NCX1 Protein Research Articles

Caveolar Compartmentalization of Pacemaker Signaling is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure.

Heart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND). Biochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking. In both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca2+ release events (LCRs), which diffuse across ~15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca2+ current, a negative shift of the HCN current (I f) activation curve, and a 40% reduction of Na+/Ca2+-exchanger function, along with ~2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND. SAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

Cardiac function is regulated by the sodium-dependent inhibition of the sodium-calcium exchanger NCX1

The Na+-Ca2+ exchanger (NCX1) is the dominant Ca2+ extrusion mechanism in cardiac myocytes. NCX1 activity is inhibited by intracellular Na+ via a process known as Na+-dependent inactivation. A central question is whether this inactivation plays a physiological role in heart function. Using CRISPR/Cas9, we inserted the K229Q mutation in the gene (Slc8a1) encoding for NCX1. This mutation removes the Na+-dependent inactivation while preserving transport properties and other allosteric regulations. NCX1 mRNA levels, protein expression, and protein localization are unchanged in K229Q male mice. However, they exhibit reduced left ventricular ejection fraction and fractional shortening, while displaying a prolonged QT interval. K229Q ventricular myocytes show enhanced NCX1 activity, resulting in action potential prolongation, higher incidence of aberrant action potentials, a faster decline of Ca2+ transients, and depressed cell shortening. The results demonstrate that NCX1 Na+-dependent inactivation plays an essential role in heart function by affecting both cardiac excitability and contractility.

MicroRNA-129-1-3p protects chicken granulosa cells from cadmium-induced apoptosis by down-regulating the MCU-mediated Ca2+ signaling pathway

Cadmium (Cd) is known as a female reproductive toxicant. Our previous study has shown that Cd can influence the proliferation and cell cycle of granulosa cells and induce apoptosis. MicroRNAs (miRNAs) play an important role in the regulation of Cd-induced granulosa cell damage in chickens. However, the mechanism remains unclear. In this study, we investigated the mechanisms by which microRNA-129–1-3p (miR-129–1-3p) regulates Cd-induced cytotoxicity in chicken granulosa cells. As anticipated, exposure to Cd resulted in the induction of oxidative stress in granulosa cells, accompanied by the downregulation of antioxidant molecules and/or enzymes of Nrf2, Mn-SOD, Cu-Zn SOD and CAT, and the upregulation of Keap1, GST, GSH-Px, GCLM, MDA, hydrogen peroxide and mitochondrial reactive oxygen species (mtROS). Further studies found that Cd exposure causes mitochondrial calcium ions (Ca2+) overload, provoking mitochondrial damage and apoptosis by upregulating IP3R, GRP75, VDAC1, MCU, CALM1, MFF, caspase 3, and caspase 9 gene and/or protein expressions and mitochondrial Ca2+ levels, while downregulating NCX1, NCLX and MFN2 gene and/or protein expressions and mitochondrial membrane potential (MMP). The Ca2+ chelator BAPTA-AM or the MCU inhibitor MCU-i4 significantly rescued Cd-induced mitochondrial dysfunction, thereby attenuating apoptosis. Additionally, a luciferase reported assay and western blot analysis confirmed that miR-129–1-3p directly target MCU. MiR-129–1-3p overexpression almost completely inhibited protein expression of MCU, increased the gene and protein expressions of NCLX and MFN2 downregulated by Cd, and attenuated mitochondrial Ca2+ overload, MMP depression and mitochondria damage induced by Cd. Moreover, the overexpression of miR-129–1-3p led to a reduction in mtROS and cell apoptosis levels, and a suppression of the gene and protein expressions of caspase 3 and caspase 9. As above, these results provided the evidence that IP3R-MCU signaling pathway activated by Cd plays a significant role in inducing mitochondrial Ca2+ overload, mitochondrial damage, and apoptosis. MiR-129–1-3p exerts a protective effect against Cd-induced granulosa cell apoptosis through the direct inhibition of MCU expression in the ovary of laying hens.

Hamster Sperm Possess Functional Na+/Ca2+-Exchanger 1: Its Implication in Hyperactivation.

Previous studies demonstrated that hamster sperm hyperactivation is suppressed by extracellular Na+ by lowering intracellular Ca2+ levels, and Na+/Ca2+-exchanger (NCX) specific inhibitors canceled the suppressive effects of extracellular Na+. These results suggest the involvement of NCX in the regulation of hyperactivation. However, direct evidence of the presence and functionality of NCX in hamster spermatozoa is still lacking. This study aimed to reveal that NCX is present and is functional in hamster spermatozoa. First, NCX1 and NCX2 transcripts were detected via RNA-seq analyses of hamster testis mRNAs, but only the NCX1 protein was detected. Next, NCX activity was determined by measuring the Na+-dependent Ca2+ influx using the Ca2+ indicator Fura-2. The Na+-dependent Ca2+ influx was detected in hamster spermatozoa, notably in the tail region. The Na+-dependent Ca2+ influx was inhibited by the NCX inhibitor SEA0400 at NCX1-specific concentrations. NCX1 activity was reduced after 3 h of incubation in capacitating conditions. These results, together with authors' previous study, showed that hamster spermatozoa possesses functional NCX1 and that its activity was downregulated upon capacitation to trigger hyperactivation. This is the first study to successfully reveal the presence of NCX1 and its physiological function as a hyperactivation brake.

NCX1 disturbs calcium homeostasis and promotes RANKL-induced osteoclast differentiation by regulating JNK/c-Fos/NFATc1 signaling pathway in multiple myeloma

Although several types of calcium channels abnormalities have been shown to promote myeloma bone disease (MBD), the relationship between Na+/Ca2+ exchanger 1 (NCX1) and MBD remains unexplored. Here, we examined the role of NCX1 in the development of multiple myeloma (MM), with a special focus on the underlying effects involved osteoclast differentiation. Firstly, we detected NCX1 protein highly expressed in BM tissues of MM patients, and its expression was positively correlated with serum calcium and the percentage of BM CD138+ cells. In vitro, NCX1 suppression with the inhibitor KB-R7943 reduced cell viability of MM cells and caused apoptosis. Extracellular high Ca2+ environment increased the level of intracellular Ca2+ in MM cells through gating the calcium influx, with subsequently promoting the expression of NCX1 and osteoclastogenesis-related genes (receptor activator of nuclear factor-κB (RANKL), nuclear factor of activated T cell cytoplasmic 1 (NFATc1), and proto-oncogene Fos (c-Fos). This phenomenon could be reversed by KB-R7943 or calcium chelation. Furthermore, NCX1 overexpression in MM cells accelerated osteoclastogenesis, while NCX1 knockdown or suppression resulted in the opposite effect. Mechanistically, we further investigated the related mechanisms of NCX1 regulating osteoclast differentiation using RNA sequencing, western blotting and Enzyme linked immunosorbent assay, and found that NCX1 modulated osteoclast differentiation in MM though JNK/c-Fos/NFATc1 signaling pathway. In conclusion, the Ca2+/NCX1-mediated signaling participates in the osteoclasts-myeloma cell interactions, which represents a promising target for future therapeutic intervention in MBD.

GATA3 (GATA-Binding Protein 3)/KMT2A (Lysine-Methyltransferase-2A) Complex by Increasing H3K4-3me (Trimethylated Lysine-4 of Histone-3) Upregulates NCX3 (Na+-Ca2+ Exchanger 3) Transcription and Contributes to Ischemic Preconditioning Neuroprotection.

NCX3 (Na+-Ca2+ exchanger 3) plays a relevant role in stroke; indeed its pharmacological blockade or its genetic ablation exacerbates brain ischemic damage, whereas its upregulation takes part in the neuroprotection elicited by ischemic preconditioning. To identify an effective strategy to induce an overexpression of NCX3, we examined transcription factors and epigenetic mechanisms potentially involved in NCX3 gene regulation. Brain ischemia and ischemic preconditioning were induced in vitro by exposure of cortical neurons to oxygen and glucose deprivation plus reoxygenation (OGD/Reoxy) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcripts and proteins of GATA3 (GATA-binding protein 3), KMT2A (lysine-methyltransferase-2A), and NCX3. GATA3 and KMT2A binding on NCX3 gene was evaluated by chromatin immunoprecipitation and Rechromatin immunoprecipitation experiments. Among the putative transcription factors sharing a consensus sequence on the ncx3 brain promoter region, GATA3 was the only able to up-regulate ncx3. Interestingly, GATA3 physically interacted with KMT2A, and their overexpression or knocking-down increased or downregulated NCX3 mRNA and protein, respectively. Notably, site-direct mutagenesis of GATA site on ncx3 brain promoter region counteracted GATA3 and KMT2A binding on NCX3 gene. More importantly, we found that in the perischemic cortical regions of preconditioned rats GATA3 recruited KMT2A and the complex H3K4-3me (trimethylated lysine-4 of histone-3) on ncx3 brain promoter region, thus reducing transient middle cerebral artery occlusion–induced damage. Consistently, in vivo silencing of either GATA3 or KMT2A prevented NCX3 upregulation and consequently the neuroprotective effect of preconditioning stimulus. The involvement of GATA3/KMT2A complex in neuroprotection elicited by ischemic preconditioning was further confirmed by in vitro experiments in which the knocking-down of GATA3 and KMT2A reverted the neuroprotection induced by NCX3 overexpression in cortical neurons exposed to anoxic preconditioning followed by oxygen and glucose deprivation plus reoxygenation. Collectively, our results revealed that GATA3/KMT2A complex epigenetically activates NCX3 gene transcription during ischemic preconditioning.

Acute Genetic Ablation of Cardiac Sodium/Calcium Exchange in Adult Mice: Implications for Cardiomyocyte Calcium Regulation, Cardioprotection, and Arrhythmia.

BackgroundSodium‐calcium (Ca2+) exchanger isoform 1 (NCX1) is the dominant Ca2+ efflux mechanism in cardiomyocytes and is critical to maintaining Ca2+ homeostasis during excitation‐contraction coupling. NCX1 activity has been implicated in the pathogenesis of cardiovascular diseases, but a lack of specific NCX1 blockers complicates experimental interpretation. Our aim was to develop a tamoxifen‐inducible NCX1 knockout (KO) mouse to investigate compensatory adaptations of acute ablation of NCX1 on excitation‐contraction coupling and intracellular Ca2+ regulation, and to examine whether acute KO of NCX1 confers resistance to triggered arrhythmia and ischemia/reperfusion injury.Methods and ResultsWe used the α‐myosin heavy chain promoter (Myh6)‐MerCreMer promoter to create a tamoxifen‐inducible cardiac‐specific NCX1 KO mouse. Within 1 week of tamoxifen injection, NCX1 protein expression and current were dramatically reduced. Diastolic Ca2+ increased despite adaptive reductions in Ca2+ current and action potential duration and compensatory increases in excitation‐contraction coupling gain, sarcoplasmic reticulum Ca2+ ATPase 2 and plasma membrane Ca2+ ATPase. As these adaptations progressed over 4 weeks, diastolic Ca2+ normalized and SR Ca2+ load increased. Left ventricular function remained normal, but mild fibrosis and hypertrophy developed. Transcriptomics revealed modification of cardiovascular‐related gene networks including cell growth and fibrosis. NCX1 KO reduced spontaneous action potentials triggered by delayed afterdepolarizations and reduced scar size in response to ischemia/reperfusion.ConclusionsTamoxifen‐inducible NCX1 KO mice adapt to acute genetic ablation of NCX1 by reducing Ca2+ influx, increasing alternative Ca2+ efflux pathways, and increasing excitation‐contraction coupling gain to maintain contractility at the cost of mild Ca2+‐activated hypertrophy and fibrosis and decreased survival. Nevertheless, KO myocytes are protected against spontaneous action potentials and ischemia/reperfusion injury.

Ncx3-Induced Mitochondrial Dysfunction in Midbrain Leads to Neuroinflammation in Striatum of A53t-α-Synuclein Transgenic Old Mice.

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca2+-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson’s disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging.

Modulated Calcium Homeostasis and Release Events Under Atrial Fibrillation and Its Risk Factors: A Meta-Analysis

Background: Atrial fibrillation (AF) is associated with calcium (Ca2+) handling remodeling and increased spontaneous calcium release events (SCaEs). Nevertheless, its exact mechanism remains unclear, resulting in suboptimal primary and secondary preventative strategies.Methods: We searched the PubMed database for studies that investigated the relationship between SCaEs and AF and/or its risk factors. Meta-analysis was used to examine the Ca2+ mechanisms involved in the primary and secondary AF preventative groups.Results: We included a total of 74 studies, out of the identified 446 publications from inception (1982) until March 31, 2020. Forty-five were primary and 29 were secondary prevention studies for AF. The main Ca2+ release events, calcium transient (standardized mean difference (SMD) = 0.49; I2 = 35%; confidence interval (CI) = 0.33–0.66; p < 0.0001), and spark amplitude (SMD = 0.48; I2 = 0%; CI = −0.98–1.93; p = 0.054) were enhanced in the primary diseased group, while calcium transient frequency was increased in the secondary group. Calcium spark frequency was elevated in both the primary diseased and secondary AF groups. One of the key cardiac currents, the L-type calcium current (ICaL) was significantly downregulated in primary diseased (SMD = −1.07; I2 = 88%; CI = −1.94 to −0.20; p < 0.0001) and secondary AF groups (SMD = −1.28; I2 = 91%; CI = −2.04 to −0.52; p < 0.0001). Furthermore, the sodium–calcium exchanger (INCX) and NCX1 protein expression were significantly enhanced in the primary diseased group, while only NCX1 protein expression was shown to increase in the secondary AF studies. The phosphorylation of the ryanodine receptor at S2808 (pRyR-S2808) was significantly elevated in both the primary and secondary groups. It was increased in the primary diseased and proarrhythmic subgroups (SMD = 0.95; I2 = 64%; CI = 0.12–1.79; p = 0.074) and secondary AF group (SMD = 0.66; I2 = 63%; CI = 0.01–1.31; p < 0.0001). Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression was elevated in the primary diseased and proarrhythmic drug subgroups but substantially reduced in the secondary paroxysmal AF subgroup.Conclusions: Our study identified that ICaL is reduced in both the primary and secondary diseased groups. Furthermore, pRyR-S2808 and NCX1 protein expression are enhanced. The remodeling leads to elevated Ca2+ functional activities, such as increased frequencies or amplitude of Ca2+ spark and Ca2+ transient. The main difference identified between the primary and secondary diseased groups is SERCA expression, which is elevated in the primary diseased group and substantially reduced in the secondary paroxysmal AF subgroup. We believe our study will add new evidence to AF mechanisms and treatment targets.

The hypoxia sensitive metal transcription factor MTF-1 activates NCX1 brain promoter and participates in remote postconditioning neuroprotection in stroke

Remote limb ischemic postconditioning (RLIP) is an experimental strategy in which short femoral artery ischemia reduces brain damage induced by a previous harmful ischemic insult. Ionic homeostasis maintenance in the CNS seems to play a relevant role in mediating RLIP neuroprotection and among the effectors, the sodium-calcium exchanger 1 (NCX1) may give an important contribution, being expressed in all CNS cells involved in brain ischemic pathophysiology. The aim of this work was to investigate whether the metal responsive transcription factor 1 (MTF-1), an important hypoxia sensitive transcription factor, may (i) interact and regulate NCX1, and (ii) play a role in the neuroprotective effect mediated by RLIP through NCX1 activation. Here we demonstrated that in brain ischemia induced by transient middle cerebral occlusion (tMCAO), MTF-1 is triggered by a subsequent temporary femoral artery occlusion (FAO) and represents a mediator of endogenous neuroprotection. More importantly, we showed that MTF-1 translocates to the nucleus where it binds the metal responsive element (MRE) located at −23/−17 bp of Ncx1 brain promoter thus activating its transcription and inducing an upregulation of NCX1 that has been demonstrated to be neuroprotective. Furthermore, RLIP restored MTF-1 and NCX1 protein levels in the ischemic rat brain cortex and the silencing of MTF-1 prevented the increase of NCX1 observed in RLIP protected rats, thus demonstrating a direct regulation of NCX1 by MTF-1 in the ischemic cortex of rat exposed to tMCAO followed by FAO. Moreover, silencing of MTF-1 significantly reduced the neuroprotective effect elicited by RLIP as demonstrated by the enlargement of brain infarct volume observed in rats subjected to RLIP and treated with MTF-1 silencing. Overall, MTF-dependent activation of NCX1 and their upregulation elicited by RLIP, besides unraveling a new molecular pathway of neuroprotection during brain ischemia, might represent an additional mechanism to intervene in stroke pathophysiology.

Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling.

Ischaemia/reperfusion (I/R) injury is a common clinical condition that results in apoptosis and oxidative stress injury. Thyroid hormone was previously reported to elicit cardiac myocyte hypertrophy and promote cardiac function after cardiac injury. We used an in vivo mouse model of I/R injury and in vitro primary cardiomyocyte culture assays to investigate the effects of thyroid hormone on cardiomyocytes during hypoxia/reoxygenation (H/R) injury. The results showed that T3 pretreatment in vivo significantly improved left ventricular function after I/R injury. In vitro, T3 pretreatment decreased cell apoptosis rate, inhibited caspase‐3 activity and decreased the Bax/Bcl‐2 ration induced by H/R injury. T3 pretreatment significantly attenuated the loss of mitochondrial membrane potential. Furthermore, it was observed that T3 diminished the expression of NCX1 protein and decreased SERCA2a protein expression in H/R‐induced cardiomyocytes, and T3 prevented intracellular Ca2+ increase during H/R injury. Also, T3 increased the expression of IGF‐1, and PI3K/Akt signalling in cardiomyocytes under H/R‐induced injury, and that the protective effect of T3 against H/R‐induced injury was blocked by the PI3K inhibitor LY294002. IGF‐1 receptor (IGF‐1R) inhibitor GSK1904529A significantly inhibited the expression of IGF‐1R and PI3K/Akt signalling. In summary, T3 pretreatment protects cardiomyocytes against H/R‐induced injury by activating the IGF‐1‐mediated PI3K/Akt signalling pathway.

NCX3 alleviates ethanol-induced apoptosis of SK-N-SH cells via the elimination of intracellular calcium ions

Long-term alcohol intake may cause nerve cell apoptosis and induce various encephalopathies. Previously, we have shown that the expression of Na+/Ca2+ exchanger 3 (NCX3) was associated with the intracellular calcium concentration ([Ca2+]i) and apoptosis, involved in the spatial memory impairment in male C57BL/6 mice with chronic ethanol (EtOH) exposure. However, the mechanism involved is unclear. Here, we investigated the expression of NCX3 and its protective effect on SK-N-SH cells (a nerve cell line) after EtOH exposure. [Ca2+]i was measured using Fluo-3 AM reagent. Cell viability and the apoptotic rate were assayed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and flow cytometry, respectively. The expression of p-cAMP-responsive element binding protein1(p-CREB 1), NCX3 protein, and mRNA were observed using Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Cleaved-caspase-3, caspase-3, rabbit anti- poly (ADP-ribose) polymerase-1 (PARP-1) and calpain-1 proteins were used to assess the degree of apoptosis. Our results showed that EtOH increased [Ca2+]i and apoptosis of SK-N-SH cells in a concentration- and time-dependent manner. The expression of NCX3 protein and mRNA was up regulated obviously after SK-N-SH cells were treated with EtOH. The phosphorylation levels of Akt and CREB 1 were up regulated in cells treated with EtOH. The expression of NCX3 protein was reduced in the SK-N-SH cells treated with Akt phosphorylation inhibitor (LY294002). The [Ca2+]i and apoptosis rate of SK-N-SH cells increased 1.31-fold and 1.52-fold after silencing NCX3 compared with those treated with 200 mM EtOH alone for 2 d. In contrast, the [Ca2+]i and apoptosis rate of SK-N-SH cells decreased 0.26-fold and 0.35-fold after overexpression of NCX3 in the 2 d-200 mM EtOH treatment group. These results suggest that NCX3 plays a critical role in neuronal protection via the elimination of intracellular Ca2+, which may be a promising target for the prevention and treatment of encephalopathy after ethanol exposure.

Effect of electroacupuncture on intestinal calcium absorption and expression of transmembrane calcium transport-related receptors in ovariectomized rats

To observe the effect of electroacupuncture (EA) on bone mineral density (BMD) and expression of calcium transport-related receptors in ovariectomized rats, so as to explore its mechanisms underlying improvement of osteoporosis. Thirty-two three-month female SD rats were randomly divided into control, sham operation, model and EA groups (n=8 in each group). The ovariectomy model was established by resection of bilateral ovaries. EA (2 Hz/15 Hz, 2 mA) was applied to "Guanyuan"(CV4)+"Sanyinjiao"(SP6) or "Shenshu"(BL23)+"Housanli"(ST36) alternatively for 20 min, once daily (except the weekends) for 12 weeks. BMD of the tibia and femur were detected by using a bone densitometer. The expression levels of transient receptor potential vanillic receptor 5 (TRPV5), transient receptor potential vanillic receptor 6 (TRPV6), sodium calcium exchanger 1 (NCX1), membrane calcium adenosine triphosphate hydrolase (PMCA1b), zonula occludens 1 (ZO-1), occludin and claudin in intestinal mucosa were detected by quantitative real-time PCR and Western blot, respectively. Following modeling, BMD of both tibia and femur, and expression levels of TRPV5, PMCA1b, ZO-1, occludin and claudin mRNAs were significantly decreased in the model group compared with the control and sham operation groups (P<0.05, P<0.01)，while the mRNAs levels of TRPV6 and NCX1 in the model group were increased (P<0.01). After the treatment, BMD of both tibia and femur, and expression levels of TRPV5, PMCA1b, ZO-1 and occludin mRNAs were significantly increased(P<0.01，P<0.05)，and TRPV6 and NCX1 were significantly decreased(P<0.01，P<0.05) in the EA group than those in the model group. Compared with the control and sham operation groups, protein expression levels of TRPV5, PMCA1b, ZO-1, occludin and claudin in the model group were significantly decreased (P<0.05), while the protein levels of TRPV6 and NCX1 in the model group were increased (P<0.05). After EA intervention, modeling-induced decrease of TRPV5, PMCA1b, ZO-1, occludin and claudin protein levels and increase of TRPV6 and NCX1 protein levels were all completely reversed in the EA group relevant to the model group (P<0.05). EA intervention can improve bone mineral density and up-regulate the mRNA and protein expression of TRPV5, PMCA1b, ZO-1 and occludin, which may be one of the mechanisms of acupuncture in the treatment of osteoporosis.

The effect of steroid hormone on the expression of the calcium-processing proteins in the immature female rat brain

The cytosolic calcium concentration is regulated by calcium-processing proteins such as transient receptor potential cation channel subfamily V member 5 (TRPV5), TRPV6, sodium-calcium exchanger 1 (NCX1), and plasma membrane Ca2+ ATPase 1 (PMCA1). Those calcium-processing proteins are important for physiological functions in the brain. The effects of steroid hormones on calcium-processing protein expressions in the brains are unknown. Thus, the effects of steroid hormones on the distribution, localization, and expressions of calcium-processing proteins in the brain were analyzed. Immature female rats were injected with estrogen (E2), progesterone (P4), dexamethasone (DEX), and their antagonists (ICI 182,780 and RU486). We found that TRPV5 and TRPV6 proteins were highly expressed in the cerebral cortex (CT), hypothalamus (HY), and brain stem (BS) compared to that in the olfactory bulb (OB) and cerebellum (CB). Also, the NCX1 protein was highly expressed in CT and BS compared to that in OB, HY, and CB, and PMCA1 protein was highly expressed in CT compared to that in other brain regions. Furthermore, expression levels of TRPV5, TRPV6, NCX1, and PMCA1 proteins were regulated by E2, P4, and/or DEX in the CT and HY. In summary, calcium-processing proteins are widely expressed in the immature rat brain, and expressions of calcium-processing proteins in CT and HY indicated that they may regulate by E2, P4, and/or DEX and can be attenuated by antagonist treatment. These results indicate that steroid hormone regulation of TRPV5, TRPV6, NCX1, and PMCA1 proteins may serve as a critical regulator of cytosolic calcium absorption and release in the brain.

Acute Genetic Ablation of Cardiac Sodium-Calcium Exchange Suppresses Arrhythmogenic Delayed after Depolarizations

Sodium-calcium exchange (NCX) is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. As an electrogenic transporter, NCX is thought to generate the inward current responsible for Ca-dependent arrhythmogenic delayed afterdepolarizations (DADs). To test this hypothesis, we patch-clamped ventricular myocytes isolated from our novel tamoxifen-inducible cardiac-specific NCX knockout (KO) mouse (αMHC-MerCreMer promotor). Tamoxifen (40mg/kg/day IP) was injected for 5 consecutive days in 10 to 12-week-old mice. 4 weeks after injection, we found a 96% decrease of NCX protein expression and an 87% reduction of caffeine-induced NCX current (p< 0.001), with 25% of cells showing no NCX current whatsoever. Sarcoplasmic reticulum Ca load was similar to control. Transient outward current (ITO) and Kv4.2 protein expression were significantly increased (by 60 and 34% respectively, p < 0.01) and ICa was reduced by 47% (p < 0.001). The action potential duration (APD) was shortened when measured at either 10 % (8.6 ± 0.7 ms in control vs 5.5 ± 0.5 ms in KO, p < 0.05) or 90 % (43.9 ± 4.9 ms in control vs 11.1 ± 0.9 ms in KO, p < 0.001) repolarization. In response to a pro-arrhythmic stimulation protocol, spontaneous APs triggered by DADs occurred in 95% of control cells, but only in 35% of NCX KO myocytes, even though we observed no change in the mean number of DADs per cell. Notably, DAD amplitude was significantly reduced in NCX KO mice: 14% of KO cells exhibited DADs with an amplitude ≥ 3mV versus 78 % in control cells. In the 25% of KO cells with no detectable DADs, there were no spontaneous APs. Most likely these were the cells with no NCX activity. Thus, acute genetic reduction/ablation of NCX suppresses DAD frequency/amplitude and subsequent spontaneous AP generation.

The Role of Adenosine A2b Receptor in Mediating the Cardioprotection of Electroacupuncture Pretreatment via Influencing Ca2+ Key Regulators.

Objective To investigate the roles played by A2b receptor and the key Ca2+ signaling components in the mediation of the cardioprotection of electroacupuncture pretreatment in the rats subjected to myocardial ischemia and reperfusion. Methods SD rats were randomly divided into a normal control (NC) group, ischemia/reperfusion model (M) group, electroacupuncture pretreatment (EA) group, and electroacupuncture pretreatment plus A2b antagonist (EAG) group. The ischemia/reperfusion model was made by ligation and loosening of the left descending branch of the coronary artery in all groups except the NC group. The EA group was pretreated with electroacupuncture at the Neiguan (PC6) point once a day for three consecutive days before the modeling. The elevation of the ST segment, arrhythmia scores, and myocardial infarction size of each group was measured. The relative expression levels of A2b, RyR2, SERCA2a, NCX1, P-PLB(S16)/PLB, and Troponin C/Troponin I proteins in the injured myocardium were detected by multiple fluorescence western blot. Results The level of ST segment, arrhythmia scores, and infarct size in the M group was significantly higher/larger than that in the NC group after ischemia and reperfusion, while all the three indices mentioned above in the EA group were significantly lower/smaller than those in the M group after reperfusion. The expression of the proteins of adenosine receptor 2b(A2b), ryanodine receptor 2(RyR2), and sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) in the EA group was significantly enhanced as compared with the M group, while in the EAG group, the contents of A2b were significantly lower than those in the EA group, and RyR2 was higher in the EAG group. In comparison with the NC group, the relative expression of NCX1 protein in M, EA, and EAG groups was not changed significantly. The ratio of phosphorylated phospholamban (P-PLB) over phospholamban (PLB) in the M group was significantly lower than that in the NC group, and the ratio in the EA group was significantly increased as compared with the M group, while the ratio of Troponin C/Troponin I in the EA group was significantly decreased in comparison with that in other groups. Conclusion Electroacupuncture pretreatment could reduce ischemia and reperfusion-induced myocardial injury via possibly increasing the A2b content and regulating the key Ca2+ signaling components, namely inhibiting RyR2 and enhancing P-PLB(S16)/PLB ratio and SERCA2a proteins, so as to diminish the intracellular Ca2+ overload and consequently lessen the myocardial injury.

HDAC4 and HDAC5 form a complex with DREAM that epigenetically down-regulates NCX3 gene and its pharmacological inhibition reduces neuronal stroke damage

The histone deacetylases (HDACs)-dependent mechanisms regulating gene transcription of the Na+/Ca+ exchanger isoform 3 (ncx3) after stroke are still unknown. Overexpression or knocking-down of HDAC4/HDAC5 down-regulates or increases, respectively, NCX3 mRNA and protein. Likewise, MC1568 (class IIa HDACs inhibitor), but not MS-275 (class I HDACs inhibitor) increased NCX3 promoter activity, gene and protein expression. Furthermore, HDAC4 and HDAC5 physically interacted with the transcription factor downstream regulatory element antagonist modulator (DREAM). As MC1568, DREAM knocking-down prevented HDAC4 and HDAC5 recruitment to the ncx3 promoter. Importantly, DREAM, HDAC4, and HDAC5 recruitment to the ncx3 gene was increased in the temporoparietal cortex of rats subjected to transient middle cerebral artery occlusion (tMCAO), with a consequent histone-deacetylation of ncx3 promoter. Conversely, the tMCAO-induced NCX3 reduction was prevented by intracerebroventricular injection of siDREAM, siHDAC4, and siHDAC5. Notably, MC1568 prevented oxygen glucose deprivation plus reoxygenation and tMCAO-induced neuronal damage, whereas its neuroprotective effect was abolished by ncx3 knockdown. Collectively, we found that: (1) DREAM/HDAC4/HDAC5 complex epigenetically down-regulates ncx3 gene transcription after stroke, and (2) pharmacological inhibition of class IIa HDACs reduces stroke-induced neurodetrimental effects.

The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy.

Background: Mounting evidence indicate that reducing the sinoatrial node (SAN) activity may be a useful therapeutic strategy to control of heart failure. Purines, like ATP and its metabolite adenosine, consistently reduce the SAN spontaneous activity leading to negative cardiac chronotropy, with variable effects on the force of myocardial contraction (inotropy). Apart from adenosine A1 receptors, the human SAN expresses high levels of ATP-sensitive ionotropic P2X4 receptors (P2X4R), yet their cardiac role is unexplored. Methods: Here, we investigated the activity of P2 purinoceptors on isolated spontaneously beating atria (chronotropy) and on 2 Hz-paced right ventricular (RV, inotropy) strips from Wistar rats. Results: ATP (pEC 50 = 4.05) and its stable analogue ATPγS (pEC 50 = 4.69) concentration-dependently reduced atrial chronotropy. Inhibition of ATP breakdown into adenosine by NTPDases with POM-1 failed to modify ATP-induced negative chronotropy. The effect of ATP on atrial rate was attenuated by a broad-spectrum P2 antagonist, PPADS, as well as by 5-BDBD, which selectively blocks the P2X4R subtype; however, no effect was observed upon blocking the A1 receptor with DPCPX. The P2X4R positive allosteric modulator, ivermectin, increased the negative chronotropic response of ATP. Likewise, CTP, a P2X agonist that does not generate adenosine, replicated the P2X4R-mediated negative chronotropism of ATP. Inhibition of the Na+/Ca2+ exchanger (NCX) with KB-R7943 and ORM-10103, but not blockage of the HCN channel with ZD7288, mimicked the effect of the P2X4R blocker, 5-BDBD. In paced RV strips, ATP caused a mild negative inotropic effect, which magnitude was 2 to 3-fold increased by 5-BDBD and KB-R7943. Immunofluorescence confocal microscopy studies confirm that cardiomyocytes of the rat SAN and RV co-express P2X4R and NCX1 proteins. Conclusions: Data suggest that activation of ATP-sensitive P2X4R slows down heart rate by reducing the SAN activity while increasing the magnitude of ventricular contractions. The mechanism underlying the dual effect of ATP in the heart may involve inhibition of intracellular Ca2+-extrusion by bolstering NCX function in the reverse mode. Thus, targeting the P2X4R activation may create novel well-tolerated heart-rate lowering drugs with potential benefits in patients with deteriorated ventricular function.

Abstract P105: Sex Hormonal Changes of Calcium-Sensing Receptor and Renin-Angiotensin-Aldosterone System in Salt-Sensitive Rats via Exposure of High Salt Diets

The CaSR regulates Ca 2+ -mediated release of parathyroid hormone (PTH) from parathyroid cells by sensing small changes in serum Ca 2+ concentration. The receptor is also involved in the regulation of cell Ca 2+ levels independent of PTH secretion. The renin-angiotensin-aldosterone system (RAAS) plays vital roles in the development of hypertension, which is associated with vascular and renal dysfunction, and treatment is difficult because the mechanisms are not fully understood. Studies show that adequate Ca 2+ intake (1.0 to 1.5 g/day) is critical for optimal BP regulation and randomized controlled trials have revealed significant reductions in hypertension risk and BP levels in humans. In the kidney, the CaSR is expressed along the nephron and plays an important role, with the Na + /Ca 2+ exchanger (NCX), in the regulation of tubular Ca 2+ , Na + and water reabsorption. In the present study, we analyzed the effects of mutation, sex hormones and high salt diets on the expression of the CaSR, its downstream signaling complements and proteins in the RAAS in both female and male salt-sensitive (SS) rats. Our data indicate that CaSR gene mutation and high salt diets modulate the expression of the CaSR, GRK2, PKCα, NCX1 and RAAS proteins in the kidney of SS female rats than male rats. And also showed statistically significant difference in electrolytes as shown in the data below. Thus, CaSR signaling is linked to RAAS to control hypertension.

Chronic ethanol exposure reduces the expression of NCX3 in the hippocampus of male C57BL/6 mice.

Chronic ethanol (EtOH) exposure can cause intracellular Ca overload by stimulating calcium channel receptors and trigger apoptosis of neurons. NCX3 may play a cytoprotective role in intracellular Ca excretion. In this study, the effect of EtOH on NCX3 was analyzed by observing NCX3 expression in the hippocampus of chronic EtOH-exposed male C57BL/6 mice. Mice were divided into a control group, a 10% EtOH group, and a 20% EtOH group for 30, 60, and 90 days. Behavioral changes were observed using the Morris water maze. The protein and mRNA expressions of NCX3 and their distribution in the hippocampus were observed by western blotting, quantitative PCR, and immunohistochemistry staining. The results showed that EtOH exposure exerted a significant adverse effect on the spatial memory capacity of mice. Increased expression of calpain-1 and cleaved caspase-3 proteins indicated increased apoptosis. The expression of NCX3 in the hippocampus was downregulated after exposure to EtOH (except 10% EtOH for 30 days) and this inhibition was time and dose dependent with EtOH exposure. The level of p-Akt, which is an upstream regulation factor of NCX3, showed a trend similar to that of NCX3 protein. Chronic EtOH exposure reduced the expression of NCX3 in the hippocampus of male C57BL/6 mice, increasing intracellular calcium and apoptosis, resulting in spatial memory impairment in mice.