This work gives evidence for a differential recruitment of human PKC isoforms in various forms of myocardial hypertrophy and heart failure.

The activity of the adrenergic system plays an important role in the genesis of malignant arrhythmias and the spreading of the infarcted zone in acute myocardial ischemia. Acute myocardial ischemia induces an increased activity of adenylyl cyclase. This sensitization at the enzyme level as shown in the isolated perfused rat heart occurs rapidly after the onset of ischemia (5-15 minutes) and is rapidly reversible on reperfusion. With prolonged ischemia, it is only transient and is followed by a gradual loss of the adenylyl cyclase activity. The increased activity of adenylyl cyclase is even retained after partial purification, suggesting a covalent modification of the enzyme. Blockade of alpha 1-adrenergic receptors does not prevent this sensitization, demonstrating that it occurs independently of alpha 1-adrenergic receptor activation. Only blockade of protein kinase C by various inhibitors, such as polymyxin B or staurosporine, is able to completely prevent this sensitization process. Moreover, in acute myocardial ischemia an activation of protein kinase C could be identified using its translocation from the cytosol to the particulate fraction as an indicator. Blockade of alpha 1-adrenergic receptors using prazosin fails to prevent the activation of protein kinase C and consequently the sensitization of the adenylyl cyclase system, indicating that the ischemia-induced translocation of protein kinase C occurs independently of alpha 1-adrenergic receptors. These data characterize for the first time an important interaction of two effector enzymes of two distinct signal transduction pathways, i.e., the adenylyl cyclase system and the protein kinase C system in acute myocardial ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

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AMP deaminase (myoadenylate deaminase; EC 3.5.4.6) is an integral part of the myofibril in skeletal muscle, and this enzyme plays an important role in energy metabolism in this tissue. We report here the identification of three AMP deaminase isoforms during skeletal muscle development in the rat. An embryonic isoform is expressed in the developing hindlimb of the rat between 7 and 14 days of gestation. This isoform is not unique to skeletal muscle or the embryo as it is also expressed in many nonmuscle tissues of the perinatal and adult rat. A perinatal isoform of AMP deaminase that is restricted to skeletal muscle is produced 4-6 days before birth and persists for 2-3 weeks of postnatal life. An adult, skeletal muscle-specific isoform of AMP deaminase appears at birth and reaches maximal levels after 3 weeks of postnatal development. We conclude from these studies there is a developmentally controlled program that leads to the sequential expression of AMP deaminase isoforms during the transition from embryonic to adult skeletal muscle.AMP deaminase (myoadenylate deaminase; EC 3.5.4.6) is the point of entry for AMP into the purine nucleotide cycle, a series of reactions that are important for energy production in skeletal muscle (1). Deficiency of this enzyme activity is associated with an exercise-related myopathy (2-4). Multiple isoforms of AMP deaminase have been identified in tissue surveys of many species (5-28) , and biochemical (5-9, 18, 19) and genetic (6, 18) data suggest there are muscle-specific isoforms of AMP deaminase. During differentiation of myoblasts into myotubes there is a 10-to 100-fold increase in AMP deaminase activity (ref. 29; unpublished data). These observations point to an important role for AMP deaminase in skeletal muscle function and suggest there may be tissuespecific expression of one or more AMP deaminase isozymes in skeletal muscle.Studies in avian (5-10) and rodent (11-22) systems suggest there is more than one muscle-specific isozyme of AMP deaminase and there may be a switch in isozyme expression during muscle development in vivo (5, 6) and differentiation in vitro (6). There is precedent for isozyme switching in the expression of other proteins during muscle developmenti.e., myosin heavy and light chains (30-32), actin (33-36), tropomyosin (55,56), troponin (37)(38)(39)(40), and creatine kinase (36, 41). The present study was undertaken to determine if there is more than one AMP deaminase polypeptide produced in skeletal muscle and whether there is differential expression ofAMP deaminase polypeptides during muscle development. Rat was selected as a model system for these studies because of the information already available on isozyme expression during muscle development in vivo and myocyte differentiation in vitro in this species. MATERIALSMale Sprague-Dawley LD strain rats were obtained from Charles River Breeding Laboratories. The protease inhibitors benzamidine, phenylmethylsulfonyl fluoride, leupeptin, soybean trypsin inhibitor, pepstatin, a2-macroglobulin, chym...

An activation of protein kinase C (PKC) in acute myocardial ischemia has been shown previously using its translocation to the plasma membrane as an indirect parameter. However, whether PKC remains activated or whether other mechanisms such as altered gene expression may mediate an isozyme-specific regulation in prolonged ischemia have not been investigated. In isolated perfused rat hearts, PKC activity and the expression of PKC cardiac isozymes were determined on the protein level using enzyme activities and Western blot analyses and on the mRNA level using reverse transcriptase-polymerase chain reaction after various periods of global ischemia (1 to 60 minutes). As early as 1 minute after the onset of ischemia, PKC activity is translocated from the cytosol to the particulate fraction without change in total cardiac enzyme activity. This translocation involves all major cardiac isozymes of PKC (ie, PKCalpha, PKCdelta, PKCepsilon, and PKCzeta). This rapid, nonselective activation of PKCs is only transient. In contrast, prolonged ischemia (>/=15 minutes) leads to an increased cardiac PKC activity (119+/-7 versus 190+/-8 pmol/min per mg protein) residing in the cytosol. This is associated with an augmented, subtype-selective isozyme expression of PKCdelta and PKCvarepsilon (163% and 199%, respectively). The specific mRNAs for PKCdelta (948+/-83 versus 1501+/-138 ag/ng total RNA, 30 minutes of ischemia) and PKCepsilon (1597+/-166 versus 2611+/-252 ag/ng total RNA) are selectively increased. PKCalpha and PKCzeta remain unaltered. In conclusion, two distinct activation and regulation processes of PKC are characterized in acute myocardial ischemia. The early, but transient, translocation involves all constitutively expressed cardiac isozymes of PKC, whereas in prolonged ischemia an increased total PKC activity is associated with an isozyme-selective induction of PKCepsilon and PKCdelta. Whether these fundamentally different activation processes interact remains to be elucidated.