Effect of thrombin fragment (TP508) on myocardial ischemia reperfusion injury in a model of type 1 diabetes mellitus

Abstract

Background: We investigated the efficacy of novel thrombin fragment TP508 on ischemia-reperfusion injury using a porcine model of type 1 diabetes mellitus.

Methods and results: Alloxan-induced diabetic male Yucatan swine underwent 60 minutes of mid-left anterior descending coronary artery occlusion, followed by 120 minutes of reperfusion. Fifty minutes into ischemia, animals received either placebo (DM; n=8) or TP508 as a bolus of 1 mg/kg followed by infusion at 2.5 mg/kg per hour (DMT; n=8). Hemodynamic parameters and myocardial function were monitored. Monastryl blue/triphenyl tetrazolium chloride staining was used to assess sizes of the areas at risk and infarction. Coronary microvascular reactivity was measured and expression of cell survival and proapoptotic proteins quantified. Preoperative serum glucose values were similar between groups (309±57 mg/dL in DM versus 318±67 mg/dL in DMT; P=0.92). Infarct size was smaller in the TP508-treated group (5.3±1.9% in DMT versus 19.4±5.6% in DM; P=0.03). There was no statistically significant difference in global or regional left ventricular function between groups. Endothelium-dependent microvessel relaxation was moderately improved in the DMT group (P=0.09), whereas endothelium-independent relaxation was similar between groups. The expression of cell survival proteins Akt, phospho-p38, and mammalian target of rapamycin was higher in the areas at risk of DMT animals compared with DM animals (P<0.05), and expressions of proapoptotic glycogen synthase kinase 3β and caspase 3 were lower in the DMT group (P<0.05).

Conclusions: This study demonstrates that, in type 1 diabetic swine, TP508 reduces infarct size after ischemia-reperfusion. Thus, TP508 may offer a novel approach in cardioprotection from ischemia-reperfusion injury in diabetic patients.

Figure 1

Serum glucose values. Shown are glucose values for DM (n=8) and DMT (n=8) groups at different timepoints in the experiment. Pre = baseline, O1 = 30 minutes of ischemia, O2 = 60 minutes of ischemia, R1 = 30 minutes of reperfusion, R2 = 60 minutes of reperfusion, R3 = 90 minutes of reperfusion, R4 = 120 minutes of reperfusion. Data are presented as means ± SEM. P group = 0.9 (two-way repeated measures ANOVA).

Figure 2

Hemodynamics and myocardial function in DM (n=8) and DMT (n=8) animals. A: mean arterial pressure. B: LAD blood flow, expressed as fold change from baseline blood flow. C: developed left ventricular pressure. D: positive first-derivative of left ventricular pressure. E: longitudinal axis segmental shortening. F: horizontal axis segmental shortening. Segmental shortening (SS) expressed as percentage decrease from maximal myocardial stretch on the longitudinal or horizontal axes. Pre = baseline, O1 = 30 minutes of ischemia, O2 = 60 minutes of ischemia, R1 = 30 minutes of reperfusion, R2 = 60 minutes of reperfusion, R3 = 90 minutes of reperfusion, R4 = 120 minutes of reperfusion. Data are presented as means ± SEM. *P <0.05.

Figure 3

AAR and infarct size in DM (n=8) and DMT (n=8) animals. A: size of AAR as a percentage of LV surface area. B: Size of infarct area as a percentage of AAR. Also shown are representative slices of DM (C) and DMT (D) ventricle, with black arrows indicating nonischemic area, red arrows indicating AAR, and white arrows to infarct area. Data presented as means ± SEM. *P = 0.03.

Figure 4

Microvessel relaxation responses in DM (n=8) and DMT (n=8) animals. A: response to endothelium-dependent ADP. B: response to endothelium-independent sodium nitroprusside. Drug concentrations are expressed as log [drug]. Vessel relaxation expressed as % increase from preconstricted diameter. Data presented as means ± SEM.

Figure 5

Selected protein immunoblot results. Akt (A), phospho-Akt(Ser473) (B), phospho-Akt(Thr308) (C), phospho-eNOS (D), caspase-3 (E), and phospho-p38 (F) expression was assessed in the AAR of DM (n=8) and DMT (n=8) animals. Data presented as mean ± SEM in arbitrary units (AU). *P < 0.05.

Figure 6

Selected protein immunoblot results. GSK-3β (A), phospho-GSK-3β (B), AIF (C), and mTOR (D) expression was assessed in the AAR of DM (n=8) and DMT (n=8) animals. Data presented as mean ± SEM in arbitrary units (AU). *P < 0.05.