Molecular regulation of sarcomere function
Authors:   , Robinett Joel 
Published: August 2022

Molecular regulation of striated muscle contraction is widely thought to be regulated through canonical thin filament mechanisms, i.e., Ca2+ activation of thin filaments. Dual filament regulation is a novel model of striated muscle contraction, in which thick filament activation is regulated by the stress, i.e., thick filaments are activated when the muscle bears a relatively high external load. This study aimed to provide insight on the molecular regulation of multiple biophysical properties of striated muscle. Myosin binding protein-C (MyBP-C) is capable of binding both thin and thick filaments rendering it a well suited regulatory molecule. These interactions are modulated by MyBP-C phosphorylation. MyBP-C's interaction with myosin has the potential to sequester myosin heads in the OFF state and genetic mutations of three myosin binding residues in cMyBP-C causes cardiac hypertrophy. We investigated MyBP-C's regulation of myofilament/sarcomere function by performing slack-restretch protocol to test force development and transient force overshoot and step-stretch protocol to test stretch activation using PKA and lambda phosphatase to modulate sMyBP-C phosphorylation. PKA doubled the transient force overshoot at low (~25 percent) Ca2+ activations in slow-twitch skeletal muscle fibers, while lambda phosphatase decreased transient force overshoot at low (~25 percent) and medium (~50 percent) Ca2+ activation levels. Rates of force development were increased following PKA and decreased following lambda phosphatase at all Ca2+ activation levels. Similarly, PKA increased stretch activation at low (~25 percent) Ca2+ activation levels, while rates of delayed force development (kdf) increased at all Ca2+ activation levels. Additionally, MyBP-C is localized in striated muscle throughout the inner third of each half thick filament, a region known as the C-zone. MyBP-C is capable of binding actin with micromolar affinity. In isolated filaments, thin filament sliding slows as they transverse the C-zone of thick filaments. In support of this, extraction or genetic ablation of MyBP-C sped rates of sarcomere shortening, i.e., MyBP-C has the potential to impose an internal drag that impedes loaded shortening. However, this has not been shown at the sarcomere level; therefore, we designed experiments whereby permeabilized slow-twitch skeletal muscle fibers were stretched to SLs at which the thin filaments were outside of the C-zone (SL ~3.08-3.04 [mu letter]m), then sub-isometric force clamps were performed to elicit sarcomere shortening to propel thin filaments into the C-zone. PKA increased sarcomere shortening into the C-zone, while dephosphorylation of sMyBP-C by lambda phosphatase resulted in a deceleration of shortening and a brief recoil at sarcomere lengths near where the thin filaments should enter the C-zone implicating the potential for a substantive internal load that can oppose myofilament sliding. Beat-to-beat heart function is modulated by [beta]-adrenergic stimulation, which elicits PKA phosphorylation of several cardiac myocyte proteins including cardiac troponin I (cTnI). PKA phosphorylation of the cTnI is known to regulate myofilament Ca2+ sensitivity of force, augment cardiac myofilament power, transient force overshoot, and cross-bridge cycling kinetics. However, it is unknown if cTnI N-terminal phosphorylation per se affects myofilament power and stretch activation. We tested the hypothesis that cTnI N-terminal pseudo-phosphorylation will increase transient force overshoot, stretch activation, rates of force development, and power output consistent with a thin filament molecular mechanism regulating sarcomere function. Molecular sufficiency was investigated by measuring power and stretch activation after cTn exchange into rat permeabilized slow-twitch skeletal muscle fibers, since slow-skeletal TnI lacks N-terminal phosphorylation sites. cTnI pseudo-phosphorylation increased stretch activation and fiber power output generating capacity. Combinatorial S22/23D + Y26E cTnI synergistically increased rates of delayed force development. Taken together, these results suggest MyBP-C and its phosphorylation state regulates sarcomere contraction by modified cross-bridge cycling kinetics, cross-bridge recruitment, and altered internal drag forces that tend to oppose force generation, myofilament sliding, and power output. TnI and its phosphorylation state appears to regulate stretch activation and power output by a combination of redundant and synergistic biophysical mechanisms, which may complement each other to tune beat-to-beat myocardial output demand.