Orientation of the Myosin Regulatory Light Chain in Cardiac Muscle Determined by Polarized Fluorescence

Abstract

The myosin regulatory light chain (RLC) is a component of the myosin lever arm, and the cardiac RLC isoforms are common sites of mutations associated with familial hypertrophic cardiomyopathy. Phosphorylation of the RLC can modulate cardiac contractility independently of troponin/tropomyosin-mediated regulation, but the molecular mechanism of such modulation is poorly understood. We are investigating structure-function relationships in the cardiac myosin RLC in the native environment of cardiac muscle cells. Pairs of cysteine residues were genetically introduced at six different pairs of surface-accessible sites in the RLC, each of which are 10-15 A apart. Three pairs of cysteines were introduced in the N-terminal lobe of the RLC, and three in the C-terminal lobe. Each pair of cysteines was cross-linked with a bifunctional rhodamine: bis-[N-iodoacetyl(piperazyl)]-sulforhodamine (BSR; B-10621, Invitrogen) and the labelled protein purified to 95% homogeneity. The pure 1:1 BSR-RLC conjugates are exchanged into demembranated cardiac muscle cells and the orientation of the BSR fluorescence dipole determined by polarized fluorescence.With three probes on each lobe of the RLC, the orientation of the lobe with respect to the actin filament axis and the bend between the lobes can be determined. We are measuring these orientations under various physiological conditions including relaxation, active contraction, and rigor, and investigating the effects of RLC phosphorylation by myosin light chain kinase. These experiments should provide insight into RLC function in cardiac muscle and the mechanism of modulation of cardiac contractility by RLC phosphorylation.Supported by the British Heart Foundation.