Cyclosporine preserves mitochondrial morphology after myocardial ischemia/reperfusion independent of calcineurin inhibition

Abstract

Background: Opening of the mitochondrial permeability transition pore (MPTP) has been shown to contribute to myocardial ischemia/reperfusion injury. We sought to demonstrate that the myocardial protective effect of inhibiting MPTP opening with cyclosporine A (CsA) results in stabilization of mitochondrial morphology and is independent of CsA-induced calcineurin inhibition.

Methods: Thirty-seven rabbits were divided into three groups: control (n = 15), CsA (MPTP and calcineurin inhibitor, n = 12), or FK506 (calcineurin inhibitor, n = 10). Each group received a 1-hour infusion of either a saline vehicle, 25 mg/kg CsA or 1 mg/kg FK506. All animals underwent 30 minutes of regional ischemia and 3 hours of reperfusion. Myocardial infarct size was determined using Evans blue dye and triphenyltetrazolium chloride. In situ oligo ligation was used to assess apoptotic cell death. Transmission electron microscopy was used to quantitatively evaluate morphologic differences in the mitochondria between groups.

Results: Infarct size in the CsA group (39% +/- 3%) was significantly reduced compared with the control group (60% +/- 2%, p < 0.001) and FK506 group (55% +/- 3%, p = 0.001). Apoptotic cell death was also attenuated in the CsA group (1.2% +/- 0.5%) compared with the control group (4.3% +/- 0.8%, p = 0.01) and FK506 group (4.1% +/- 0.9%, p = 0.05). Transmission electron microscopy revealed a preservation of normal mitochondrial morphology and a reduction in the percentage of disrupted mitochondria in the CsA group (20% +/- 7%) compared with the control group (53% +/- 12%) and FK506 group (47% +/- 9%).

Conclusions: Cyclosporine A-induced MPTP inhibition preserves mitochondrial morphology after myocardial ischemia/reperfusion and limits myocyte necrosis and apoptosis. These effects are independent of calcineurin inhibition.

Fig. 1

Photographs of rabbit left ventricle sliced perpendicular to its long axis. (A) Evans blue dye staining to delineate the ischemic area at risk (AR). The AR is the unstained myocardium. (B) Triphenyltetrazolium chloride (TTC) staining to delineate viable myocardium (brick red) from nonviable myocardium (pale) in the AR. (C) Comparisons of area at risk (AR) and infarct size measured as a percentage of the area at risk (I/AR) in rabbits receiving either a phosphate- buffered saline vehicle (control [C], black bars; n = 15), cyclosporine A (CsA [light gray bars; n= 12]), or FK506 (dark gray bars; n = 10). *p < 0.001 from the control group.

Fig. 2

Comparisons of apoptotic index in rabbits receiving either a phosphate-buffered saline vehicle (control [C]), cyclosporine A (CsA), or FK506 and undergoing the ischemia/reperfusion protocol. *p < 0.05 from the control and FK506 groups.

Fig. 3

Transmission electron micrographs of rabbit myocardium. Representative images at × 12,000 compare the differences among the mitochondria of the (A) control, (B) FK506-treated, (C) CsA-treated, and (D) normal rabbit groups. At ×30,000, mitochondrial differences are more evident. The mitochondria of the (G) CsA-treated and (H) normal group are tightly packed, possessing an intact mitochondrial membrane and well-organized cristae, unlike the (E) control group and (F) FK506-treated group that are characterized by swollen, loosely packed, vacuolated, and completely disrupted mitochondria.