Molecular Mechanisms of Myofilament Dysfunction in Heart Failure

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Congestive heart failure (CHF) is associated with an abnormality in cardiac cell function. The molecular mechanisms that underlie this depressed function in CHF are unknown. In previous work we have shown that myofilament function is depressed in CHF in terms of depressed maximum force generating capacity, calcium responsiveness, and cross-bridge cycle kinetics. Experimental mechanical/biochemical data suggest that dys-regulated myofilament contractile protein phosphorylation causes myofilament dysfunction in CHF, possibly via altered phosphorylation of myosin light chain (MLC), myosin binding protein C (MyoBPC), and Troponin-l (Tnl). However, the precise structure-function relationship has not been determined. In this proposal for continued support we will employ a well-established model of CHF in the guinea-pig secondary to pressure overload. The guinea-pig model allows study of control, compensatory hypertrophy, and CHF in a model that closely resembles the myofilament and EC-coupling parameters as found in the human. Biochemical proteomics analysis will be used to identify pathways and proteins that are targeted in CHF and the impact of identified post-translational modifications on contractile function will be determined using a variety of biophysical techniques ranging from intact electrically stimulated isolated muscle to single myofibrils (aim 1). Contractile protein post-translational modifications and signal pathways will be manipulated via adenoviral gene delivery, kinase/phosphatase treatment and recombinant protein contractile protein exchange in permeabilized isolated myocardium (aim 1). As we recently demonstrated, regional myofilament function is not uniform in the heart and this distribution is significantly altered in heart failure. Experiments proposed in specific aim 2 will determine the signal pathways and contractile protein posttranslational modifications that underlie these phenomena. Finally, experiments proposed in aim3 will determine the dynamic and temporal coupling between the driving Ca2+ transient and the mechanical dynamic contractile protein force production; these experiments will be performed in single cardiac myofibrils. Overall, our aim is to determine the mechanisms that underlie contractile protein dysfunction in CHF. Our research will aid in the development of new therapeutic strargies to combat OHF in patients.