Familial Hypertrophic Cardiomyopathy-linked Alterations in Ca2+ Binding of Human Cardiac Myosin Regulatory Light Chain Affect Cardiac Muscle Contraction

Abstract

The ventricular isoform of human cardiac regulatory light chain (HCRLC) has been shown to be one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular and/or septal hypertrophy, myofibrillar disarray, and sudden cardiac death. Our recent studies have demonstrated that the properties of isolated HCRLC could be significantly altered by the FHC mutations and that their detrimental effects depend upon the specific position of the missense mutation. This report reveals that the Ca 2؉ sensitivity of myofibrillar ATPase activity and steady-state force development are also likely to change with the location of the specific FHC HCRLC mutation. The largest effect was seen for the two FHC mutations, N47K and R58Q, located directly in or near the single Ca 2؉ -Mg 2؉ binding site of HCRLC, which demonstrated no Ca 2؉ binding compared with wild-type and other FHC mutants (A13T, F18L, E22K, P95A). These two mutants when reconstituted in porcine cardiac muscle preparations increased Ca 2؉ sensitivity of myofibrillar ATPase activity and force development. These results suggest the importance of the intact Ca 2؉ binding site of HCRLC in the regulation of cardiac muscle contraction and imply its possible role in the regulatory light chain-linked pathogenesis of FHC.The regulatory light chain (RLC) 1 of myosin is a major regulatory subunit of smooth-muscle and non-muscle myosins and a modulator of the troponin (Tn) -controlled regulation of the striated muscle contraction. The crystal structures of chicken skeletal S1 (1) and the regulatory domain of scallop myosin, consisting of one RLC, one essential light chain (ELC), and a part of the myosin heavy chain (2), have revealed that the RLC is localized at the head-rod junction of the myosin heavy chain and, together with the ELC, stabilizes the ␣-helical neck of the myosin head. The N terminus of RLC is noncovalently bound to the myosin heavy chain between Asn-825 and Leu-842, whereas its C terminus wraps around the region located between Glu-808 and Val-826 of the myosin heavy chain (1). The N-terminal domain of the RLC contains a divalent cationbinding site, located in the first helix-loop-helix motif, which binds both Ca 2ϩ and Mg 2ϩ (Fig. 1A). The N-terminal region of RLC also contains the myosin light chain kinase-specific phosphorylation site (Ser-15), which is located in the proximity of the cation-binding site (3).Recent studies have revealed that the ventricular RLC is one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (4 -6). FHC is an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray, and sudden cardiac death. It is caused by missense mutations in various genes that encode for ␤-myosin heavy chain (7), myosin-binding protein C (8), ventricular RLC and ELC (4 -6, 9), troponin T (10), troponin I (11), troponin C (TnC) (12), ␣-tropomyosin (13), actin (14), and titin (15). Depending on the affecte...

Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry

“…As mentioned earlier, the observed reduction in force in the mutant myosins is consistent with a mutation-induced reduction in the stiffness of the lever arm. Consistent with the idea that the mutations change the mechanical properties of the lever arm, both R58Q and N47K disrupt cationic binding to the RLC (12,13). Disruption of the calcium binding site has been shown to cause structural changes in the RLC, possibly impairing its role in mechanically supporting the MHC α-helix (3, 4) and transmitting strain to the active site.…”

“…Interestingly, cationic binding to R58Q RLC can be restored with phosphorylation of the RLC (12,13), raising the intriguing possibility that a phosphorylation-induced stiffening of the myosin lever arm could rescue the mutant phenotypes. Our previous research with skeletal muscle myosin suggests that myosin with phosphorylated RLC exhibits a greater strain sensitivity than dephosphorylated myosin (23,24).…”

“…Population studies have identified two FHCassociated mutations located near the RLC cation binding site, R58Q and N47K (10,11), that were shown to disrupt Ca 2+ binding to RLC and its phosphorylation (12,13). Several biochemical, structural, and physiological defects have been shown to correlate with the R58Q and N47K mutations when studied in isolation (12), exchanged in skinned porcine papillary muscles (13) or in transgenic mouse models (14,15).…”

A public resource facilitating clinical use of genomes

“…Focused follow-up was performed for one of the first variants found, MYL2-A13T in PGP6, which has been reported to cause familial hypertrophic cardiomyopathy in a dominant manner (40)(41)(42)(43)(44). Because this disease is potentially lethal and because there were several publications supporting a pathogenic effect for the variant (SI Appendix, Fig.…”

Biochemical and Physiological Regulation of Cardiac Myocyte Contraction by Cardiac-Specific Myosin Light Chain Kinase

“…10 The MLC2 also contains a Ca 2+ /Mg 2+ binding site at its N-terminus from Asp-37 to Asp-48, located in the first helix-loop-helix motif, and the binding of divalent cation alters the structural and contractile properties. 28, 29 The neck region of the myosin head has been proposed to act as a lever arm. The phosphorylation of MLC2 at Ser-15 results in the addition of a negative charge to the N-terminal region of MLC2, which induces the myosin head to swing out from a position close to the thick filament's backbone toward the actin filament, and this structural change increases the rate through which the myosin-actin interaction occurs and promotes force generation at a given level of Ca 2+ (Figures 1,3).…”