Familial hypertrophic cardiomyopathy is a disease caused by single mutations in several sarcomeric proteins, including the human myosin ventricular regulatory light chain (vRLC). The effects of four of these mutations (A13T, F18L, E22K, and P95A) in vRLC on force generation were determined as a function of Ca2+ concentration. The endogenous RLC was removed from skinned rabbit psoas muscle fibers, and replaced with either rat wildtype vRLC or recombinant rat vRLC (G13T, F18L, E22K, and P95A). Compared to fibers with wildtype rat vRLC, the E22K mutant increased Ca sensitivity of force generation, whereas the G13T and F18L mutants decreased the Ca sensitivity, and the P95A mutant had no significant effect. None of the RLC mutants affected the maximal tension (observed at saturating Ca2+ concentrations), except for F18L, which decreased the maximal tension to 69 ± 10% of the wildtype value. Of the mutant RLCs, only F18L decreased the cooperativity of activation of force generation. These results suggest that the primary cause of familial hypertrophic cardiomyopathy, in some cases, is perturbation in the Ca sensitivity of force generation, in which Ca-sensitizing or Ca-desensitizing effects can lead to similar disease phenotypes.
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The present results show that ∼50% mutant RLC is sufficient to perturb the tension at subsaturating Ca concentration ( Fig. 5 D ), Ca sensitivity, and cooperativity of force generation of fibers ( Fig. 6 ), supporting the hypothesis that the mechanical dysfunction of the fibers may be directly linked to the mechanism for FHC. The enhanced (E22K) and diminished (G13T and F18L) function of these FHC-RLC mutations fibers suggest that the FHC disease may be caused by fibers that are in either a hypercontractile (E22K) or hypocontractile (G13T and F18L) state. Previous reports also suggest that different FHC mutations can induce either a gain or loss of function ( Roopnarine, 2002 ; Bonne et al., 1998 ; Seidman and Seidman, 2001 ; Hernandez et al., 2001 ; Michele and Metzger, 2000 ). The dysfunction caused by the FHC-RLC mutants suggest that the mutant protein will alter the force generated in myocytes and lead to mechanical stress within the heart, which is likely to cause myocyte disarray and remodeling of the heart via hypertrophy. This study shows that wildtype ventricular RLC confers Ca-sensitive force in skeletal muscle similar to that of cardiac muscle. Of the four FHC-RLC mutants tested, three caused immediate and substantial changes in the Ca sensitivity of force production, suggesting that these mechanical abnormalities initiate the FHC disease process. The author thanks Dr. Leslie A. Leinwand for the wildtype rat vRLC cDNA; Dr. LaDora Thompson for use of Muscle Research System for the single fiber mechanics; Dr. Dawn Lowe for technical instruction on using the Muscle Research System; and Dr. David D. Thomas for research facilities and scientific advice. The author also thanks Bilal Anwer (University of Minnesota); Richard Timm, Laura Gingras, and Melanie Thomas (Breck High School, Minnesota), for assistance in protein purification and muscle fiber preparation. This work was supported by grants from the American Heart Association Northland Affiliate, the Muscular Dystrophy Association, the Minnesota Medical Foundation, and the National Institutes of Health (AR32961).