Increased Protein Kinase C Activity in Myotrophin-Induced Myocyte Growth

From the Department of Molecular Cardiology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio.

Correspondence to Subha Sen, PhD, DSc, Department of Molecular Cardiology/FF40, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail sens{at}ccsmtp.ccf.org

Abstract—Myotrophin, a novel protein that has been shown to stimulate myocyte growth, has been isolated, purified, and sequenced from the hearts of spontaneously hypertensive rats and dilated cardiomyopathic human tissue. Recently, the cDNA clones encoding myotrophin have been isolated and expressed in Escherichia coli, and the recombinant myotrophin was found to be as biologically and immunologically active as natural myotrophin. The mechanism by which myotrophin stimulates protein synthesis and initiates myocardial hypertrophy is not known. To evaluate the involvement of protein kinase C (PKC) in myotrophin-induced hypertrophy, PKC activity and its distribution in the subcellular fraction were determined in cultured neonatal and adult myocytes. PKC activity was determined by measuring the incorporation of ³²P into histone type III-S and PKC substrate peptide (_pep) from [-³²P]ATP in neonatal myocytes. Myotrophin significantly stimulated PKC activity in neonatal myocytes and was associated with a significant increase in protein synthesis. The effect of myotrophin on the stimulation of PKC activity and [³H]leucine incorporation was abolished by pretreatment with either staurosporine or H-7, two selective, pharmacological PKC inhibitors. Pretreatment of myocytes with staurosporine also reduced the myotrophin-induced mRNA levels of c-fos and ß-myosin heavy chain. To evaluate the subcellular events whose occurrence was due to myotrophin and translocation of PKC, we studied the effect of genistein, a tyrosine kinase inhibitor, on myotrophin-induced neonatal myocyte growth. Genistein attenuated the [³H]leucine incorporation induced by myotrophin. To define the specificity of the PKC isoform(s) involved in myotrophin-stimulated myocyte growth, both neonatal and adult myocytes were treated with myotrophin, and Western blot analyses were performed by using the antibodies of different PKC isoforms. Results showed that both PKC and PKC isoforms participated in the myotrophin-induced neonatal myocyte growth, whereas only the PKC isoform was involved in myotrophin-induced adult myocyte hypertrophy. PKC and PKC do not seem to participate in either neonatal or adult myocyte growth induced by myotrophin. Treatment with antisense oligonucleotides specific for PKC and PKC isoforms further supported this result. PKC is the major PKC isoform in neonatal myocytes and needs Ca²⁺ and phospholipids for its activation, and PKC (the Ca²⁺-independent PKC isoform) is present in both neonatal and adult myocytes; the 15-mer antisense oligodeoxynucleotides of each were used for this study. Treatment of neonatal myocytes with the PKC and PKC antisense oligodeoxynucleotides for 5 days significantly reduced Ca²⁺-dependent and Ca²⁺-independent PKC activity, respectively, as well as the [³H]leucine incorporation induced by myotrophin. Furthermore, myotrophin-induced PKC activity was primarily located in the particulate fraction and did not result in a concomitant decrease in the cytosolic fraction. Myotrophin does not change PKC isoform expression (both Ca²⁺ dependent and independent PKC isoforms used in this study) in rat neonatal cardiac fibroblasts. Our data suggest that myotrophin exerts its action on protein synthesis, possibly through a tyrosine kinase–coupled pathway and translocation of PKC from the cytosol to the cell membrane.

Key Words: myotrophin • PKC • ß-myosin heavy chain • myocytes • hypertrophy

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