scholarly journals A Troponin T Variant Linked with Pediatric Dilated Cardiomyopathy Reduces the Coupling of Thin Filament Activation to Myosin and Calcium Binding

2021 ◽  
pp. mbc.E21-02-0082
Author(s):  
Samantha K. Barrick ◽  
Lina Greenberg ◽  
Michael J. Greenberg
Keyword(s):  
Dilated Cardiomyopathy ◽  
Calcium Binding ◽  
Thin Filament ◽  
Troponin T ◽  
Calcium Sensitivity ◽  
Cellular Level ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Fluorescence Measurements

Dilated cardiomyopathy (DCM) is a significant cause of pediatric heart failure. Mutations in proteins that regulate cardiac muscle contraction can cause DCM; however, the mechanisms by which molecular-level mutations contribute to cellular dysfunction are not well-understood. Better understanding of these mechanisms might enable the development of targeted therapeutics that benefit patient subpopulations with mutations that cause common biophysical defects. We examined the molecular- and cellular-level impacts of a troponin T variant associated with pediatric-onset DCM, R134G. The R134G variant decreased calcium sensitivity in an in vitro motility assay. Using stopped-flow and steady-state fluorescence measurements, we determined the molecular mechanism of the altered calcium sensitivity: R134G decouples calcium binding by troponin from the closed-to-open transition of the thin filament and decreases the cooperativity of myosin binding to regulated thin filaments. Consistent with the prediction that these effects would cause reduced force per sarcomere, cardiomyocytes carrying the R134G mutation are hypocontractile. They also show hallmarks of DCM that lie downstream of the initial insult, including disorganized sarcomeres and cellular hypertrophy. These results reinforce the importance of multiscale studies to fully understand mechanisms underlying human disease and highlight the value of mechanism-based precision medicine approaches for DCM.

Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Saiti S Halder ◽  
Lorenzo R Sewanan ◽  
Michael J Rynkiewicz ◽  
Jeffrey R Moore ◽  
William J Lehman ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Calcium Sensitivity ◽  
Heart Tissue ◽  
Missense Mutations ◽  
Wild Type ◽  
Twitch Force ◽  
In Vitro Motility ◽  
Isometric Twitch ◽  
Engineered Heart Tissues

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

scholarly journals Atomic resolution probe for allostery in the regulatory thin filament

2016 ◽  
Vol 113 (12) ◽  
pp. 3257-3262 ◽  
Author(s):  
Michael R. Williams ◽  
Sarah J. Lehman ◽  
Jil C. Tardiff ◽  
Steven D. Schwartz
Keyword(s):  
Binding Site ◽  
Human Disease ◽  
Calcium Binding ◽  
Cardiac Troponin ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin T ◽  
Calcium Binding Site

Calcium binding and dissociation within the cardiac thin filament (CTF) is a fundamental regulator of normal contraction and relaxation. Although the disruption of this complex, allosterically mediated process has long been implicated in human disease, the precise atomic-level mechanisms remain opaque, greatly hampering the development of novel targeted therapies. To address this question, we used a fully atomistic CTF model to test both Ca2+ binding strength and the energy required to remove Ca2+ from the N-lobe binding site in WT and mutant troponin complexes that have been linked to genetic cardiomyopathies. This computational approach is combined with measurements of in vitro Ca2+ dissociation rates in fully reconstituted WT and cardiac troponin T R92L and R92W thin filaments. These human disease mutations represent known substitutions at the same residue, reside at a significant distance from the calcium binding site in cardiac troponin C, and do not affect either the binding pocket affinity or EF-hand structure of the binding domain. Both have been shown to have significantly different effects on cardiac function in vivo. We now show that these mutations independently alter the interaction between the Ca2+ ion and cardiac troponin I subunit. This interaction is a previously unidentified mechanism, in which mutations in one protein of a complex indirectly affect a third via structural and dynamic changes in a second to yield a pathogenic change in thin filament function that results in mutation-specific disease states. We can now provide atom-level insight that is potentially highly actionable in drug design.

scholarly journals Calcium regulation of thin filament movement in an in vitro motility assay

1996 ◽  
Vol 70 (4) ◽  
pp. 1881-1892 ◽  
Author(s):  
E. Homsher ◽  
B. Kim ◽  
A. Bobkova ◽  
L.S. Tobacman
Keyword(s):  
Thin Filament ◽  
Calcium Regulation ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility

scholarly journals Mavacamten rescues increased myofilament calcium sensitivity and dysregulation of Ca2+ flux caused by thin filament hypertrophic cardiomyopathy mutations

2020 ◽  
Vol 318 (3) ◽  
pp. H715-H722 ◽  
Author(s):  
Alexander J. Sparrow ◽  
Hugh Watkins ◽  
Matthew J. Daniels ◽  
Charles Redwood ◽  
Paul Robinson
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Cardiac Troponin ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin T ◽  
Thick Filament ◽  
Calcium Sensitivity ◽  
Calcium Handling ◽  
Cellular Models

Thin filament hypertrophic cardiomyopathy (HCM) mutations increase myofilament Ca2+ sensitivity and alter Ca2+ handling and buffering. The myosin inhibitor mavacamten reverses the increased contractility caused by HCM thick filament mutations, and we here test its effect on HCM thin filament mutations where the mode of action is not known. Mavacamten (250 nM) partially reversed the increased Ca2+ sensitivity caused by HCM mutations Cardiac troponin T (cTnT) R92Q, and cardiac troponin I (cTnI) R145G in in vitro ATPase assays. The effect of mavacamten was also analyzed in cardiomyocyte models of cTnT R92Q and cTnI R145G containing cytoplasmic and myofilament specific Ca2+ sensors. While mavacamten rescued the hypercontracted basal sarcomere length, the reduced fractional shortening did not improve with mavacamten. Both mutations caused an increase in peak systolic Ca2+ detected at the myofilament, and this was completely rescued by 250 nM mavacamten. Systolic Ca2+ detected by the cytoplasmic sensor was also reduced by mavacamten treatment, although only R145G increased cytoplasmic Ca2+. There was also a reversal of Ca2+ decay time prolongation caused by both mutations at the myofilament but not in the cytoplasm. We thus show that mavacamten reverses some of the Ca2+-sensitive molecular and cellular changes caused by the HCM mutations, particularly altered Ca2+ flux at the myofilament. The reduction of peak systolic Ca2+ as a consequence of mavacamten treatment represents a novel mechanism by which the compound is able to reduce contractility, working synergistically with its direct effect on the myosin motor. NEW & NOTEWORTHY Mavacamten, a myosin inhibitor, is currently in phase-3 clinical trials as a pharmacotherapy for hypertrophic cardiomyopathy (HCM). Its efficacy in HCM caused by mutations in thin filament proteins is not known. We show in reductionist and cellular models that mavacamten can rescue the effects of thin filament mutations on calcium sensitivity and calcium handling although it only partially rescues the contractile cellular phenotype and, in some cases, exacerbates the effect of the mutation.

A new in vitro motility assay technique to evaluate calcium sensitivity of the cardiac contractile proteins

1995 ◽  
Vol 429 (3) ◽  
pp. 443-445 ◽  
Author(s):  
Masataka Sata ◽  
Hiroshi Yamashita ◽  
Seiryo Sugiura ◽  
Hideo Fujita ◽  
Shin -ichi Momomura ◽  
...  
Keyword(s):  
Calcium Sensitivity ◽  
Contractile Proteins ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Cardiac Contractile ◽  
Cardiac Contractile Proteins ◽  
Assay Technique

scholarly journals Ca++-sensitizing mutations in troponin, Pi, and 2-deoxyATP alter the depressive effect of acidosis on regulated thin-filament velocity

2014 ◽  
Vol 116 (9) ◽  
pp. 1165-1174 ◽  
Author(s):  
Thomas J. Longyear ◽  
Matthew A. Turner ◽  
Jonathan P. Davis ◽  
Joseph Lopez ◽  
Brandon Biesiadecki ◽  
...  
Keyword(s):  
Thin Filament ◽  
Contractile Activity ◽  
Dependent Manner ◽  
In Vitro Motility Assay ◽  
Depressive Effect ◽  
In Vitro Motility ◽  
Acidic Conditions ◽  
Underlying Mechanisms ◽  
Regulated Thin Filament

Repeated, intense contractile activity compromises the ability of skeletal muscle to generate force and velocity, resulting in fatigue. The decrease in velocity is thought to be due, in part, to the intracellular build-up of acidosis inhibiting the function of the contractile proteins myosin and troponin; however, the underlying molecular basis of this process remains poorly understood. We sought to gain novel insight into the decrease in velocity by determining whether the depressive effect of acidosis could be altered by 1) introducing Ca++-sensitizing mutations into troponin (Tn) or 2) by agents that directly affect myosin function, including inorganic phosphate (Pi) and 2-deoxy-ATP (dATP) in an in vitro motility assay. Acidosis reduced regulated thin-filament velocity ( VRTF) at both maximal and submaximal Ca++ levels in a pH-dependent manner. A truncated construct of the inhibitory subunit of Tn (TnI) and a Ca++-sensitizing mutation in the Ca++-binding subunit of Tn (TnC) increased VRTF at submaximal Ca++ under acidic conditions but had no effect on VRTF at maximal Ca++ levels. In contrast, both Pi and replacement of ATP with dATP reversed much of the acidosis-induced depression of VRTF at saturating Ca++. Interestingly, despite producing similar magnitude increases in VRTF, the combined effects of Pi and dATP were additive, suggesting different underlying mechanisms of action. These findings suggest that acidosis depresses velocity by slowing the detachment rate from actin but also by possibly slowing the attachment rate.

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