This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heart disease and consequent heart failure contibutes to high mortality in Western society. A greater understanding of the regulatory proteins associated with this disease is required. In heart, the anti-apoptotic factor, Bcl-xL attenuates cell death during ischemia-reperfusion. This is observed at the single cell and whole organ level. Though the pro-survival mechanism of Bcl-xl in unclear, Bcl-xl is thought to inhibit pro-apoptotic factors such as Bax and Bak while also regulating ATP-ADP exchange across the inner mitochondrial membranes. For example, anti-apoptotic Bcl-2 proteins (of which Bcl-xL is a member) inhibits the glycolytic consumption of ATP thereby maintaining the ATP pool during ischemia and preventing metabolic breakdown. Overexpression of these proteins in cardiac myocytes also maintains mitochondrial membrane potential and prevents the release of mitochondrial proteins during ischemic interventions. Prior studies on Bcl-xL in the heart have focused on mitochondrial transport kinetics, free radical production and cell death/growth assays. There is a lack of information concerning the metabolic changes associated with Bcl-xL, unexpected since Bcl-xL interacts directly with mitochondrial membrane proteins associated with the regulation of oxidative phosphorylation. In addition, there is limited information on Ca2+ homeostasis during cardioprotective and/or ischemic interventions. Bcl-xL-dependent modulation of IP3-receptors and mitochondrial Na+-Ca2+ exchange by Bcl-xL has been proposed and may contribute to the proposed cardioprotective qualities of Bcl-xL. The BRC aims to characterize the effect of Bcl-xl overepxression on both cellular energy metabolism and contractile function in cardiac myocytes. Metabolism will be assessed using self-referencing oxygen microsensors that will assess the oxygen uptake of control and Bcl-xL+ myocytes. In addition, myocytes will be co-infected with adenovirus containing constructs for cytosolic and mitochondrial luciferase in order to assess intracellular ATP concentration via the luciferin/luciferase reaction. Ca2+ homeostasis will be measured using AM ester Ca2+ dyes and confocal microscopy. It is the aim of the BRC to address whether there is a metabolic basis to the cardioprotective action of Bcl-2 proteins. In addition, it is our objective to assess Ca2+ transport and Ca2+-dependent signal transduction during Bcl-xL upregulation and to address whether this contributes to the observed changes in cardiac bioenergetics and cell survival. It is anticipated that integrated technologies available at the BRC will generate invaluable information regarding cardiac bioenergetics and identify potential therapeutic targets for the implementation of cardioprotection.
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