Ischemic preconditioning-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contraction-induced myocardial injury

Abstract

Dystrophin is an integral membrane protein involved in the stabilization of the sarcolemmal membrane in cardiac muscle. We hypothesized that the loss of membrane dystrophin during ischemia and reperfusion is responsible for contractile force-induced myocardial injury and that cardioprotection afforded by ischemic preconditioning (IPC) is related to the preservation of membrane dystrophin. Isolated and perfused rat hearts were subjected to 30 min of global ischemia, followed by reperfusion with or without the contractile blocker 2,3-butanedione monoxime (BDM). IPC was introduced by three cycles of 5-min ischemia and 5-min reperfusion before the global ischemia. Dystrophin was distributed exclusively in the membrane of myocytes in the normally perfused heart but was redistributed to the myofibril fraction after 30 min of ischemia and was lost from both of these compartments during reperfusion in the presence or absence of BDM. The loss of dystrophin preceded uptake of the membrane-impermeable Evans blue dye by myocytes that occurred after the withdrawal of BDM and was associated with creatine kinase release and the development of contracture. Although IPC did not alter the redistribution of membrane dystrophin induced by 30 min of ischemia, it facilitated the restoration of membrane dystrophin during reperfusion. Also, myocyte necrosis was not observed when BDM was withdrawn after complete restoration of membrane dystrophin. These results demonstrate that IPC-mediated restoration of membrane dystrophin during reperfusion correlates with protection against contractile force-induced myocardial injury and suggest that the cardioprotection conferred by IPC can be enhanced by the temporary blockade of contractile activity until restoration of membrane dystrophin during reperfusion.

Role of Mechanical Stress in the Form of Cardiomyocyte Death During the Early Phase of Reperfusion

“…Several recent studies have indeed demonstrated that the intracellular ATP level is a critical determinant of the form of cell death. [17][18][19] Although our study was unable to evaluate ATP levels in individual myocytes undergoing apoptosis and oncosis because of the inherent difficulty in measuring ATP in individual myocytes, Terminella et al 25 found that the critical value of ATP required for completion of apoptotic signaling was 12-50% of the normal cellular ATP concentration. Provided that the average ∆ m in individual myocytes is proportional to the intracellular ATP level, the average TMRE uptake of myocytes undergoing apoptosis (31.9±1.8% of non-ischemic myocytes) observed in the present study is consistent with such a critical ATP concentration in determining the form of cell death.…”

Inhibition of Contractile Activity During Postconditioning Enhances Cardioprotection by Restoring Sarcolemmal Dystrophin Through Phosphatidylinositol 3-Kinase

“…The isolated and buffer-perfused rat heart preparation was established as described previously, 23 and the heart was subjected to 30 min ischemia followed by 120 min reperfusion. IPost Figure 1.…”

“…23 During the stabilization period, a latex balloon was inserted into the left ventricle through the left atrium to measure the isovolumic LV function. The balloon was filled with saline solution to produce an LV end-diastolic pressure (LVEDP) of 5-10 mmHg at the baseline, and the balloon volume was kept constant throughout the experiment.…”

“…23 Briefly, frozen samples were cut into ~6 μm sections and were mounted on glass slides, incubated in acetone and hydrogen peroxide, rinsed with PBS, and blocked with 10% normal rabbit serum. The sections were incubated for 1 h at room temperature with mouse monoclonal anti-dystrophin antibodies (MANDRA-1; Sigma) at a dilution of 1:100 and washed with PBS.…”

“…17, 18 Dystrophin provides a mechanically strong physical linkage between the sarcolemmal membrane and the costameric cytoskeleton in the cardiac muscle, 19 thereby stabilizing the sarcolemmal membrane against the shear stresses imposed during eccentric muscle contraction. 20, 21 We have previously reported that dystrophin is translocated from the sarcolemma during ischemia, 22, 23 and that redistribution of dystrophin to the sarcolemma during reperfusion depends on mitochondrial ATP generation. 24 Therefore, despite the fact that IPost protects the mitochondria, it is anticipated that the inhibition of aerobic ATP generation during IPost is detrimental for the recovery of sarcolemmal integrity and renders cardiomyocytes susceptible to oncosis.…”