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 (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<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.
Integrative Biology and Physiology, University of Minnesota School of Medicine, 6-125 Jackson Hall, 321 Church St. E, Minneapolis, MN 55455, USA.
Rene
R
Vandenboom
Todd
T
Herron
Elizabeth
E
Favre
Faris P
FP
Albayya
Joseph M
JM
Metzger
D008841:Actin Cytoskeleton / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D020013:Calcium Signaling / Q000235:genetics / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology; D002469:Cell Separation; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D009200:Myocardial Contraction / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D009218:Myosins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D051381:Rats; D012518:Sarcomeres / Q000378:metabolism
D018076:DNA, Complementary; D018995:Myosin Heavy Chains; D009218:Myosins