2؉sensitivity of the myofilament.It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca 2؉ sensitivity of the myofilament, and that phosphorylation of Ser-43/ Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca 2؉ sensitivity, respectively, of actomyosin S-1 MgATPase.In cardiac myocytes, the activation of several types of receptors, such as ␣ 1 -adrenergic (1-5), muscarinic (1, 6), and purinergic (6) dynorphin A (7), endothelin-1 (8, 9), and angiotensin (14 -18). However, the complex molecular events mediated by PKC (or more precisely, by the individual isozymes) that are responsible for cardiac contractility regulation, for example, remain largely unclear. It has been reported that phenylephrine (␣ 1 -adrenergic receptor agonist) elicited transient negative inotropy followed by sustained positive inotropy (3, 19 -21), endothelin-1 caused monotonic positive inotropy (22), whereas dynorphin A (-opioid receptor agonist) induced negative inotropy (7). All three of these distinct receptor agonists are believed to act, at least in part, through PKC activation. Furthermore, phorbol esters (such as TPA), potent and long-acting PKC activators, produced predominantly negative inotropic effects in various cardiac preparations (23-27). These seemingly paradoxical observations might reflect certain opposing factors of PKC activation which include the net effects of intracellular pH change, the size of intracellular Ca 2ϩ transient, and the states and species of cellular proteins being phosphorylated.One target for PKC in the heart is the contractile apparatus itself. Cardiac TnI and TnT from the thin filament have been shown to be effective substrates for PKC (28), and some of the phosphorylation sites in these proteins have been determined (29,30). Phosphorylation of TnI and/or TnT by PKC resulted in an inhibition of Ca 2ϩ -stimulated MgATPase of the reconstituted actomyosin complex (31-33) or in native myofibril preparations (32, 34), an effect associated with altered interactions among the contractile protein components (32,33). PKC also phosphorylated MLC2 (34 -36) and C-protein (34 -36) in myofibrillar and thick filament preparations. Phosphorylation of

Protein kinase C (PKC) isozymes alpha, delta, epsilon, and zeta, shown to be expressed in adult rat cardiomyocytes, displayed distinct substrate specificities in phosphorylating troponin I and troponin T subunits in the bovine cardiac troponin complex. Thus, because they have different substrate affinities, PKC-alpha, -delta, and -epsilon phosphorylated troponin I more than troponin T, but PKC-zeta conversely phosphorylated the latter more than the former. Furthermore, PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in these proteins as free subunits or in the troponin complex. Unlike other isozymes, PKC-delta was uniquely able to phosphorylate Ser-23/Ser-24 in troponin I, the bona fide phosphorylation sites for protein kinase A (PKA); and consequently, like PKA, it reduced Ca2+ sensitivity of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. In addition, PKC-delta, like PKC-alpha, readily phosphorylated Ser-43/Ser-45 (sites common for all PKC isozymes) and reduced maximal activity of MgATPase. In this respect, PKC-delta functioned as a hybrid of PKC-alpha and PKA. In contrast to PKC-alpha, -delta, and -epsilon, PKC-zeta exclusively phosphorylated two previously unknown sites in troponin T. Phosphorylation of troponin T by PKC-alpha resulted in decreases in both Ca2+ sensitivity and maximal activity, whereas phosphorylation by PKC-zeta resulted in a slight increase of the Ca2+ sensitivity without affecting the maximal activity of MgATPase. Most of the in vitro phosphorylation sites in troponin I and troponin T were confirmed in situ in adult rat cardiomyocytes. The present study has demonstrated for the first time distinct specificities of PKC isozymes for phosphorylation of two physiological substrates in the myocardium, with functional consequences.

The role of protein kinase C (PKC) in the phosphorylation of myosin light chain 2 (MLC2) in adult rat heart cells has been investigated. PKC-mediated phosphorylation of MLC2 in adult rat cardiac myofibrils in vitro occurs with a stoichiometry (0.7 mol of phosphate/mol of protein) similar to that mediated by myosin light chain kinase (MLCK). Two-dimensional tryptic phosphopeptide mapping of MLC2 following phosphorylation by PKC or MLCK in vitro yields the same major phosphopeptides for each protein kinase. These sites are also 32P-labelled in situ when isolated cardiomyocytes are incubated with [32P]P(i). 32P labelling of MLC2 in cardiomyocytes is increased by 5-fold in 10 min upon incubation with the phosphatase inhibitor calyculin A, demonstrating the existence of a rapidly turning over component of MLC2 phosphorylation in these cells. 32P label is completely removed from MLC2 when myocytes are exposed to 2,3-butanedione monoxime, an inhibitor of cardiac contraction known to desensitize the myofilaments to activation by Ca2+. 32P labelling of MLC2 is also decreased by 50-100% following exposure to the PKC-selective inhibitors calphostin C and chelerythrine, suggesting that PKC, and not MLCK, is primarily responsible for incorporation of rapidly turning over phosphate into MLC2 in situ. Taken together, these data implicate PKC in the phosphorylation of MLC2 in heart cells and support the hypothesis that phosphorylation of cardiac MLC2 has a role in determining myofibrillar Ca2+ sensitivity.

Effects of phosphorylation of bovine cardiac troponin T (TnT) by protein kinase C on the Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin complex and the binding of TnT to tropomyosin(Tm)-F-actin were investigated. The Ca(2+)-stimulated MgATPase of actomyosin containing phosphorylated TnT (1.8 mol of P/mol), compared with that containing unphosphorylated TnT, was decreased by up to 48%. Phosphorylation of TnT also decreased (up to 48%) its maximum binding to Tm-F-actin, which was accompanied by a decrease (up to 3.5-fold) in its apparent binding affinity. The findings indicate that the effects of phosphorylated TnT in decreasing actomyosin MgATPase might be secondary to its decreased interactions with the other components of the thin filament, representing a new mechanism underlying the negative inotropic responses of various cardiac preparations to protein kinase C-activating phorbol esters.

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