The amelioration of cardioprotective effect of estrogen in diabetes suggests potential interactive action of estrogen and insulin on myofilament activation. We compared Ca²⁺-dependent Mg²⁺-ATPase activity of isolated myofibrillar preparations from hearts of sham and 10-week ovariectomized rats with or without simultaneous 8 week-induction of diabetes, and from diabetic-ovariectomized rats with estrogen and/or insulin supplementation. Similar magnitude of suppressed maximum myofibrillar ATPase activity was demonstrated in ovariectomized, diabetic, and diabetic-ovariectomized rat hearts. Such suppressed activity and the relative suppression in -MHC level in ovariectomy combined with diabetes could be completely restored by estrogen and insulin supplementation. Conversely, the myofilament Ca²⁺ hypersensitivity detected only in ovariectomized but not diabetic group was also observed in diabetic-ovariectomized rat, which was restored upon estrogen supplementation. Binding kinetics of ₁-adrenergic receptors and immunoblots of ₁-adrenoceptors as well as heat shock 72 (HSP72) were analyzed to determine the association of changes in receptors and HSP72 to that of myofilament response to Ca²⁺. The amount of ₁-adrenoceptors significantly increased concomitant with Ca²⁺ hypersensitivity of the myofilament, without differences in the receptor binding affinity among the groups. In contrast, changes in HSP72 paralleled that of maximum myofibrillar ATPase activity. These results indicate that hypersensitivity of cardiac myofilament to Ca²⁺ is specifically induced in ovariectomized rats even under diabetes complication, and that alterations in the expression of ₁-adrenoceptors may, in part, play a mechanistic role underlying the cardioprotective effects of estrogen that act together with Ca²⁺ hypersensitivity of the myofilament in determining the gender difference in cardiac activation.