TROPONIN MODULATION IN HEART FAILURE

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DESCRIPTION (adapted from the applicant's description): Experiments proposed here test hypothesis that alterations in the structure, function, and regulation of cardiac troponin I (cTnI) and cTnT contribute to the evolution and end-stage pathology of heart failure. The long term objective is know how myofilament remodeling and covalent modulation play a role in the evolution of hypertrophy/failure in human hearts. Aims #1, #2, and #3 address the questions: Can altered tension and economy of myofilaments from human hearts in end-stage failure be rescued by replacing components of the Tn complex with recombinant proteins? What is the nature of the thin filament alterations in human heart failure? Are there changes in protein phosphorylation of specific sites on cTnI or isoform population of cTnT? How do these changes alter force and ATPase rate in reconstituted preparations? Aim #4 addresses the question: What is the specific role of protein kinase C (PKC) sites on cTnI in the development of hypertrophy and failure in response to hemodynamic stress of pressure overload? The approach to these objectives involves the use of procedures for exchanging thin filament proteins in the intact force generating lattice, and for isolating Tn complex from small heart tissue samples. Recombinant proteins are prepared in an unphosphorylated state and specifically phosphorylated at PKC and PKA sites. Gel electrophoresis, immunoblotting and antibodies that recognize phosphorylated forms of cTnl are used to detect changes in the Tn complex. To test the role of specific PKC sites on cTnI in the evolution of hypertrophy and failure, mice harboring transgenes expressing a mutant form of cTnl (lacking PKC sites at Ser 43 and Ser 45) and slow skeletal TnI (lacking a PKC site at Thr 144 in the inhibitory peptide) are stressed by pressure overload. These experiments provide crucial information on the mechanisms of heart failure and on the potential value of developing pharmacological approaches to the inhibition of the PKC pathway.