Role of mitochondria in cardiac protection

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Despite many investigations establishing the role of mitochondrial permeability transition (MPT)-an opening of non-selective pores spanning the matrix to the cytosol that leads to mitochondrial dysfunction and cell death-in ischemic injury, the discrete mechanisms governing modulation of a proposed multiprotein complex that regulates MPT (i.e. the MPT pore), remain poorly defined. Two proteins, the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT) have been implicated as the core pore-forming units of the MPT pore by virtue of their independent abilities to form non-selective channels in response to stresses associated with ischemia/reperfusion injury. The focus of this application is to define the fundamental roles of these molecules in protection against myocardial ischemic insult. Previous studies have mapped both VDAC and ANT localization to contact sites between outer and inner mitochondrial membranes. Pharmacological interventions targeting VDAC and ANT show effective modulation of MPT in cell and organelle settings. Despite this, the sub-organelle distribution and isoform-specific function(s) of distinct VDAC and ANT isoforms-critical to understand the regulatory mechanisms of the MPT-have been virtually unexplored, especially in cardiac research. Our recent preliminary findings suggest that targeted inhibition of VDAC is sufficient to protect the heart against ischemic insult and that induction of pore-formation by ANT blocks the protective effects of pharmacologic and genetic cardioprotective regiments. Furthermore, our preliminary data demonstrate for the first time that multiple isoforms of VDAC (VDAC1-3) and ANT (ANT1-2) are expressed in the adult mouse heart as detected by both immunoblotting (using antibodies we developed since the original submission) and LC/MS/MS. These findings are very exciting, as they dispel the widely-held belief that all actions performed by myocardial VDAC and ANT can be attributed to VDAC1 and ANT1. This A1 application has been completely revised in response to critiques by the review panel. Stimulated by our new data as well as other developments in the field, the revised proposal addresses a number of key questions that are critical to the role of MPT in myocardial ischemic injury. Our central hypothesis is that isoforms of VDAC and ANT participate in distinct manners in the phenomenon of MPT, in part owing to their differential suborganellar localization, pore-forming properties, and interactions with regulatory proteins (e.g., PKC(, Bcl-2, and Bax). We will define the sub-organelle distribution of distinct isoforms of VDAC and ANT in the normal heart and during cardioprotection and will rigorously elucidate the pore-forming properties of these molecules in the reconstituted liposome format in response to known injurious factors present during ischemia/reperfusion injury (including ROS and Ca2+). We will examine the expression, localization and interactions among critical components of the MPT pore subproteome using confocal and electron microscopy. Lastly, we will delineate post-translational modification of MPT pore components using mass spectrometry and interrogate the role of these modifications to regulate MPT. These studies will provide novel and mechanistic information to aid our understanding of mitochondrial function in myocardial ischemic injury.