Abstract
Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.
Highlights
Muscle contraction is achieved by the cyclic interaction between myosin crossbridges of the thick filament and actin on the thin filament, during which the actin-myosin-ATP complex undergoes several different states to achieve the transduction of chemical energy stored in ATP to mechanical work
CMyBP-C Phosphorylation and Cross-Bridge Kinetics cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases
With the importance of cardiac myosin binding protein C (cMyBP-C) phosphorylation in contraction, regulation, and the hierarchically functional S273, S282 and S302 sites in mind, we have recently found that phospho-aberrant Ala282 combined with phosphomimetic residues Asp273 and/or Asp302 is detrimental to cardiomyocytes as evidenced by the lower active isometric tension and altered cross-bridge kinetics with decreased 2pc and increased 2pb
Summary
Muscle contraction is achieved by the cyclic interaction between myosin crossbridges of the thick filament and actin on the thin filament, during which the actin-myosin-ATP complex undergoes several different states to achieve the transduction of chemical energy stored in ATP to mechanical work. This is called the cross-bridge cycle, and each step within the cycle is called an elementary step. The phosphorylation status of cMyBP-C is associated with various pathological conditions in the heart, and plays a critical role in regulating force generation by modulating the thick-tothin filament interaction [6,7,8,9]. S282 is phosphorylated first before phosphorylation takes place at S273 and S302 [10], S282 is thought to play a critical role in the phosphorylation of cMyBP-C [13]
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