Combination of Cyclosporine A and Levosimendan Induces Cardioprotection under Acute Hyperglycemia

Abstract

Prognosis of patients with myocardial infarction is detrimentally affected by comorbidities like diabetes mellitus. In the experimental setting, not only diabetes mellitus but also acute hyperglycemia is shown to hamper cardioprotective properties by multiple pharmacological agents. For Levosimendan-induced postconditioning, a strong infarct size reducing effect is demonstrated in healthy myocardium. However, acute hyperglycemia is suggested to block this protective effect. In the present study, we investigated whether (1) Levosimendan-induced postconditioning exerts a concentration-dependent effect under hyperglycemic conditions and (2) whether a combination with the mitochondrial permeability transition pore (mPTP) blocker cyclosporine A (CsA) restores the cardioprotective properties of Levosimendan under hyperglycemia. For this experimental investigation, hearts of male Wistar rats were randomized and mounted onto a Langendorff system, perfused with Krebs-Henseleit buffer with a constant pressure of 80 mmHg. All isolated hearts were subjected to 33 min of global ischemia and 60 min of reperfusion under hyperglycemic conditions. (1) Hearts were perfused with various concentrations of Levosimendan (Lev) (0.3-10 μM) for 10 min at the onset of reperfusion, in order to investigate a concentration-response relationship. In the second set of experiments (2), 0.3 μM Levosimendan was administered in combination with the mPTP blocker CsA, to elucidate the underlying mechanism of blocked cardioprotection under hyperglycemia. Infarct size was determined by tetrazolium chloride (TTC) staining. (1) Control (Con) hearts showed an infarct size of 52 ± 12%. None of the administered Levosimendan concentrations reduced the infarct size (Lev0.3: 49 ± 9%; Lev1: 57 ± 9%; Lev3: 47 ± 11%; Lev10: 50 ± 7%; all ns vs. Con). (2) Infarct size of Con and Lev0.3 hearts were 53 ± 4% and 56 ± 2%, respectively. CsA alone had no effect on infarct size (CsA: 50 ± 10%; ns vs. Con). The combination of Lev0.3 and CsA (Lev0.3 ± CsA) induced a significant infarct size reduction compared to Lev0.3 (Lev0.3+CsA: 35 ± 4%; p < 0.05 vs. Lev0.3). We demonstrated that (1) hyperglycemia blocks the infarct size reducing effects of Levosimendan-induced postconditioning and cannot be overcome by an increased concentration. (2) Furthermore, cardioprotection under hyperglycemia can be restored by combining Levosimendan and the mPTP blocker CsA.

Keywords: Levosimendan; cardioprotection; hyperglycemia; myocardial infarction.

Figure 1

Infarct size measurement part 1. Histogram shows all infarct sizes of the study. Data are presented as means ± SD.

Figure 2

Infarct size measurement part 2. Histogram shows all infarct sizes of the study. Data are presented as means ± SD, * p < 0.05 vs. Lev0.3.

Figure 3

Experimental protocol. Con = Control; Lev = Levosimendan, KHB = Krebs-Henseleit-Buffer; Vehicle = Krebs-Henseleit Buffer (KHB). Green bar: Hearts were perfused with a total of 22 mmol/L glucose concentration by combining 11 mmol/L glucose solution with KHB (containing 11 mmol/L glucose). Control (Con): Hearts were perfused with Krebs-Henseleit-Buffer (KHB) as vehicle for 10 min. Levosimendan 0.3 μM (Lev0.3): Hearts were perfused with 0.3 μM Lev for 10 min. Levosimendan 1 μM (Lev1): Hearts were perfused with 1 μM Lev for 10 min. Levosimendan 3 μM (Lev3): Hearts were perfused with 3 μM Lev for 10 min. Levosimendan 10 μM (Lev10): Hearts were perfused with 10 μM Lev for 10 min.

Figure 4

Experimental protocol. Con = Control; Lev = Levosimendan, KHB = Krebs-Henseleit-Buffer; Vehicle = Krebs-Henseleit Buffer (KHB); CsA = Cyclosporine A (mPTP inhibitor). Green bar: Hearts were perfused with a total of 22 mmol/L glucose concentration by combining 11 mmol/L glucose solution with KHB (containing 11 mmol/L glucose). Control (Con): Hearts were perfused with Krebs-Henseleit-Buffer as vehicle for 10 min. Levosimendan 0.3 µM (Lev0.3): Hearts were perfused with 0.3 μM Lev for 10 min. Cyclosporine A (CsA): Hearts were perfused with 0.2 μM CsA for 10 min. Levosimendan 0.3 μM + Cyclosporine A (Lev0.3+CsA): Hearts were perfused with 0.3 μM Lev and 0.2 μM CsA for 10 min.