MECHANISMS OF CONTRACTION IN THE HEART

The long-term goal of the proposed research is to determine the mechanism of Ca2+ activated force generation and filament sliding in cardiac muscle. The basis of Ca2+ activated contraction will be determined utilizing the permeabilized single cardiac myocyte preparation. The cardiac myocyte preparation has several advantages over the multicellular papillary or trabecular muscle preparations including: (1) simplified viscoelastic properties, and (2) the ability to relate contractile function to the protein content determined from the same myocyte. Parallel studies will be performed using skinned single slow and fast skeletal muscle fibers. The parallel studies will be done to take advantage of the wealth of biochemical and contractile information available on skeletal muscle. The comparative approach will facilitate exposing the differences in contractility between cardiac and skeletal muscles and will provide interpretive insights regarding the fundamental processes of force generation and filament sliding in cardiac myocytes. The experimental strategy is to use inorganic phosphate, pH and a newly discovered actin peptide fragment as probes to dissect out the specific cross-bridge state transitions and the functional domains of the actin- myosin bond that underlie force generation and filament sliding in heart. From skeletal muscle studies it appears that phosphate release from actomyosin is closely associated with force generation. the hypothesis tested here is that in cardiac myocytes the effects of phosphate on chemomechanical coupling include alterations in the rate of cross-bridge detachment which would limit the velocity of filament sliding in heart. Further, synthesized actin and myosin peptide fragments will be used to probe the functional domains of the binding sites of these molecules during contraction. For example, it will be determined if the functional binding domain(s) of weakly bound cross-bridges differs from the strongly bound, force-bearing cross-bridge domain(s). The specific aims are: 1. To use inorganic phosphate and pH as probes of the chemomechanical coupling mechanism with the aim of dissecting out cross-bridge state transitions and protein-protein interactions that underlie force generation and filament sliding in heart. 2. To use synthesized peptide fragments that correspond to regions of the putative myosin binding domain of the actin molecule to test the hypothesis that the highly conserved N-terminal domain of the actin molecule is, or is in close proximity to, a myosin binding site in striated muscle. 3. To probe chemomechanical coupling mechanisms and actin-myosin binding domains by manipulating the expression of the myosin heavy chain isoform in cardiac myocytes. The hypothesis tested is that the functional domains of actin and myosin in terms of force generation and filament sliding are myosin isoform-dependent in heart. The experiments proposed here will provide new insights into the fundamental mechanism by which Ca2+ activates the contractile event in normal heart muscle, and shall provide a foundation on which to pursue the long-term objective of understanding mechanisms of contractile dysfunction in diseased heart.