Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain...

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Bibliographic Details
Published in:American journal of physiology. Heart and circulatory physiology 2011-02, Vol.300 (2), p.H574-H582
Main Authors: Vandenboom, Rene, Herron, Todd, Favre, Elizabeth, Albayya, Faris P, Metzger, Joseph M
Format: Article
Language:English
Subjects:
Actin Cytoskeleton - physiology
Animals
Blotting, Western
Calcium Signaling - genetics
Calcium Signaling - physiology
Cardiac Excitation and Contraction
Cardiomyocytes
Cell Membrane Permeability - physiology
Cell Separation
DNA, Complementary - biosynthesis
DNA, Complementary - genetics
Electrophoresis, Polyacrylamide Gel
Fluorescent Antibody Technique
Gene expression
Gene Transfer Techniques
Genetic Vectors
Humans
Immunohistochemistry
Immunoprecipitation
Molecules
Myocardial Contraction - physiology
Myocardium - metabolism
Myocytes, Cardiac - metabolism
Myosin Heavy Chains - biosynthesis
Myosin Heavy Chains - genetics
Myosins - biosynthesis
Myosins - chemistry
Myosins - genetics
Physiology
Protein Conformation
Rats
Sarcomeres - metabolism
Studies
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Summary:The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P
ISSN:0363-6135
1522-1539
DOI:10.1152/ajpheart.00786.2009