E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium

Effect on cross-bridge kinetics

D. Dumka, J. Talent, I. Akopova, G. Guzman, Danuta Szczesna-Cordary, J. Borejdo

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Abstract

Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca 2+-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (τ1) of myosin heads from thin filaments, 2) rebinding time (τ2) of the cross bridges to actin, and 3) dissociation time (τ3) of ADP from the active site of myosin. τ1 was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. τ2 was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. τ3 was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. τ1 was statistically greater in Tg-m than in controls. τ2 was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. τ3 was the same in Tg-m and controls. To determine whether the difference in τ1 was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in τ1 was probably caused by myofibrillar disarray. The decrease in τ2 of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.

Original languageEnglish
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume291
Issue number5
DOIs
StatePublished - Nov 24 2006

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Familial Hypertrophic Cardiomyopathy
Myosins
Myocardium
Myofibrils
Anisotropy
Light
Mutation
Actins
Myosin Light Chains
Adenosine Diphosphate
Transgenic Mice
Protein Isoforms
Ventricular Myosins
Muscles
Rhodamines
Hypertrophy
Catalytic Domain
Binding Sites
Proteins

Keywords

  • Anisotropy
  • Confocal microscopy

ASJC Scopus subject areas

  • Physiology

Cite this

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title = "E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: Effect on cross-bridge kinetics",
abstract = "Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca 2+-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (τ1) of myosin heads from thin filaments, 2) rebinding time (τ2) of the cross bridges to actin, and 3) dissociation time (τ3) of ADP from the active site of myosin. τ1 was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. τ2 was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. τ3 was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. τ1 was statistically greater in Tg-m than in controls. τ2 was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. τ3 was the same in Tg-m and controls. To determine whether the difference in τ1 was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in τ1 was probably caused by myofibrillar disarray. The decrease in τ2 of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.",
keywords = "Anisotropy, Confocal microscopy",
author = "D. Dumka and J. Talent and I. Akopova and G. Guzman and Danuta Szczesna-Cordary and J. Borejdo",
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T1 - E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium

T2 - Effect on cross-bridge kinetics

AU - Dumka, D.

AU - Talent, J.

AU - Akopova, I.

AU - Guzman, G.

AU - Szczesna-Cordary, Danuta

AU - Borejdo, J.

PY - 2006/11/24

Y1 - 2006/11/24

N2 - Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca 2+-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (τ1) of myosin heads from thin filaments, 2) rebinding time (τ2) of the cross bridges to actin, and 3) dissociation time (τ3) of ADP from the active site of myosin. τ1 was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. τ2 was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. τ3 was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. τ1 was statistically greater in Tg-m than in controls. τ2 was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. τ3 was the same in Tg-m and controls. To determine whether the difference in τ1 was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in τ1 was probably caused by myofibrillar disarray. The decrease in τ2 of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.

AB - Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca 2+-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (τ1) of myosin heads from thin filaments, 2) rebinding time (τ2) of the cross bridges to actin, and 3) dissociation time (τ3) of ADP from the active site of myosin. τ1 was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. τ2 was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. τ3 was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. τ1 was statistically greater in Tg-m than in controls. τ2 was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. τ3 was the same in Tg-m and controls. To determine whether the difference in τ1 was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in τ1 was probably caused by myofibrillar disarray. The decrease in τ2 of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.

KW - Anisotropy

KW - Confocal microscopy

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