Modulators of Cardiomyopathic Diseases

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 DESCRIPTION (provided by applicant): The long-term goal of this work is to identify components inside the cardiac cell that are involved with the development of cardiomyopathic diseases. This proposal focuses on cardiac troponin C (cTnC), the on-off switch of the contractile apparatus and a major cardiomyocyte intracellular Ca2+ buffer. Aimed at understanding the regulatory properties of the troponin complex in vivo and its link to abnormal intracellular Ca2+ handling governing heart disease, this proposal is of considerable health relevance. Mutations in the regulatory complex of the thin filament (tropomyosin, troponin T and troponin I) associated with phenotypic outcomes of hypertrophic (HCM) and dilated (DCM) cardiomyopathies are suggested to indirectly disrupt cardiac muscle contraction by altering the Ca2+-binding properties of cTnC. However, effects of cTnC mutants that influence Ca2+-sensitive contractile responses have yet to be tested for their pathogenic capacity in living organisms. The central hypothesis guiding this proposal is that changes in cTnC N-terminus Ca2+-binding affinity, alone, can evoke cardiac remodeling in vivo. We further posit that ablation of a dedicated high-fidelity kinase has the potential to reverse the hypercontractile state imposed by Ca2+-sensitizing HCM-linked cTnC mutants. Aim 1 will evaluate direct changes in cTnC Ca2+-binding affinity in the thin filament as a critical determinant underlying cardiomyopathic development. This Aim tests the hypothesis that cTnC mutants increasing Ca2+-binding affinity in the N-domain (regulatory) can instigate diastolic dysfunction, leading to HCM; while a designed mutant decreasing cTnC Ca2+- binding affinity will recapitulate a DCM-reminiscent phenotype. Cardiac patho-physiological, biophysical and biochemical approaches will be used to dissect the role of cTnC mutations in our newly developed knock-in (KI) mice. The antithetical effects that these cTnC mutants exert on Ca2+-binding dynamics will be investigated early, prior to development of distinctive cardiac remodeling. In addition, this projet will further define TNNC1 (cTnC- encoding gene) as a cardiomyopathy-susceptibility gene. Aim 2 will establish mechanistic and potential therapeutic links regarding normalization of myofilament Ca2+-sensitivity. This Aim examines whether conditional removal of a dedicated sarcomeric kinase will correct the myofilament Ca2+ response, diminish the hypercontractile phenotype and improve cardiac relaxation, thus reversing post-symptomatic HCM disease. The consequences of conditional removal of this kinase in KI cTnC-HCM hearts will be monitored as a function of time. The last aim can serve as a proof-of-concept for the development of targeted therapies aimed at modulating the activity of dedicated sarcomeric kinases. The novel concepts generated here will define the role of cTnC in initiating and modulating one class of cardiomyopathies, thus opening avenues for development of new tailored therapeutic approaches.