Effects of Troponin T Cardiomyopathy Mutations on the Calcium Sensitivity of the Regulated Thin Filament and the Actomyosin Cross-Bridge Kinetics of Human β-Cardiac Myosin

The rate constant of tension redevelopment (ktr; 1986. Proc. Natl. Acad. Sci. USA. 83:3542-3546) was determined at various levels of thin filament activation in skinned single fibers from mammalian fast twitch muscles. Activation was altered by (a) varying the concentration of free Ca2+ in the activating solution, or (b) extracting various amounts of troponin C (TnC) from whole troponin complexes while keeping the concentration of Ca2+ constant. TnC was extracted by bathing the fiber in a solution containing 5 mM EDTA, 10 mM HEPES, and 0.5 mM trifluoperazine dihydrochloride. Partial extraction of TnC resulted in a decrease in the Ca2+ sensitivity of isometric tension, presumably due to disruption of near-neighbor molecular cooperativity between functional groups (i.e., seven actin monomers plus associated troponin and tropomyosin) within the thin filament. Altering the level of thin filament activation by partial extraction of TnC while keeping Ca2+ concentration constant tested whether the Ca2+ sensitivity of ktr results from a direct effect of Ca2+ on cross-bridge state transitions or, alternatively, an indirect effect of Ca2+ on these transitions due to varying extents of thin filament activation. Results showed that the ktr-pCa relation was unaffected by partial extraction of TnC, while steady-state isometric tension exhibited the expected reduction in Ca2+ sensitivity. This finding provides evidence for a direct effect of Ca2+ on an apparent rate constant that limits the formation of force-bearing cross-bridge states in muscle fibers. Further, the kinetics of this transition are unaffected by disruption of near-neighbor thin filament cooperativity subsequent to extraction of TnC. Finally, the results support the idea that the steepness of the steady-state isometric tension-calcium relationship is at least in part due to mechanisms involving molecular cooperativity among thin filament regulatory proteins.

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The Effect of HCM and RCM Causing Mutations of Troponin T on Force Generation and Cross-Bridge Kinetics in Thin-Filament Reconstituted Bovine Cardiac Muscle Fibers

Biophysical Journal ◽

10.1016/j.bpj.2011.11.853 ◽

2012 ◽

Vol 102 (3) ◽

pp. 156a

Author(s):

Fan Bai ◽

Hannah M. Caster ◽

Jose R. Pinto ◽

James D. Potter ◽

Masataka Kawai

Keyword(s):

Cardiac Muscle ◽

Thin Filament ◽

Troponin T ◽

Muscle Fibers ◽

Force Generation ◽

Cross Bridge ◽

Cardiac Muscle Fibers

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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

Molecular Biology of the Cell ◽

10.1091/mbc.e21-02-0082 ◽

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.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00023.2020 ◽

2020 ◽

Vol 318 (3) ◽

pp. H715-H722 ◽

Cited By ~ 4

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.

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Nebulin and titin modulate cross-bridge cycling and length-dependent calcium sensitivity

The Journal of General Physiology ◽

10.1085/jgp.201812165 ◽

2019 ◽

Vol 151 (5) ◽

pp. 680-704 ◽

Cited By ~ 10

Author(s):

Srboljub M. Mijailovich ◽

Boban Stojanovic ◽

Djordje Nedic ◽

Marina Svicevic ◽

Michael A. Geeves ◽

...

Keyword(s):

Muscle Function ◽

Thin Filament ◽

Structural Changes ◽

Sarcomere Length ◽

Muscle Force ◽

Calcium Sensitivity ◽

Filament Length ◽

Simulation Code ◽

Cross Bridge ◽

Myosin Binding

Various mutations in the structural proteins nebulin and titin that are present in human disease are known to affect the contractility of striated muscle. Loss of nebulin is associated with reduced actin filament length and impairment of myosin binding to actin, whereas titin is thought to regulate muscle passive elasticity and is likely involved in length-dependent activation. Here, we sought to assess the modulation of muscle function by these sarcomeric proteins by using the computational platform muscle simulation code (MUSICO) to quantitatively separate the effects of structural changes, kinetics of cross-bridge cycling, and calcium sensitivity of the thin filaments. The simulations show that variation in thin filament length cannot by itself account for experimental observations of the contractility in nebulin-deficient muscle, but instead must be accompanied by a decreased myosin binding rate. Additionally, to match the observed calcium sensitivity, the rate of TnI detachment from actin needed to be increased. Simulations for cardiac muscle provided quantitative estimates of the effects of different titin-based passive elasticities on muscle force and activation in response to changes in sarcomere length and interfilament lattice spacing. Predicted force–pCa relations showed a decrease in both active tension and sensitivity to calcium with a decrease in passive tension and sarcomere length. We conclude that this behavior is caused by partial redistribution of the muscle load between active muscle force and titin-dependent passive force, and also by redistribution of stretch along the thin filament, which together modulate the release of TnI from actin. These data help advance understanding of how nebulin and titin mutations affect muscle function.

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Protein Kinase A Catalyzed Phosphorylation of Cardiac Myosin Binding Protein C Decreases Calcium Sensitivity of Force and Increases Cross-Bridge Cycling Kinetics in Murine Myocardium

Biophysical Journal ◽

10.1016/j.bpj.2008.12.2579 ◽

2009 ◽

Vol 96 (3) ◽

pp. 500a

Author(s):

Peter P. Chen ◽

Jitandrakumar R. Patel ◽

Inna N. Rybakova ◽

Jeffery W. Walker ◽

Richard L. Moss

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Protein C ◽

Calcium Sensitivity ◽

Cardiac Myosin ◽

Cross Bridge ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding ◽

Kinase A

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Overexpression of troponin T in Drosophila muscles causes a decrease in the levels of thin-filament proteins

Biochemical Journal ◽

10.1042/bj20041240 ◽

2005 ◽

Vol 386 (1) ◽

pp. 145-152 ◽

Cited By ~ 9

Author(s):

Raquel MARCO-FERRERES ◽

Juan J. ARREDONDO ◽

Benito FRAILE ◽

Margarita CERVERA

Keyword(s):

Thin Filament ◽

Troponin I ◽

Troponin T ◽

Thin Filaments ◽

Muscle Formation ◽

Thin Filament Proteins ◽

Flight Muscles ◽

Transgenic Drosophila ◽

Regulated Thin Filament

Formation of the contractile apparatus in muscle cells requires co-ordinated activation of several genes and the proper assembly of their products. To investigate the role of TnT (troponin T) in the mechanisms that control and co-ordinate thin-filament formation, we generated transgenic Drosophila lines that overexpress TnT in their indirect flight muscles. All flies that overexpress TnT were unable to fly, and the loss of thin filaments themselves was coupled with ultrastructural perturbations of the sarcomere. In contrast, thick filaments remained largely unaffected. Biochemical analysis of these lines revealed that the increase in TnT levels could be detected only during the early stages of adult muscle formation and was followed by a profound decrease in the amount of this protein as well as that of other thin-filament proteins such as tropomyosin, troponin I and actin. The decrease in thin-filament proteins is not only due to degradation but also due to a decrease in their synthesis, since accumulation of their mRNA transcripts was also severely diminished. This decrease in expression levels of the distinct thin-filament components led us to postulate that any change in the amount of TnT transcripts might trigger the down-regulation of other co-regulated thin-filament components. Taken together, these results suggest the existence of a mechanism that tightly co-ordinates the expression of thin-filament genes and controls the correct stoichiometry of these proteins. We propose that the high levels of unassembled protein might act as a sensor in this process.

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Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00172.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. H1756-H1761 ◽

Cited By ~ 19

Author(s):

Julian E. Stelzer ◽

Jitandrakumar R. Patel ◽

M. Charlotte Olsson ◽

Daniel P. Fitzsimons ◽

Leslie A. Leinwand ◽

...

Keyword(s):

Cardiac Troponin ◽

Thin Filament ◽

Troponin T ◽

Systolic Function ◽

Systolic Dysfunction ◽

Hill Coefficient ◽

Cardiac Troponin T ◽

Cross Bridge ◽

Interaction Kinetics ◽

Filament Activation

Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnTtrunc) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnTtrunc (i.e., <5%) is due to slowed cross-bridge kinetics and reduced thin filament activation as a consequence of reduced cross-bridge binding. We measured the Ca2+ sensitivity of force development [pCa for half-maximal tension generation (pCa50)] and the rate constant of force redevelopment ( ktr) in cTnTtrunc and wild-type (WT) skinned myocardium both in the absence and in the presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1). Compared with WT mice, cTnTtrunc mice exhibited greater pCa50, reduced steepness of the force-pCa relationship [Hill coefficient ( nH)], and faster ktr at submaximal Ca2+ concentration ([Ca2+]), i.e., reduced activation dependence of ktr. Treatment with NEM-S1 elicited similar increases in pCa50 and similar reductions in nH in WT and cTnTtrunc myocardium but elicited greater increases in ktr at submaximal activation in cTnTtrunc myocardium. Contrary to our initial hypothesis, cTnTtrunc appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca2+-activated force and the rate of cross-bridge attachment at submaximal [Ca2+]. Although these mechanisms would not be expected to depress systolic function per se in cTnTtrunc hearts, they would account for slowed rates of myocardial relaxation during early diastole.

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