A troponin T variant linked with pediatric dilated cardiomyopathy reduces the coupling of thin filament activation to myosin and calcium binding

Barrick, Samantha K.; Greenberg, Michael J.

doi:10.1091/mbc.e21-02-0082

Cited by 11 publications

(16 citation statements)

References 73 publications

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“…4b ). Consistent with previous studies (7,9,10,45), our stopped-flow measurements show that at physiologically relevant ATP concentrations, the rate of ADP release is much slower than the rate of ATP-induced actomyosin dissociation ( Figs. 3b-c ), and therefore the muscle shortening speed will be limited by the rate of ADP release.…”

Section: Discussionsupporting

confidence: 93%

“…ADP release experiments were performed as described previously (7,45,61). Briefly, 1 μM phalloidin-stabilized, pyrene-labeled actin, 1.5 μM tropomyosin (when appropriate), 1.5 μM troponin (when appropriate), 1 μM S1 myosin, and 100 μM Mg*ADP were rapidly mixed with 5 mM Mg*ATP.…”

Section: Methodsmentioning

confidence: 99%

“…At the molecular scale, these regulatory mechanisms include the calcium-dependent gating of the interactions between myosin and the thin filament by the proteins troponin and tropomyosin (3), load-dependent effects on myosin kinetics (4,5), and thick filament dependent regulation (6). Dysfunction of these regulatory mechanisms can lead to cardiomyopathy, arrhythmias, and heart failure (7)(8)(9)(10), and these regulatory mechanisms have emerged as therapeutic targets (11)(12)(13).…”

Section: Introductionmentioning

confidence: 99%

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Single Molecule Mechanics and Kinetics of Cardiac Myosin Interacting with Regulated Thin Filaments

Schulte

Scott

Barrick

et al. 2023

Preprint

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The cardiac cycle is a tightly regulated process wherein the heart generates force to pump blood to the body during systole and then relaxes during diastole. Disruption of this finely tuned cycle can lead to a range of diseases including cardiomyopathies and heart failure. Cardiac contraction is driven by the molecular motor myosin, which pulls regulated thin filaments in a calcium-dependent manner. In some muscle and non-muscle myosins, regulatory proteins on actin tune the kinetics, mechanics, and load dependence of the myosin working stroke; however, it is not well understood whether or how thin filament regulatory proteins tune the mechanics of the cardiac myosin motor. To address this critical gap in knowledge, we used single-molecule techniques to measure the kinetics and mechanics of the substeps of the cardiac myosin working stroke in the presence and absence of thin filament regulatory proteins. We found that regulatory proteins gate the calcium-dependent interactions between myosin and the thin filament. At physiologically relevant ATP concentrations, cardiac myosin’s mechanics and unloaded kinetics are not affected by thin filament regulatory proteins. We also measured the load-dependent kinetics of cardiac myosin at physiologically relevant ATP concentrations using an isometric optical clamp, and we found that thin filament regulatory proteins do not affect either the identity or magnitude of myosin’s primary load-dependent transition. Interestingly, at low ATP concentrations, thin filament regulatory proteins have a small effect on actomyosin dissociation kinetics, suggesting a mechanism beyond simple steric blocking. These results have important implications for both disease modeling and computational models of muscle contraction.Significance StatementHuman heart contraction is powered by the molecular motor β-cardiac myosin, which pulls on thin filaments consisting of actin and the regulatory proteins troponin and tropomyosin. In some muscle and non-muscle systems, these regulatory proteins tune the kinetics, mechanics, and load dependence of the myosin working stroke. Despite having a central role in health and disease, it is not well understood whether the mechanics or kinetics of β-cardiac myosin are affected by regulatory proteins. We show that regulatory proteins do not affect the mechanics or load-dependent kinetics of the working stroke at physiologically relevant ATP concentrations; however, they can affect the kinetics at low ATP concentrations, suggesting a mechanism beyond simple steric blocking. This has important implications for modeling of cardiac physiology and diseases.

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Section: Discussionsupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Single Molecule Mechanics and Kinetics of Cardiac Myosin Interacting with Regulated Thin Filaments

Schulte

Scott

Barrick

et al. 2023

Preprint

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“…We speculate that mutation-induced changes in sarcomeric tension affect the tension transmitted between the extracellular matrix and nucleus, leading to altered sarcomeric organization and signaling. Consistent with this notion, cardiomyopathy mutations that affect force production, drug-induced changes in contractility, and genetic manipulation of proteins involved in linking the cytoskeleton to the extracellular matrix can all cause altered sarcomeric organization (13,33,41,42).…”

Section: Mutation-induced Molecular Dysfunction Affects Mechanics Dri...mentioning

confidence: 81%

Harnessing molecular mechanism for precision medicine in dilated cardiomyopathy caused by a mutation in troponin T

Greenberg,

Stump,

Lin

et al. 2024

Preprint

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Familial dilated cardiomyopathy (DCM) is frequently caused by autosomal dominant point mutations in genes involved in diverse cellular processes, including sarcomeric contraction. While patient studies have defined the genetic landscape of DCM, genetics are not currently used in patient care, and patients receive similar treatments regardless of the underlying mutation. It has been suggested that a precision medicine approach based on the molecular mechanism of the underlying mutation could improve outcomes; however, realizing this approach has been challenging due to difficulties linking genotype and phenotype and then leveraging this information to identify therapeutic approaches. Here, we used multiscale experimental and computational approaches to test whether knowledge of molecular mechanism could be harnessed to connect genotype, phenotype, and drug response for a DCM mutation in troponin T, deletion of K210. Previously, we showed that at the molecular scale, the mutation reduces thin filament activation. Here, we used computational modeling of this molecular defect to predict that the mutant will reduce cellular and tissue contractility, and we validated this prediction in human cardiomyocytes and engineered heart tissues. We then used our knowledge of molecular mechanism to computationally model the effects of a small molecule that can activate the thin filament. We demonstrate experimentally that the modeling correctly predicts that the small molecule can partially rescue systolic dysfunction at the expense of diastolic function. Taken together, our results demonstrate how molecular mechanism can be harnessed to connect genotype and phenotype and inspire strategies to optimize mechanism-based therapeutics for DCM.

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“…Traction force microscopy. Traction force microscopy was performed as previously described (38,56,70). Details can be found in the supplemental methods.…”

Section: Methodsmentioning

confidence: 99%

Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility

Garg,

Jansen,

Zhang

et al. 2024

Preprint

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Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1's high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only ~20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 mutation, R256H. We previously identified this mutation in multiple family members with dilated cardiomyopathy (DCM), who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent functional effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using Cryo-EM, and we see alterations in actin's structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that ACTA1R256H/+human induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric disorganization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy.

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A troponin T variant linked with pediatric dilated cardiomyopathy reduces the coupling of thin filament activation to myosin and calcium binding

Cited by 11 publications

References 73 publications

Single Molecule Mechanics and Kinetics of Cardiac Myosin Interacting with Regulated Thin Filaments

Single Molecule Mechanics and Kinetics of Cardiac Myosin Interacting with Regulated Thin Filaments

Harnessing molecular mechanism for precision medicine in dilated cardiomyopathy caused by a mutation in troponin T

Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility

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