Mutations in myosin S2 alter cardiac myosin-binding protein-C interaction in hypertrophic cardiomyopathy in a phosphorylation-dependent manner

Abstract

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disorder primarily caused by mutations in the β-myosin heavy-chain gene. The proximal subfragment 2 region (S2), 126 amino acids of myosin, binds with the C0-C2 region of cardiac myosin-binding protein-C to regulate cardiac muscle contractility in a manner dependent on PKA-mediated phosphorylation. However, it is unknown if HCM-associated mutations within S2 dysregulate actomyosin dynamics by disrupting its interaction with C0-C2, ultimately leading to HCM. Herein, we study three S2 mutations known to cause HCM: R870H, E924K, and E930Δ. First, experiments using recombinant proteins, solid-phase binding, and isothermal titrating calorimetry assays independently revealed that mutant S2 proteins displayed significantly reduced binding with C0-C2. In addition, CD revealed greater instability of the coiled-coil structure in mutant S2 proteins compared with S2 Wt proteins. Second, mutant S2 exhibited 5-fold greater affinity for PKA-treated C0-C2 proteins. Third, skinned papillary muscle fibers treated with mutant S2 proteins showed no change in the rate of force redevelopment as a measure of actin–myosin cross-bridge kinetics, whereas S2 Wt showed increased the rate of force redevelopment. In summary, S2 and C0-C2 interaction mediated by phosphorylation is altered by mutations in S2, which augment the speed and force of contraction observed in HCM. Modulating this interaction could be a potential strategy to treat HCM in the future.

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin-Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy-Causing Double Mutation MYBPC3Δ25bp/D389V

“…The K d value of the interaction of β-CM with C0–C2 wt has been calculated to be 12.7 ± 4.7 µM, whereas C0–C2 D389V has an affinity of 14.8 ± 7.7 µM. The values are consistent with prior studies [ 16 , 18 , 58 , 59 ] and indicate no effect of D389V on the interaction of the N-terminal region of MyBPC with F-actin and β-CM. Since the binding of C0–C2 to β-CM and F-actin is at least two orders of magnitude stronger, it is unlikely that the interactions of the C2 domain alone are of physiological importance.…”

“…The affinities determined for the interaction of C0–C2 with β-CM and actin are at least two orders of magnitude greater, which implies that potential interactions of C2 make a minor contribution compared to those of the other N-terminal domains. The values are unchanged by the D389V mutation and consistent with those determined in previous studies [ 16 , 18 , 58 , 59 ], thus excluding altered affinities as a pathomechanism.…”

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin–Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy–causing Double Mutation MYBPC3Δ25bp/D389V

“…The affinities determined for the interaction of C0-C2 with myosin and actin are at least two orders of magnitude greater, which implies that potential interactions of C2 make a minor contribution compared to those of the other N-terminal domains. The values are unchanged by the D389V mutation and consistent with those determined in previous studies [16,18,57,58], thus excluding altered affinities as a pathomechanism.…”

“…The Kd value of the interaction of myosin with C0-C2 wt has been calculated to be 12.7 ± 4.7 µM, whereas C0-C2 has an affinity of 14.8 ± 7.7 µM. The values are consistent with prior studies [16,18,57,58] and indicate no effect of D389V on the interaction of the N-terminal region of MyBPC with F-actin and myosin.…”

Structural and Phenotypic Correction of K210del Genetic Cardiomyopathy by an FDA Approved Drug

“…A pair of papillary muscles were isolated and placed in the skinning buffer, to skin overnight at 4 0 C. Skinning buffer comprised of 1% Triton X-100 in buffer containing; 55.74 mM potassium propionate, 7 mM ethylene glycol bis(2-aminoethyl) tetra acetic acid, 100 mM N,N-bis(2-hydroxyethyl)-2-amino ethane sulfonic acid, 20 µM calcium chloride, 5.5 mM magnesium chloride, 5 mM dithiothreitol, 15 mM creatine phosphate and 4.7 mM adenosine triphosphate with pH adjusted to 7.0 with 4 M potassium hydroxide and ionic strength maintained at 180 with potassium propionate. The calcium strength in the above buffer was minimal with pCa 9.0 also termed as relaxing buffer since the muscles don't contract in the pCa 9.0 buffer [47][48][49] . After overnight skinning, the bers were ready for experimental use.…”