Time-Resolved Fluorescence Quenching Measurements to Study the Structural Defects Induced by Myosin Essential Light Chain Cardiomyopathy Mutations in Muscle Fibers

Abstract

We used time-resolved fluorescence quenching measurements to determine the effects of three cardiomyopathy-causing myosin essential light chain (ELC) mutations (E143K-, M149V, and R154H) on the structure of myosin in muscle fibers. These ELC mutations cause familial hypertrophic cardiomyopathy (FHC) in humans. A cysteine at C187 in the ventricular ELC was labeled in the absence (wildtype) and presence of the ELC mutations, with a fluorescent probe, called IAEDANS. The labeled ELCs were then be separately reconstituted into ELC-depleted skeletal muscle fibers. Acrylamide was used as a “quencher” to determine whether the ELC mutation changes the environment of the probe in rigor, relaxing, and isometrically contracting muscle fiber bundles. Time-resolved emission lifetimes were acquired as a function of [acrylamide] for the wildtype and the ELC mutants in the muscle fibers. The fluorescence lifetime of the probe will decay faster when it is exposed to the acrylamide on the surface of the protein than when it is more-buried in the protein interior. We determined that the ELC mutants perturbed the extent of acrylamide quenching in rigor, relaxing, and contracting muscle fiber bundles compared to fiber bundles with the wildtype ELC, suggesting that the ELC mutations induced structural changes to expose the probe to the solvent. This research is significant to understanding how myosin dysfunction is correlated with the phenotype of the FHC disease. Fluorescence spectroscopy was performed at the Biophysical Spectroscopy Center at the University of Minnesota, with assistance from Fluorescence Innovations, Inc. (Greg Gillispie, President). This research was supported by a NIH grant (AR052360) to OR and UofMn UROP fellowships to RJP.