Vol. 62, Issue 6, 1482-1491, December 2002

Identification of PKC Isoform-Specific Effects in Cardiac Myocytes Using Antisense Phosphorothioate Oligonucleotides

Risto Kerkelä, Mika Ilves, Sampsa Pikkarainen, Heikki Tokola, Jarkko Ronkainen, Olli Vuolteenaho, Juhani Leppäluoto, and Heikki Ruskoaho

Departments of Pharmacology and Toxicology (R.K., S.P., H.T., H.R.) and Physiology (M.I., J.R., O.V., J.L.), Biocenter Oulu, University of Oulu, Finland

Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in multiple cellular processes. In the heart, PKC signaling is involved in hypertrophic agonist-induced gene expression and hypertrophic growth. To investigate the specific function of PKC signaling in regulating cardiomyocyte growth, we used antisense oligonucleotides to inhibit PKC , the major isozyme present in the neonatal heart. Transfection of cultured neonatal cardiomyocytes with antisense PKC oligonucleotides resulted in a marked reduction in both PKC mRNA and protein levels. PKC antisense treatment also reduced phenylephrine (PE)-induced PKC activity and perinuclear translocation of PKC. Antisense inhibition of PKC led to reduction of PE-induced increase in skeletal -actin mRNA levels and atrial natriuretic peptide (ANP) secretion but had no significant effects on PE-induced -myosin heavy chain, ANP, or B-type natriuretic peptide (BNP) gene expression. On the other hand, antisense PKC treatment attenuated endothelin-1-induced increase in ANP and BNP peptide secretion, whereas endothelin-1-induced gene expression of ANP and BNP remained unchanged. The hypertrophic agonist-induced growth of cardiomyocytes, characterized by increased [³H]leucine incorporation, was not affected with antisense PKC treatment. Furthermore, we found that PE-induced increase in extracellular signal-regulated kinase (ERK) activity was partially inhibited by antisense PKC treatment, implicating ERK as a downstream mediator for PKC signaling. These results indicate that PKC isozyme is involved in hypertrophic signaling in cardiomyocytes and provide novel strategies for future studies to identify other cellular targets controlled selectively by PKC or other PKC isozymes.