Regulatory Light Chain Mutants Linked to Heart Disease affect the Cardiac Myosin Lever-Arm

Abstract

Myosin is the chemomechanical energy transducer in striated muscle working in the heart to circulate blood. Transduction occurs by cyclical actomyosin interaction causing myosin thick filament sliding relative to the actin thin filament while consuming chemical energy from ATP. Transduction begins in the myosin head domain or cross-bridge with ATP hydrolysis driving rotary movement of a lever-arm converting rotation into linear translation. Myosin regulatory light chain (RLC) binds to the lever-arm and affects its ability to translate actin. Gene sequencing implicated several RLC mutations in human heart disease and three of them are investigated here using single molecule detection of photoactivatable GFP tagged RLC (RLC-PAGFP) exchanged into permeabilized porcine papillary muscle fibers. The RLC-PAGFP detects single lever-arm orientation. Single molecule orientation detection benefits from new analysis tools to deduce PAGFP orientation from the 3 spatial dimensional fluorescence emission pattern. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence single molecule detection, and for 2-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports discretized lever-arm orientation intermediates in active isometric fibers characteristic to the wild type (WT) RLC where lever-arms occupy intermediates on the free-energy gradient that on average correspond to the cross-bridge stall force. Two disease linked mutants introduced to RLC move state occupancy further down the free energy gradient implying lever-arms rotate more to reach cross-bridge stall force because mutant RLC increases lever-arm shear strain. Lower free energy states could involve strong binding that will inhibit filament sliding in contraction. This research was supported by NIH Institutes: NIAMS R01 AR049277 and NHLBI HL095572 and by the Mayo Foundation.