Tachypacing-induced CREB/CD44 signaling contributes to the suppression of L-type calcium channel expression and the development of atrial remodeling.

BACKGROUND: Atrial fibrillation (AF), a common arrhythmia in clinics, is characterized as downregulation of L-type calcium channel (LTCC) and shortening of atrial action potential duration (APD). Our prior studies have shown the association of CD44 with AF genesis. OBJECTIVE: The purpose of this study was to explore the potential role of CD44 and its related signaling in tachypacing-induced downregulation of LTCC. METHODS AND RESULTS: In vitro, tachypacing in atrium-derived myocytes (HL-1 cell line) induced activation (phosphorylation) of cyclic adenosine monophosphate response element-binding protein (CREB). Furthermore, tachypacing promoted an association between CREB and CD44 in HL-1 myocytes, which was documented in atrial tissues from patients with AF. Deletion and mutational analysis of the LTCC promoter along with chromatin immunoprecipitation revealed that cyclic adenosine monophosphate response element is essential for tachypacing-inhibited LTCC transcription. Tachypacing also hindered the binding of p-CREB to the promoter of LTCC. Blockade of CREB/CD44 signaling in HL-1 cells attenuated tachypacing-triggered downregulation of LTCC and shortening of APD. Atrial myocytes isolated from CD44-/- mice exhibited higher LTCC current and longer APD than did those from wild-type mice. Ex vivo, tachypacing caused less activation of CREB in CD44-/- mice than in wild-type mice. In vivo, burst atrial pacing stimulated less inducibility of AF in CREB inhibitor-treated mice than in controls. CONCLUSION: Tachypacing-induced CREB/CD44 signaling contributes to the suppression of LTCC, which provides valuable information about the pathogenesis of atrial modeling and AF.

Antenatal Hypoxia Affects Pulmonary Artery Contractile Functions via Downregulating L-type Ca2+ Channels Subunit Alpha1 C in Adult Male Offspring.

Background Antenatal intrauterine fetal hypoxia is a common pregnancy complication that has profound adverse effects on an individual's vascular health later in life. Pulmonary arteries are sensitive to hypoxia, but adverse effects of antenatal hypoxia on pulmonary vasoreactivities in the offspring remain unknown. This study aimed to determine the effects and related mechanisms of antenatal hypoxia on pulmonary artery functions in adult male offspring. Methods and Results Pregnant Sprague-Dawley rats were housed in a normoxic or hypoxic (10.5% O2) chamber from gestation days 10 to 20. Male offspring were euthanized at 16 weeks old (adult offspring). Pulmonary arteries were collected for vascular function, electrophysiology, target gene expression, and promoter methylation studies. In pulmonary artery rings, contractions to serotonin hydrochloride, angiotensin II, or phenylephrine were reduced in the antenatal hypoxic offspring, which resulted from inactivated L-type Ca2+ channels. In pulmonary artery smooth muscle cells, the basal whole-cell Ca2+ currents, as well as vasoconstrictor-induced Ca2+ transients were significantly reduced in antenatal hypoxic offspring. In addition, increased promoter methylations within L-type Ca2+ channel subunit alpha1 C were compatible with its reduced expressions. Conclusions This study indicated that antenatal hypoxia programmed long-lasting vascular hypocontractility in the male offspring that is linked to decreases of L-type Ca2+ channel subunit alpha1 C in the pulmonary arteries. Antenatal hypoxia resulted in pulmonary artery adverse outcomes in postnatal offspring, was strongly associated with reprogrammed L-type Ca2+ channel subunit alpha1 C expression via a DNA methylation-mediated epigenetic mechanism, advancing understanding toward the effect of antenatal hypoxia in early life on long-term vascular health.

Interleukin-17 upregulation participates in the pathogenesis of heart failure in mice via NF-κB-dependent suppression of SERCA2a and Cav1.2 expression.

Interleukin-17 (IL-17), also called IL-17A, is an important regulator of cardiac diseases, but its role in calcium-related cardiac dysfunction remains to be explored. Thus, we investigated the influence of IL-17 on calcium handling process and its contribution to the development of heart failure. Mice were subjected to transaortic constriction (TAC) to induce heart failure. In these mice, the levels of IL-17 in the plasma and cardiac tissue were significantly increased compared with the sham group. In 77 heart failure patients, the plasma level of IL-17 was significantly higher than 49 non-failing subjects, and was negatively correlated with cardiac ejection fraction and fractional shortening. In IL-17 knockout mice, the shortening of isolated ventricular myocytes was increased compared with that in wild-type mice, which was accompanied by significantly increased amplitude of calcium transient and the upregulation of SERCA2a and Cav1.2. In cultured neonatal cardiac myocytes, treatment of with IL-17 (0.1, 1 ng/mL) concentration-dependently suppressed the amplitude of calcium transient and reduced the expression of SERCA2a and Cav1.2. Furthermore, IL-17 treatment increased the expression of the NF-κB subunits p50 and p65, whereas knockdown of p50 reversed the inhibitory effects of IL-17 on SERCA2a and Cav1.2 expression. In mice with TAC-induced mouse heart, IL-17 knockout restored the expression of SERCA2a and Cav1.2, increased the amplitude of calcium transient and cell shortening, and in turn improved cardiac function. In addition, IL-17 knockout attenuated cardiac hypertrophy with inhibition of calcium-related signaling pathway. In conclusion, upregulation of IL-17 impairs cardiac function through NF-κB-mediated disturbance of calcium handling and cardiac remodeling. Inhibition of IL-17 represents a potential therapeutic strategy for the treatment of heart failure.

A New Zebrafish Model for CACNA2D4-Dysfunction.

Purpose: Mutations in CACNA2D4, encoding the α2δ4 subunit of retinal voltage-gated calcium channels (Cav), cause a rare type of retinal dysfunction in human, mainly affecting cone vision. Here, we investigate the role of CACNA2D4 in targeting of Cav, its influence on cone-mediated signal transmission, and the cellular and subcellular changes upon loss of α2δ4 by exploiting the advantages of the cone-dominant zebrafish as model system. Methods: We identified two zebrafish CACNA2D4 paralogs (cacna2d4a and cacna2d4b), analyzed their expression by RNA in situ hybridization and introduced truncating frameshift mutations through CRISPR/Cas9-mediated mutagenesis. We analyzed retinal function and morphology of the single and double mutant lines by electroretinography, immunohistochemistry, light- and electron microscopy. Results: Knockout of cacna2d4b reduces the expression of Cacna1fa, the pore-forming subunit of retinal Cav1.4, whereas loss of cacna2d4a did not. Only knockout of both paralogs impaired cone-mediated ERG b-wave amplitude. The number of "floating" ribbons is increased in double-KO, while retinal morphology and expression of postsynaptic mGluR6b remain largely unaffected. Both Cacna1fa and Ribeyeb show ectopic punctate expression in cacna2d4b-KO and double-KO photoreceptors. Conclusions: We find that increasing the expression of Cav at the synaptic membrane is an evolutionarily conserved function of Cacna2d4b. Yet, since both paralogs participate in cone synaptic transmission, we propose partial subfunctionalization in zebrafish. Similar to human patients, our double KO zebrafish model shows mild cone dysfunction, which was not associated with signs of retinal degeneration. Therefore, cacna2d4-KO zebrafish is a suitable model to study the pathophysiological mechanisms underlying CACNA2D4 dysfunction in human.

Adenylyl cyclase 5-generated cAMP controls cerebral vascular reactivity during diabetic hyperglycemia.

Elevated blood glucose (hyperglycemia) is a hallmark metabolic abnormality in diabetes. Hyperglycemia is associated with protein kinase A (PKA)-mediated stimulation of L-type Ca2+ channels in arterial myocytes resulting in increased vasoconstriction. However, the mechanisms by which glucose activates PKA remain unclear. Here, we showed that elevating extracellular glucose stimulates cAMP production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity. Super-resolution imaging suggested nanometer proximity between subpopulations of AC5 and the L-type Ca2+ channel pore-forming subunit CaV1.2. In vitro, in silico, ex vivo and in vivo experiments revealed that this close association is critical for stimulation of L-type Ca2+ channels in arterial myocytes and increased myogenic tone upon acute hyperglycemia. This pathway supported the increase in L-type Ca2+ channel activity and myogenic tone in two animal models of diabetes. Our collective findings demonstrate a unique role for AC5 in PKA-dependent modulation of L-type Ca2+ channel activity and vascular reactivity during acute hyperglycemia and diabetes.

LRP5 controls cardiac QT interval by modulating the metabolic homeostasis of L-type calcium channel.

BACKGROUND: Low-density lipoprotein receptor-related protein 5 (LRP5) has been intensively studied as a co-receptor for ß-catenin-dependent Wnt signaling. Emerging evidences have demonstrated ß-catenin-independent functions of LRP5. However, the biological role of LRP5 in the mammalian heart is largely unknown. METHODS AND RESULTS: Conditional cardiac-specific Lrp5 knockout (Lrp5-CKO) mice were generated by crossing Lrp5flox/flox mice with αMHC/MerCreMer mice. Lrp5-CKO mice consistently displayed normal cardiac structure and function. Telemetric electrocardiogram recordings revealed a short QT interval in Lrp5-CKO mice, which was tightly linked to the striking abbreviation of action potential duration (APD) in ventricular myocytes. The analysis of whole-cell currents indicated that a reduction in activity and protein expression of L-type calcium channel (LTCC), rather than other ion channels, contributed to the abnormality in APD. Furthermore, we showed that Lrp5 ablation induced a significant convergence of CaV1.2α1c proteins to the endoplasmic reticulum. Consequently, increased proteasomal degradation of these proteins was observed, which was independent of the Wnt/ß-catenin signaling pathway. CONCLUSIONS: LRP5 directly modulates the degradation of LTCC to control cardiac QT interval. These findings provide compelling evidence for the potential role of LRPs in cardiac electrophysiology.

Stac Proteins Suppress Ca2+-Dependent Inactivation of Neuronal l-type Ca2+ Channels.

Stac protein (named for its SH3- and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca2+ channels. Overexpression of the three Stac isoforms eliminated Ca2+-dependent inactivation (CDI) of l-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-l-type current. Using heterologous expression in tsA201 cells (together with ß and α2-δ1 auxiliary subunits), we found that CDI for CaV1.2 and CaV1.3 (the predominant, neuronal l-type Ca2+ channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, CaV2.1, was not. For CaV1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel's C terminus. Within the Stac proteins, a weakly conserved segment containing ∼100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI for l-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca2+ entry via neuronal l-type channels.SIGNIFICANCE STATEMENT Stac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal muscle l-type Ca2+ channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here, we report that one likely function of neuronal Stac proteins is tuning Ca2+ entry via l-type, but not that via non-l-type, Ca2+ channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation of l-type channel Ca2+ signaling in these brain regions.

Participation of voltage-gated sodium and calcium channels in the acute cardiac effects of toluene.

Inhaling solvents can lead to occurrence of cardiac arrhythmias and sudden sniffing death. Mechanisms related to this phenomenon are not fully understood. The purpose of this study was to investigate the effect of acute toluene exposure on heart reactivity to epinephrine and the participation of voltage-gated sodium and calcium channels. We found that acute toluene exposure increased perfusion pressure, left ventricular developed pressure, and heart rate. These actions were inhibited by lidocaine and nifedipine. Our results suggest that acute toluene exposure modify voltage-gated sodium and calcium channel function and expression likely due to a cardiac adrenergic mechanism and these effects could be participating, at least in part, in the presence of cardiac arrhythmias. To our best knowledge, this is the first report to establish a direct participation of voltage-gated Na+ and Ca2+ channels, toluene and epinephrine on cardiac function in rats.

Relaxin-2 therapy reverses radiation-induced fibrosis and restores bladder function in mice.

AIM: To determine the efficacy of human relaxin-2 (hRLX2) in reversing radiation-induced bladder fibrosis and lower urinary tract dysfunction (LUTD). Radiation cystitis is a consequence of radiotherapy for pelvic malignancies. Acutely, irradiation leads to reactive oxygen/nitrogen species in urothelial cells, apoptosis, barrier disruption, and inflammation. Chronically, this results in collagen deposition, bladder fibrosis, and attenuated storage and voiding functions. In severe cases, cystectomies are performed as current therapies do not reverse fibrosis. METHODS: We developed a mouse model for selective bladder irradiation (10 Gray; 1 Gy = 100 rads) resulting in chronic fibrosis within 6 weeks, with decreased bladder compliance, contractility, and overflow incontinence. Seven weeks post-irradiation, female C57Bl/6 mice were continuously infused with hRLX2 (400 µg/kg/day/14 days) or vehicle (saline) via subcutaneous osmotic pumps. Mice were evaluated in vivo using urine spot analysis, cystometrograms and external urethral sphincter electromyograms; and in vitro using length-tension measurements, Western blots, histology, and immunohistochemistry. RESULTS: hRLX2 reversed fibrosis, decreased collagen content, improved bladder wall architecture, and increased bladder compliance, detrusor smooth muscle Cav1.2 expression and detrusor contractility in mice with chronic radiation cystitis. hRLX2 treatment outcomes were likely caused by the activation of RXFP1/2 receptors which are expressed on the detrusor. CONCLUSION: hRLX2 may be a new therapeutic option for rescuing bladders with chronic radiation cystitis.

Alterations of L-type voltage dependent calcium channel alpha 1 subunit in the hippocampal CA3 region during and after pilocarpine-induced epilepsy.

Voltage-dependent calcium channels (VDCC) have been shown to regulate neuronal excitability and their antagonists have been used clinically for the control of seizures. While functional studies of VDCC in epileptogenesis in the CA1 area of hippocampus or the dentate gyrus have been done, few studies were carried out in the CA3 area. Given the bursting characteristics of the CA3 neurons, we speculated that VDCC in the CA3 area might play an important role in the epileptogenesis. In the present study in the mouse pilocarpine model of temporal lobe epilepsy, we investigated the alterations of alpha 1 subunits of L-type VDCC in the CA3 area of the hippocampus at different stages of epileptogenesis, i.e., acute stage from 10 min to 1 day during and after pilocapine-induced status epilepticus (SE), latent period at 1 week, and chronic stage with spontaneous recurrent seizures at 2 months after SE. We found that an immediate redistribution of alpha 1 subunits in the CA3 area occurred during SE which might be involved in the seizure occurrence indicated by the Racine score record. Alterations of alpha 1 subunits were also demonstrated in the latent period and chronic stage, which might be related to the epileptogenesis and occurrence of epilepsy. Cav1.3, but not Cav1.2, was expressed in reactive astrocytes of the CA3 area, indicating the involvement of Cav1.3 in the modulation of astrocytic Ca2+ homeostasis during epileptogenesis.

Estradiol up-regulates L-type Ca2+ channels via membrane-bound estrogen receptor/phosphoinositide-3-kinase/Akt/cAMP response element-binding protein signaling pathway.

BACKGROUND: In long QT syndrome type 2, women are more prone than men to the lethal arrhythmia torsades de pointes. We previously reported that 17ß-estradiol (E2) up-regulates L-type Ca2+ channels and current (ICa,L) (∼30%) in rabbit ventricular myocytes by a classic genomic mechanism mediated by estrogen receptor-α (ERα). In long QT syndrome type 2 (IKr blockade or bradycardia), the higher Ca2+ influx via ICa,L causes Ca2+ overload, spontaneous sarcoplasmic reticulum Ca2+ release, and reactivation of ICa,L that triggers early afterdepolarizations and torsades de pointes. OBJECTIVE: The purpose of this study was to investigate the molecular mechanisms whereby E2 up-regulates ICa,L, which are poorly understood. METHODS: H9C2 and rat myocytes were incubated with E2 ± ER antagonist, or inhibitors of downstream transcription factors, for 24 hours, followed by western blots of Cav1.2α1C and voltage-clamp measurements of ICa,L. RESULTS: Incubation of H9C2 cells with E2 (10-100 nM) increased ICa,L density and Cav1.2α1C expression, which were suppressed by the ER antagonist ICI182,780 (1 µM). Enhanced ICa,L and Cav1.2α1C expression by E2 was suppressed by inhibitors of phosphoinositide-3-kinase (Pi3K) (30 µM LY294002; P <.05) and Akt (5 µM MK2206) but not of mitogen-activated protein kinase (5 µM U0126) or protein kinase A (1 µM KT5720). E2 incubation increased p-CREB via the Pi3K/Akt pathway, reached a peak in 20 minutes (3-fold), and leveled off to 1.5-fold 24 hours later. Furthermore, a CREB decoy oligonucleotide inhibited E2-induced Cav1.2α1C expression, whereas membrane-impermeable E2 (E2-bovine serum albumin) was equally effective at Cav1.2α1C up-regulation as E2. CONCLUSION: Estradiol up-regulates Cav1.2α1C and ICa,L via plasma membrane ER and by activating Pi3K, Akt, and CREB signaling. The promoter regions of the CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding sites for p-CREB and ERα, which suggests a synergistic regulation by these pathways.

Protein kinase C enhances plasma membrane expression of cardiac L-type calcium channel, CaV1.2.

L-type-voltage-dependent Ca2+ channels (L-VDCCs; CaV1.2, α1C), crucial in cardiovascular physiology and pathology, are modulated via activation of G-protein-coupled receptors and subsequently protein kinase C (PKC). Despite extensive study, key aspects of the mechanisms leading to PKC-induced Ca2+ current increase are unresolved. A notable residue, Ser1928, located in the distal C-terminus (dCT) of α1C was shown to be phosphorylated by PKC. CaV1.2 undergoes posttranslational modifications yielding full-length and proteolytically cleaved CT-truncated forms. We have previously shown that, in Xenopus oocytes, activation of PKC enhances α1C macroscopic currents. This increase depended on the isoform of α1C expressed. Only isoforms containing the cardiac, long N-terminus (L-NT), were upregulated by PKC. Ser1928 was also crucial for the full effect of PKC. Here we report that, in Xenopus oocytes, following PKC activation the amount of α1C protein expressed in the plasma membrane (PM) increases within minutes. The increase in PM content is greater with full-length α1C than in dCT-truncated α1C, and requires Ser1928. The same was observed in HL-1 cells, a mouse atrium cell line natively expressing cardiac α1C, which undergoes the proteolytic cleavage of the dCT, thus providing a native setting for exploring the effects of PKC in cardiomyocytes. Interestingly, activation of PKC preferentially increased the PM levels of full-length, L-NT α1C. Our findings suggest that part of PKC regulation of CaV1.2 in the heart involves changes in channel's cellular fate. The mechanism of this PKC regulation appears to involve the C-terminus of α1C, possibly corroborating the previously proposed role of NT-CT interactions within α1C.

Verapamil augments carmustine- and irradiation-induced senescence in glioma cells by reducing intracellular reactive oxygen species and calcium ion levels.

Resistance to conventional therapies and frequent recurrence are the major obstacles to the treatment of high-grade gliomas, including glioblastoma. Thus, the development of new therapeutic strategies to overcome these obstacles is necessary to improve the treatment outcomes. In this study, we found that verapamil, a pan-adenosine triphosphate-binding cassette transporter and L-type voltage-dependent calcium channel inhibitor, sensitized U87MG glioma cells to carmustine- and irradiation-induced senescence. Furthermore, our results indicated that verapamil treatment, in combination with carmustine and irradiation, rendered U87MG glioma cells and several patient-derived glioma stem cells more sensitive to therapy-induced senescence than individual or dual-combination treatments. When investigating the underlying mechanism, we found that verapamil treatment markedly decreased intracellular reactive oxygen species and calcium ion levels. Reactive oxygen species reduction with N-acetylcysteine, a reactive oxygen species scavenger, rendered U87MG glioma cells more sensitive to carmustine and irradiation whereas the protein kinase C agonist, phorbol 12-myristate 13-acetate, mitigated the effects of carmustine and irradiation. Taken together, our results indicate that verapamil may be a potent therapeutic sensitizer for increasing the effectiveness of glioblastoma treatment.

STIM1 Ca2+ Sensor Control of L-type Ca2+-Channel-Dependent Dendritic Spine Structural Plasticity and Nuclear Signaling.

Potentiation of synaptic strength relies on postsynaptic Ca2+ signals, modification of dendritic spine structure, and changes in gene expression. One Ca2+ signaling pathway supporting these processes routes through L-type Ca2+ channels (LTCC), whose activity is subject to tuning by multiple mechanisms. Here, we show in hippocampal neurons that LTCC inhibition by the endoplasmic reticulum (ER) Ca2+ sensor, stromal interaction molecule 1 (STIM1), is engaged by the neurotransmitter glutamate, resulting in regulation of spine ER structure and nuclear signaling by the NFATc3 transcription factor. In this mechanism, depolarization by glutamate activates LTCC Ca2+ influx, releases Ca2+ from the ER, and consequently drives STIM1 aggregation and an inhibitory interaction with LTCCs that increases spine ER content but decreases NFATc3 nuclear translocation. These findings of negative feedback control of LTCC signaling by STIM1 reveal interplay between Ca2+ influx and release from stores that controls both postsynaptic structural plasticity and downstream nuclear signaling.

Decreased intracellular [Ca2+ ] coincides with reduced expression of Dhprα1s, RyR1, and diaphragmatic dysfunction in a rat model of sepsis.

INTRODUCTION: Sepsis can cause decreased diaphragmatic contractility. Intracellular calcium as a second messenger is central to diaphragmatic contractility. However, changes in intracellular calcium concentration ([Ca2+ ]) and the distribution and co-localization of relevant calcium channels [dihydropyridine receptors, (DHPRα1s) and ryanodine receptors (RyR1)] remain unclear during sepsis. In this study we investigated the effect of changed intracellular [Ca2+ ] and expression and distribution of DHPRα1s and RyR1 on diaphragm function during sepsis. METHODS: We measured diaphragm contractility and isolated diaphragm muscle cells in a rat model of sepsis. The distribution and co-localization of DHPRα1s and RyR1 were determined using immunohistochemistry and immunofluorescence, whereas intracellular [Ca2+ ] was measured by confocal microscopy and fluorescence spectrophotometry. RESULTS: Septic rat diaphragm contractility, expression of DHPRα1s and RyR1, and intracellular [Ca2+ ] were significantly decreased in the rat sepsis model compared with controls. DISCUSSION: Decreased intracellular [Ca2+ ] coincides with diaphragmatic contractility and decreased expression of DHPRα1s and RyR1 in sepsis. Muscle Nerve 56: 1128-1136, 2017.

Splice variants of the CaV1.3 L-type calcium channel regulate dendritic spine morphology.

Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson's disease. The L-type calcium channel CaV1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length CaV1.3L and two C-terminally truncated splice variants (CaV1.342A and CaV1.343S) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged CaV1.3L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in CaV1.3L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with CaV1.3L and correlated with increased CaV1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of CaV1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of CaV1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Calcium handling precedes cardiac differentiation to initiate the first heartbeat.

The mammalian heartbeat is thought to begin just prior to the linear heart tube stage of development. How the initial contractions are established and the downstream consequences of the earliest contractile function on cardiac differentiation and morphogenesis have not been described. Using high-resolution live imaging of mouse embryos, we observed randomly distributed spontaneous asynchronous Ca2+-oscillations (SACOs) in the forming cardiac crescent (stage E7.75) prior to overt beating. Nascent contraction initiated at around E8.0 and was associated with sarcomeric assembly and rapid Ca2+ transients, underpinned by sequential expression of the Na+-Ca2+ exchanger (NCX1) and L-type Ca2+ channel (LTCC). Pharmacological inhibition of NCX1 and LTCC revealed rapid development of Ca2+ handling in the early heart and an essential early role for NCX1 in establishing SACOs through to the initiation of beating. NCX1 blockade impacted on CaMKII signalling to down-regulate cardiac gene expression, leading to impaired differentiation and failed crescent maturation.

Resistance to pathologic cardiac hypertrophy and reduced expression of CaV1.2 in Trpc3-depleted mice.

Sustained elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) reprograms cardiovascular cell fate, leading to cellular hypertrophy via Ca(2+)-calmodulin/calcineurin (Cn)/NFAT activation. Accumulating evidence suggests that transient receptor potential canonical (Trpc) channels play important roles in the development of pathologic cardiac hypertrophy. Here, we demonstrated that Trpc3 mediates pathologic cardiac hypertrophy in neurohumoral elevation via direct regulation of CaV1.2 expressions. Elevated PE (phenylephrine) was maintained in mice by continuous infusion using an osmotic pump. Wild-type (WT) mice, but not Trpc3 (-/-) showed a sudden decrease in blood pressure (BP) or death following elevation of BP under conditions of elevated PE. Trpc3 (-/-) mesenteric artery showed decreased PE-stimulated vasoconstriction. Analysis of morphology, function, and pathologic marker expression revealed that PE elevation caused pathologic cardiac hypertrophy in WT mice, which was prevented by deletion of Trpc3. Interestingly, protection by Trpc3 deletion seemed to be a result of reduced cardiac CaV1.2 expressions. Basal and PE induced increased expression of protein and mRNA of CaV1.2 was decreased in Trpc3 (-/-) heart. Accordingly, altered expression of CaV1.2 was observed by knockdown or stimulation of Trpc3 in cardiomyocytes. These findings suggest that Trpc3 is a mediator of pathologic cardiac hypertrophy not only through mediating part of the Ca(2+) influx, but also through control of CaV1.2 expressions.