Basal and β-adrenergic regulation of the cardiac calcium channel Ca _V 1.2 requires phosphorylation of serine 1700

Abstract

L-type calcium (Ca 2+ ) currents conducted by voltage-gated Ca 2+ channel Ca V 1.2 initiate excitation-contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of Ca V 1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca 2+ 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 Ca V 1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type Ca V 1.2 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 fightor-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca 2+ current and regulation by β-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.U pon membrane depolarization, Ca V 1.2 channels conduct L-type calcium (Ca 2+ ) current into cardiomyocytes and initiate excitation-contraction coupling (1, 2). Ca 2+ influx through Cav1.2 channels activates Ca 2+ release from the sarcoplasmic reticulum, which leads to contraction of myofilaments. As the initiator of excitation-contraction coupling, Ca 2+ influx via Ca V 1.2 channels is tightly regulated. Under conditions of fear, stress, and exercise, the sympathetic nervous system activates the fight-or-flight response, in which the marked increase in contractile force of the heart is caused by epinephrine and norepinephrine acting through β-adrenergic receptors, activation of adenylyl cyclase, increased cAMP, activation of cAMP-dependent protein kinase (PKA), and phosphorylation of the Ca V 1.2 channel (1, 3). Phosphorylation of the Ca V 1.2 channel leads to a threefold to fourfold increase in peak current amplitude in mammalian cardiomyocytes. Regulation of the Ca V 1.2 channel by the cAMP signaling pathway is altered in cardiac hypertrophy and heart failure (4-6). Under those pathological conditions, responsiveness of Ca V 1.2 channel activit...

Loss of β-adrenergic–stimulated phosphorylation of Ca V 1.2 channels on Ser1700 leads to heart failure

“…1 D-F) and reduced BW resulting from illness. These results show that loss of Ser1700 phosphorylation on Ca V 1.2 causes spontaneous cardiac hypertrophy and heart failure, without changes in the expression, localization, or function of Ca V 1.2 (25,26) and further indicate that loss of Thr1704 phosphorylation plays a synergistic role. In further studies, we focused primarily on the healthier SA mice, in which only PKA phosphorylation of Ser1700 is prevented.…”

“…The running activity and circadian patterns were similar in SA and age-matched WT mice ( Fig. 3 B and C), suggesting that SA mice have normal voluntary wheel-running activity even though they had reduced maximal exercise capacity on a forced-treadmill test (25). Exercised SA mice had significantly increased HW/BW ratios compared with nonexercised age-matched SA control mice (Fig.…”

“…The protein levels of Ca V 1.2 are similar in these genotypes at 2 mo of age (Fig. 1A) (25,26), although slightly reduced expression of Ca V 1.2 has been observed in older, healthy STAA and SA mice (25,26). Both STAA and SA mice died prematurely as they reached ages of 6-8 mo (Fig.…”

“…We have shown previously that the contraction of isolated SA cardiomyocytes in response to β-adrenergic stimulation is only mildly decreased, suggesting that compensatory mechanisms enhance contractility despite substantially reduced Ca V 1.2 channel activity (25). We used echocardiography to assess cardiac structure and function in vivo in 3-mo-old WT, SA Het, and SA mice.…”

“…In reconstituted nonmuscle cells, Ser1700 and Thr1704 are required for normal basal and PKA-regulated Ca V 1.2 channel activity (14). Either the Ca V 1.2/S1700A (SA) mutation or the Ca V 1.2/S1700A/ T1704A (STAA) mutation (25,26) reduced basal Ca V 1.2 channel activity in cardiomyocytes, decreased β-adrenergic stimulation of channel activity and cellular contractility, and impaired exercise capacity (25,26). Here we report that the SA and STAA mutations lead to dramatic hypertrophy with increasing age and to 100% lethal heart failure, accompanied by compensatory increases in PKA expression, hyperphosphorylation of RyR2 PLB, and troponin-I at PKA sites, increased Ca 2+ load in the SR, and activation of a calcineurin-dependent hypertrophic pathway.…”