Molecular Regulation of Mitochondrial Permeability Transition and its Role in Regulated Necrosis

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Project Summary/Abstract Regulated necrotic death of cardiomyocytes is the direct cause of mortality during a myocardial infarction. One crucial step that is required for regulated necrosis to occur is the opening of the mitochondrial permeability transition pore (MPTP). Opening of the MPTP, leads to loss of ATP production, mitochondrial dysfunction, and eventual necrotic cell death. Identification of the components of the MPTP has plagued the scientific community for over 30 years. The identification of the inner mitochondrial membrane pore-forming components and regulators of the MPTP is vital for the mechanistic understanding of regulated necrosis. The adenine nucleotide translocator (ANT) family was previously thought to be the pore-forming component of the MPTP within the inner mitochondrial membrane, but this was genetically disproven when mitochondria lacking ANT1 and ANT2 still underwent mitochondrial permeability transition. However, these mitochondria are significantly desensitized to MPTP opening. Recently, we generated mice lacking all three isoforms of the ANT family in liver and showed that the MPTP in mitochondria isolated from ANT null livers are even more desensitized, but the pore eventually opens in the presence of very high Ca2+. Surprisingly, when we treated the ANT null mitochondria with cyclosporine A (CsA), a cyclophilin D (CypD) inhibitor, the MPTP was inhibited. Treatment of CsA normally desensitizes the MPTP, but with enough Ca2+ CsA can be overcome and the MPTP still engages. We now have the ability to definitively determine the full therapeutic potential of complete MPTP inhibition during I/R injury by generating mice lacking the predominant isoforms of ANT and CypD in the heart. In addition to the ANT family and CypD, the MPTP is also regulated from from the outer mitochondrial membrane by Bax and Bak. The relationship between the ANTs, CypD, and Bax/Bak in regards to MPTP regulation is undefined. In this proposal, we aim to elucidate how they function in concert with one another to lead to MPTP opening. Our preliminary data suggest the existence of multiple inner membrane pore-forming components of the MPTP including the ANT family and some other unidentified pore. We will utilize ANT null mitochondria to explore this hypothesis in further detail and aim to identify the novel pore-forming component by using targeted and unbiased approaches. Together this proposal will provide crucial mechanistic insight in the composition and regulation of the MPTP that will reveal potential strategies to inhibit MPTP opening to prolong cardiomyocyte survival in the face of an ischemic event.