β-adrenergic Stimulation Activates PKCε Inducing ERK Phosphorylation And Cardiomyocyte Hypertrophy

Background:Myocardial hypertrophy, which is a reaction of cardiac myocytes to a variety of pathological stimuli, is a common complication of hypertension, coronary heart disease, valvular heart disease, congenital heart disease and other cardiovascular diseases. Cardiac hypertrophy is an independent risk factor of cardiovascular morbidity and mortality. Activating various signaling pathways by prohypertrophic stimuli including stretch stimulation, neurohumoral factors, and local humoral factors is involved in the induction of cardiac hypertrophy. Understanding the signaling mechanisms that mediate the growth of cardiac myocytes may lead to better treatment for patients with cardiac hypertrophy. Cardiac adrenergic signaling pathway includes a and β adrenergic receptors。 α and (3adrenergic receptors are activated, which results in stimulation of phospholipase C/protein kinase C (PKC) or adenylate cyclase/protein kinase A (PKA) signaling pathways, respectively. Evidence from cultured cardiac myocytes and transgenic mouse studies demonstrated the critical contribution of the adrenergic signaling pathway to cardiac hypertrophy.The protein kinase C pathway is classically considered as independent of the ρ-adrenergic receptor (βAR) pathway. However, it was recently shown that the exchange factor Epac (Exchange protein directly activated by cAMP) which is activated by cAMP activates in turn phospholipase Cs, suggesting that PAR stimulation may activate PKC. Epac and phospholipase C could mediate the interconnection between ρ-adrenergic and PKC pathways.Objective:The aim of this study is to examine PKCs translocation after PAR stimulation in isolated cardiomyocytes, evaluate the role of Epac and phospholipase C in the cross-talk of PAR and PKCs, and investigate the effect of this signaling pathway on ERK phosphorylation and cardiomyocyte hypertrophy.Methods:Rat neonatal cardiomyocytes were cultured and treated with isoproterenol (Iso, lμmol/L for l~30min), Epac specific activator8-CPT (lμmol/L for10min), and phospholipase C inhibitor U73122(2μmol/L for30min). After infected with adenovirus coding for the green fluorescent protein (GFP), dominant negative (DN) form of Epac (Epac R279K), and adenovirus coding for rabbit muscle cAMP-dependent protein kinase inhibitor (Ad.PKI), cells were subjected to Iso (lμmol/L for lmin). Translocation of PKCs was evaluated by Western blot analysis of PKCε isoform expression in particulate fractions of cell lysates. The localization of translocation of PKCε was studied by confocal microscopy. Iso is known to induce cell hypertrophy. The physiological role of translocated PKCs was shown by the use of a specific PKCs inhibitor peptide and a scramble peptide (negative control) treated with Iso (lOμmol/L for48h, andl μmol/L for lOmin). Cell surface area and protein content were measured, and pERK1/2expression was assessed by Western blot.Results:(1) In cultured rat neonatal cardiomyocytes it was shown that, in response to Iso, PKCε content was increased in particulate fractions of cell lysates and, by confocal microscopic studies, PKCε was translocated to the perinuclear area. The PKCε content in particulate fractions increased as early as1min after the onset of Iso incubation. Compared with control, this increase persisted for15min and decreased at30min.(2) After incubation with8-CPT, an activator of Epac, both particulate PKCε content and PKCε perinulear accumulation increased. Using Epac R279K (dominant negative) to down-regulating or inhibiting Epac, it blocked Iso-induced PKCs activation.(3) After infected with Ad.PKI that specifically inhibiting PKA expression, PKCs content was not decreased in particulate fractions by Iso stimulation, and Iso-induced PKCε translocation to the perinuclear was not inhibited by Ad.PKI.(4) Iso-induced PKCs activation was blocked after incubating with phospholipase C inhibitor U73122(2μmol/L for30min). After pretreatment with U73122, there was no significant increase in particulate PKCs content and PKCs perinuclear accumulation by Iso stimulation.(5) The functional role of Iso-induced PKCε activation was evidenced by the effect of Iso on ERK phosphorylation and cardiomyocyte hypertrophy. Activation of PKCε by Iso was associated with an increased pERKl/2expression. Iso-induced ERK phosphorylation was blocked by the specific PKCs inhibitor peptide. Compared with control, both cell surface area and protein content increased after incubating with Iso for48h,(1319.79±460.00)μm2vs.(1874.36±479.52)μm2(P<0.05) and0.64±0.06vs.0.92±0.11(P<0.01). However, after transfecting PKCε inhibitor peptide, Iso-induced increases of cell surface area and protein content were blocked. The cell surface area and protein content were control (1268.78±501.63) μm2,0.73±0.12vs. Iso group (1604.85±489.88) μm2,0.62±0.07(P>0.05), respectively.Conclusion:These data demonstrate a new connection between the ρ-adrenergic and PKCs pathways in cardiomyocytes. ρ-adrenergic receptors are able to activate PKCε dependent of Epac and phospholipase C, but independent of PKA. Isoproterenol activate PKCs induces ERK phosphorylation and cardiomyocyte hypertrophy.