Altered PKA-dependent phosphorylation of Cav1.2 in left ventricular myocardium from Cacna1c haploinsufficient rats

Introduction

CACNA1C encodes Cav1.2, the major L-type calcium channel in the heart. Previously, it was shown that Cacna1c haploinsufficient rats (Cacna1c+/-) exhibit a cardiac phenotype with remodelling of calcium handling proteins in left ventricular (LV) myocardium (reduced expression of Cav1.2, increased expression of NCX and SERCA2a, and increased phosphorylation of RyR2 at Ser2808) and an impaired response to beta-adrenergic stimulation by isoprenaline (ISO) when compared to wild-type (WT) littermates. Surprisingly, however, despite reduced expression of Cav1.2, L-type calcium current and sarcolemmal calcium influx were unaltered in Cacna1c+/-, whereas the ISO-mediated increase in sarcolemmal calcium influx and contraction was impaired in Cacna1c+/- ventricular myocytes (versus WT myocytes). Here, we tested the hypothesis that altered protein kinase A (PKA)-dependent phosphorylation of Cav1.2 may contribute to the observed impaired regulation of calcium influx in ventricular myocytes from Cacna1c+/-.

Methods and Results

Hearts were isolated from Cacna1c+/- rats (N=24) and WT littermates (N=24). Hearts were either immediately shock-frozen for later analysis of basal phosphorylation (N=8 for each genotype) or mounted on a Langendorff setup for retrograde perfusion with modified Tyrode’s solution with (N=8 for each genotype) or without (N=8 for each genotype) 100 nM ISO for 5 minutes before being shock-frozen for later analysis. A commercially available antibody raised against a PKA-dependent phosphorylation site in Cav1.2 (Ser1981 in human, Ser1927 in rat) was used to quantify phosphorylation in homogenates from LV myocardium by means of standard immunoblot techniques. First, however, we tested the validity of this antibody. To this end, Langendorff hearts from control rats were treated with solutions designed to induce either maximum PKA-dependent phosphorylation (solution containing 100 nM ISO, 0.1 mM IBMX and two protein phosphatase inhibitors) or dephosphorylation (solution containing 1 mg/ml BDM and two protein kinase inhibitors) of proteins (N=4 hearts for each condition). Under dephosphorylation conditions, no signal could be detected, whereas under maximum phosphorylation conditions a strong band was detected at approx. 200 kDa. Using this validated antibody, we observed that basal PKA-dependent phosphorylation of Cav1.2 in LV myocardium from Cacna1c+/- was about doubled compared to WT (2.2±0.3 vs 1.0±0.1; N=8 each; P<0.01). In LV myocardium from Langendorff hearts treated with ISO, PKA-dependent phosphorylation of Cav1.2 was elevated to 7.5±0.7 (normalized to untreated WT control = 1) in WT, but only to 5.1±0.6 in Cacna1c+/- (N=8 each; P<0.01 vs WT). Thus, the ISO-induced increase in PKA-dependent phosphorylation of Cav1.2 was impaired in Cacna1c+/- compared to WT.

Conclusions

LV myocardium from Cacan1c+/- exhibits elevated baseline PKA-dependent phosphorylation of Cav1.2, which may explain unaltered sarcolemmal calcium influx in ventricular myocytes despite reduced expression of Cav1.2. Beta-adrenergic stimulation with ISO reveals impaired PKA-dependent phosphorylation of Cav1.2 in Cacna1c+/-, however, which may contribute to the impaired ISO-mediated increase in sarcolemmal calcium influx and calcium transients observed in ventricular myocytes from these animals.