Cardiac CaV1 Research Articles

Temporal expression of calcium channel subunits in satellite cells and bone marrow mesenchymal cells.

Bone marrow-derived mesenchymal stem cells (MSC) can be differentiated into myocytes, as well as adipocytes, chondrocytes, and osteocytes in culture. Calcium channels mediate excitation-contraction coupling and are essential for the function of muscle. However, little is known about the expression of calcium channel subunits and calcium handling in stem cells. We examined whether the expression of calcium channel subunits in MSC is similar to that of skeletal muscle satellite cells and if their levels of expression are modified after treatment with bone morphogenetic protein-4 (BMP4). We found that during myogenic differentiation, MSC first express the α2δ1 subunit and the cardiac channel subunit Cav1.2. In contrast to the α2δ1 subunit levels, the Cav1.2 subunit decreases rapidly with time. The skeletal channel subunit Cav1.1 is detected at day 3 but its expression increases considerably, resembling more closely the expression of the subunits in satellite cells. Treatment of MSC with BMP4 caused a significant increase in expression of Cav1.2, a delay in expression of Cav1.1, and a reduction in the duration of calcium transients when extracellular calcium was removed. Calcium currents and transients followed a pattern related to the expression of the cardiac (Cav1.2) or skeletal (Cav1.1) α1subunits. These results indicate that differentiation of untreated MSC resembles differentiation of skeletal muscle and that BMP4 reduces skeletal muscle calcium channel expression and promotes the expression of cardiac calcium channels during myogenic differentiation.

L-Type CaV1.2 Calcium Channels: From In Vitro Findings to In Vivo Function

The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca(2+) itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that β-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaVβ2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca(2+)-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2α1 gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results.

Multiple Regions Inhibit Expression of Cav1.1 Ca2+ Channels in Non-Muscle Cells

Compared to the cardiac CaV1.2, the skeletal CaV1.1 Ca2+ channel has been very difficult to express in non-muscle cells. To identify domains of CaV1.1 that inhibit such expression, we have co-transfected tsA201 cells with β1a and chimeric constructs of CaV1.1 and CaV1.2 tagged with YFP. A tandem construct of CaV1.1 linked to CaV1.2 expressed robustly in tsA201 cells, suggesting that either CaV1.2 masks a sequence of CaV1.1 which causes ER retention in non-muscle cells, or that CaV1.1 lacks a signal important for ER export. Replacement of individual transmembrane repeats of CaV1.1 (in non-tandem constructs) with the corresponding repeat of CaV1.2 was ineffective at increasing functional expression as indicated by slowly activating, L-type Ca2+ current and/or membrane-bound charge movement. However, simultaneous replacement of all four CaV1.1 repeats with those of CaV1.2, while retaining CaV1.1 sequence for the cytoplasmic domains (construct “CSk9”), resulted in increased expression, although not to the level of CaV1.2. By contrast, expression levels of CSk9 and CaV1.2 were equivalent in dysgenic myotubes. Thus, both the transmembrane and cytoplasmic domains appear to play a role in suppressing expression of CaV1.1 in non-muscle cells. To investigate which cytoplasmic domains might be particularly important at suppressing expression, we tested CaV1.2 constructs in which the I-II loop, II-III loop, I-II and II-III loops, or C-terminus were replaced with CaV1.1 sequence and found expression not to differ for any of these constructs from that of the entirely CaV1.2 construct. We are currently testing constructs of CaV1.2 with other combinations of cytoplasmic domains replaced by CaV1.1.

Leucine-Rich Repeat Containing 10 (Lrrc10) Protein is a Novel Regulator of Cardiac Cav1.2 L-Type Calcium Channels

Leucine-rich repeat containing 10 (LRRC10) is a cardiac-specific protein that plays a critical role in cardiac function. We have demonstrated that the Lrrc10-null (Lrrc10-/-) mice develop dilated cardiomyopathy. Our recent data indicate that Lrrc10-/- cardiomyocytes exhibit reduced L-type Ca2+ channel (LTCC) current (ICa,L). However, it is unclear how LRRC10 regulates ICa,L in the heart. To investigate the role of LRRC10 in the regulation of LTCCs, we co-expressed the Myc tagged LRRC10 (LRRC10-Myc), heamagglutinin tagged Cav1.2 (Cav1.2-HA) and the auxiliary Cavβ2C subunit in HEK293 cells and performed co-immunoprecipitation (co-IP) on lysates using either anti-HA, anti-Myc antibody or control IgG. Western blot analysis demonstrated that Cav1.2 and LRRC10 associated with one another without the co-expression of Cavβ2C subunit. Also, the Cavβ2C and LRRC10 did not co-IP with one another suggesting that the LRRC10 may directly interact with Cav1.2 subunit. We then tested if a single point mutation H150A or triple point mutations Y104A, W127A and H150A would alter putative functional interaction sites in the LRRC10 and investigated if these mutations disrupt LRRC10 association with Cav1.2. Both LRRC10 mutants did not associate with Cav1.2. Additionally, co-IP analysis using mouse ventricular homogenates demonstrated that LRRC10 and Cav1.2 subunit are associated with one another. Finally, whole-cell patch clamp experiments performed in ventricular myocytes from Lrrc10-/- mice demonstrated a significant reduction in the ICa,L density (−2.5 0.2 pA/pF) and delayed inactivation, compared to WT myocytes (−6 0.6 pA/pF). In summary, we demonstrate that the LRRC10 and Cav1.2 subunit of LTCC may directly interact with one another and that mutations in LRRC10 residues, likely important for protein-protein interactions, disrupts this association. We conclude that LRRC10 is a novel and essential regulator of the LTCC function in ventricular myocytes.

Coxsackievirus B3 modulates cardiac ion channels

Infections with coxsackieviruses of type B (CVBs), which are known to induce severe forms of acute and chronic myocarditis, are often accompanied by ventricular arrhythmias and sudden cardiac death. The mechanisms underlying the development of virus-induced, life-threatening arrhythmias, which are phenotypically similar to those observed in patients having functionally impaired cardiac ion channels, remain, however, enigmatic. In the present study, we show, for the first time, modulating time-dependent effects of CVB3 on the cardiac ion channels KCNQ1, hERG1, and Cav1.2 in heterologous expression. Channel protein abundance in cellular plasma membrane and patterns of their subcellular distribution were altered in infected murine hearts. The antiviral compound AG7088 did not prevent these effects on channels. In silico analyses of infected human myocytes suggest pronounced alterations of electrical and calcium signaling and increased risk of arrhythmogenesis. These modifications are attenuated by the common Asian polymorphism KCNQ1 P448R, a genetic determinant preventing coxsackievirus-induced effects in vitro. This study provides a previously unknown explanation for the development of arrhythmias in enteroviral myocarditis, which will help to develop therapeutic strategies for arrhythmia treatment.

Truncation of murine CaV1.2 at Asp 1904 increases CaV1.3 expression in embryonic atrial cardiomyocytes

Cardiac CaV1.2 channels play a critical role in cardiac function. It has been proposed that the carboxyl-terminal intracellular tail of the CaV1.2 channel is the target of Ca(2+)-dependent and Ca(2+)-independent regulation of the channel. Recent studies on C-terminal truncated forms of the CaV1.2 channel reported neonatal death, reduced CaV1.2 current, and failure of β-adrenergic stimulation of these channels in ventricular cardiomyocytes (CMs). Here, we used atrial CMs at embryonic day 18.5 that expressed a C-terminal truncated form of the CaV1.2 channel (Stop/Stop). Surprisingly, the atrial CMs showed robust L-type Ca(2+) currents which could be stimulated by forskolin, an activator of adenylyl cyclase. These currents exhibited a left-ward shift in the voltage-dependent activation curve and a reduced sensitivity to the Ca(2+) channel blocker isradipine as compared to currents in wild-type atrial CMs. RT-PCR analysis revealed normal levels of mRNA for the CaV1.2 channel but a twofold increase in the level of mRNA for the CaV1.3 channel in the Stop/Stop atrium as compared to wild-type atrium. A Western blot analysis indicated an increase of CaV1.3 protein in the Stop/Stop atrium. We suggest that, in contrast to Stop/Stop ventricular CMs, Stop/Stop atrial CMs can compensate the functional loss of the truncated CaV1.2 channel with an upregulation of the CaV1.3 channel.

Increased intracellular magnesium attenuates b-adrenergic stimulation of the cardiac CaV1.2 channel

Increased intracellular magnesium attenuates b-adrenergic stimulation of the cardiac Ca_V1.2 channel

Increased intracellular magnesium attenuates β-adrenergic stimulation of the cardiac CaV1.2 channel

Increases in intracellular Mg2+ (Mg2+i), as observed in transient cardiac ischemia, decrease L-type Ca2+ current of mammalian ventricular myocytes (VMs). However, cardiac ischemia is associated with an increase in sympathetic tone, which could stimulate L-type Ca2+ current. Therefore, the effect of Mg2+i on L-type Ca2+ current in the context of increased sympathetic tone was unclear. We tested the impact of increased Mg2+i on the β-adrenergic stimulation of L-type Ca2+ current. Exposure of acutely dissociated adult VMs to higher Mg2+i concentrations decreased isoproterenol stimulation of the L-type Ca2+ current from 75 ± 13% with 0.8 mM Mg2+i to 20 ± 8% with 2.4 mM Mg2+i. We activated this signaling cascade at different steps to determine the site or sites of Mg2+i action. Exposure of VMs to increased Mg2+i attenuated the stimulation of L-type Ca2+ current induced by activation of adenylyl cyclase with forskolin, inhibition of cyclic nucleotide phosphodiesterases with isobutylmethylxanthine, and inhibition of phosphoprotein phosphatases I and IIA with calyculin A. These experiments ruled out significant effects of Mg2+i on these upstream steps in the signaling cascade and suggested that Mg2+i acts directly on CaV1.2 channels. One possible site of action is the EF-hand in the proximal C-terminal domain, just downstream in the signaling cascade from the site of regulation of CaV1.2 channels by protein phosphorylation on the C terminus. Consistent with this hypothesis, Mg2+i had no effect on enhancement of CaV1.2 channel activity by the dihydropyridine agonist (S)-BayK8644, which activates CaV1.2 channels by binding to a site formed by the transmembrane domains of the channel. Collectively, our results suggest that, in transient ischemia, increased Mg2+i reduces stimulation of L-type Ca2+ current by the β-adrenergic receptor by directly acting on CaV1.2 channels in a cell-autonomous manner, effectively decreasing the metabolic stress imposed on VMs until blood flow can be reestablished.

Protein phosphatase 2A effectively modulates basal L-type Ca2+ current by dephosphorylating Cav1.2 at serine 1866 in mouse cardiac myocytes

Calcium (Ca2+) influx through Cav1.2 L-type Ca2+ channels is an important event for cardiac excitation–contraction (E–C) coupling. The functional regulation of Cav1.2 is controlled by multiple kinases and phosphatases. It has been well documented that phosphorylation of Cav1.2 by PKA or other kinases is sufficient for the upregulation of channel activity. However, little is known about the role of protein phosphatases in counterbalancing the phosphorylation of Cav1.2, especially the degree to which protein phosphatase 2A (PP2A)-mediated dephosphorylation is involved in the regulation of Cav1.2 in the mouse heart. Here, we report a physical interaction between PP2A and the C-terminus of Cav1.2 in mouse heart extracts as revealed by coimmunoprecipitation. This interaction was further confirmed by the observation that PP2A and Cav1.2 are colocalized in isolated mouse cardiomyocytes. Specifically, PP2A was bound at serine 1866 in the C-terminus of Cav1.2, and PP2A-induced Cav1.2 dephosphorylation at serine 1866 was observed in mouse cardiomyocytes. Importantly, the density of L-type calcium current increased in line with the increase in the phosphorylation at serine 1866 of Cav1.2 in cardiac-specific PP2A Cα knockout mice. These phenomena were reproduced by treatment with okadaic acid, a PP2A inhibitor, in H9c2 cells. In summary, our data reveal the functional role of PP2A in cardiac Cav1.2 regulation.

The Brugada syndrome mutation A39V does not affect surface expression of neuronal rat Cav1.2 channels

BackgroundA loss of function of the L-type calcium channel, Cav1.2, results in a cardiac specific disease known as Brugada syndrome. Although many Brugada syndrome channelopathies reduce channel function, one point mutation in the N-terminus of Cav1.2 (A39V) has been shown to elicit disease a phenotype because of a loss of surface trafficking of the channel. This lack of cell membrane expression could not be rescued by the trafficking chaperone Cavβ.FindingsWe report that despite the striking loss of trafficking described previously in the cardiac Cav1.2 channel, the A39V mutation while in the background of the brain isoform traffics and functions normally. We detected no differences in biophysical properties between wild type Cav1.2 and A39V-Cav1.2 in the presence of either a cardiac (Cavβ2b), or a neuronal beta subunit (Cavβ1b). In addition, the A39V-Cav1.2 mutant showed a normal Cavβ2b mediated increase in surface expression in tsA-201 cells.ConclusionsThe Brugada syndrome mutation A39V when introduced into rat brain Cav1.2 does not trigger the loss-of-trafficking phenotype seen in a previous study on the human heart isoform of the channel.

Arrhythmic Intracellular Ca2+ Signaling and Electrocardiogram in the Heart of the TRIC-A-/- Mice

Trimeric intracellular cation (TRIC) channel subtypes are present in the endo/sarcoplasmic reticulum (SR) of muscle, heart and other tissues, which contribute to the movement of counter ions associated with the rapid release of Ca2+ from intracellular stores (Yazawa et al., Nature 448: 78–82). We recently showed that skeletal muscle derived from the TRIC-A knockout mice develop overload of Ca2+ inside the SR that leads to instability of ryanodine receptor (RyR1)-mediated Ca2+ release and muscle contraction (Zhao et al., JBC, 285: 37370-6). Here we examine the function of TRIC-A in regulation of Ca2+ signaling in the cardiac muscle. Cardiomyocytes isolated from the TRIC-A knockout mice show reduced Ca2+ spark frequency under resting condition. Ca2+ transients in response to voltage stimulation display arrhythmic patterns compared with the wild type controls. While electrocardiogram (ECG) of TRIC-A knockout mice reveal minor arrhythmic patterns under resting condition, infusion of isoproterenol (80 mg/kg) leads to drastic and acute elevation of arrhythmic ECG patterns that are rarely seen in the wild type mice. Chronic treatments with isoproterenol (60 mg/kg/day for 14 days) induce development of cardiac hypertrophy and fibrosis that are significantly more evident in the TRIC-A knockout mice compared with the wild type controls. Biochemical studies show that the major Ca2+ regulatory proteins in the cardiac muscle, e.g. RyR2, NCX, SERCA, Cav1.2 and JP2 are not changed. Our data suggest an important role for TRIC-A in regulation of Ca2+ signaling in heart function. Since the absence of TRIC-A caused overload of Ca2+ inside the SR, the stress-induced arrhythmic Ca2+ signaling and ECG of the TRIC-A knockout heart could reflect the store-overload induced Ca2+ release mechanism, which has been demonstrated for the RyR2 Ca2+ release channel in the cardiac muscle.

Transcriptional Up-Regulation by Aldosterone of the Cardiac Cav1.2 Encoding Gene CACNA1C

Although the effect of aldosterone beyond the epithelia is now well recognized, specific action through the mineralocorticoid receptor (MR) in heart is still matter of debate. In the past decade, we have accumulated evidence, both ex vivo and in vivo, that modulation of Ca2+ signaling, especially Ca2+ influx via L-type Ca2+ channel (Cav1.2), is a central factor in the cardiac action of aldosterone. Even if our results established that a specific transcriptional upregulation is involved in the effect of aldosterone on Cav1.2, it cannot be concluded whether this genomic effect is direct or indirect. The effect of the mineralocorticoid, aldosterone, on the regulation of the cardiac Cav1.2 expression was investigated in primary cultures of neonatal rat ventricular myocytes (nRVM). By use of quantitative real time RT-PCR, we found that aldosterone increased the Cav1.2 mRNA amount in time- and dose-dependent manner. This up-regulation appeared as early as 1hour of incubation even with 1 nM aldosterone. Inspection of the upstream promoter region from rat Cav1.2 encoding gene, revealed one putative hormone response element, GRE. The activity of the promoter-luciferase reporter gene constructs in transfected nRVMs showed similar time- and dose-dependent stimulation with aldosterone. We will examine the intracellular trafficking of MR in nRVMs using a fusion protein of green fluorescent protein and human MR. Selectivity of these effects will be tested as well as electrophoretic mobility shift assays to identify the region of the cacna1c promoter that can bind MRs. The data will provide new insights in the molecular mechanisms underlying the regulation of Cav1.2 channel expression by mineralocorticoids.

Restoration of Defective L-type Ca2+ Current in Cardiac Myocytes of Type 2 Diabetic db/db Mice by Akt and PKC-ι

Diabetes is associated with an increased risk of heart failure and the development of a cardiomyopathy whose etiology is only partially understood. Ca entry through the voltage-dependent L-type Ca channel CaV1.2 initiates the contractile cycle in cardiac myocytes. Decreased cardiac contractility and depressed CaV1.2 function have been reported in obese type 2 diabetic db/db mice. Here, we demonstrate that a reduction in phosphoinositide 3-kinase (PI3K) signaling is a major contributor to the altered function of CaV1.2 in db/db cardiac myocytes. Using the whole-cell patch clamp technique, we determined that intracellular infusion of cardiac myocytes from db/db mice with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K, increased the L-type Ca current (ICa,L) density nearly to the level seen in wild-type cells. PIP3 also reversed the positive shift in the voltage dependence of the steady-state current activation observed in db/db myocytes. Infusion of protein kinases that act downstream of PI3K also affected ICa,L. Akt1 and Akt2 were as effective as PIP3 in restoring the ICa,L density in db/db myocytes but did not affect the voltage dependence of current activation. The infusion of atypical PKC-ι (the human homolog of mouse PKC-λ) caused a small but significant increase in the ICa,L density and completely reversed the shift in voltage dependence of steady-state current activation. These results indicate that a defect in PI3K/PIP3/Akt/PKC-λ signaling is mainly responsible for the depressed CaV1.2 function in the hearts of db/db mice with type 2 diabetes.

Caveolin-1 modulates TGF-β1 signaling in cardiac remodeling

The cardiac response to myocardial injury includes fibrotic and hypertrophic processes and a key mediator in this response is transforming growth factor-β1 (TGF-β1). Caveolin-1 (cav1), the main structural protein of caveolae, is an inhibitor of the TGF-β1 signaling pathway. To examine the role of cav1 in cardiac repair, cav1 deficient (Cav1−/−) and wild type (WT) mice were subjected to cryoinjury of the left ventricle (LV). At baseline the two groups exhibited no inflammation, similar collagen content, and similar cardiac function. After injury, Cav1−/− animals displayed enhanced TGF-β1 signaling, as reflected by a 3-fold increase in the activation of the Smad2-dependent pathway and more widespread collagen deposition in the heart. Qualitative and quantitative analyses indicated that collagen deposition peaked in the WT LV 14days after injury, accompanied by increased mRNA abundance for procol1a2 (2-fold) and procol3a1 (3-fold). Collagen deposition was further enhanced in Cav1−/− mice, which was accompanied by reduced expression of matrix metalloproteinases MMP-8 (3-fold) and -13 mRNA (2-fold). The levels of expression of inflammatory markers of acute phase were similar between the strains However, macrophage clearance in the damaged region was delayed in Cav1−/− mice. We observed a 4-fold decrease in collagen deposition in Cav1−/− mice injected with a cav1 scaffolding domain peptide (CSD) and a 2-fold decrease in WT mice treated with the CSD. We conclude that cav1 has a direct role in reducing TGF-β1 signaling and as such might be an appropriate target for therapies to influence cardiac remodeling.

Functional Roles of a C-terminal Signaling Complex of CaV1 Channels and A-kinase Anchoring Protein 15 in Brain Neurons

Regulation of CaV1.2 channels in cardiac myocytes by the β-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the β-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for CaV1.2 and CaV1.3 channels in neurons. AKAP15 co-immunoprecipitates with CaV1.2 and CaV1.3 channels. AKAP15 has overlapping localization with CaV1.2 and CaV1.3 channels in cell bodies and proximal dendrites and is closely co-localized with CaV1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with CaV1.2 and CaV1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of CaV1.2 channels (CaV1.2ΔDCT) reveal that AKAP15 interacts with neuronal CaV1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of CaV1.2 channels via that interaction. CaV1.2ΔDCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from CaV1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of CaV1.2 and CaV1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal CaV1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.

Targeting of Protein Phosphatases PP2A and PP2B to the C Terminus of L-Type Calcium Channel Ca V1.2

The L-type Ca2+ channel CaV1.2 lies within a macromolecular complex including β2- adrenergic receptor, trimeric GS protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA). This complex mediates highly spatially and temporally regulated signaling, both acute (cardiac excitation contraction coupling, synaptic transmission, neurosecretion) and long-term (gene expression). Protein phosphatases PP2A (PPP2CA) and PP2B (PPP3CA; calcineurin) are also constitutive complex members. Acutely, either phosphatase counteracts PKA-dependent increase in Ca current by PKA. We here report that PP2A binds independently to two separate specific regions in the C-terminus of the central pore-forming α1 subunit of CaV1.2: one region spans residues 1795-1818 and the other residues 1965-1971. PP2B binds independently to a distinct region immediately downstream of residue 1971. To determine the binding regions for these phosphatases we assayed their ability to bind α1 C-terminal fragments with appropriate subregions cleaved. Further, we assayed a pallette of small peptides and identified those able to compete with PP2A and PP2B binding in pull-down assays. With these interfering peptides, we investigated PP2A and PP2B function, assayed by recording ICa(L) in acutely isolated rabbit cardiomyocytes. Using whole-cell β-escin perforated patch, which allowed pipette introduction of peptides while limiting rundown, we found that the PP2B competing peptide increased both basal and isoproterenol-stimulated ICa(L), while a PP2A competing peptide had no effect vs a control peptide or no peptide. Together, the biochemical and electrophysiological results suggest that while PP2A and PP2B are anchored at their respective binding sites on cardiac CaV1.2 α1 and negatively regulate ICa(L) (in this case by counterbalancing PKA-mediated phosphorylation), a fine dynamic balance between phosphorylation and dephosphorylation responses exists. This would be important to the ability of ICa(L) flux to efficiently regulate end effects.

β-Adrenergic–regulated phosphorylation of the skeletal muscle Ca V 1.1 channel in the fight-or-flight response

Ca(V)1 channels initiate excitation-contraction coupling in skeletal and cardiac muscle. During the fight-or-flight response, epinephrine released by the adrenal medulla and norepinephrine released from sympathetic nerves increase muscle contractility by activation of the β-adrenergic receptor/cAMP-dependent protein kinase pathway and up-regulation of Ca(V)1 channels in skeletal and cardiac muscle. Although the physiological mechanism of this pathway is well defined, the molecular mechanism and the sites of protein phosphorylation required for Ca(V)1 channel regulation are unknown. To identify the regulatory sites of phosphorylation under physiologically relevant conditions, Ca(V)1.1 channels were purified from skeletal muscle and sites of phosphorylation on the α1 subunit were identified by mass spectrometry. Two phosphorylation sites were identified in the proximal C-terminal domain, serine 1575 (S1575) and threonine 1579 (T1579), which are conserved in cardiac Ca(V)1.2 channels (S1700 and T1704, respectively). In vitro phosphorylation revealed that Ca(V)1.1-S1575 is a substrate for both cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II, whereas Ca(V)1.1-T1579 is a substrate for casein kinase 2. Treatment of rabbits with isoproterenol to activate β-adrenergic receptors increased phosphorylation of S1575 in skeletal muscle Ca(V)1.1 channels in vivo, and treatment with propranolol to inhibit β-adrenergic receptors reduced phosphorylation. As S1575 and T1579 in Ca(V)1.1 channels and their homologs in Ca(V)1.2 channels are located at a key regulatory interface between the distal and proximal C-terminal domains, it is likely that phosphorylation of these sites in skeletal and cardiac muscle is directly involved in calcium channel regulation in response to the sympathetic nervous system in the fight-or-flight response.

Facilitation of murine cardiac L-type Ca v 1.2 channel is modulated by Calmodulin kinase II–dependent phosphorylation of S1512 and S1570

Activity-dependent means of altering calcium (Ca(2)(+)) influx are assumed to be of great physiological consequence, although definitive tests of this assumption have only begun to emerge. Facilitation and inactivation offer two opposing, activity-dependent means of altering Ca(2+) influx via cardiac Ca(v)1.2 calcium channels. Voltage- and frequency-dependent facilitation of Ca(v)1.2 has been reported to depend on Calmodulin (CaM) and/or the activity of Calmodulin kinase II (CaMKII). Several sites within the cardiac L-type calcium channel complex have been proposed as the targets of CaMKII. Here, we generated mice with knockin mutations of alpha(1)1.2 S1512 and S1570 phosphorylation sites [sine facilitation (SF) mice]. Homocygote SF mice were viable and reproduced in a Mendelian ratio. Voltage-dependent facilitation in ventricular cardiomyocytes carrying the SF mutation was decreased from 1.58- to 1.18-fold. The CaMKII inhibitor KN-93 reduced facilitation to 1.28 in control cardiomyocytes. SF mutation negatively shifted the voltage-dependent inactivation and slowed recovery from inactivation, thereby making fewer channels available for activation. Telemetric ECG recordings at different heart rates showed that QT time decreased significantly more in SF than in control mice at higher rates. Our results strongly support the notion that CaMKII-dependent phosphorylation of Cav1.2 at S1512 and S1570 mediates Ca(2+) current facilitation in the murine heart.

Apocynin inhibits and its removal augments L‐type Ca2+ currents in coronary artery smooth muscle cells and ventricular myocytes

Exogenously applied H2O2 augments L‐type Ca2+ current (ICa,L). NADPH oxidase (NOX) initiates sequential reactions to generate reactive oxygen species (ROS) including H2O2. We studied the regulatory role of NOX‐generated ROS on ICa,L by applying apocynin (APO), a NOX inhibitor, and diphenyleneiodonium (DPI), a flavoprotein inhibitor. Whole cell ICa,L was recorded from bovine coronary arterial smooth muscle (BCASMC), rat ventricular myocytes (RVMC) and HEK 293 cells transiently expressing human cardiac CaV1.2. APO (0.1–10 mM) rapidly and dose‐dependently inhibited ICa,L: 10‐mM APO decreased ICa,L to 27, 34 and 16% in BCASMC, RVMC and 293 cells, respectively, while DPI (10 μM) inhibited more slowly and less intensively (to 64% in RVMC). The inhibitory effects of both inhibitors were mainly additive. Surprisingly, washout of high concentrations of APO rapidly recovered ICa,L to slightly above control in BCASMC and increased to 150% of control with prolonged current decay in RVMC. ROS levels measured simultaneously with ICa,L by increase rate of CM‐H2DCF fluorescence were decreased by APO and markedly increased by its removal in BCAVSM and RVMC. Possible accumulation of NADPH during NOX inhibition by APO explains the increased generation of ROS. We conclude that decrease of ROS inhibits and its increase augments cardiovascular ICa,L and NOX is involved in the regulation of ICa,L. This study is supported by NIH grant #R01HL85352

Apocynin Reversibly Inhibits L-type Ca2+ Channel Current Involvement of Reactive Oxygen Species

L-type Ca2+ channel current (ICa,L) is a major Ca2+ entry mechanism and contraction of arterial smooth muscle (ASM) is regulated by reactive oxygen species (ROS). We utilized apocynin (APO) as a tool to clarify the contribution of ROS in the regulation of ICa,L. APO, a natural organic compound contained in a variety of plants, is widely used as inhibitor of NADPH oxidase (NOX) that reduces oxygen to superoxide in the presence of NADPH to generate ROS including H2O2. We recorded whole cell ICa,L with Ba2+ as charge carrier from isolated bovine coronary ASM (BCASM) and HEK293 cells transiently expressing human cardiac CaV1.2. APO was introduced and washed out during continuous application of depolarization step to 0 mV from HP −80 mV at 1/min. We found that APO inhibited ICa,L in a dose-dependent manner between 0.1 and 10 mM decreasing its amplitude to ∼20% of control. APO also accelerated ICa,L decay during depolarization. Surprisingly, washout of the high concentration of APO caused rapid recovery of ICa,L. It could even produce a rebound increase of ICa,L with its peak at ∼3 min in BCASM. We performed fluorometric analysis of APO-induced change of cellular ROS by loading cells with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Oxidation of CM-H2DCF to CM-DCF by ROS induces an increase of fluorescence. APO markedly inhibited the fluorescence increase, while washout of APO caused more intensive increase of fluorescence than control. The results are explained by the inhibition of NOX by APO which results in decrease of ROS and overproduction of ROS during washout utilizing accumulated NADPH produced by prior NOX inhibition. ROS which changes dynamically in situ, e.g. hypoxia and reoxygenation, seems to be vital to sustain ICa,L.