Abstract 1032: PKC-ε and Calcineurin Interactions in Cardiomyocyte Stretch
Myocardial stretch activates a number of interconnected pathways including the protein kinase C (PKC) pathway that in turn activates mitogen activated protein kinases (MAPK), leading to gene expression stimulation and ventricular hypertrophy. A role of calcineurin has also been shown during hypertrophy. The goal of our study was to look for a possible interaction between PKC and calcineurin in myocardial stretch. In a first step, the activation of MAPK was evaluated by Western blot analysis of phosphorylated forms of ERK and JNK in neonatal rat cardiomyocytes cultured for 5 days. A 15% stretch increased pERK1 and 2 by 77.5 +/− 10.0% and 118.9 +/− 11.5% respectively and pJNK1 and 2 by 67.6 +/− 10.0% and 62.0 +/− 8.5% respectively. Stretch-induced MAPK activation was decreased by more than 50% when PKC was down-regulated by PMA for 24 hours or when calcineurin was blocked by ciclosporin A. The addition of the 2 drugs did not induce a larger reduction in MAPK activations, suggesting a cooperativity between calcineurin and PKC. The involved PKC isoform in stretch-induced ERK and JNK activations was shown to be PKC-ε since it was translocated by stretch from the cytosolic to the particulate fraction and stretch-induced MAPK activation was blocked by transfection of an isoform-specific inhibitory peptide. In a second step, we found that calcineurin and PKC-ε are in a complex. After stretch, PKC-ε was found in the immunoprecipitate of calcineurin without the presence of the other isoforms. Immunofluorescence staining showed a diffuse cytoplasmic localization of the enzymes in basal conditions. During stretch, calcineurin and PKC-ε were co-localized in the perinuclear region. A direct interaction between PKC-ε and calcineurin was strongly suggested by overlay experiments and confirmed by Surface Plasmon Resonance (BIACORE ): there was a significantly larger interaction between calcineurin fixed on the chip and cell lysates containing PKC-ε (5.22+/−1.98 RU/μg prot) than that with the supernatant of the PKC-ε immunoprecipitate of the lysate containing less PKC-ε(3.43 +/− 1.60 RU/μg prot; p<0.05). These data show thus that PKC-ε and calcineurin that are known as independent stimuli of hypertrophy co-operate during stretch with a direct physical interaction.