Danicamtiv affected isometric force and cross-bridge kinetics similarly in skinned myocardial strips from male and female rats.

Myotropes are pharmaceuticals that have recently been developed or are under investigation for the treatment of heart diseases. Myotropes have had varied success in clinical trials. Initial research into myotropes have widely focused on animal models of cardiac dysfunction in comparison with normal animal cardiac physiology-primarily using males. In this study we examined the effect of danicamtiv, which is one type of myotrope within the class of myosin activators, on contractile function in permeabilized (skinned) myocardial strips from male and female Sprague-Dawley rats. We found that danicamtiv increased steady-state isometric force production at sub-maximal calcium levels, leading to greater Ca2+-sensitivity of contraction for both sexes. Danicamtiv did not affect maximal Ca2+-activated force for either sex. Sinusoidal length-perturbation analysis was used to assess viscoelastic myocardial stiffness and cross-bridge cycling kinetics. Data from these measurements did not vary with sex, and the data suggest that danicamtiv slows cross-bridge cycling kinetics. These findings imply that danicamtiv increases force production via increasing cross-bridge contributions to activation of contraction, especially at sub-maximal Ca2+-activation. The inclusion of both sexes in animal models during the formative stages of drug development could be helpful for understanding the efficacy or limitation of a drug's therapeutic impact on cardiac function.

Sarcomere length affects Ca2+ sensitivity of contraction in ischemic but not non-ischemic myocardium.

In healthy hearts, myofilaments become more sensitive to Ca2+ as the myocardium is stretched. This effect is known as length-dependent activation and is an important cellular-level component of the Frank-Starling mechanism. Few studies have measured length-dependent activation in the myocardium from failing human hearts. We investigated whether ischemic and non-ischemic heart failure results in different length-dependent activation responses at physiological temperature (37°C). Myocardial strips from the left ventricular free wall were chemically permeabilized and Ca2+-activated at sarcomere lengths (SLs) of 1.9 and 2.3 µm. Data were acquired from 12 hearts that were explanted from patients receiving cardiac transplants; 6 had ischemic heart failure and 6 had non-ischemic heart failure. Another 6 hearts were obtained from organ donors. Maximal Ca2+-activated force increased at longer SL for all groups. Ca2+ sensitivity increased with SL in samples from donors (P < 0.001) and patients with ischemic heart failure (P = 0.003) but did not change with SL in samples from patients with non-ischemic heart failure. Compared with donors, troponin I phosphorylation decreased in ischemic samples and even more so in non-ischemic samples; cardiac myosin binding protein-C (cMyBP-C) phosphorylation also decreased with heart failure. These findings support the idea that troponin I and cMyBP-C phosphorylation promote length-dependent activation and show that length-dependent activation of contraction is blunted, yet extant, in the myocardium from patients with ischemic heart failure and further reduced in the myocardium from patients with non-ischemic heart failure. Patients who have a non-ischemic disease may exhibit a diminished contractile response to increased ventricular filling.

RLC phosphorylation amplifies Ca2+ sensitivity of force in myocardium from cMyBP-C knockout mice.

Hypertrophic cardiomyopathy (HCM) is the leading genetic cause of heart disease. The heart comprises several proteins that work together to properly facilitate force production and pump blood throughout the body. Cardiac myosin binding protein-C (cMyBP-C) is a thick-filament protein, and mutations in cMyBP-C are frequently linked with clinical cases of HCM. Within the sarcomere, the N-terminus of cMyBP-C likely interacts with the myosin regulatory light chain (RLC); RLC is a subunit of myosin located within the myosin neck region that modulates contractile dynamics via its phosphorylation state. Phosphorylation of RLC is thought to influence myosin head position along the thick-filament backbone, making it more favorable to bind the thin filament of actin and facilitate force production. However, little is known about how these two proteins interact. We tested the effects of RLC phosphorylation on Ca2+-regulated contractility using biomechanical assays on skinned papillary muscle strips isolated from cMyBP-C KO mice and WT mice. RLC phosphorylation increased Ca2+ sensitivity of contraction (i.e., pCa50) from 5.80 ± 0.02 to 5.95 ± 0.03 in WT strips, whereas RLC phosphorylation increased Ca2+ sensitivity of contraction from 5.86 ± 0.02 to 6.15 ± 0.03 in cMyBP-C KO strips. These data suggest that the effects of RLC phosphorylation on Ca2+ sensitivity of contraction are amplified when cMyBP-C is absent from the sarcomere. This implies that cMyBP-C and RLC act in concert to regulate contractility in healthy hearts, and mutations to these proteins that lead to HCM (or a loss of phosphorylation with disease progression) may disrupt important interactions between these thick-filament regulatory proteins.

MgADP Promotes Myosin Head Movement toward Actin at Low [Ca2+] to Increase Force Production and Ca2+-Sensitivity of Contraction in Permeabilized Porcine Myocardial Strips.

Myosin cross-bridges dissociate from actin following Mg2+-adenosine triphosphate (MgATP) binding. Myosin hydrolyses MgATP into inorganic phosphate (Pi) and Mg2+-adenosine diphosphate (ADP), and release of these hydrolysis products drives chemo-mechanical energy transitions within the cross-bridge cycle to power muscle contraction. Some forms of heart disease are associated with metabolic or enzymatic dysregulation of the MgATP-MgADP nucleotide pool, resulting in elevated cytosolic [MgADP] and impaired muscle relaxation. We investigated the mechanical and structural effects of increasing [MgADP] in permeabilized myocardial strips from porcine left ventricle samples. Sarcomere length was set to 2.0 µm at 28 °C, and all solutions contained 3% dextran T-500 to compress myofilament lattice spacing to near-physiological values. Under relaxing low [Ca2+] conditions (pCa 8.0, where pCa = -log10[Ca2+]), tension increased as [MgADP] increased from 0-5 mM. Complementary small-angle X-ray diffraction measurements show that the equatorial intensity ratio, I1,1/I1,0, also increased as [MgADP] increased from 0 to 5 mM, indicating myosin head movement away from the thick-filament backbone towards the thin-filament. Ca2+-activated force-pCa measurements show that Ca2+-sensitivity of contraction increased with 5 mM MgADP, compared to 0 mM MgADP. These data show that MgADP augments tension at low [Ca2+] and Ca2+-sensitivity of contraction, suggesting that MgADP destabilizes the quasi-helically ordered myosin OFF state, thereby shifting the cross-bridge population towards the disordered myosin ON state. Together, these results indicate that MgADP enhances the probability of cross-bridge binding to actin due to enhancement of both thick and thin filament-based activation mechanisms.

Mavacamten decreases maximal force and Ca2+ sensitivity in the N47K-myosin regulatory light chain mouse model of hypertrophic cardiomyopathy.

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 Ca2+ sensitivity of contraction for both genotypes, but this reduction in pCa50 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 Ca2+ 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 Ca2+ 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.

Effects of mavacamten on Ca2+ sensitivity of contraction as sarcomere length varied in human myocardium.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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). CONCLUSION AND IMPLICATIONS: 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.

Demembranated skeletal and cardiac fibers produce less force with altered cross-bridge kinetics in a mouse model for limb-girdle muscular dystrophy 2i.

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 Ca2+-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.

Omecamtiv Mecarbil Slows Myosin Kinetics in Skinned Rat Myocardium at Physiological Temperature.

Heart failure is a life-threatening condition that occurs when the heart muscle becomes weakened and cannot adequately circulate blood and nutrients around the body. Omecamtiv mecarbil (OM) is a compound that has been developed to treat systolic heart failure via targeting the cardiac myosin heavy chain to increase myocardial contractility. Biophysical and biochemical studies have found that OM increases calcium (Ca2+) sensitivity of contraction by prolonging the myosin working stroke and increasing the actin-myosin cross-bridge duty ratio. Most in vitro studies probing the effects of OM on cross-bridge kinetics and muscle force production have been conducted at subphysiological temperature, even though temperature plays a critical role in enzyme activity and cross-bridge function. Herein, we used skinned, ventricular papillary muscle strips from rats to investigate the effects of [OM] on Ca2+-activated force production, cross-bridge kinetics, and myocardial viscoelasticity at physiological temperature (37°C). We find that OM only increases myocardial contractility at submaximal Ca2+ activation levels and not maximal Ca2+ activation levels. As [OM] increased, the kinetic rate constants for cross-bridge recruitment and detachment slowed for both submaximal and maximal Ca2+-activated conditions. These findings support a mechanism by which OM increases cardiac contractility at physiological temperature via increasing cross-bridge contributions to thin-filament activation as cross-bridge kinetics slow and the duration of cross-bridge attachment increases. Thus, force only increases at submaximal Ca2+ activation due to cooperative recruitment of neighboring cross-bridges, because thin-filament activation is not already saturated. In contrast, OM does not increase myocardial force production for maximal Ca2+-activated conditions at physiological temperature because cooperative activation of thin filaments may already be saturated.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower ß-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases ß-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of ß-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and ß-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that ß-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm (20ΔRRM) Mice.

Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20 (ΔRRM) ) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20 (ΔRRM) animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20 (ΔRRM) on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca(2+)-activated, skinned papillary muscle strips from Rbm20 (ΔRRM) and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca(2+)-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20 (ΔRRM) vs. WT mice. As sarcomere length increased from 1.9 to 2.2 µm, length-dependent activation of contraction was eliminated in the Rbm20 (ΔRRM) myocardium, even though myosin MgADP release rate decreased ~20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.

Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 µm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 µm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles.

Myosin MgADP release rate decreases at longer sarcomere length to prolong myosin attachment time in skinned rat myocardium.

Cardiac contractility increases as sarcomere length increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. The capacity of myosin to bind with actin and generate force in a muscle cell is Ca(2+) regulated by thin-filament proteins and spatially regulated by sarcomere length as thick-to-thin filament overlap varies. One mechanism underlying greater cardiac contractility as sarcomere length increases could involve longer myosin attachment time (ton) due to slowed myosin kinetics at longer sarcomere length. To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 µm sarcomere lengths. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate and MgATP binding rates. As MgATP increased, ton decreased for both sarcomere lengths, but ton was roughly 70% longer for 2.2 vs. 1.9 µm sarcomere length at maximally activated conditions. These ton differences were driven by a slower MgADP release rate at 2.2 µm sarcomere length (41 ± 3 vs. 74 ± 7 s(-1)), since MgATP binding rate was not different between the two sarcomere lengths. At submaximal activation levels near the pCa50 value of the tension-pCa relationship for each sarcomere length, length-dependent increases in ton were roughly 15% longer for 2.2 vs. 1.9 µm sarcomere length. These changes in cross-bridge kinetics could amplify cooperative cross-bridge contributions to force production and thin-filament activation at longer sarcomere length and suggest that length-dependent changes in myosin MgADP release rate may contribute to the Frank-Starling relationship in the heart.

Serum antibodies from a subset of horses positive for Babesia caballi by competitive enzyme-linked immunosorbent assay demonstrate a protein recognition pattern that is not consistent with infection.

Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)-competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1-cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi.