ABSTRACT PROJECT SUMMARY Muscular Dystrophy (MD) is a family of genetic disorders characterized by progressive muscle wasting, loss of muscle function, and premature death. MD pathology is driven by sarcolemma instability resulting in membrane rupture, necrosis, and myofiber death. Research has demonstrated that MD myofibers have increased cellular Ca2+ levels and that mitochondrial membrane permeability pore (MPTP) dependent myofiber death contributes to MD. There is evidence that MD myofiber mitochondria have elevated Ca2+ levels, but isolated MD mitochondria also have reduced Ca2+ uptake rates and increased expression of MCUb, a negative regulator of Ca2+ uptake. In the heart limiting mitochondrial Ca2+ uptake protects from MPTP-dependent cell death, while genetically promoting Ca2+ overload leads to increased MPTP-activation and cell death. We will therefore test the role of mitochondrial Ca2+ homeostasis in MD (Aim #1). We will disrupt the main mitochondrial Ca2+ efflux mechanism by deleting the myofiber mitochondrial Na+/Ca2+ exchanger (Nclx) in the Mdx model of MD to test the hypothesis that Ca2+ overload promotes MPTP-dependent myofiber death and pathology in MD. We will also disrupt the acute mitochondrial Ca2+ uptake mechanism by deleting the mitochondrial Ca2+ uniporter (Mcu) in myofbers in the Mdx model of MD to test the hypothesis that inhibiting Ca2+ uptake will reduce MPTP-dependent myofiber death pathology in MD. We will delete myofiber Mcub in the Mdx model of MD to test the hypothesis that increased MCUb expression in MD myofibers is protective in MD disease. To better understand the role of MPTP in MD disease we will directly target the MPTP genetically. We have recently demonstrated that adenine nucleotide translocator (ANT) proteins represent one of at least two protein species that comprise the MPTP. It has recently been observed that isolated MD myofiber mitochondria have increased sensitivity to MPTP- activation as well as a specific increase in ANT2 expression. We will therefore test the role of ANT-dependent MPTP activation in MD (Aim #2). We will study Ant1 knockout mice in the Sgcd-/- model of MD, which lack the main muscle ANT isoform, to test the hypothesis that ANT-dependent MPTP (MPTPANT) contributes to MD pathology. We will delete myofiber Ant2 in the Sgcd-/- model of MD to test the hypothesis that ANT2 upreglation in MD mitochondria specifically promotes MPTP activation and pathology in MD. Additionally, we will generate mice lacking all murine isoforms of ANT in the Sgcd-/- model of MD to measure the total contribution of MPTPANT to MD. Since we have demonstrated that inhibition of cyclophilin D (CypD) in the context of total ANT knockout is sufficient to completely inhibit MPTP, we will treat Sgcd-/- mice lacking all ANT isoforms with the CypD inhibitor Debio-025 to test what proportion of total myofiber death and MD pathology is dependent on MPTP-activation. This research will be the first test of the role of ...