Cardiac troponin T isoforms demonstrate similar effects on mechanical performance in a regulated contractile system

2002 ◽  
Vol 282 (5) ◽  
pp. H1665-H1671 ◽  
Author(s):  
Peter VanBuren ◽  
Shari L. Alix ◽  
Joseph A. Gorga ◽  
Kelly J. Begin ◽  
Martin M. LeWinter ◽  
...  
Keyword(s):  
Mechanical Performance ◽  
Troponin T ◽  
Motility Assay ◽  
Free Calcium ◽  
In Vitro Motility Assay ◽  
Small Magnitude ◽  
Thin Filaments ◽  
Myocardial Failure ◽  
Isoform Expression

Alteration of troponin T (TnT) isoform expression has been reported in human and animal models of myocardial failure. The two adult beef cardiac TnT isoforms (TnT3 and TnT4) were isolated for comparative functional analysis. Thin filaments were reconstituted containing pure populations of the isoforms. The in vitro motility assay was used to directly compare the effect of the two TnT isoforms on force and unloaded shortening as a function of free calcium. We found no significant differences between the two isoforms in terms of calcium sensitivity, cooperativity, or maximal activation (velocity and force) as assessed in a fully calcium-regulated system. Activation by myosin strong binding was similar for thin filaments containing either of the two TnT isoforms. Whereas maximally activated velocity and cooperativity was depressed at pH 6.5, no difference between thin filaments containing the two isoforms was detected. From the small magnitude of the TnT isoform shifts detected in myocardial failure and the lack of significant mechanical effect detected in the motility assay, variable TnT isoform expression is unlikely to be any functional significance in heart failure.

Abstract 1464: Cardiac Myosin Binding Protein-C (cMyBP-C) Directly Modulates Actomyosin Binding and Kinetics Independent of LMM and S2 Binding.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Walid Saber ◽  
Kelly J Begin ◽  
David M Warshaw ◽  
Peter VanBuren
Keyword(s):  
Actin Filament ◽  
Thin Filament ◽  
Motility Assay ◽  
Actin Binding ◽  
Maximal Velocity ◽  
In Vitro Motility Assay ◽  
Thin Filaments ◽  
Myosin Binding ◽  
Cross Bridges

BACKGROUND: While mutations in cMyBP-C constitute a common cause of FHC, its role in sarcomere contraction remains unclear. cMyBP-C binds to actin, titin and the S2 and LMM proteolytic domains of myosin. Through its numerous binding interactions cMyBP-C may act as a tether, restricting myosin and/or actomyosin function. We directly tested this hypothesis in the in vitro motility assay using either whole myosin or the myosin subfragments, HMM and S1 (which lack LMM and LMM-S2, respectively). METHODS AND RESULTS: The motility assay is an in vitro model of muscle contraction in which thin filaments are propelled across a myosin coated surface. The addition of cMyBP-C to the motility assay resulted in a concentration dependent reduction in actin filament velocity when using either whole myosin, HMM or S1, demonstrating that cMyBP-C inhibits thin filament velocity independent of LMM or S2 binding. Using whole myosin and thin filaments reconstituted with troponin/tropomyosin, the addition of cMyBP-C resulted in a 29% reduction in maximal velocity (P=0.002) with no effect on maximal force. At sub-maximal calcium, the pCa50 for velocity was increased (6.64 ± 0.06 vs. control, 6.44 ± 0.03, P=0.003) whereas the pCa50 for force was decreased (6.25 ± 0.09 vs. control, 6.55 ± 0.02, P=0.008). Thin filament activation by myosin strong-binding demonstrated an increased amount of myosin required to half maximally activate the thin filament in the presence of cMyBP-C, indicating that myosin binding to the thin filament is reduced with cMyBP-C. These findings were supported by co-sedimentation experiments which demonstrate that cMyBP-C competes with S1 for actin binding in the presence of ATP, with no effect on S1/actin binding in the absence of ATP. Finally, while the number of cross-bridges interacting with the thin filament is rate limiting for velocity at shorter filament lengths, this was not observed at longer filament lengths indicating that cMyBP- C directly modulates the kinetics of actomyosin. CONCLUSIONS: The effects of cMyBP-C on velocity and force demonstrate that cMyBP-C does not simply act as a tether but likely affects both the kinetics and the recruitment of myosin cross-bridges through its direct interaction with the myosin head and/or the actin filament.

scholarly journals The HCM-Associated Cardiac Troponin T Mutation K273N in a Human Heart Sample Studied by in Vitro Motility Assay

2011 ◽  
Vol 100 (3) ◽  
pp. 115a
Author(s):  
Christopher Bayliss ◽  
Andrew Messer ◽  
Christobal Dos Remedios ◽  
Jolanda Van der Velden ◽  
Steven Marston
Keyword(s):  
Cardiac Troponin ◽  
Human Heart ◽  
Troponin T ◽  
Motility Assay ◽  
Cardiac Troponin T ◽  
In Vitro Motility Assay ◽  
In Vitro Motility

scholarly journals Cardioinotropic Effects in Subchronic Intoxication of Rats with Lead and/or Cadmium Oxide Nanoparticles

2021 ◽  
Vol 22 (7) ◽  
pp. 3466
Author(s):  
Svetlana V. Klinova ◽  
Boris A. Katsnelson ◽  
Ilzira A. Minigalieva ◽  
Oksana P. Gerzen ◽  
Alexander A. Balakin ◽  
...  
Keyword(s):  
Cadmium Oxide ◽  
Male Rats ◽  
Sliding Velocity ◽  
Muscle Type ◽  
In Vitro Motility Assay ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Toxic Impact ◽  
Cdo Nanoparticles

Subchronic intoxication was induced in outbred male rats by repeated intraperitoneal injections with lead oxide (PbO) and/or cadmium oxide (CdO) nanoparticles (NPs) 3 times a week during 6 weeks for the purpose of examining its effects on the contractile characteristics of isolated right ventricle trabeculae and papillary muscles in isometric and afterload contractions. Isolated and combined intoxication with these NPs was observed to reduce the mechanical work produced by both types of myocardial preparation. Using the in vitro motility assay, we showed that the sliding velocity of regulated thin filaments drops under both isolated and combined intoxication with CdO–NP and PbO–NP. These results correlate with a shift in the expression of myosin heavy chain (MHC) isoforms towards slowly cycling β–MHC. The type of CdO–NP + PbO–NP combined cardiotoxicity depends on the effect of the toxic impact, the extent of this effect, the ratio of toxicant doses, and the degree of stretching of cardiomyocytes and muscle type studied. Some indices of combined Pb–NP and CdO–NP cardiotoxicity and general toxicity (genotoxicity included) became fully or partly normalized if intoxication developed against background administration of a bioprotective complex.

Caged FEDA-ATP: a new tool in the measurement of ATP turnover during the in vitro motility assay

1995 ◽  
Vol 23 (3) ◽  
pp. 401S-401S ◽  
Author(s):  
Daren S. Jeffreys ◽  
Robert J. Eaton ◽  
Clive R. Bagshaw
Keyword(s):  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Atp Turnover

scholarly journals An automated in vitro motility assay for high-throughput studies of molecular motors

Lab on a Chip ◽  
2018 ◽  
Vol 18 (20) ◽  
pp. 3196-3206 ◽  
Author(s):  
Till Korten ◽  
Elena Tavkin ◽  
Lara Scharrel ◽  
Vandana Singh Kushwaha ◽  
Stefan Diez
Keyword(s):  
High Throughput ◽  
Molecular Motors ◽  
Lab On A Chip ◽  
Force Generation ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Cargo Transport

Molecular motors, essential to force-generation and cargo transport within cells, are invaluable tools for powering nanobiotechnological lab-on-a-chip devices.

scholarly journals Determination of the Maximum Velocity of Filaments in the in vitro Motility Assay

2019 ◽  
Vol 10 ◽  
Author(s):  
Nasrin Bopp ◽  
Lisa-Mareike Scheid ◽  
Rainer H. A. Fink ◽  
Karl Rohr
Keyword(s):  
Maximum Velocity ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility
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