Mechanisms of cardiac myosin replacement.

Abstract

A recent mass spectrometry-based proteomic study from our lab demonstrated that newly synthesized myosin molecules are randomly inserted into preexisting thick filaments in adult mouse hearts in vivo. Therefore, we hypothesized that myosin molecules exist in a dynamic equilibrium between a filamentous pool required for contractility, and a monomeric pool required for myosin replacement. To test this hypothesis, we replaced 27 ± 7% of the cardiac myosin regulatory light chain (RLC) in adult mice with fluorescent RLC (GFP-RLC), via adeno-associated viral transfection. We visualized the random distribution of GFP-RLC within thick filaments in intact hearts with 2-photon microscopy, and the exchange of GFP-RLC between these filaments by quantifying fluorescence recovery after photobleaching (FRAP). Rapid FRAP occurred within minutes, when photobleaching small, ∼1-µm3 areas, of partial sarcomeres. These data demonstrate that the GFP-RLC exchanges between the thick filaments and a monomeric pool on a timescale that is orders of magnitude faster than that of protein replacement (t1/2 = 10.8 days). To determine whether the mechanism of this exchange may involve the folding of monomeric myosin, GFP-labeled myosin molecules were extracted from the transfected hearts and their Stokes radii were measured over a range of ionic strengths, using microfluidic diffusional sizing. At low ionic strengths (150 mM KCl), filament formation was favored, but the monomeric fraction of GFP-labeled myosin demonstrated a radius of 12.6±1.1 nm. At higher ionic strengths (400-600 mM KCl), the radius was increased to 18.0 nm. Similar radii have been reported for smooth muscle myosin monomers in folded and extended conformations. Together, these data suggest that myosin molecules exist in rapid equilibrium between a filamentous and monomeric pool within the sarcomere, and individual monomers may adopt a folded, compact conformation to regulate their exchange into, and out of filaments.