Opening of mitochondrial permeability transition pore induces hypercontracture in Ca2+ overloaded cardiac myocytes

Abstract

After myocardial ischemia, necrotic cell death occurs mainly during the first minutes of reperfusion through ATP-dependent hypercontracture leading to sarcolemmal rupture. Recent studies indicate that opening of a mitochondrial permeability transition pore (mPTP) is a critical event in reperfusion-induced necrosis. We investigated the hypothesis that mPTP can induce hypercontracture. Both intact and digitonin-permeabilized rat cardiac myocytes were loaded with TMRE and submitted to oxidative damage (intermittent 568 nm laser illumination) to promote mPTP, detected as mitochondrial depolarization. The effect of cytosolic Ca(2+) overload (5 mmol/L extracellular Ca(2+)) and ATP availability on mPTP-induced cell shortening were analyzed, and changes in cytosolic and mitochondrial Ca(2+) were simultaneously monitored by confocal microscopy (Fluo-4 and Rhod-2). In the absence of Ca(2+) overload, induction of mPTP was consistently followed by mitochondrial depolarization and rigor shortening that, in permeabilized cells, was prevented by ATP. Exposure of intact cardiac myocytes to 5 mmol/L Ca(2+) induced an increase in cytosolic and mitochondrial Ca(2+) content. In Ca(2+) overloaded myocytes, induction of mPTP resulted in a further increase in cytosolic Ca(2+) and hypercontracture (> 50% reduction in length with distortion of cell geometry) that started before depolarization involved all mitochondria within the cell and could be prevented by the mPTP inhibitor cyclosporin A. In permeabilized myocytes, mPTP could promote hypercontracture when cytosolic Ca(2+) overload was mimicked in the presence of ATP, and was prevented when ATP was removed from the intracellular-like medium. mPTP opening may induce ATP-dependent hypercontracture in Ca(2+) overloaded myocytes. This phenomenon could reconcile the apparently contradictory hypotheses of hypercontracture and mPTP opening as main determinants of necrosis during the first minutes of reperfusion.