This competing renewal encompasses six collaborative investigations, supported by four integrated core facilities, to address issues fundamental to the etiology, pathogenesis and treatment of cardiac failure. The studies are focused on an attempt to unravel molecular mechanisms underlying cardiac growth, hypertrophy and failure in both acquired and inherited disorders. In Projects 1-2, we will investigate the molecular basis for hypertrophic cardiomyopathy (HCM) and myotonic dystrophy (DM). Complementary DNA constructs with three known beta-myosin heavy chain (betaMHC) mutations will be expressed via E1-deficient adenovirus in feline cardiocytes and packaged into minigenes for in vivo expression in the transgenic rabbit. Cardiac structure and function will be assessed serially in vivo by echocardiography and subsequently by light and electron microscopy to elucidate the consequences of beta MHC mutations. In Project 2, utilizing yeast interaction cloning and immobilized recombinant myotonin protein kinase (MtPK) as an affinity matrix, substrates for MtPK will be identified as an initial step toward identifying the defect in the phosphorylation cascade presumed responsible for DM. In other studies of Projects 1 and 2, large informative pedigrees will be analyzed to identify novel genes for both diseases in families not linked to established loci. In Project 3, using an exceptionally large pedigree (>400) with familial dilated cardiomyopathy (DCM), linkage analysis and positional cloning will be employed to identify the responsible gene. In Project 4, the postulated role of TGFbeta1 in cardiac hypertrophy will be assessed with the use of novel dominant- negative mutants of the TGFbeta receptor in adult ventricular myocytes, using adenoviral gene transfer and in the intact myocardium in transgenic mice. In Project 5, wild-type and dominant-negative mutants of the P55 and P75 TNFalpha receptor will be expressed via adenovirus in adult ventricular myocytes to determine the form of TNFalpha receptor responsible for depressed contractility. A soluble form of the receptor will be given as a TNFalpha inhibitor in an attempt to improve cardiac function in patients with failure and evaluate the role of endogenous TNFalpha in vivo. In Project 6, studies will be performed to determine the role of insulin-like growth factor-I (IGF-l) in cardiac growth and hypertrophy, utilizing transgenic mice overexpressing IGF-l or its binding protein. Adenoviral gene transfer and dominant-negative mutations of postulated IGF signalling proteins will be used to identify the responsible cytoplasmic transducers and transcription factors that mediate the effect of IGF on cardiac hypertrophy. Together, these six studies should provide significant new insights into the molecular foundations of cardiac hypertrophy and failure and contribute to a rational basis for more effective therapy.
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