scholarly journals Characterization of α-Tropomyosin (TM) Mutants that Cause Hypertrophic Cardiomyopathy (HCM) in Humans: In vitro Motility Assays with a Microscopic Heat Pulse

2015 ◽  
Vol 108 (2) ◽  
pp. 445a-446a
Author(s):  
Shuya Ishii ◽  
Kotaro Oyama ◽  
Madoka Suzuki ◽  
Masataka Kawai ◽  
Shin’ichi Ishiwata
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Heat Pulse ◽  
In Vitro Motility

scholarly journals Facilitated Cross-Bridge Interactions with Thin Filaments by Familial Hypertrophic Cardiomyopathy Mutations inα-Tropomyosin

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Fang Wang ◽  
Nicolas M. Brunet ◽  
Justin R. Grubich ◽  
Ewa A. Bienkiewicz ◽  
Thomas M. Asbury ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Molecular Mechanisms ◽  
Helical Structure ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Maximum Speed ◽  
Sliding Speed ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Cardiac Sarcomeres

Familial hypertrophic cardiomyopathy (FHC) is a disease of cardiac sarcomeres. To identify molecular mechanisms underlying FHC pathology, functional and structural differences in three FHC-related mutations in recombinantα-Tm (V95A, D175N, and E180G) were characterized using both conventional and modified in vitro motility assays and circular dichroism spectroscopy. Mutant Tm's exhibited reducedα-helical structure and increased unordered structure. When thin filaments were fully occupied by regulatory proteins, little or no motion was detected at pCa 9, and maximum speed (pCa 5) was similar for all tropomyosins. Ca2+-responsiveness of filament sliding speed was increased either by increasedpCa50(V95A), reduced cooperativityn(D175N), or both (E180G). When temperature was increased, thin filaments with E180G exhibited dysregulation at temperatures ~10°C lower, and much closer to body temperature, than WT. When HMM density was reduced, thin filaments with D175N required fewer motors to initiate sliding or achieve maximum sliding speed.

scholarly journals Impact of familial hypertrophic cardiomyopathy-linked mutations in the NH2 terminus of the RLC on β-myosin cross-bridge mechanics

2014 ◽  
Vol 117 (12) ◽  
pp. 1471-1477 ◽  
Author(s):  
Gerrie P. Farman ◽  
Priya Muthu ◽  
Katarzyna Kazmierczak ◽  
Danuta Szczesna-Cordary ◽  
Jeffrey R. Moore
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Sarcomeric Proteins ◽  
Motion Generation ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Cross Bridge ◽  
Significant Difference ◽  
The Impact

Familial hypertrophic cardiomyopathy (HCM) is associated with mutations in sarcomeric proteins, including the myosin regulatory light chain (RLC). Here we studied the impact of three HCM mutations located in the NH2 terminus of the RLC on the molecular mechanism of β-myosin heavy chain (MHC) cross-bridge mechanics using the in vitro motility assay. To generate mutant β-myosin, native RLC was depleted from porcine cardiac MHC and reconstituted with mutant (A13T, F18L, and E22K) or wild-type (WT) human cardiac RLC. We characterized the mutant myosin force and motion generation capability in the presence of a frictional load. Compared with WT, all three mutants exhibited reductions in maximal actin filament velocity when tested under low or no frictional load. The actin-activated ATPase showed no significant difference between WT and HCM-mutant-reconstituted myosins. The decrease in velocity has been attributed to a significantly increased duty cycle, as was measured by the dependence of actin sliding velocity on myosin surface density, for all three mutant myosins. These results demonstrate a mutation-induced alteration in acto-myosin interactions that may contribute to the pathogenesis of HCM.

scholarly journals Cardiac actin changes in the actomyosin interface have different effects on myosin duty ratio

2018 ◽  
Vol 96 (1) ◽  
pp. 26-31 ◽  
Author(s):  
Haidun Liu ◽  
Mary Henein ◽  
Maria Anillo ◽  
John F. Dawson
Keyword(s):  
Cardiovascular Disease ◽  
Hypertrophic Cardiomyopathy ◽  
Left Ventricle ◽  
Duty Ratio ◽  
Actomyosin Atpase ◽  
In Vitro Motility ◽  
Cardiac Actin ◽  
Myosin Binding ◽  
Cardiac Sarcomeres

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disease (CD) that commonly causes an increased size of cardiomyocytes in the left ventricle. The proteins myosin and actin interact in the myocardium to produce contraction through the actomyosin ATPase cycle. The duty ratio (r) of myosin is the proportion of the actomyosin ATPase cycle that myosin is bound to actin and does work. A common hypothesis is that HCM mutations increase contraction in cardiac sarcomeres; however, the available data are not clear on this connection. Based on previous work with human α-cardiac actin (ACTC), we hypothesize that HCM-linked ACTC variants with alterations near the myosin binding site have an increased r, producing more force. Myosin duty ratios using human ACTC variant proteins were calculated with myosin ATPase activity and in-vitro motility data. We found no consistent changes in the duty ratio of the ACTC variants, suggesting that other factors are involved in the development of HCM when ACTC variants are present.

scholarly journals Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function

2015 ◽  
Vol 1 (9) ◽  
pp. e1500511 ◽  
Author(s):  
Suman Nag ◽  
Ruth F. Sommese ◽  
Zoltan Ujfalusi ◽  
Ariana Combs ◽  
Stephen Langer ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Actin Filaments ◽  
Duty Ratio ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Genes Encoding ◽  
New Methodologies ◽  
Unloaded Velocity

Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation.

Familial hypertrophic cardiomyopathy mutations in troponin I (K183Δ, G203S, K206Q) enhance filament sliding

2003 ◽  
Vol 14 (2) ◽  
pp. 117-128 ◽  
Author(s):  
Jan Köhler ◽  
Ying Chen ◽  
Bernhard Brenner ◽  
Albert M. Gordon ◽  
Theresia Kraft ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Troponin I ◽  
Isometric Force ◽  
Familial Hypertrophic Cardiomyopathy ◽  
In Vitro Assays ◽  
Maximum Speed ◽  
Exchange Method ◽  
In Vitro Motility ◽  
Maximum Isometric Force

A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac sarcomeric genes. Linkage studies identified FHC-related mutations in the COOH terminus of cardiac troponin I (cTnI), a region with unknown function in Ca2+ regulation of the heart. Using in vitro assays with recombinant rat troponin subunits, we tested the hypothesis that mutations K183Δ, G203S, and K206Q in cTnI affect Ca2+ regulation. All three mutants enhanced Ca2+ sensitivity and maximum speed ( smax) of filament sliding of in vitro motility assays. Enhanced smax (pCa 5) was observed with rabbit skeletal and rat cardiac (α-MHC or β-MHC) heavy meromyosin (HMM). We developed a passive exchange method for replacing endogenous cTn in permeabilized rat cardiac trabeculae. Ca2+ sensitivity and maximum isometric force did not differ between preparations exchanged with cTn(cTnI,K206Q) or wild-type cTn. In both trabeculae and motility assays, there was no loss of inhibition at pCa 9. These results are consistent with COOH terminus of TnI modulating actomyosin kinetics during unloaded sliding, but not during isometric force generation, and implicate enhanced cross-bridge cycling in the cTnI-related pathway(s) to hypertrophy.

Two mutations in troponin I that cause hypertrophic cardiomyopathy have contrasting effects on cardiac muscle contractility

2002 ◽  
Vol 362 (2) ◽  
pp. 443-451 ◽  
Author(s):  
David BURTON ◽  
Hassan ABDULRAZZAK ◽  
Adam KNOTT ◽  
Kathryn ELLIOTT ◽  
Charles REDWOOD ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Wild Type ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Inhibitory Function ◽  
Type Protein ◽  
Wild Type Protein

We investigated the effects of two mutations in human cardiac troponin I, Arg145 → Gly and Gly203 → Ser, that are reported to cause familial hypertrophic cardiomyopathy. Mutant and wild-type troponin I, overexpressed in Escherichia coli, were used to reconstitute troponin complexes in vanadate-treated guinea pig cardiac trabeculae skinned fibres, and thin filaments were reconstituted with human cardiac troponin and tropomyosin along with rabbit skeletal muscle actin for in vitro motility and actomyosin ATPase assays. Troponin containing the Arg145 → Gly mutation inhibited force in skinned trabeculae less than did the wild-type, and had almost no inhibitory function in the in vitro motility assay. There was an enhanced inhibitory function with mixtures of 10–30% [Gly145]troponin I with the wild-type protein. Skinned trabeculae reconstituted with troponin I containing the Gly203 → Ser mutation and troponin C produced less Ca2+-activated force (64±8% of wild-type) and demonstrated lower Ca2+ sensitivity [ΔpCa50 (log of the Ca2+ concentration that gave 50% of maximal activation) 0.25 unit (P < 0.05)] compared with wild-type troponin I, but thin filaments containing [Ser203]-troponin I were indistinguishable from those containing the wild-type protein in in vitro motility and ATPase assays. Thus these two mutations each result in hypertrophic cardiomyopathy, but have opposite effects on the overall contractility of the muscle in the systems we investigated, indicating either that we have not yet identified the relevant alteration in contractility for the Gly203 → Ser mutation, or that the disease does not result directly from any particular alteration in contractility.

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