Suppression of poly (ADP-ribose) polymerase activation by 3-aminobenzamide in a rat model of myocardial infarction: long-term morphological and functional consequences

Abstract

1. Recent studies demonstrated that inhibition or genetic inactivation of the enzyme poly (ADP-ribose) polymerase (PARP) is beneficial in myocardial reperfusion injury. PARP activation in the reperfused myocardium has been assumed, but not directly demonstrated. Furthermore, the issue whether pharmacological PARP inhibition affords long-term functional benefit in the reperfused myocardium has not been explored. These questions were addressed in the present study. 2. In a rat model of myocardial ischemia (1 h) and reperfusion (up to 24 h), there was a marked and significant activation of PARP in the ischemic borderzone, as determined by poly(ADP-ribose) (PAR) immunohistochemistry. PAR localized to the nuclei of myocytes and infiltrating mononuclear cells. In the core of the infarction, necrotic tissues and diffuse PAR staining were observed. PARP activation remained markedly detectable 24 h after reperfusion. The PARP inhibitor 3-aminobenzamide (20 mg kg(-1) intraperitoneally 10 min before reperfusion, and every 2 h thereafter for 6 h) markedly reduced the activation of the enzyme in myocytes. 3. 3-aminobenzamide significantly protected against myocardial morphological and functional alterations at 24 h post-reperfusion. Notably, infarct size was reduced, circulating creatine kinase activity was attenuated, and myocardial contractility (dP dt(-1)) was restored by 3-aminobenzamide. 4. Our results demonstrate a significant and prolonged activation of PARP in the reperfused myocardium, localizing to the necrotic area and the ischaemic borderzone. Furthermore, the studies demonstrate that PARP inhibition affords long-term beneficial morphological and functional effects in the reperfused myocardium. These data strengthen the notion that pharmacological PARP inhibition is a viable novel experimental approach for protection against myocardial reperfusion injury.

Figures

Figure 1

Immunohistochemical localization of PARP activation. Poly(ADP-ribose) formation, an indicator of PARP activation, as determined in whole heart sections (a) from rats exposed to 1 h ischaemia and either 2 or 23 h reperfusion. In both conditions, a massive staining is evident in the left ventricular free wall of control animals. The staining was clearly reduced in rats treated with 3-aminobenzamide. Higher magnification (×40) shows that PARP activation was mainly located in the nuclei of myocytes in the peri-infarction (border) zone. In the infarcted myocardium, severe architectural alterations are coexisting with a more diffuse pattern of PAR staining. Treatment with 3-AB attenuated PARP activation in the border zone, while limiting PAR formation in the infarcted myocardium. Immunohistochemical pictures represent n⩾4 sections per group.

Figure 2

3-aminobenzamide reduces myocardial infarct size. Rats were exposed to 1 h LAD occlusion followed by 2 h (a) or 23 h (b) reperfusion. Area at risk (AAR) and infarct size were determined using the triphenyl-tetrazolium chloride (TTC)-Evans blue technique. Treatment of the animals with 3-AB (20 mg kg−1 i.p. 10 min before reperfusion, 2 h later, and in the 24 h group every 2 h thereafter for 6 h) significantly reduced infarct size (expressed as a percentage of the area at risk), both after 2 or 23 h reperfusion. *P<0.05 control vs 3-AB (unpaired t-test; n=10–11 animals in each group).

Figure 3

Effect of 3-aminobenzamide on serum CKMB after myocardial infarction. Serum creatine-phosphokinase (myocardial specific MB fraction, CKMB) was measured in sham rats (n=10) as well as in rats exposed to 1 h LAD occlusion and 23 h reperfusion. A significant increase in CKMB activity was observed after myocardial infarction in control rats (n=15), which was significantly reduced by treatment with 3-AB (20 mg kg−1 i.p. 10 min before reperfusion and every 2 h thereafter for 6 h, n=15). †P<0.05 vs sham; *P<0.05 control vs 3-AB (ANOVA followed by Bonferroni).

Figure 4

Effect of 3-AB on haemodynamic parameters after myocardial infarction. Control rats (n=15) exposed to 1 h LAD occlusion and 23 h reperfusion had a severe reduction in dP dt−1 max, left ventricular systolic pressure (LVSP) and mean blood pressure (mean BP), as well as a significant increase in left ventricular end-diastolic pressure (LVEDP) when compared to sham-operated animals (n=10). Treatment with 3-AB (20 mg kg−1 i.p. 10 min before reperfusion and every 2 h thereafter for 6 h, n=15) significantly reduced the left ventricular systolic dysfunction, restored normal blood pressure, and blunted the increase in LVEDP. †P<0.05 vs sham *P<0.05 control vs 3-AB (ANOVA followed by Bonferroni).