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AbstractPrecise regulation of sarcomeric contraction is essential for normal cardiac function. The heart must generate sufficient force to pump blood throughout the body, but either inadequate or excessive force can lead to dysregulation and disease. Myosin regulatory light chain (RLC) is a thick‐filament protein that binds to the neck of the myosin heavy chain. Post‐translational phosphorylation of RLC (RLC‐P) by myosin light chain kinase is known to influence acto‐myosin interactions, thereby increasing force production and Ca²⁺‐sensitivity of contraction. Here, we investigated the role of RLC‐P on cardiac structure and function as sarcomere length and [Ca²⁺] were altered. We found that at low, non‐activating levels of Ca²⁺, RLC‐P contributed to myosin head disorder, though there were no effects on isometric stress production and viscoelastic stiffness. With increases in sarcomere length and Ca²⁺‐activation, the structural changes due to RLC‐P become greater, which translates into greater force production, greater viscoelastic stiffness, slowed myosin detachment rates and altered nucleotide handling. Altogether, these data suggest that RLC‐P may alter thick‐filament structure by releasing ordered, off‐state myosin. These more disordered myosin heads are available to bind actin, which could result in greater force production as Ca²⁺levels increase. However, prolonged cross‐bridge attachment duration due to slower ADP release could delay relaxation long enough to enable cross‐bridge rebinding. Together, this work further elucidates the effects of RLC‐P in regulating muscle function, thereby promoting a better understanding of thick‐filament regulatory contributions to cardiac function in health and disease.image Key pointsMyosin regulatory light chain (RLC) is a thick‐filament protein in the cardiac sarcomere that can be phosphorylated (RLC‐P), and changes in RLC‐P are associated with cardiac dysfunction and disease.This study assesses how RLC‐P alters cardiac muscle structure and function at different sarcomere lengths and calcium concentrations.At low, non‐activating levels of Ca²⁺, RLC‐P contributed to myofilament disorder, though there were no effects on isometric stress production and viscoelastic stiffness.With increases in sarcomere length and Ca²⁺‐activation, the structural changes due to RLC‐P become greater, which translates into greater force production, greater viscoelastic stiffness, slower myosin detachment rate and altered cross‐bridge nucleotide handling rates.This work elucidates the role of RLC‐P in regulating muscle function and facilitates understanding of thick‐filament regulatory protein contributions to cardiac function in health and disease. 

Mechanical Control of Relaxation refers to the dependence of myocardial relaxation on the strain rate just prior to relaxation, but the mechanisms of enhanced relaxation are not well characterized. This study aimed to characterize how crossbridge kinetics varied with strain rate and time-to-stretch as the myocardium relaxed in early diastole. Ramp-stretches of varying rates (amplitude = 1% muscle length) were applied to intact rat cardiac trabeculae following a load-clamp at 50% of the maximal developed twitch force, which provides a first-order estimate of ejection and coupling to an afterload. The resultant stress-response was calculated as the difference between the time-dependent stress profile between load-clamped twitches with and without a ramp-stretch. The stress-response exhibited features of the step-stretch response of activated, permeabilized myocardium, such as distortion-dependent peak stress, rapid force decay related to crossbridge detachment, and stress recovery related to crossbridge recruitment. The peak stress was strain rate dependent, but the minimum stress and the time-to-minimum stress values were not. The initial rapid change in the stress-response indicates enhanced crossbridge detachment at higher strain rates during relaxation in intact cardiac trabeculae. Physiologic considerations, such as time-varying calcium, are discussed as potential limitations to fitting these data with traditional distortion-recruitment models of crossbridge activity. 

Muscle contraction results from force-generating cross-bridge interactions between myosin and actin. Cross-bridge cycling kinetics underlie fundamental contractile properties, such as active force production and energy utilization. Factors that influence cross-bridge kinetics at the molecular level propagate through the sarcomeres, cells and tissue to modulate whole-muscle function. Conversely, movement and changes in the muscle length can influence cross-bridge kinetics on the molecular level. Reduced, single-molecule and single-fibre experiments have shown that increasing the strain on cross-bridges may slow their cycling rate and prolong their attachment duration. However, whether these strain-dependent cycling mechanisms persist in the intact muscle tissue, which encompasses more complex organization and passive elements, remains unclear. To investigate this multi-scale relationship, we adapted traditional step-stretch protocols for use with mouse soleus muscle during isometric tetanic contractions, enabling novel estimates of length-dependent cross-bridge kinetics in the intact skeletal muscle. Compared to rates at the optimal muscle length ( L o ), we found that cross-bridge detachment rates increased by approximately 20% at 90% of L o (shorter) and decreased by approximately 20% at 110% of L o (longer). These data indicate that cross-bridge kinetics vary with whole-muscle length during intact, isometric contraction, which could intrinsically modulate force generation and energetics, and suggests a multi-scale feedback pathway between whole-muscle function and cross-bridge activity. 

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that influences sarcomere stiffness and modulates cardiac contraction-relaxation through its phosphorylation. Phosphorylation of cMyBP-C and ablation of cMyBP-C have been shown to increase the rate of MgADP release in the acto-myosin cross-bridge cycle in the intact sarcomere. The influence of cMyBP-C on Pi-dependent myosin kinetics has not yet been examined. We investigated the effect of cMyBP-C, and its phosphorylation, on myosin kinetics in demembranated papillary muscle strips bearing the β-cardiac myosin isoform from nontransgenic and homozygous transgenic mice lacking cMyBP-C. We used quick stretch and stochastic length-perturbation analysis to characterize rates of myosin detachment and force development over 0–12 mM Pi and at maximal (pCa 4.8) and near-half maximal (pCa 5.75) Ca 2+ activation. Protein kinase A (PKA) treatment was applied to half the strips to probe the effect of cMyBP-C phosphorylation on Pi sensitivity of myosin kinetics. Increasing Pi increased myosin cross-bridge detachment rate similarly for muscles with and without cMyBP-C, although these rates were higher in muscle without cMyBP-C. Treating myocardial strips with PKA accelerated detachment rate when cMyBP-C was present over all Pi, but not when cMyBP-C was absent. The rate of force development increased with Pi in all muscles. However, Pi sensitivity of the rate force development was reduced when cMyBP-C was present versus absent, suggesting that cMyBP-C inhibits Pi-dependent reversal of the power stroke or stabilizes cross-bridge attachment to enhance the probability of completing the power stroke. These results support a functional role for cMyBP-C in slowing myosin detachment rate, possibly through a direct interaction with myosin or by altering strain-dependent myosin detachment via cMyBP-C-dependent stiffness of the thick filament and myofilament lattice. PKA treatment reduces the role for cMyBP-C to slow myosin detachment and thus effectively accelerates β-myosin detachment in the intact myofilament lattice. NEW & NOTEWORTHY Length perturbation analysis was used to demonstrate that β-cardiac myosin characteristic rates of detachment and recruitment in the intact myofilament lattice are accelerated by Pi, phosphorylation of cMyBP-C, and the absence of cMyBP-C. The results suggest that cMyBP-C normally slows myosin detachment, including Pi-dependent detachment, and that this inhibition is released with phosphorylation or absence of cMyBP-C. 

Morbidity and mortality associated with heart disease is a growing threat to the global population, and novel therapies are needed. Mavacamten (formerly called MYK-461) is a small molecule that binds to cardiac myosin and inhibits myosin ATPase. Mavacamten is currently in clinical trials for the treatment of obstructive hypertrophic cardiomyopathy (HCM), and it may provide benefits for treating other forms of heart disease. We investigated the effect of mavacamten on cardiac muscle contraction in two transgenic mouse lines expressing the human isoform of cardiac myosin regulatory light chain (RLC) in their hearts. Control mice expressed wild-type RLC (WT-RLC), and HCM mice expressed the N47K RLC mutation. In the absence of mavacamten, skinned papillary muscle strips from WT-RLC mice produced greater isometric force than strips from N47K mice. Adding 0.3 µM mavacamten decreased maximal isometric force and reduced Ca 2+ sensitivity of contraction for both genotypes, but this reduction in pCa 50 was nearly twice as large for WT-RLC versus N47K. We also used stochastic length-perturbation analysis to characterize cross-bridge kinetics. The cross-bridge detachment rate was measured as a function of [MgATP] to determine the effect of mavacamten on myosin nucleotide handling rates. Mavacamten increased the MgADP release and MgATP binding rates for both genotypes, thereby contributing to faster cross-bridge detachment, which could speed up myocardial relaxation during diastole. Our data suggest that mavacamten reduces isometric tension and Ca 2+ sensitivity of contraction via decreased strong cross-bridge binding. Mavacamten may become a useful therapy for patients with heart disease, including some forms of HCM. NEW & NOTEWORTHY Mavacamten is a pharmaceutical that binds to myosin, and it is under investigation as a therapy for some forms of heart disease. We show that mavacamten reduces isometric tension and Ca 2+ sensitivity of contraction in skinned myocardial strips from a mouse model of hypertrophic cardiomyopathy that expresses the N47K mutation in cardiac myosin regulatory light chain. Mavacamten reduces contractility by decreasing strong cross-bridge binding, partially due to faster cross-bridge nucleotide handling rates that speed up myosin detachment. 

Abstract Background The pia arachnoid complex (PAC) is a cerebrospinal fluid-filled tissue conglomerate that surrounds the brain and spinal cord. Pia mater adheres directly to the surface of the brain while the arachnoid mater adheres to the deep surface of the dura mater. Collagen fibers, known as subarachnoid trabeculae (SAT) fibers, and microvascular structure lie intermediately to the pia and arachnoid meninges. Due to its structural role, alterations to the biomechanical properties of the PAC may change surface stress loading in traumatic brain injury (TBI) caused by sub-concussive hits. The aim of this study was to quantify the mechanical and morphological properties of ovine PAC. Methods Ovine brain samples (n = 10) were removed from the skull and tissue was harvested within 30 min post-mortem. To access the PAC, ovine skulls were split medially from the occipital region down the nasal bone on the superior and inferior aspects of the skull. A template was used to remove arachnoid samples from the left and right sides of the frontal and occipital regions of the brain. 10 ex-vivo samples were tested with uniaxial tension at 2 mm s −1 , average strain rate of 0.59 s −1 , until failure at < 5 h post extraction. The force and displacement data were acquired at 100 Hz. PAC tissue collagen fiber microstructure was characterized using second-harmonic generation (SHG) imaging on a subset of n = 4 stained tissue samples. To differentiate transverse blood vessels from SAT by visualization of cell nuclei and endothelial cells, samples were stained with DAPI and anti-von Willebrand Factor, respectively. The Mooney-Rivlin model for average stress–strain curve fit was used to model PAC material properties. Results The elastic modulus, ultimate stress, and ultimate strain were found to be 7.7 ± 3.0, 2.7 ± 0.76 MPa, and 0.60 ± 0.13, respectively. No statistical significance was found across brain dissection locations in terms of biomechanical properties. SHG images were post-processed to obtain average SAT fiber intersection density, concentration, porosity, tortuosity, segment length, orientation, radial counts, and diameter as 0.23, 26.14, 73.86%, 1.07 ± 0.28, 17.33 ± 15.25 µm, 84.66 ± 49.18°, 8.15%, 3.46 ± 1.62 µm, respectively. Conclusion For the sizes, strain, and strain rates tested, our results suggest that ovine PAC mechanical behavior is isotropic, and that the Mooney-Rivlin model is an appropriate curve-fitting constitutive equation for obtaining material parameters of PAC tissues. 

The force response of cardiac muscle undergoing a quick stretch is conventionally interpreted to represent stretching of attached myosin crossbridges (phase 1) and detachment of these stretched crossbridges at an exponential rate (phase 2), followed by crossbridges reattaching in increased numbers due to an enhanced activation of the thin filament (phases 3 and 4). We propose that, at least in mammalian cardiac muscle, phase 2 instead represents an enhanced detachment rate of myosin crossbridges due to stretch, phase 3 represents the reattachment of those same crossbridges, and phase 4 is a passive-like viscoelastic response with power-law relaxation. To test this idea, we developed a two-state model of crossbridge attachment and detachment. Unitary force was assigned when a crossbridge was attached, and an elastic force was generated when an attached crossbridge was displaced. Attachment rate, f(x), was spatially distributed with a total magnitude f0. Detachment rate was modeled as g(x) = g0+ g1x, where g0 is a constant and g1 indicates sensitivity to displacement. The analytical solution suggested that the exponential decay rate of phase 2 represents (f0 + g0) and the exponential rise rate of phase 3 represents g0. The depth of the nadir between phases 2 and 3 is proportional to g1. We prepared skinned mouse myocardium and applied a 1% stretch under varying concentrations of inorganic phosphate (Pi). The resulting force responses fitted the analytical solution well. The interpretations of phases 2 and 3 were consistent with lower f0 and higher g0 with increasing Pi. This novel scheme of interpreting the force response to a quick stretch does not require enhanced thin-filament activation and suggests that the myosin detachment rate is sensitive to stretch. Furthermore, the enhanced detachment rate is likely not due to the typical detachment mechanism following MgATP binding, but rather before MgADP release, and may involve reversal of the myosin power stroke.