Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy

K146N is a dominant mutation in human β-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical, physiological and mechanical foundations of the disease. ATPase assays, actin motility, and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment, yielding prolonged binding. This increases isometric force generation, but also resistive force and work absorption during cyclical contractions, resulting in decreased work, power output, flight ability and degeneration of flight muscle sarcomere morphology. Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes, 146N/+ hearts are hypercontractile with increased tension generation periods, decreased diastolic/systolic diameters and myofibrillar disarray. This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.

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Mavacamten Decreases Maximal Force and Ca2+-sensitivity in the N47K-Myosin Regulatory Light Chain Mouse Model of Hypertrophic Cardiomyopathy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00345.2020 ◽

2020 ◽

Author(s):

Peter O Awinda ◽

Marissa Watanabe ◽

Yemeserach M. Bishaw ◽

Anna M Huckabee ◽

Keinan B Agonias ◽

...

Keyword(s):

Heart Disease ◽

Hypertrophic Cardiomyopathy ◽

Light Chain ◽

Isometric Force ◽

Regulatory Light Chain ◽

Isometric Tension ◽

Myosin Regulatory Light Chain ◽

Cardiac Myosin ◽

Cross Bridge ◽

Obstructive 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 vs. 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 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.

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Author response: Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy

10.7554/elife.38064.025 ◽

2018 ◽

Cited By ~ 1

Author(s):

William A Kronert ◽

Kaylyn M Bell ◽

Meera C Viswanathan ◽

Girish C Melkani ◽

Adriana S Trujillo ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Author Response ◽

Muscle Dysfunction ◽

Cross Bridge ◽

Drosophila Model

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Decision letter: Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy

10.7554/elife.38064.024 ◽

2018 ◽

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Muscle Dysfunction ◽

Cross Bridge ◽

Drosophila Model

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Cross-bridge kinetics and shortening in smooth muscle

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y94-192 ◽

1994 ◽

Vol 72 (11) ◽

pp. 1334-1337 ◽

Cited By ~ 2

Author(s):

Per Hellstrand

Keyword(s):

Skeletal Muscle ◽

Smooth Muscle ◽

Isometric Force ◽

Muscle Mechanics ◽

Cross Bridge ◽

Intracellular Ca ◽

Cross Bridges ◽

Latch State ◽

Smooth Muscle Mechanics ◽

Low Rate

Stiffness measurements were performed on smooth muscle preparations from guinea-pig taenia coli to obtain information on the number of attached cross bridges under varying contractile conditions. The normalized stiffness of the cross-bridge system in smooth muscle may be of a magnitude similar to that assumed in skeletal muscle. Transition from isometric contraction to unloaded shortening was associated with a decrease in stiffness to 50% or less of the isometric value, slightly higher than that found in skeletal muscle fibers. Comparison of phasic (5 s) and tonic (5 min) contractions showed lower Vmax, intracellular [Ca2+], and myosin 20 kDa light chain phosphorylation at 5 min, indicating development of a latch state. Isometric force and stiffness were identical in the two types of contraction. However, stiffness during unloaded shortening was greater in the latch state, which may be the result of the presence of a population of cross bridges with a low rate constant for detachment.Key words: smooth muscle mechanics, cross bridges, latch, myosin phosphorylation.

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Oxytocin-mediated recruitment of slowly cycling cross bridges and isometric force in rat myometrium

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1996.270.2.e203 ◽

1996 ◽

Vol 270 (2) ◽

pp. E203-E208

Author(s):

A. L. Ruzycky ◽

B. T. Ameredes

Keyword(s):

Isometric Force ◽

Additional Stress ◽

Shortening Velocity ◽

Cycling Rate ◽

Quick Release ◽

Cross Bridge ◽

Cross Bridges ◽

Unit Stress ◽

Release Method ◽

The Relationship

The relationship between cross-bridge cycling rate and isometric stress was investigated in rat myometrium. Stress production by myometrial strips was measured under resting, K+ depolarization, and oxytocin-stimulated conditions. Cross-bridge cycling rates were determined from measurements of maximal unloaded shortening velocity, using the quick-release method. Force redevelopment after the quick release was used as an index of cross-bridge attachment. With maximal K+ stimulation, stress increased with increased cross-bridge cycling (+76%; P < 0.05) and attached cross bridges (+112%; P < 0.05). Addition of oxytocin during K+ stimulation further increased stress (+30%; P < 0.05). With this force component, the cross-bridge cycling rate decreased (-60%; P < 0.05) similar to that under resting conditions. Attached cross-bridges did not increase with this additional stress. The results suggest two distinct mechanisms mediating myometrial contractions. One requires elevated intracellular calcium and rapidly cycling cross bridges. The other mechanism may be independent of calcium and appears to be mediated by slowly cycling cross bridges, supporting greater unit stress.

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Phosphorylation of cardiac myosin binding protein C releases myosin heads from the surface of cardiac thick filaments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614020114 ◽

2017 ◽

Vol 114 (8) ◽

pp. E1355-E1364 ◽

Cited By ~ 35

Author(s):

Robert W. Kensler ◽

Roger Craig ◽

Richard L. Moss

Keyword(s):

Protein C ◽

Structural Changes ◽

Binding Protein ◽

Cardiac Contraction ◽

Cardiac Myosin ◽

Cross Bridge ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Thick Filaments ◽

Myosin Binding

Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of cross-bridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of β1-adrenergic receptors in myocardium.

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Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction

Biochemical Journal ◽

10.1042/bcj20180685 ◽

2018 ◽

Vol 475 (24) ◽

pp. 3933-3948 ◽

Cited By ~ 4

Author(s):

Sahar I. Da'as ◽

Khalid Fakhro ◽

Angelos Thanassoulas ◽

Navaneethakrishnan Krishnamoorthy ◽

Alaaeldin Saleh ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Binding Protein ◽

Clinical Severity ◽

Actin Binding ◽

Cardiac Myosin ◽

C2 Domains ◽

Cardiac Phenotype ◽

Myosin Binding Protein ◽

Myosin Binding ◽

The Impact

The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0–C2 protein fragments revealed that c-MYBPC3 variants alter the C0–C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0–C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations.

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Metabolic heat production during fatigue from voluntary repetitive isometric contractions in humans

Journal of Applied Physiology ◽

10.1152/jappl.1996.81.3.1323 ◽

1996 ◽

Vol 81 (3) ◽

pp. 1323-1330 ◽

Cited By ~ 5

Author(s):

E. Saugen ◽

N. K. Vollestad

Keyword(s):

Energy Cost ◽

Isometric Force ◽

Recovery Period ◽

Isometric Exercise ◽

Muscle Mechanics ◽

Force Production ◽

Cellular Level ◽

Voluntary Contraction ◽

O2 Uptake ◽

Postexercise Recovery

The effect of repetitive isometric knee extensions on the energy cost of contraction was examined. The rate of temperature rise (dT/dt) was determined in test contractions at 30 and 50% of maximal voluntary contraction (MVC) force before and during 30% MVC repetitive isometric exercise (RIE) to exhaustion and regularly in a 30-min postexercise recovery period (n = 9). Pulmonary O2 uptake and muscle temperature (Tmus) were determined at regular intervals. During the 30% MVC test contractions, dT/dt was 5.6 +/- 0.6 mK/s in unfatigued muscle, increasing linearly by 68% during exercise. In the 50% MVC test contractions, dT/dt rose by 84% from 9.8 +/- 1.1 mK/s. dT/dt determined during test contractions at both force levels did not decrease significantly throughout the 30-min postexercise recovery period. The rise in dT/dt was paralleled by 76% increased in O2 uptake. In contrast, Tmus rose initially and then leveled off. The present data indicate that RIE induced a gradual rise in the rate of energy turnover associated with isometric force production. Neither increased Tmus nor recruitment of less economic type II fibers can fully explain the increased energy cost. We suggest that energetic changes may occur at the cellular level and argue that this may be associated with the changes in muscle mechanics occurring during fatigue from submaximal voluntary RIE.

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