World Journal of Cardiovascular Diseases, 2013, 3, 21-25 WJCD
http://dx.doi.org/10.4236/wjcd.2013.35A004 Published Online August 2013 (http://www.scirp.org/journal/wjcd/)
Dual effect of pre-ischemic administration of TNF-alph a on
myocardial infarct size
Thuy Tran Quang1, Raja Hatem2, Guy Rousseau1,3, Audrey-Anne Gosselin1, Erick Schampaert1,2,
Thierry Charron1,2
1Centre de Recherche, Hôpital du Sacré-Cœur de Montréal, Montréal, Canada
2Département de Cardiologie, Hôpital du Sacré-Cœur de Montréal, Montréal, Canada
3Département de Pharmacologie, Université de Montréal, Montréal, Canada
Received 26 June 2013; revised 28 July 2013; accepted 8 August 2013
Copyright © 2013 Thuy Tran Quang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Tumour necrosis factor-α is a cytokine released dur-
ing myocardial infarction. According to the literature,
the effect of TNFα on myocardial infarction is contro-
versial, especially when administered before the is-
chemic period. The deleterious effects of TNFα seem
to be related to the triggering of apoptosis. This study
has been designed to determine if different doses of
TNFα, administered before the ischemic period, have
the same effect on infarct size and on activation of ca-
spase-3 and -8, two enzymes involved in apoptosis.
Four groups, using a porcine model of myocardial in-
farction, have been used: placebo and TNFα (0.1 µg/kg;
1 µg/kg and 3 µg/kg). All administered 15 minutes
before a 50 minutes occlusion of the left anterior de-
scending artery. Myocardial infarct size has been de-
termined at 3 hours of reperfusion. In a subgroup of
animals, reperfusion period has been limited to 15
min to determine the activity of caspase-3 and -8 by
spectrofluorometry. Results indicated that infarct size
is significantly smaller in groups 0.1 µg/kg and 1 µg/
kg as compared to the placebo group. In contrast, the
3 µg/kg group presented an infarct size similar to the
placebo group. Activity of caspase-3 and -8 is reduc-
ed in the ischemic region in groups 0.1 and 1 µg/ kg as
compared to the placebo group whereas activity in
the 3 µg/kg group was similar to the placebo. The re-
sults obtained indicated that a low dose of TNFα ad-
ministered before the ischemic period reduces infarct
size, whereas the cardioprotection is lost with the high
dose.
Keywords: TNF-Alpha; Myocardial Infarct Size;
Protection; Apoptosis; Caspase-8
1. INTRODUCTION
TNFα is a multifunctional 26 kDa cytokine involved as a
mediator of diverse physiologic and pathophysiologic
events including inflammation, growth, differentiation and
apoptosis. TNFα is thought to contribute to the deve-
lopment of myocardial disease, with direct correlation
between plasmatic concentrations of TNFα and the se-
verity and progression of heart failure [1-3]. However,
recent clinical trials of anti-TNFα therapy for cardiovas-
cular disease have been disappointing [4] possibly due to
the dual role of TNFα in attenuation and aggravation of
cardiac injury [5]. Indeed, some studies have shown that
TNFα was cardioprotective [6,7] whereas others have
shown that TNFα has either no effect or possibly dele-
terious effects [8,9].
One of the best known effect of TNFα is its invol-
vement in apoptosis [1]. When activated by TNFα, the
TNF receptor 1 can activate the formation of a complex
that leads to the activation of caspase-8. Nonetheless, it
seems also possible that the stimulation of the TNFR1
induces the activation of enzymes involved in different
cardioprotective pathways [10].
We have speculated that depending on the dose of
TNFα administered before the onset of ischemia, the ef-
fect on myocardial infarct size could be altered. To deter-
mine the mechanism by which the different doses of
TNFα can modulate the myocardium damage, activation
of the caspase-3 and -8 was measured.
2. MATERIALS AND METHODS
2.1. Experimental Model
Experiments were performed in accordance with the Ca-
nadian Council for Animal Care guidelines and were ap-
proved by the Institutional Animal Care Committee.

T. T. Quang et al. / World Journal of Cardiovascular Diseases 3 (2013) 21-25 22
Male pigs weighing from 35 to 40 kg were used to create
a porcine model of myocardial infarction. Animals were
anaesthetized with intramuscular ketamine-xylazine (20
mg/kg - 2 mg/kg; Wyeth Pharmaceutical, Montreal, Ca-
nada), intubated and ventilated with positive pressure
ventilation (Ohmeda 7800 anaesthesia ventilator, DRE
Medical, Louisville, KY) at a fraction of inspired oxygen
maintained constant at 20% - 30%. Anaesthesia was main-
tained with 2% isoflurane (Abbott Laboratory, Montreal,
Canada) mixed in O2:N2O (2:1). The left internal jugular
vein was cannulated with a 9F vascular sheath and used
as an access for intravenous fluid. Fluid repletion was
maintained by an I.V. infusion of 0.9% sodium chloride
solution at a rate of 10 ml/kg/h. The right carotid artery
was cannulated with an 8F vascular sheath and used for
systemic artery pressure monitoring (8-channel poly-
graph; Nihon Kohden America, Foothill Ranch, CA).
2.2. Induction of Infarct Size
The procedure was performed via the right femoral ar-
tery with an 8F vascular sheath using an 8F AL1 coro-
nary guiding catheter. An IQ angioplasty guide wire (Bo-
ston Scientific, Natick, Massachusetts, USA) was advan-
ced in the left anterior descending (LAD), distally to the
second diagonal, followed by the insertion of a 3.0 × 15
mm balloon catheter (ApexTM, over-the-wire) (Boston Sci-
entific). The balloon catheter was inflated at 4 atm for a
total period of 50 min simulating a myocardial infarction.
After this period, the balloon was deflated to allow a
period of reperfusion.
After the reperfusion period (15 min or 3 hours), the
catheter balloon was reinflated. The coronary angiogra-
phy was performed to ensure complete occlusion of the
LAD. We then injected Evan Blue via the 8F AL1 coro-
nary guiding catheter in order to stain the heart in vivo.
All pigs were euthanized, with a solution of saturated
KCl, at the end of procedures.
Four different groups were performed during that
study. In the placebo group (P), normal saline was injec-
ted 15 minutes before creating the myocardial infarction
period in the coronary artery just at the level of occlusion.
Three different doses (0.1 µg/kg, 1 µg/kg and 3 µg/kg) of
TNF were also given 15 minutes before the myocardial
infarction period in the coronary artery just at the level of
occlusion.
During the period of occlusion, if ventricular fibrilla-
tion, detected by ECG, occurred, we would immediately
defibrillate (up to a maximum of 10 shocks) with an au-
tomated external defibrillator.
2.3. Angiography (TIMI Frame Count)
Angiography was performed after every important step
of the procedure. Cine digital image sequences of every
angiography were recorded and treated at 30 frames/s.
2.4. Myocardial Infarct Size
The heart was removed after 3 hours of reperfusion, fro-
zen (–80˚C for 10 min), and then sliced into about 7
transverse sections of 1 cm thick, and placed in 2,3,5-
triphenyltetrazolium chloride at 37˚C for 10 min. The
area of necrosis (I) was expressed as a percentage of the
area at risk (I/AR). AR was represented as a percentage
of the left ventricular (LV), area (AR/LV). This method
was described previously [11,12].
2.5. Biochemical Analysis
For the biochemical analysis, a subgroup of pigs was
performed with only 15 min of reperfusion. Their heart
tissues were not stained, to avoid alteration of enzymatic
activity under biochemical analysis. Instead, the ische-
mic area (AR) was separated from the nonischemic area
of the myocardium (area not at risk, ANR). Each region
was divided into epi-(Epi) and subendocardial (Endo)
areas, defined as the upper and lower part of the myocar-
dium. Heart tissues were snap frozen in liquid nitrogen
and stored at –80˚C.
Caspase-3 and -8 activities were measured according
to the caspase-3 protocol described previously [13]. Brie-
fly, tissues were homogenized by sonification in lysis
buffer and incubated for 30 min on ice. The tissue homo-
genates were centrifuged at 4˚C for 10 min. Enzymatic
reactions were undertaken in reaction buffer with 25 µg
protein and attested by the Bradford method and fluo-
rescent substrate (Ac-DEVD-AMC or Ac-IETD-AMC, re-
spectively, for caspase-3 and -8) (40 mmol/L). Reactions
were studied after incubation in the dark for 3 h at 37˚C
and stopped with the addition of 0.4 mol/L NaOH and
0.4 mol/L glycine buffer. Fluorescence was quantified by
spectrofluorometry (Photon Technology International,
Lawrenceville, N.J., USA) at an excitation wavelength of
365 nm and emission wavelength of 465 nm for caspase-
3 or 430 nm for caspase-8.
2.6. Statistical Analysis
Results are reported as mean ± standard error of the
mean (S.E.M). Comparisons were made using analysis of
variance followed by Dunnett’s t test correction. p < 0.05
was considered statistically significant. All statistics was
performed with SPSS 13.0 (SPSS, Chicago, IL).
3. RESULTS
Infarct size, estimated in percent of the area at risk,
showed a significant reduction in the presence of the
lower doses of TNFα (0.1 and 1 µg/kg) as compared to

T. T. Quang et al. / World Journal of Cardiovascular Diseases 3 (2013) 21-25 23
the placebo group (Figure 1; p < 0.05). However, when
the dose of 3 µg/kg was administered, no further differ-
ence was observed as compared to the placebo group.
Area at risk, which represented around 40% of the left
ventricle, shows no difference among groups. These data
indicate that lower doses of TNFα have a beneficial ef-
fect on infarct size when administered before the ische-
mic period.
Activity of the caspase-3 in the ischemic endocardial
region, is significantly lower in the 0.1 and 1 µg/kg groups
as compared to the placebo group (Figure 2). However
as it is for the myocardial infarct size, activity of the cas-
pase-3 in the 3 µg/kg group is similar to the one meas-
ured in the placebo group. No significant difference was
observed in the ischemic epicardial region.
In presence of TNFα, caspase-8 may be activated by
the TNF receptor. For this reason, we have measured the
activity of the caspase-8 in the endocardial and epicardial
ischemic region. Again, the results indicate a significant
reduction of the activity of the caspase-8 in the 0.1 and 1
µg/kg as compared to the placebo group (Figure 3) in
both regions. No significant difference was observed for
the 3 µg/kg group as compared to the placebo group.
Placebo 0.1 μg/kg 1 μg/kg 3 μg/kg
50
40
30
20
10
0
Myocardial infarct size
(% of the area ar risk)
* *
Figure 1. Myocardial infarct size (I), expressed as a percentage
of the area at risk (AR), shows a significant difference between
0.1 µg/kg and 1 µg/kg as compared to placebo. No difference
was observed between 3 µg/kg group and placebo. *p < 0.05 vs
placebo group.
Placebo 0.1 μg/kg 1 μg/kg 3 μg/kg
150
100
50
0
Activity of caspase-3
(% of placebo)
*
*
Figure 2. Caspase-3 activity in ischemic endocardial regions
assessed by spectrofluorometry after 15 min reperfusion. n = 4
- 6/group: *p < 0.05 between the placebo and 0.1 µg/kg or 1
µg/kg groups.
150
100
50
0
Activity of caspase-8
(% of placebo)
*
*
Endo Epi
*
*
Placebo
0.1 μg/kg
1 μg/kg
3 μg/kg
Figure 3. Caspase-8 activity in ischemic endocardial (Endo)
and epicardial (Epi) regions assessed by spectrofluorometry
after 15-min reperfusion. n = 4 - 6/group: *p < 0.05 between the
placebo and 0.1 µg/kg or 1 µg/kg groups.
4. DISCUSSION
Our data indicate that pretreatment with TNFα results in
a significant reduction of infarct size when low doses
(0.1 and 1 µg/kg) are used. In contrast, higher dose of
TNFα (3 µg/kg) did not afford any protection as com-
pared to our placebo group. The protection is probably
related to a lower activity of the caspase-3 and -8, known
to be involved in apoptosis.
The effects of TNFα on myocardial infarct size have
been controversial. For example, when TNFα is reduced
before ischemia with the use of a TNFα antibody, studies
indicated a significant reduction of infarct size [14,15].
However, when the level of TNFα is modulated by solu-
ble TNF receptor, some studies observed a significant
reduction of infarct size [16] whereas others detected no
significant effects [8]. Using TNFα KO mice, Maekawa
et al. observed a reduction in infarct size [9] but in con-
trast, KO of TNF p75 receptor induces a loss in cardio-
protection, suggesting a protective role of TNFα in some
circumstances. According to the results obtained by Deu-
char et al. [6], administration of different doses of TNFα,
administered 24-min before the ischemia results in an
inverse bell shape effect, maximal protection observed
with the dosage of 0.1 µg/kg whereas with dosage of 0.2
µg/kg or 4 µg/kg results in an infarct size similar to pla-
cebo group. These results are similar to the one observed
with the present study and suggest, as proposed by Le-
cour et al. [17], that the dosage and the timing are critical
for the protection afforded by TNFα. The difference in
the effect of the dosage may be due to the species used
(rat against pig) as well as the way the TNFα was ad-
ministered. In the study of Deuchar et al. TNFα was in-
jected 24 min before the ischemic period i.v. whereas in
the present protocol, TNFα was administered i.c. 15 min
before the occlusion of the coronary artery.
These results also reflect the duality of the TNFα ef-
fects in the context of an ischemia-reperfusion protocol
as reported by Duran [18]. Different cell types, such as

T. T. Quang et al. / World Journal of Cardiovascular Diseases 3 (2013) 21-25 24
myocytes or vascular smooth muscle cells, can synthe-
size TNFα. In normal conditions the level of synthesis of
TNFα is low but increases rapidly during ischemia/re-
perfusion [19]. These levels then diminish during the
first hours of reperfusion [20]. TNFα exerts its effects
through 2 different receptors: TNFR1 and TNFR2. The
binding of the TNFα to the TNFR1 induces the trimeri-
zation of the receptor and promotes the dissociation of
the silencer death domain from the receptor. The death
domain of the activated receptor recruits the TNFR1-as-
sociated death domain (TRADD), RIP1 and TRAF2,
which then becomes modified and dissociate from TNFR1.
The released death domain of TRADD is now able to
bind to Fas-associated protein with death domain, result-
ing in the activation of the caspase-8 and then caspase-3
[1]. In contrast, TNFR1 can also activate the NF-kB
pathway, through the phosphorylation and ubiquity-na-
tion of different proteins that bind the TNFR1-TRADD-
TRAF-RIP1 complex. When NF-kB is activated, its trans-
location to the nucleus mediates the transcription of dif-
ferent proteins such as TNFα.
TNFR1 can also activate other signaling pathway in-
cluding c-Jun N-terminal kinase (JNK), p38 mitogen-
activated protein kinase and PI3k/Akt [10]. These en-
zymes have been identified to be members of two im-
portant signaling pathways that can induce cardiopro-
tection: the RISK [21] and SAFE pathways [22]. Our da-
ta indicate that the activity of both caspase-3 and -8 is
elevated in the control and 3 µg/kg groups whereas the
activation is significantly less in the 0.1 µg/kg and 1 µg/
kg groups. This difference in the activation suggests that
the lower doses of TNFα can induce the activation of the
protective pathways, when activated before the ischemic
period, whereas other pathways are activated with the
higher doses. The difference in the myocardial infarct
size between placebo and the other groups (0.1 µg/kg
and 1 µg/kg) can be due to the activation of the protec-
tive pathway induced by the activation of the TNFR1
before the ischemic period, but other experiments are
needed to confirm this hypothesis.
We have measured the activity of the caspase-3 and -8
after 15 minutes of reperfusion because previous data
indicate that this activity is high during the first minute
of reperfusion to reduce, and it is also basal at 5 hours of
reperfusion [23].
One major limitation in this protocol is the availability
of antibodies to perform biochemical analysis that is
compatible with a porcine model. For this reason, we
have been limited in the number of measures that can be
performed.
In conclusion, we have documented that low doses of
TNFα can induce cardioprotection when administered
before the ischemic period. However, with high doses,
this protection is lost probably due to the activation of
the caspase-3 and -8.
5. ACKNOWLEDGEMENTS
This work was supported by the Centre de recherche, Hôpital du Sacré-
Coeur de Montréal and Fonds de la recherche du Québec-Santé (FRQS).
We thank Louis Chiocchio and Caroline Bouchard for their help in
animal care.
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