STRONG BINDING OF MYOSIN MODULATES LENGTH-DEPENDENT CA2+ ACTIVATION OF RAT VENTRICULAR MYOCYTES

Reductions in sarcomere length (SL) and concomitant increases in inter filament lattice spacing have been shown to decrease the Ca2+ sensitiv ity of tension in myocardium. We tested the idea that increased lattic e spacing influences the SL dependence of isometric tension by reducin g the probability of strong interactions of myosin crossbridges with a ctin, thereby decreasing cooperative activation of the thin filament. Single ventricular myocytes were isolated by enzymatic digestion of ra t hearts and were subsequently rapidly skinned. Maximal tension and Ca 2+ sensitivity of tension (ie, pCa(50)) were measured in the absence a nd presence of N-ethylmaleimide-modified myosin subfragment 1 (NEM-S1) at both short and long SLs. NEM-S1, a strong-binding non-tension-gene rating derivative of the myosin head, was applied to single skinned my ocytes to cooperatively promote strong binding of endogenous myosin cr ossbridges, Compared with control myocytes at SL of approximate to 1.9 0 pm, application of NEM-SI markedly increased submaximal Ca2+-activat ed tensions and thereby increased Ca2+ sensitivity; ie, pCa(50) increa sed from 5.40+/-0.02 to 5.52+/-0.02 pCa units in the presence of NEM-S 1, Furthermore, NEM-S 1 treatment reversibly eliminated the SL depende nce of the Ca2+ sensitivity of tension, in that the Delta pCa(50) betw een short and long lengths was 0.02+/-0.01 pCa units in the presence o f NEM-S1 compared with a Delta pCa(50) of 0.10+/-0.01 pCa units in con trol myocytes. From these results we conclude that the decrease in the Ca2+ sensitivity of tension at short SL results predominantly from de creased cooperative activation of the thin filament due to reductions in the number of strong-binding crossbridges.