The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This ...therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI, Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of ß myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activitys role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.
The a and b isoforms of keratin 6 (K6), a type II intermediate filament (IF) protein, are robustly induced upon injury to interfollicular epidermis. We previously showed that complete loss of K6a/K6b ...stimulates keratinocyte migration, correlating with enhanced Src activity. In this study, we demonstrate that this property is cell autonomous, depends on the ECM, and results from elevated speed, enhanced directionality, and an increased rate of focal adhesion disassembly. We show that myosin IIA interacts with K6a/K6b, that its levels are markedly reduced in
-null keratinocytes, and that inhibiting myosin ATPase activity normalizes the enhanced migration potential of
-null cells. Desmoplakin, which mediates attachment of IFs to desmosomes, is also expressed at reduced levels and is mislocalized to the nucleus in
-null cells, correlating with defects in cell adhesion. These findings reveal that K6a/K6b modulate keratinocyte migration by regulating cell-matrix and cell-cell adhesion and highlight a role for keratins in collective cell migration.
The mechanisms by which truncating mutations in
(encoding cardiac myosin-binding protein C; cMyBPC) or myosin missense mutations cause hypercontractility and poor relaxation in hypertrophic ...cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches, we explored how depletion of cMyBPC altered sarcomere function. We demonstrated that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM), normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation, enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with
mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across the cardiac cycle as the pathophysiologic mechanisms by which
truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in patients with DCM, whereas inhibiting myosin by MYK-461 should benefit the substantial proportion of patients with HCM with
mutations.
In 1964 Michael Bárány and colleagues published a paper ((M. Bárány, E. Gaetjens, K. Bárány, Karp E. Arch Biochem Biophys 106(1964)280-93. http://10.1016/0003-9861(64)90,189-4)) that has been one of ...the most cited papers in Archives of Biochemistry and Biophysics. This was followed in 1967 by another most cited paper (M. Bárány. J Gen Physiol 50(1967)197–218. https://doi.org/10.1085/jgp.50.6.197). I have commemorated these achievements as tipping points in the understanding of myosin motors in muscle function. Tipping points are generally defined as a temporal point in which a series of progressive advances (in this case the understanding of the relations between myosin ATP hydrolysis and muscle function) inspire more expansive, wide-ranging, significant changes. I first concisely summarize the background against which the papers came to publication as well as the unimaginable personal challenges faced by Michael and Kate Bárány. A final section summarizes the impact of these publications as key steps in the progression of contemporary understanding of diverse control of myosin ATPase activity with focus on the thick filaments in cardiac homeostasis, disorders, and as targets for therapeutic applications in translational investigations.
Key points
Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle.
Omecamtiv mecarbil increases contractility at low levels of activation by ...stabilizing the ON state of the thick filament.
Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto‐myosin ATPase cycle.
Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto‐myosin ATPase.
Thick filament regulation is a promising target for novel therapeutics in heart disease.
Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin‐containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules – omecamtiv mecarbil (OM) and blebbistatin (BS) – that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small‐molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin‐myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin‐myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co‐operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments.
Key points
Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle.
Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament.
Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto‐myosin ATPase cycle.
Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto‐myosin ATPase.
Thick filament regulation is a promising target for novel therapeutics in heart disease.
Mavacamten is a FDA-approved small-molecule therapeutic designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It ...shifts myosin toward ordered
states close to the thick filament backbone. It remains elusive whether these myosin heads in the
state(s) can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these
state(s) by mavacamten are recruitable by 1) Ca
, 2) increased chronotropy heart rate (HR), 3) stretch, and 4) β-adrenergic (β-AR) stimulation, all known physiological inotropic interventions. At the molecular level, we show that Ca
increases myosin ATPase activity by shifting mavacamten-stabilized myosin heads from the inactive super-relaxed state to the active disordered relaxed state. At the myofilament level, both Ca
and passive lengthening can shift mavacamten-ordered
myosin heads from positions close to the thick filament backbone to disordered
states closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dt
an index of inotropy, increased with HR in mavacamten-treated animals. Finally, we show that β-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted
states of myosin in the thick filament are at least partially activable, thus preserving cardiac reserve mechanisms.
Tropomyosin (Tpm) is an actin‐binding protein central to muscle contraction regulation. The Tpm sequence consists of periodic repeats corresponding to seven actin‐binding sites, further divided in ...two functionally distinct halves. To clarify the importance of the first and second halves of the actin‐binding periods in regulating the interaction of myosin with actin, we introduced hypercontractile mutations D20H, E181K located in the N‐terminal halves of periods 1 and 5 and hypocontractile mutations E41K, N202K located in the C‐terminal halves of periods 1 and 5 of the skeletal muscle Tpm isoform Tpm2.2. Wild‐type and mutant Tpms displayed similar actin‐binding properties, however, as revealed by FRET experiments, the hypercontractile mutations affected the binding geometry and orientation of Tpm2.2 on actin, causing a stimulation of myosin motor performance. Contrary, the hypocontractile mutations led to an inhibition of both, actin activation of the myosin ATPase and motor activity, that was more pronounced than with wild‐type Tpm2.2. Single ATP turnover kinetic experiments indicate that the introduced mutations have opposite effects on product release kinetics. While the hypercontractile Tpm2.2 mutants accelerated product release, the hypocontractile mutants decelerated product release from myosin, thus having either an activating or inhibitory influence on myosin motor performance, which agrees with the muscle disease phenotypes caused by these mutations.
Myopathy‐linked mutations in Tpm2.2, an isoform of striated muscle tropomyosin, can lead to hypercontractile or hypocontractile molecular phenotypes. Depending on the localization of the mutations in Tpm2.2 sequence, they have different impacts on the association of Tpm2.2 with actin and the regulation of the actin–myosin cross‐bridge cycle.
Congenital myopathies are a heterogeneous group of human skeletal muscle disorders characterized by muscle hypotonia and weakness. The clinical picture of myopathies differs significantly in ...different patients, which makes it extremely difficult to diagnose the myopathy variant and develop treatment approaches. There are several pharmacological agents in clinical use or under clinical investigation for the treatment of cardiomyopathies, the mechanism of action of which can also be used to treat congenital myopathies. One such agent is 2,3-butanedione monoxime (BDM), a noncompetitive inhibitor of myosin ATPase activity used to suppress acute myocardial injury. Molecular mechanisms of myosin inhibition in the presence of BDM in skeletal muscle have not been studied, so the aim of this work was to study the effect of this agent on the interaction of myosin with actin in modeling several stages of the ATPase cycle in skeletal muscle fiber in order to assess the prospects for the use of BDM for the treatment of congenital myopathies. We found that BDM, in the absence of the regulatory proteins tropomyosin and troponin, enhances the rigidity of myosin binding to actin while modeling the weak binding of these muscle proteins, which can slow down the transition of actomyosin from the A∙M∙ADP∙Pi state to the A∙M∙ADP state and is one of the reasons for the decrease in the ATPase activity of myosin. When modeling the successive stages of the ATPase cycle using ADP, AMPPNP, ATPγS, and ATP, the myosin heads gradually switch to a state of weak interaction with actin. In the presence of tropomyosin and troponin in the muscle fiber, BDM does not affect the formation of a weak form of actomyosin binding, but increases the number of myosin heads essential for force generation. BDM can be used to increase the efficiency of myosin conformational rearrangements in the presence of tropomyosin with the R90P mutation associated with congenital myopathy, since this reagent increases the number of myosin heads in the muscle fiber capable of effective conformational rearrangements in the ATPase cycle and partially inhibits the pathological effects of the mutation.
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