, NY: Futura Publishing Co;
1994:644.
9. Vanoli E, De Ferrari GM, Stramba-Badiale M, et al. Vagal stimulation and
prevention of sudden death in conscious dogs with a healed myocardial
infarction. Circ Res. 1991;68:1471–1481.
10. Schwartz PJ, Vanoli E, Stramba-Badiale M, et al. Autonomic mechanisms
and sudden death. New insights from analysis of baroreceptor reflexes in
conscious dogs with and without a myocardial infarction. Circulation.
1988;78:969–979.
11. La Rovere MT, Bigger JT Jr, Marcus FI, et al; for the ATRAMI (Autonomic
Tone and Reflexes After Myocardial Infarction) Investigators. Baroreflex
sensitivity and heart-rate variability in prediction of total cardiac mortality
after myocardial infarction. Lancet. 1998;351:478–484.
12. Katare RG, Ando M, Kakinuma Y, et al. Differential regulation of TNF
receptors by vagal nerve stimulation protects heart against acute ischemic
injury. J Mol Cell Cardiol. 2010;49:234–244.
13. Uemura K, Zheng C, Li M, et al. Early short-term vagal nerve stimulation
attenuates cardiac remodeling after reperfused myocardial infarction.
J Card Fail. 2010;16:689–699.
14. Li W, Olshanky B. Inflammatory cytokines and nitric oxide in heart failure
and potential modulation by vagus nerve stimulation. Heart Fail Rev.
2011;16:137–145.
15. Nicklas W. FELASA guidelines for the accreditation of health monitoring
programmes and for testing laboratories involved in health monitoring.
Lab Anim. 2010;44:69.
16. Li XW, Wang H. Non-neural nicotinic alpha 7 receptor, a new endothelial
target for revascularization. Life Sci. 2006;78:1863–1870.
17. Michael LH, Entman ML, Hartley CJ, et al. Myocardial ischemia and
reperfusion: a murine model. Am J Physiol. 1995;269:H2147–H2154.
18. Xu Y, Kulkosky J, Acheampong E, et al. HIV-1-mediated apoptosis of
neuronal cells: proximal molecular mechanisms of HIV-1-induced
encephalopathy. Proc Natl Acad Sci U S A. 2004;101:7070–7075.
19. Pinckard RN, Olson MS, Kelley RE, et al. Antibody-independent activation
of human C1 after interaction with heart subcellular membranes. J Immunol.
1973;110:1376–1382.
20. Ley K. Histamine can induce leukocyte rolling in rat mesenteric venules.
Am J Physiol. 1994;267:H1017–H1023.
2011 Lippincott Williams & Wilkins
VS Protects From Ischemia/Reperfusion
21. Frangogiannis NG, Youker KA, Rossen RD, et al. Cytokines and the
microcirculation in ischemia and reperfusion. J Mol Cell Cardiol. 1998;
30:2567–2576.
22. Frangogiannis NG. Chemokines in the ischemic myocardium: from
inflammation to fibrosis. Inflamm Res. 2004;53:585–595.
23. Baldus S, Heeschen C, Meinertz T, et al; CAPTURE Investigators.
Myeloperoxidase serum levels predict risk in patients with acute coronary
syndromes. Circulation. 2003;108:1440–1445.
24. de Lemos JA, Morrow DA, Sabatine MS, et al. Association between
plasma levels of monocyte chemoattractant protein-1 and long-term
clinical outcomes in patients with acute coronary syndromes. Circulation.
2003;107:690–695.
25. Aukrust P, Ueland T, Müller F, et al. Elevated circulating levels of C-C
chemokines in patients with congestive heart failure. Circulation. 1998;
97:1136–1143.
26. Kakinuma Y, Ando M, Kuwabara M, et al. Acetylcholine from vagal
stimulation protects cardiomyocytes against ischemia and hypoxia
involving additive non-hypoxic induction of HIF-1a. FEBS Lett. 2005;
579:2111–2118.
27. Tracey KJ. The inflammatory reflex. Nature. 2002;420:853–859.
28. Pavlov VA, Tracey KJ. The cholinergic anti-inflammatory pathway. Brain
Behav Immun. 2005;19:493–499.
29. Wang H, Yu M, Ochani M, et al. Nicotinic acetylcholine receptor a7
subunit is an essential regulator of inflammation. Nature. 2003;421:
384–388.
30. Pavlov VA, Wang H, Czura CJ, et al. The cholinergic anti-inflammatory
pathway: a missing link in neuroimmunomodulation. Mol Med. 2003;9:
125–134.
31. Mann DL, McMurray JJ, Packer M, et al. Targeted anticytokine therapy in
patients with chronic heart failure: results of the Randomized Etanercept
Worldwide Evaluation (RENEWAL). Circulation. 2004;109:1594–1602.
32. Heymans S, Hirsch E, Anker SD, et al. Inflammation as a therapeutic
target in heart failure? A scientific statement from the Translational
Research Committee of the Heart Failure Association of the European
Society of Cardiology. Eur J Heart Fail. 2009;11:119–129.
33. De Ferrari GM, Schwartz PJ. Vagus nerve stimulation: from pre-clinical to
clinical application: challenges and future directions. Heart Fail Rev.
2011;16:195–203.
www.jcvp.org |
507