Thick filament dynamics: A mechanism for protein replacement within the sarcomere.

Abstract

The heart contracts continually throughout one's lifetime through antiparallel sliding of actin-based thin filaments along myosin-based thick filaments, organized within muscle sarcomeres. A recent biochemical study from our lab demonstrated that the half-life of cardiac thick filament proteins is ∼10 days in adult mice, suggesting that continual protein replacement supports structural and functional fidelity. These data further suggest that newly synthesized molecules are randomly incorporated into preexisting thick filaments by a stochastic mechanism. Therefore, we hypothesize that myosin replacement involves the dynamic exchange of single molecules into thick filaments in vivo. To test this hypothesis, we generated an adeno-associated virus that replaced 27±7% of the endogenous regulatory light chain (RLC) on myosin molecules with a fluorescent GFP-RLC. We then visualized myosin within intact hearts using two-photon microscopy. At high magnification, individual sarcomeres were observed with GFP-RLC incorporated into thick filaments. Next, we measured the movement of fluorescently labeled myosin within the heart by irreversibly photobleaching limited areas of the cells and quantifying fluorescence recovery after photobleaching (FRAP). No FRAP occurred within 30 minutes, when photobleaching regions encompassed entire sarcomeres. However, rapid FRAP occurred after photobleaching small portions of single sarcomeres at rates orders of magnitude faster than protein synthesis. Recovery appeared to come from the exchange of molecules in the unbleached portion of the sarcomere and was fastest near the tips of the thick filaments, suggesting enhanced exchange where myosin is less densely packed in the filament. Collectively, these data demonstrate dynamic thick filament structure in vivo with myosin molecules freely exchanging between thick filaments within single sarcomeres. Therefore, these large macromolecular complexes appear to be designed to allow for the rapid incorporation and release of myosin molecules while maintaining the structural integrity that is essential to support contractility.