期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：46
页码：16598-16603
DOI：10.1073/pnas.1419129111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe heart contracts more forcefully in response to fear, stress, or exercise through the fight-or-flight response. This physiological process is mediated by {beta}-adrenergic receptors acting through adenylyl cyclase, cAMP, and cAMP-dependent protein kinase (PKA), which phosphorylates the cardiac calcium channel CaV1.2 and increases its activity. We show that mutation of a single amino acid residue, Ser-1700, in a PKA phosphorylation site at the interface between the distal and proximal C-terminal domains substantially disrupts this regulatory mechanism in mice. Basal and {beta}-adrenergic-stimulated calcium currents, myocyte contractility, and stress-induced exercise capacity are all reduced. Moreover, these mice develop cardiac hypertrophy, an indication of failure of cardiac homeostasis in vivo. Evidently, phosphorylation of Ser-1700 is a primary event in cardiovascular regulation. L-type calcium (Ca2+) currents conducted by voltage-gated Ca2+ channel CaV1.2 initiate excitation-contraction coupling in cardiomyocytes. Upon activation of {beta}-adrenergic receptors, phosphorylation of CaV1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca2+ entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700-Ala in the CaV1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type CaV1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3-4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca2+ current and regulation by {beta}-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.
