Replacement of Myosin Molecules Within Cardiac Thick Filaments in Intact Mouse Hearts

A mouse heart contracts ten times per second with exquisite fidelity, while myosin, the heart's most abundant molecular constituent responsible for contraction, is continually renewed through protein synthesis and degradation. To examine how myosin molecules are replaced in intact cardiac muscle, we labeled cardiac myosin with a GFP-tag in living mice using an adeno-associated virus. The GFP-labeled myosin replaced endogenous myosin within cardiac muscle sarcomeres without overexpression and had no effect on whole heart structure or myosin function as measured in an in vitro motility assay. Biochemical extraction of myosin from mouse hearts demonstrated that the majority of myosin molecules were sequestered within thick filaments in cardiac sarcomeres, with <10% of the myosin being in the sarcomere's cytosol. Multiphoton fluorescence recovery after photobleaching (FRAP) in ex vivo whole heart preparations demonstrated the myosin molecules rapidly exchange between myosin thick filaments and the cytosol. The exchange of myosin molecules was restricted to thick filaments within a single sarcomere, with an equilibration half-life of <1 hour. To determine whether the exchange of myosin molecules was coupled to the rates of myosin synthesis and degradation (i.e. myosin turnover), we quantified the incorporation of isotopically labeled amino acids into cardiac myosin invivo using mass spectrometry. The rate constant for myosin turnover was orders of magnitude slower than that of myosin exchange, with the half-life of a single myosin molecule being ∼10 days. We conclude that myosin thick filament structure is dynamic in intact mouse hearts, with myosin molecules being continually exchanged in and out of thick filaments within each sarcomere. We hypothesize that physiological perturbations which limit the rate of myosin exchange, may limit myosin's degradation, and result in the accumulation of myosin within cardiac muscle cells.