A highly specific benzimidazole pyridazinone reverses phosphate-induced changes in cardiac myofilament activation

Abstract

Both Ca2+ and the actin cross-bridge reaction itself can activate contraction in myofilaments. We are interested in identifying ligands which modify one or both mechanisms of contractile activation with high affinity and specificity. Results presented here suggest that the benzimidazole-substituted pyridazinone, UD-CG 212 Cl, potently modulates myofilament activation by the cross-bridge reaction. Cross-bridge-mediated activation was studied by varying the population of force-generating (strong) cross-bridges with inorganic phosphate (Pi). Addition of Pi to detergent-extracted (skinned) canine ventricular preparations reduces the population of strong cross-bridges, which causes reduced myofilament force and Ca2+ sensitivity. Increased Pi concentration ([Pi]) also favors cross-bridge-mediated myofilament activation, so cooperativity increases. The decreased Ca2+ sensitivity produced by Pi was reversed by a racemic mixture of 10(-10) M UD-CG 212 Cl, but this agent had no effect on maximum force or cooperativity. In experiments with stereoisomers, only (+)-UD-CG 212 Cl stimulated force. At higher doses (10(-6)-10(-4) M), submaximal but not maximal force decreased and (-)-UD-CG 212 Cl was the active stereoisomer. Neither Pi nor UD-CG 212 Cl affected Ca2+ binding to myofilament troponin C (TnC). Thus, UD-CG 212 Cl appears to reverse Pi-induced decreases in submaximal force via high-affinity binding to a myofibrillar domain not directly involved with myofibrillar TnC-Ca2+ binding. The actions of UD-CG 212 Cl were further investigated by reducing [ATP] as another means of varying the cross-bridge population.(ABSTRACT TRUNCATED AT 250 WORDS)