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Identification of PKCalpha isoform-specific effects in cardiac myocytes using antisense phosphorothioate
Risto Kerkelä1, Mika Ilves, Sampsa Pikkarainen
1Department of Pharmacology and Toxicology, Biocenter Oulu, University of Oulu, Finland.
Abstract:
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 alpha, the major isozyme present in the neonatal heart. Transfection of cultured neonatal cardiomyocytes with antisense PKCalpha oligonucleotides resulted in a marked reduction in both PKCalpha mRNA and protein levels. PKCalpha antisense treatment also reduced phenylephrine (PE)-induced PKC activity and perinuclear translocation of PKCalpha. Antisense inhibition of PKCalpha led to reduction of PE-induced increase in skeletal alpha-actin mRNA levels and atrial natriuretic peptide (ANP) secretion but had no significant effects on PE-induced beta-myosin heavy chain, ANP, or B-type natriuretic peptide (BNP) gene expression. On the other hand, antisense PKCalpha 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 [(3)H]leucine incorporation, was not affected with antisense PKCalpha treatment. Furthermore, we found that PE-induced increase in extracellular signal-regulated kinase (ERK) activity was partially inhibited by antisense PKCalpha treatment, implicating ERK as a downstream mediator for PKCalpha signaling. These results indicate that PKCalpha isozyme is involved in hypertrophic signaling in cardiomyocytes and provide novel strategies for future studies to identify other cellular targets controlled selectively by PKCalpha or other PKC isozymes.
Insights
Protein Kinase C alpha (PKCalpha) plays a role in cardiomyocyte hypertrophic signaling. Inhibiting PKCalpha reduced some but not all agonist-induced responses, suggesting specific roles in cardiac hypertrophy.
Area of Science:
- Cardiovascular Biology
- Molecular Cell Biology
- Biochemistry
Background:
- Protein Kinase C (PKC) superfamily members regulate crucial cellular functions.
- In cardiac cells, PKC signaling influences gene expression and growth in response to hypertrophic stimuli.
- PKCalpha is the predominant isozyme in neonatal hearts.
Purpose of the Study:
- To elucidate the specific role of PKCalpha in regulating cardiomyocyte growth.
- To investigate the impact of inhibiting PKCalpha on hypertrophic signaling pathways.
Main Methods:
- Utilized antisense oligonucleotides to selectively inhibit PKCalpha expression in cultured neonatal cardiomyocytes.
- Measured changes in PKCalpha mRNA and protein levels, PKC activity, and protein translocation.
- Assessed the effects of PKCalpha inhibition on agonist-induced gene expression (skeletal alpha-actin, ANP, BNP) and peptide secretion.
- Evaluated the impact on cardiomyocyte growth ([(3)H]leucine incorporation) and extracellular signal-regulated kinase (ERK) activity.
Main Results:
- Antisense inhibition of PKCalpha significantly reduced PKCalpha mRNA and protein, as well as phenylephrine (PE)-induced PKC activity and translocation.
- PKCalpha inhibition attenuated PE-induced skeletal alpha-actin mRNA and ANP secretion, but not other PE-induced gene expressions.
- Endothelin-1-induced ANP and BNP peptide secretion were reduced, while gene expression remained unaffected.
- Cardiomyocyte growth and PE-induced ERK activity were partially inhibited by antisense PKCalpha treatment.
Conclusions:
- PKCalpha is implicated in specific aspects of hypertrophic signaling in cardiomyocytes.
- PKCalpha influences agonist-induced peptide secretion and certain gene expressions, but not overall cardiomyocyte growth.
- ERK signaling is a potential downstream mediator of PKCalpha in cardiac hypertrophy.
- Findings offer insights for targeting PKCalpha or other PKC isozymes in cardiac disease research.