Dual effect of pre-ischemic administration of TNF-alpha on myocardial infarct size

doi:10.4236/wjcd.2013.35A004

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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

Email: [email protected]

Received 26 June 2013; revised 28 July 2013; accepted 8 August 2013

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.

OPEN ACCESS

T. T. Quang et al. / World Journal of Cardiovascular Diseases 3 (2013) 21-25

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

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

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

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

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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