Effects of IVS6-1 Mutation in MYl2 Associated with Cardioskeletal Myopathy and Early Cardiac Death of Infants

Abstract

The homozygous appearance of intronic mutation (IVS6-1) in MYL2 encoding myosin ventricular/slow skeletal myosin regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy. The affected infants died between 4 and 6 months of age due to dilated or hypertrophic cardiomyopathy. In this report we have investigated, the molecular mechanism and functional consequences associated with IVS6-1 using recombinant human cardiac IVS6-1-RLC reconstituted in RLC-depleted porcine cardiac muscle preparations. The results were compared with those obtained for recombinant human cardiac WT-RLC. Despite the ability of the mutant to become phosphorylated, IVS6-1 displayed a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT (0.18±0.2 s-1 vs. 0.25±0.02 s-1; n=3), indicating a slower myosin cross-bridge turnover rate of the mutant. Likewise, stopped flow kinetics of the ATP induced dissociation of the acto-myosin complex showed a significantly reduced slope of the kobs-[MgATP] relationship for IVS6-1 reconstituted myosin (4.3±0.02×105 M-1 s-1, n=5), depicting slower second-order MgATP binding rates compared with WT (5.3±0.02×105 M-1 s-1, n=5). Steady-state fluorescence binding experiments of mutant vs. WT reconstituted myosin to pyrene labeled F-actin under rigor conditions demonstrated significantly reduced binding affinity of IVS6-1 (Kd=16.7±2.8 nM, n=3) compared with WT (Kd=4.6±1.3 nM, n=3). In skinned porcine cardiac muscles, a significant decrease in maximal force was observed for IVS6-1 reconstituted fibers compared with WT (27.1±1.0 kN/m2 vs. 34.9±2.4 kN/m2, n=6). The Ca2+ sensitivity and cooperativity were significantly different in IVS6-1 (pCa50=5.66±0.01, nH=2.9±0.15) vs. WT (pCa50=5.48±0.01, nH=2.2±0.14). In conclusion, we demonstrate that IVS6-1 may lead to cardiac dysfunction by disrupting the acto-myosin interaction (steady-state and kinetics) and compromising the ability of the mutant myosin to develop contractile force.