Distinct mPTP activation mechanisms in ischaemia-reperfusion: contributions of Ca2+, ROS, pH, and inorganic polyphosphate

Aims The mitochondrial permeability transition pore (mPTP) plays a central role for tissue damage and cell death during ischaemia-reperfusion (I/R). We investigated the contribution of mitochondrial inorganic polyphosphate (polyP), a potent activator of Ca2+-induced mPTP opening, towards mPTP activation and cardiac cell death in I/R. Methods and results A significant increase in mitochondrial free calcium concentration ([Ca2+](m)), reactive oxygen species (ROS) generation, mitochondrial membrane potential depolarization (Delta Psi m), and mPTP activity, but no cell death, was observed after 20 min of ischaemia. The [Ca2+](m) increase during ischaemia was partially prevented by the mitochondrial Ca2+ uniporter (MCU) inhibitor Ru360 and completely abolished by the combination of Ru360 and the ryanodine receptor type 1 blocker dantrolene, suggesting two complimentary Ca2+ uptake mechanisms. In the absence of Ru360 and dantrolene, mPTP closing by polyP depletion or CSA decreased mitochondrial Ca2+ uptake, suggesting that during ischaemia Ca2+ can enter mitochondria through mPTP. During reperfusion, a burst of endogenous polyP production coincided with a decrease in [Ca2+](m), a decline in superoxide generation, and an acceleration of hydrogen peroxide (H2O2) production. An increase in H2O2 correlated with restoration of mitochondrial pH(m) and an increase in cell death. mPTP opening and cell death on reperfusion were prevented by antioxidants Trolox and MnTBAP [Mn (III) tetrakis (4-benzoic acid) porphyrin chloride]. Enzymatic polyP depletion did not affect mPTP opening during reperfusion, but increased ROS generation and cell death, suggesting that polyP plays a protective role in cellular stress response. Conclusions Transient Ca2+/polyP-mediated mPTP opening during ischaemia may serve to protect cells against cytosolic Ca2+ overload, whereas ROS/pH-mediated sustained mPTP opening on reperfusion induces cell death.