A 5-year research program is outlined with the broad, long-term objective of elucidating the molecular mechanisms responsible for altered Ca2+ transporter gene expression in patients with cardiac hypertrophy and heart failure. There is now substantial evidence to indicate that expression levels of SERCA2 and NCX1, the major Ca2+ transporters in cardiac muscle, are profoundly altered in the failing human ventricular myocardium. These changes may result in reduced contractile function and increased susceptibility to ventricular arrhythmias. However, the underlying intracellular me4chanisms responsible for these changes, and the signal transduction pathways involved are only now being elucidated.
Four specific aims are outlined to clarify these mechanisms in cultured cardiomyocytes, and related them to what may be occurring in hypertrophy and heart failure in experimental animals and man. First, previous work and preliminary data indicate a critical role of PKC activation in SERCA2 down-regulation during hypertrophy and heart failure. We will therefore use molecular biological techniques to over- express and down-regulate specific PKC isozymes to ascertain which PKC isozymes is responsible. Second, we will characterize the [Ca2+]i and Ras-dependent signaling pathways that regulate SERCA2 gene expression. Studies will focus on the non-receptor protein tyrosine kinase PYK2 that is activated by [Ca2+]i and PKC, and that may link G1- coupled receptor activation to the Ras-Raf-MEK-ERK protein kinase cascade. Third, preliminary data indicate that the 3' untranslated region of the SERCA2 mRNA regulates its stability in response to mechanical and neurohormonal stimuli that activate PKCs. Therefore, a series of experiments is outlined to define the cis-acting sequences and trans-acting factors that are involved. Fourth, we will test the hypothesis that activation of PKCs by either neurohormonal or mechanical stimuli (or their combination) up-regulates NXC1 mRNA and protein levels, and begin to analyze the signaling pathways responsible for these changes. The proposed experiments should substantially contribute to our understanding of the mechanisms responsible for altered Ca2+ transporter gene expression in heart failure Future therapeutic strategies targeted towards prevention or reversal of these changes require a thorough understanding of the responsible intracellular mechanisms.
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