There is little direct evidence on the role of myosin regulatory light chain phosphorylation in ejecting hearts. In studies reported here we determined the effects of regulatory light chain (RLC) phosphorylation on in situ cardiac systolic mechanics and in vitro myofibrillar mechanics. We compared data obtained from control nontransgenic mice (NTG) with a transgenic mouse model expressing a cardiac specific nonphosphorylatable RLC (TG-RLC(P؊). We also determined whether the depression in RLC phosphorylation affected phosphorylation of other sarcomeric proteins. TG-RLC(P؊) demonstrated decreases in base-line load-independent measures of contractility and power and an increase in ejection duration together with a depression in phosphorylation of myosin-binding protein-C (MyBP-C) and troponin I (TnI). Although TG-RLC(P؊) displayed a significantly reduced response to ␤ 1 -adrenergic stimulation, MyBP-C and TnI were phosphorylated to a similar level in TG-RLC(P؊) and NTG, suggesting cAMP-dependent protein kinase signaling to these proteins was not disrupted. A major finding was that NTG controls were significantly phosphorylated at RLC serine 15 following ␤ 1 -adrenergic stimulation, a mechanism prevented in TG-RLC(P؊), thus providing a biochemical difference in ␤ 1 -adrenergic responsiveness at the level of the sarcomere. Our measurements of Ca 2؉ tension and Ca 2؉ -ATPase rate relations in detergent-extracted fiber bundles from LV trabeculae demonstrated a relative decrease in maximum Ca 2؉ -activated tension and tension cost in TG-RLC(P؊) fibers, with no change in Ca 2؉ sensitivity. Our data indicate that RLC phosphorylation is critical for normal ejection and response to ␤ 1 -adrenergic stimulation. Our data also indicate that the lack of RLC phosphorylation promotes compensatory changes in MyBP-C and TnI phosphorylation, which when normalized do not restore function.Phosphorylation of sarcomeric proteins tunes the intensity and dynamics of cardiac contraction and relaxation independent of membrane Ca 2ϩ fluxes to meet physiologic demands (1, 2). We focus here on ventricular myosin regulatory light chain, which is phosphorylated in vivo (3-5) but whose functional role in control of cardiac dynamics has remained unclear. The identification of RLC 2 mutations linked to familial hypertrophic cardiomyopathy (6) underscores the importance of understanding its action as a regulator of contraction. Functionally, in vitro cardiac RLC phosphorylation by MLCK produces a sensitizing shift in the force-Ca 2ϩ relation in skinned fibers (7-11). Moreover, studies show that RLC phosphorylation manifests as a gradient across the wall of the heart, which may be important for both normalizing wall stress and for generation of torsion about the long axis of the ejecting heart (12-14). Yet there remains a lack of understanding of the in situ functional effects of RLC phosphorylation and whether phosphorylation of RLC influences other sarcomeric sites as substrates for kinases and phosphatases.Understanding the precise mechanisms by w...

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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-wk ovariectomized rats with or without simultaneous 8 wk-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 alpha-myosin heavy chain level in ovariectomy combined with diabetes could be completely restored by estrogen and insulin supplementation. Conversely, the myofilament Ca2+ hypersensitivity detected only in the ovariectomized but not diabetic group was also observed in diabetic-ovariectomized rats, which was restored upon estrogen supplementation. Binding kinetics of beta1-adrenergic receptors and immunoblots of beta1-adrenoceptors as well as heat shock 72 (HSP72) were analyzed to determine the association of changes in receptors and HSP72 to that of the myofilament response to Ca2+. The amount of beta1-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 beta1-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.

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