Evidence for significance of serine 1487 in β-adrenergic regulation of Cav1.2 channel protein function in genetically engineered mice

Abstract

Stress responses are mediated through activation of the β-adrenergic signaling pathway. Chronic sympathetic stimulation can result in the development of hypertrophy, arrhythmia and sudden cardiac death but the molecular mechanisms are still not understood. We identified a single serine, following phosphorylation by cAMP-dependent protein kinase A, responsible for the increase in function of the human cardiac Cav1.2 channel protein in an artificial cell system. To confirm whether the serine is involved in in vivo channel stimulation by β-adrenergic signaling, we created CRISPR/Cas9 mutant mouse lines with glutamate and alanine-substitution of the serine1487 on Cav1.2 protein to mimic the phosphorylated and the non-phosphorylated protein. The average litter size (6.7 ± 0.7; N = 16 and 6.1 ± 0.4; N = 24 respectively) and survival rate (48.6% vs. 42.5%) were similar in the two lines, but homozygous mutation of Cav1.2 S1487E caused 100% mortality with a median survival of ~1 day. The relative size of the hearts of homozygous S1487A mice were smaller than S1487E (heart weight/tibial length: 7.0 ± 0.1; N = 3 vs 7.8 ± 0.3; N = 3). Administration of intraperitoneal injection of 20 mg/kg isoproterenol increased the heart rate up to 30% in both glutamate and alanine-substituted heterozygous mutants in a similar manner to the negatively genotyped littermates, but did not alter the heart rate of the homozygous S1487A mutants. Chronic isoproterenol treatment (20 mg/kg isoproterenol daily injection for 2 weeks) induced hypertrophy in the wild type and heterozygous S1497A mutant mice, but there were no significant changes in the posterior wall thickness of the heart of homozygous S1487A mutants.