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Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes
Published on: September 25, 2013
E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on
1Univ. of North Texas Health Science Center, Fort Worth, TX 76107, USA. jborejdo@hsc.unt.edu
Insights
The E22K mutation in familial hypertrophic cardiomyopathy does not alter myosin cross-bridge mechanics. Myofibrillar disarray, not the mutation itself, likely explains changes in contraction dynamics.
Area of Science:
- Cardiovascular Biology
- Muscle Physiology
- Molecular Cardiology
Background:
- Familial hypertrophic cardiomyopathy (HCM) involves cardiac hypertrophy and myofibrillar disarray, often caused by sarcomeric protein mutations.
- The E22K mutation in the myosin regulatory light chain (RLC) is linked to HCM and affects the RLC's Ca(2+)-binding site.
Purpose of the Study:
- To investigate the impact of the E22K RLC mutation on the mechanical properties of cardiac myosin cross-bridges during contraction.
- To determine if the E22K mutation influences myosin head dissociation from actin (tau(1)), cross-bridge rebinding (tau(2)), or ADP dissociation (tau(3)).
Main Methods:
- Studied transgenic (Tg) mouse cardiac myofibrils with and without the E22K mutation (Tg-m) using single-turnover contraction assays.
- Measured kinetic parameters (tau(1), tau(2), tau(3)) using actin rotation, anisotropy changes of labeled myosin light chains, and fluorescent ADP binding.
- Assessed direct myosin-actin binding affinity using fluorescence lifetime and time-resolved anisotropy.
Main Results:
- The dissociation time of myosin heads from thin filaments (tau(1)) was significantly longer in Tg-m myofibrils compared to controls.
- Cross-bridge rebinding time (tau(2)) was shorter in Tg-m than non-Tg, but similar to Tg-wild-type (Tg-wt).
- ADP dissociation time (tau(3)) and direct myosin-actin binding affinity were unaffected by the E22K mutation.
Conclusions:
- The E22K mutation does not directly alter the intrinsic mechanical properties of cardiac myosin cross-bridges.
- Increased tau(1) in Tg-m hearts is likely attributable to myofibrillar disarray, a hallmark of HCM.
- Observed changes in tau(2) may result from the replacement of mouse RLC with the human isoform in Tg mice.
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 (tau(1)) of myosin heads from thin filaments, 2) rebinding time (tau(2)) of the cross bridges to actin, and 3) dissociation time (tau(3)) of ADP from the active site of myosin. tau(1) was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. tau(2) 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. tau(3) 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. tau(1) was statistically greater in Tg-m than in controls. tau(2) was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. tau(3) was the same in Tg-m and controls. To determine whether the difference in tau(1) 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 tau(1) was probably caused by myofibrillar disarray. The decrease in tau(2) of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice.
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