To eventually understand the mechanisms of cardiac hypertrophic stimulation and its subsequent progression to congestive heart failure, sense must be made of a complex cascade of hemodynamic and molecular events that affect the cardiocyte's ability to function. The preliminary data generated by the applicant establishes that among the cardiocytes initial response to pathologic hemodynamic load, both in vivo and in vitro, is an up regulation of several components of the nuclear and mitochondrially derived mitochondrial oxidative phosphorylation system at both the mRNA and protein level. Initial studies in a feline pressure- overloaded right ventricle model show upregulation within l to 4 hours of the FO6, beta-F1, and alpha-F1, subunits of FOF1-ATP synthase, as well as cytochrome b and cytochrome oxidase subunits Il and IV. These novel findings have been confirmed in two in vitro neonatal rat cardiocyte models of hypertrophic stimulation, one using electrical pacing and one with phenylephrine. These models have also demonstrated an increase in cytochrome c at the protein level. Oncemore, cloning 1480 nucleotides of the 5' flanking region of the critical beta-F1 ATPase gene followed by transient transfection of a reporter construct into neonatal cardiocytes demonstrates a cis region which upregulates in response to phenylephrine and electrical pacing. As the link of early transcriptional (and/or post-) events in the cardiocyte which may lead to physiologically relevant responses to acute load are largely unknown, this proposal offers a strategy for an understanding of the coordinated expression of the nuclear and mitochondrial genomes in this unique phenotype along 4 specific aims:
Specific Aim l: To define the response of highly selected nuclear and mitochondrially encoded oxidative phosphorylation gene mRNAs and their proteins involved in the cardiocyte's response to acute and chronic hypertrophic stress. In particular, the mitochondrially encoded FO6 and nuclear encoded beta-Fl ATPase and cytochrome c will be studied. Partial cDNA constructs and characterized antibodies (to the nuclear gene products) have been used to generate the preliminary data.
Specific Aim 2 : To determine the relative importance of transcriptional versus post-transcriptional mechanisms in the acute upregulation of mitochondrial mRNAs involved in the cardiocyte's hypertrophic response. The beta-Fl ATPase subunit contains a critical catalytic site and is an excellent candidate to study response in the cardiocyte. Preliminary data indicates much of the response is transcriptional.
Specific Aim 3 : To determine the promoter-enhancer regions of the beta- ATPase gene important in the initiation and regulation of the cardiocyte's hypertrophic response.
Specific Aim 4 : To confirm in well-characterized adult models of pressure overload the regulatory response elements determined to be involved in the cardiac hypertrophic response in vitro. Once elements have been identified using the in vitro models, the in vivo response to hypertrophic load stimulation will be determined by established methods of direct gene transfer into the feline heart before and after pulmonary artery banding. As confirmation in cultured, primary adult cardiocytes, highly selected constructs would be transfected using replication-deficient adenovirus constructs. Studying the genetic regulation of oxidative phosphorylation in cardiocytes subjected to hypertrophic stimulus should lead to important, physiologically relevant, insights into key initial molecular events.
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