The mitochondrial permeability transition and heart failure

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DESCRIPTION (provided by applicant): Mitochondria are central in both energy production and cellular death pathways, yet a variety of questions concerning structure-function relationships of mitochondria remain poorly understood. The mitochondrial outer membrane (MOM) is more than a passive barrier separating the cytosol from the inner membrane. It is the site for integrating metabolic and cell signaling events, and provides a means for compartmentalizing discrete metabolic states. The applicant's lab has been studying the function of MOM permeability using molecular genetic approaches in order to understand the functions of a small family of highly abundant channel proteins termed Voltage-dependent Anion Channels (VDACs) and how they integrate into both the bioenergetics of the cell and cell survival through their participation in apoptosis. Two aspects are relevant to this proposal: VDAC2 directly interacts with the multi-domain pro-apoptotic protein BAK and considerable evidence suggests that VDACs are a component of the mitochondrial permeability transition pore (MPTP), an incompletely characterized large conductance channel activated by calcium overload and other stimuli. We hypothesize that VDAC2 and the MPTP interact and play an important role in maintenance of cardiac function and cell death. We have preliminary evidence for this concept and are interested in pursuing this by examining the in vivo consequences of loss of function and over-expression of VDAC2 in mutant mice using the heart as a model organ. Our specific aims are to use knockout and transgenic mice to determine the consequences of loss and over-expression of VDAC2 using an ischemia-reperfusion (I/R) injury model, since it is known that I/R involves both intrinsic and extrinic apoptotic pathways. We will genetically test to what degree this is mediated by pro- and anti-apoptotic proteins and the MPTP by breeding the VDAC2 mutants to BAK or BAX deficient mice, Bcl-2 over-expressing mice and mice lacking other components of the permeability transition pore, specifically cyclophilin D. Since mitochondrial dysfunction is a feature of heart failure, these studies will delineate the mechanistic role of MOM channel proteins in this disease process. Heart failure is characterized by progressive cell death arising from multiple stressors. The mechanisms leading to cell death are being elucidated and mitochondria are central in this process. This application is intended to provide in vivo data on the function of mitochondrial outer membrane (MOM) permeability with regard to both the bioenergetics of the cell and cell survival through its role in apoptosis. Permeability is conferred by a family of channel proteins; the Voltage-dependent Anion Channels (VDACs). We have previously demonstrated that the isoform termed VDAC2 directly interacts with the multi-domain pro-apoptotic protein BAK to suppress its pro-apoptotic function. More recent data provided in this application demonstrates that there is a reciprocal relationship between VDAC2 expression and expression of BAK and BAX. Loss of VDAC2 leads to a re-distribution of BAK from the MOM to the endoplasmic reticulum. In addition, there is considerable evidence to suggest that VDACs are a component of the mitochondrial permeability transition pore (MPTP), an incompletely characterized large conductance channel activated by calcium overload and other stimuli. Since it is known that loss of another purported component of the MPTP; cyclophilin D (CypD), confers protection from cell death in an ischemia/reperfusion paradigm, it is important to understand what functions VDACs have in regard to cardiac metabolism and cell death. Hence, we are interested in pursuing this by examining the in vivo consequences of loss of function and over-expression of VDAC2 in mutant mice using the heart as a model organ. Our aim is to use knockout and transgenic mice we have generated to determine the consequences of loss and over-expression of VDAC2 using an ischemia-reperfusion (I/R) injury model. We will genetically test to what degree this is mediated by pro- and anti-apoptotic proteins and the MPTP by breeding the VDAC2 mutants to BAK or BAX deficient mice, Bcl-2 over-expressing mice and mice lacking other components of the permeability transition pore, specifically cyclophilin D. We will characterize these mutant strains both bioenergetically and with regard to susceptibility to apoptosis, both spontaneous and induced. In the first year of this two year application we will complete the generation of the various mutant strains described in the application and carry out the intial characterization of spontaneous apoptosis. In the second year we will complete the bioenergetic studies by examining mitochondrial respiration by polarography and respiratory chain enzymatic function, and quantify the responses to ischemia/reperfusion. It is expected that completion of these goals will identify targets for therapeutic interventions in heart failure.