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Heart failure can reflect impaired contractile function at the myofilament level. In healthy hearts, myofilaments become more sensitive to Ca²⁺as cells are stretched. This represents a fundamental property of the myocardium that contributes to the Frank–Starling response, although the molecular mechanisms underlying the effect remain unclear. Mavacamten, which binds to myosin, is under investigation as a potential therapy for heart disease. We investigated how mavacamten affects the sarcomere‐length dependence of Ca²⁺‐sensitive isometric contraction to determine how mavacamten might modulate the Frank–Starling mechanism.

Multicellular preparations from the left ventricular‐free wall of hearts from organ donors were chemically permeabilized and Ca²⁺activated in the presence or absence of 0.5‐μM mavacamten at 1.9 or 2.3‐μm sarcomere length (37°C). Isometric force and frequency‐dependent viscoelastic myocardial stiffness measurements were made.

At both sarcomere lengths, mavacamten reduced maximal force and Ca²⁺sensitivity of contraction. In the presence and absence of mavacamten, Ca²⁺sensitivity of force increased as sarcomere length increased. This suggests that the length‐dependent activation response was maintained in human myocardium, even though mavacamten reduced Ca²⁺sensitivity. There were subtle effects of mavacamten reducing force values under relaxed conditions (pCa 8.0), as well as slowing myosin cross‐bridge recruitment and speeding cross‐bridge detachment under maximally activated conditions (pCa 4.5).

Mavacamten did not eliminate sarcomere length‐dependent increases in the Ca²⁺sensitivity of contraction in myocardial strips from organ donors at physiological temperature. Drugs that modulate myofilament function may be useful therapies for cardiomyopathies.

 

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.

 

Limb-girdle muscular dystrophy 2i (LGMD2i) is a dystroglycanopathy that compromises myofiber integrity and primarily reduces power output in limb muscles but can influence cardiac muscle as well. Previous studies of LGMD2i made use of a transgenic mouse model in which a proline-to-leucine (P448L) mutation in fukutin-related protein severely reduces glycosylation of α-dystroglycan. Muscle function is compromised in P448L mice in a manner similar to human patients with LGMD2i. In situ studies reported lower maximal twitch force and depressed force-velocity curves in medial gastrocnemius (MG) muscles from male P448L mice. Here, we measured Ca 2+ -activated force generation and cross-bridge kinetics in both demembranated MG fibers and papillary muscle strips from P448L mice. Maximal activated tension was 37% lower in MG fibers and 18% lower in papillary strips from P448L mice than controls. We also found slightly faster rates of cross-bridge recruitment and detachment in MG fibers from P448L than control mice. These increases in skeletal cross-bridge cycling could reduce the unitary force output from individual cross bridges by lowering the ratio of time spent in a force-bearing state to total cycle time. This suggests that the decreased force production in LGMD2i may be due (at least in part) to altered cross-bridge kinetics. This finding is notable, as the majority of studies germane to muscular dystrophies have focused on sarcolemma or whole muscle properties, whereas our findings suggest that the disease pathology is also influenced by potential downstream effects on cross-bridge behavior. 

Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca 2+ concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca 2+ -induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin–actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca 2+ -induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca 2+ -troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length–dependent enhancement of troponin regulation with both Ca 2+ activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length–dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length–dependent Ca 2+ -troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca 2+ -troponin regulation of the myocardium.