Strong binding of myosin modulates length-dependent Ca2+ activation of rat ventricular myocytes.
Reductions in sarcomere length (SL) and concomitant increases in interfilament lattice spacing have been shown to decrease the Ca2+ sensitivity of tension in myocardium. We tested the idea that increased lattice spacing influences the SL dependence of isometric tension by reducing the probability of strong interactions of myosin crossbridges with actin, thereby decreasing cooperative activation of the thin filament. Single ventricular myocytes were isolated by enzymatic digestion of rat hearts and were subsequently rapidly skinned. Maximal tension and Ca2+ sensitivity of tension (ie, pCa50) were measured in the absence and presence of N-ethylmaleimide-modified myosin subfragment 1 (NEM-S1) at both short and long SLs. NEM-S1, a strong-binding non-tension-generating derivative of the myosin head, was applied to single skinned myocytes to cooperatively promote strong binding of endogenous myosin crossbridges. Compared with control myocytes at SL of approximately 1.90 microm, application of NEM-S1 markedly increased submaximal Ca2+-activated tensions and thereby increased Ca2+ sensitivity; ie, pCa50 increased from 5.40+/-0.02 to 5.52+/-0.02 pCa units in the presence of NEM-S1. Furthermore, NEM-S1 treatment reversibly eliminated the SL dependence of the Ca2+ sensitivity of tension, in that the DeltapCa50 between short and long lengths was 0. 02+/-0.01 pCa units in the presence of NEM-S1 compared with a DeltapCa50 of 0.10+/-0.01 pCa units in control myocytes. From these results we conclude that the decrease in the Ca2+ sensitivity of tension at short SL results predominantly from decreased cooperative activation of the thin filament due to reductions in the number of strong-binding crossbridges.
Fitzsimons
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From the Department of Physiology, University of Wisconsin School of Medicine, Madison, USA. fitzsimo@facstaff.wisc.edu
Moss
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D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D006352:Heart Ventricles / Q000166:cytology*; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects; D009218:Myosins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley
D009218:Myosins; D005033:Ethylmaleimide; D002118:Calcium