NCX Current Research Articles

Glucagon-like Peptide-1 Receptor Activation Reduces Pulmonary Vein Arrhythmogenesis and Regulates Calcium Homeostasis.

Glucagon-like peptide-1 (GLP-1) receptor agonists are associated with reduced atrial fibrillation risk, but the mechanisms underlying this association remain unclear. The GLP-1 receptor agonist directly impacts cardiac Ca2+ homeostasis, which is crucial in pulmonary vein (PV, the initiator of atrial fibrillation) arrhythmogenesis. This study investigated the effects of the GLP-1 receptor agonist on PV electrophysiology and Ca2+ homeostasis and elucidated the potential underlying mechanisms. Conventional microelectrodes and whole-cell patch clamp techniques were employed in rabbit PV tissues and single PV cardiomyocytes before and after GLP-1 (7-36) amide, a GLP-1 receptor agonist. Evaluations were conducted both with and without pretreatment with H89 (10 μM, an inhibitor of protein kinase A, PKA), KN93 (1 μM, an inhibitor of Ca2+/calmodulin-dependent protein kinase II, CaMKII), and KB-R7943 (10 μM, an inhibitor of Na+/Ca2+ exchanger, NCX). Results showed that GLP-1 (7-36) amide (at concentrations of 1, 10, and 100 nM) reduced PV spontaneous activity in a concentration-dependent manner without affecting sinoatrial node electrical activity. In single-cell experiments, GLP-1 (7-36) amide (at 10 nM) reduced L-type Ca2+ current, NCX current, and late Na+ current in PV cardiomyocytes without altering Na+ current. Additionally, GLP-1 (7-36) amide (at 10 nM) increased sarcoplasmic reticulum Ca2+ content in PV cardiomyocytes. Furthermore, the antiarrhythmic effects of GLP-1 (7-36) amide on PV automaticity were diminished when pretreated with H89, KN93, or KB-R7943. This suggests that the GLP-1 receptor agonist may exert its antiarrhythmic potential by regulating PKA, CaMKII, and NCX activity, as well as modulating intracellular Ca2+ homeostasis, thereby reducing PV arrhythmogenesis.

The reverse mode of the Na+/Ca2+ exchanger contributes to the pacemaker mechanism in rabbit sinus node cells

Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca2+-handling. The fundamental role of the inward Na+/Ca2+ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca2+-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 μM ORM-10962. Mechanistic simulations were performed by Maltsev–Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca2+-transient amplitude with larger SR Ca2+-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 μM strophantin the Ca2+i and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca2+i and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca2+i and heart rate with increasing Na+i. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca2+-influx that could increase SR Ca2+-content, which consequently leads to enhanced pacemaking activity.

Regulation of APD and Force by the Na+/Ca2+ Exchanger in Human-Induced Pluripotent Stem Cell-Derived Engineered Heart Tissue.

The physiological importance of NCX in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is not well characterized but may depend on the relative strength of the current, compared to adult cardiomyocytes, and on the exact spatial arrangement of proteins involved in Ca2+ extrusion. Here, we determined NCX currents and its contribution to action potential and force in hiPSC-CMs cultured in engineered heart tissue (EHT). The results were compared with data from rat and human left ventricular tissue. The NCX currents in hiPSC-CMs were larger than in ventricular cardiomyocytes isolated from human left ventricles (1.3 ± 0.2 pA/pF and 3.2 ± 0.2 pA/pF for human ventricle and EHT, respectively, p < 0.05). SEA0400 (10 µM) markedly shortened the APD90 in EHT (by 26.6 ± 5%, p < 0.05) and, to a lesser extent, in rat ventricular tissue (by 10.7 ± 1.6%, p < 0.05). Shortening in human left ventricular preparations was small and not different from time-matched controls (TMCs; p > 0.05). Force was increased by the NCX block in rat ventricle (by 31 ± 5.4%, p < 0.05) and EHT (by 20.8 ± 3.9%, p < 0.05), but not in human left ventricular preparations. In conclusion, hiPSC-CMs possess NCX currents not smaller than human left ventricular tissue. Robust NCX block-induced APD shortening and inotropy makes EHT an attractive pharmacological model.

Interaction of background Ca2+ influx, sarcoplasmic reticulum threshold and heart failure in determining propensity for Ca2+ waves in sheep heart.

Ventricular arrhythmias can cause death in heart failure (HF). A trigger is the occurrence of Ca2+ waves which activate a Na+‐Ca2+ exchange (NCX) current, leading to delayed after‐depolarisations and triggered action potentials. Waves arise when sarcoplasmic reticulum (SR) Ca2+ content reaches a threshold and are commonly induced experimentally by raising external Ca2+, although the mechanism by which this causes waves is unclear and was the focus of this study. Intracellular Ca2+ was measured in voltage‐clamped ventricular myocytes from both control sheep and those subjected to rapid pacing to produce HF. Threshold SR Ca2+ content was determined by applying caffeine (10 mM) following a wave and integrating wave and caffeine‐induced NCX currents. Raising external Ca2+ induced waves in a greater proportion of HF cells than control. The associated increase of SR Ca2+ content was smaller in HF due to a lower threshold. Raising external Ca2+ had no effect on total influx via the L‐type Ca2+ current, I Ca‐L, and increased efflux on NCX. Analysis of sarcolemmal fluxes revealed substantial background Ca2+ entry which sustains Ca2+ efflux during waves in the steady state. Wave frequency and background Ca2+ entry were decreased by Gd3+ or the TRPC6 inhibitor BI 749327. These agents also blocked Mn2+ entry. Inhibiting connexin hemi‐channels, TRPC1/4/5, L‐type channels or NCX had no effect on background entry. In conclusion, raising external Ca2+ induces waves via a background Ca2+ influx through TRPC6 channels. The greater propensity to waves in HF results from increased background entry and decreased threshold SR content.Key points Heart failure is a pro‐arrhythmic state and arrhythmias are a major cause of death.At the cellular level, Ca2+ waves resulting in delayed after‐depolarisations are a key trigger of arrhythmias. Ca2+ waves arise when the sarcoplasmic reticulum (SR) becomes overloaded with Ca2+.We investigate the mechanism by which raising external Ca2+ causes waves, and how this is modified in heart failure.We demonstrate that a novel sarcolemmal background Ca2+ influx via the TRPC6 channel is responsible for SR Ca2+ overload and Ca2+ waves.The increased propensity for Ca2+ waves in heart failure results from an increase of background influx, and a lower threshold SR content.The results of the present study highlight a novel mechanism by which Ca2+ waves may arise in heart failure, providing a basis for future work and novel therapeutic targets.

Intracellular Ca2+-Mediated Mechanisms for the Pacemaker Depolarization of the Mouse and Guinea Pig Sinus Node Tissue

Intracellular Ca2+-mediated mechanisms for pacemaker depolarization were studied in sinus node tissue preparations from mice and guinea pigs. Microelectrode recordings revealed that the sinus node of the mouse, which had a higher beating rate, had a steeper slope of the pacemaker depolarization than that of the guinea pig. BAPTA and ryanodine, agents that interfere with intracellular Ca2+, significantly decreased the slope of the pacemaker depolarization in both species. In contrast, SEA0400, a specific inhibitor of the Na+-Ca2+ exchanger (NCX), as well as change to low Na+ extracellular solution, significantly decreased the slope in the mouse, but not in the guinea pig. Niflumic acid, a blocker of the Ca2+ activated Cl− channel, decreased the slope in both species. Confocal microscopy revealed the presence of spontaneous Ca2+ oscillations during the interval between Ca2+ transients; such phenomenon was more pronounced in the mouse than in the guinea pig. Thus, although intracellular Ca2+-mediated mechanisms were involved in the pacemaker depolarization of the sinus node in both species, the NCX current was involved in the mouse but not in the guinea pig.

Calcium dysregulation increases right ventricular outflow tract arrhythmogenesis in rabbit model of chronic kidney disease.

Chronic kidney disease (CKD) increases the risk of arrhythmia. The right ventricular outflow tract (RVOT) is a crucial site of ventricular tachycardia (VT) origination. We hypothesize that CKD increases RVOT arrhythmogenesis through its effects on calcium dysregulation. We analysed measurements obtained using conventional microelectrodes, patch clamp, confocal microscopy, western blotting, immunohistochemical examination and lipid peroxidation for both control and CKD (induced by 150 mg/kg neomycin and 500 mg/kg cefazolin daily) rabbit RVOT tissues or cardiomyocytes. The RVOT of CKD rabbits exhibited a short action potential duration, high incidence of tachypacing (20 Hz)‐induced sustained VT, and long duration of isoproterenol and tachypacing‐induced sustained and non‐sustained VT. Tachypacing‐induced sustained and non‐sustained VT in isoproterenol‐treated CKD RVOT tissues were attenuated by KB‐R7943 and partially inhibited by KN93 and H89. The CKD RVOT myocytes had high levels of phosphorylated CaMKII and PKA, and an increased expression of tyrosine hydroxylase‐positive neural density. The CKD RVOT myocytes exhibited large levels of I to, I Kr, NCX and L‐type calcium currents, calcium leak and malondialdehyde but low sodium current, SERCA2a activity and SR calcium content. The RVOT in CKD with oxidative stress and autonomic neuron hyperactivity exhibited calcium handling abnormalities, which contributed to the induction of VT.

The Investigation of Combined Na+/Ca2+ Exchanger and the L-type Ca2+- Channel Inhibition in Langendorff-Perfused Isolated Guinea Pig Hearts

Abstract Objective The sodium/calcium exchanger (NCX) and the L-type Ca2+-channel (LTCC) are nowadays considered the major transmembrane transport mechanisms that control Ca2+ homeostasis. In pathophysiological conditions the altered function of these currents may influence the Ca2+ homeostasis and cardiac contractility and thereby, may enhance the development of severe tachyarrhythmias. The blockade of NCX current has been proposed as possible approach in the prevention and/or suppression of arrhythmias; however, this mechanism is not always favourable because the inhibition of both modes of NCX may induce Ca2+-overload. The decrease of the Ca2+ level by partial LTCC inhibition may be beneficial in increasing the antiarrhythmic efficacy. Therefore, the aim of our study was to investigate the antiarrhythmic effects of combined NCX and LTCC blockade in the ex vivo guinea pig arrhythmia model. Methods We have performed Langendorff experiments in isolated guinea pig hearts. We have recorded electrocardiograms (ECG) and left ventricle pressure. We have applied 1 μM ORM-10962 (ORM), a compound that block NCX current and 30 nM nisoldipine for the inhibition of LTCC. Arrhythmias have been provoked by decreasing the activity of the sodium/potassium pump with 5 μM ouabain. Results We found that neither LTCC nor NCX blockade alone increased, while the combined inhibition of the two currents significantly delayed (p&lt;0.05) the mean time of appearance of ouabain-induced ventricular fibrillation. The heart frequency was affected by none of the drugs, only the left ventricular pressure (end-systolic and diastolic difference) was significantly elevated by ORM (p&lt;0.001). Conclusion In the Langendorff-perfused guinea pig heart, specific, combined NCX and LTCC blockade may be favourable than the inhibition of NCX or LTCC alone. However, further investigations are necessary to identify the pathological settings in which this combined cardiac drug therapy may be a potential new approach.

Effects of phosphodiesterase-1 inhibitor on pulmonary vein electrophysiology and arrhythmogenesis.

Phosphodiesterase (PDE) isoform inhibitors have mechanical and electrical effects on the heart. Inhibition of PDE-1 enzymes is a novel strategy for treating heart failure. However, the electrophysiological effects of PDE-1 inhibition on the heart remain unclear. This study explored the effects of PDE-1 inhibition using ITI-214 on electrical activity in the pulmonary vein (PV), the most common trigger of atrial fibrillation, and investigated the underlying ionic mechanisms. Conventional microelectrodes or whole-cell patch clamps were employed to study the effects of ITI-214 (0.1-10μM) on PV electrical activity, mechanical responses and ionic currents in isolated rabbit PV tissue specimens and isolated single PV cardiomyocytes. ITI-214 at 1μM and 10μM (but not 0.1μM) significantly reduced PV spontaneous beating rate (10±2% and 10±3%, respectively) and PV diastolic tension (11±3% and 17±3%, respectively). ITI-24 (1μM) significantly reduced late sodium current (INa-Late ), L-type calcium current (ICa-L ) and the reverse mode of the sodium-calcium exchanger (NCX), but it did not affect peak sodium currents. ITI-214 reduces PV spontaneous activity and PV diastolic tension by reducing INa-Late , ICa-L and NCX current. Considering its therapeutic potential in heart failure, targeting PDE-1 inhibition may provide a novel strategy for managing atrial arrhythmogenesis.

Abstract 202: In Vitro Effect of Trisulfate Disaccharide on ICa (L-type), INa, IK, and the Na/Ca Exchange Current Recorded From Rat Ventricular Myocytes by Patch Clamp

Background: Ion channels are pharmacological targets for antiarrhythmic action, and drugs currently used for this purpose are generally not specific to a site of action and may act on several channels and even trigger proarrhythmic phenomena. Trisulfate disaccharide (TD) is an heparin fragment known to act on the sodium calcium exchanger (NCX), reducing intracellular calcium in overload situations and reversing arrhytmias, but its action on other ionic currents is unknown. Objective: To evaluate by patch clamp the action of TD at different concentrations in NCX and ionic currents in situations of intracellular calcium overload. Materials and Methods: Adult rat myocytes were obtained from a sample from ventricles. Currents were measured using the whole-cell variant of the patch clamp method. Creation of voltage clamp pulses and data acquisition was controlled by a computer with pClamp software. Peak inward current amplitude was measured for ion currents. For Na/Ca exchange current a ramp voltage protocol was employed. Three different concentrations of Cai (300nM, 400nM and 600nM) were used in separate experiments. One drug concentration was applied per cell (10, 30 and 100 micromolar each). The current sensitive to 5mM nickel was taken as the Na/Ca exchange current. The effects of TD on the INa, L-type Ca, and the potassium currents, transiente outward current (Ito), inwardly rectifying potassium current (IK1), and sustained current (Isus) recorded from adult rat ventricular myocytes were also examined in the same conditions. Results: TD concentration-dependently increased the inward Na/Ca exchange current in all intracellular calcium concentration. The effects of TD on the INa, L-type Ca, and the potassium currents, Ito, IK1 and Isus was associated with less than 30% mean reduction on any current at the highest concentration of TD tested (100 micromolar) and still below the positive block controls for different channels that is above 40% block. Conclusion: TD acts on NCX under different concentrations used, without affecting other ionic currents, suggesting specificity in the mechanism of action and possibly not exerting a pro-arrhythmic activity, this effect being desirable for its possible use in reversal of cardiac arrhythmias.

Genetic Ablation of NCX1.1 Na+-dependent Inactivation Impacts Cardiac Action Potential and Ca2+ Transient

The cardiac Na+-Ca2+ exchanger (NCX) influences Ca2+ homeostasis therefore cardiac contractility, by extruding one Ca2+ for every three Na+ ions entering the myocyte. Intracellular Na inhibits NCX activity causing an inactivation process known as Na-dependent inactivation. Modulation of NCX by Na is assumed to be physiologically irrelevant, as relatively high [Na] is required for inactivation (half inactivation ∼15-20 mM). To challenge this view, we used CRISPR technology to genetically modify NCX1.1 to remove the Na dependent inactivation from NCX via K229Q mutation. This mutation exclusively removes Na+-dependent inactivation without affecting other regulatory properties (Ca2+ and pH) of NCX (Matsouka et al, JGP 109:273-86, 1997). Adult ventricular myocytes were isolated from K229Q and WT mice. We used the patch clamp technique to measure action potential duration (APD) and NCX current. Compared to WT, the APD of homozygous K229Q myocytes was prolonged (APD90 ms: K229Q 104±25, n=5; WT 43±8, n=3; 35°C, 1Hz). Nickel sensitive NCX currents were measured in the presence of 25 mM intracellular Na+ to promote inactivation. NCX current density was larger in K229Q homozygous mice (pA/pF: K229Q 0.98±0.14 n=5; and WT 0.55±0.08 n=5; 30°C, 50 mV). Ca2+ transients were recorded from field stimulated WT and K229Q myocytes loaded with the Ca2+ sensitive dye Fluo-4, and paced from 0.2 to 5 Hz, 35°C. The Ca2+ transient peak to half time decay was 160±10 ms (n=8) in WT and 205±16 ms (n=11) in K229Q myocytes (1 Hz). These results suggest the Na+-dependent inactivation, heretofore thought of as physiologically irrelevant, actually has an impact on cardiac myocytes properties.

Cardioprotection Conferred by a CRISPR/Cas9 Single Amino Acid Substitution of NCX1 (H165A): the PH Insensitive NCX Mouse

The cardiac sodium calcium-exchanger (NCX1), the dominant calcium efflux mechanism of cardiomyocytes, is strongly regulated by pH. This pH regulation is thought to contribute to ischemia/reperfusion injury by allowing Na ions to accumulate to very high levels during ischemia when NCX is disabled by low pH, resulting in massive Ca overload via reverse NCX when reperfusion suddenly restores normal pH and NCX function. We recently found in a heterologous system that substitution of a single Histidine for Alanine at position 165 (H165A) of NCX1 drastically decreases its pH sensitivity (John S et al., J Gen Physiol, 2018). We then used CRISPR/Cas9 technology (Ma X et al, Sci Reports, 2017) to produce the same single amino acid substitution in mice. We confirmed the mutation by sequencing and RT-qPCR. The mice live into adulthood and are fertile with reduced LV function. In enzymatically isolated adult ventricular myocytes, we used the patch clamp technique with voltage ramps to measure nickel sensitive NCX current. At normal pH, NCX current was the same in control and H165A mice. However, when pH was lowered to 6.4 using NH4Cl, NCX current in H165A mice was minimally inhibited, while NCX in control mice was reduced severely. In voltage mapping studies of control mouse hearts, acidosis to pH 6.4 resulted in the onset of abnormal cardiac rhythm, characterized by repetitive bursts of depolarization alternating with pauses. However, in H165A mice, rhythm was unaffected by lowering the pH. In preliminary whole heart ischemia/reperfusion studies using Langendorff perfusion, pH mice had a 58% reduction in the area of necrosis compared to WT. These results suggest that a pH insensitive NCX may be able to reduce ischemia/reperfusion injury and arrythmia.

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.

Arginine vasopressin modulates electrical activity and calcium homeostasis in pulmonary vein cardiomyocytes

BackgroundAtrial fibrillation (AF) frequently coexists with congestive heart failure (HF) and arginine vasopressin (AVP) V1 receptor antagonists are used to treat hyponatremia in HF. However, the role of AVP in HF-induced AF still remains unclear. Pulmonary veins (PVs) are central in the genesis of AF. The purpose of this study was to determine if AVP is directly involved in the regulation of PV electrophysiological properties and calcium (Ca2+) homeostasis as well as the identification of the underlying mechanisms.MethodsPatch clamp, confocal microscopy with Fluo-3 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, Ca2+ homeostasis, and Ca2+ regulatory proteins in isolated rabbit single PV cardiomyocytes incubated with and without AVP (1 μM), OPC 21268 (0.1 μM, AVP V1 antagonist), or OPC 41061 (10 nM, AVP V2 antagonist) for 4–6 h.ResultsAVP (0.1 and 1 μM)-treated PV cardiomyocytes had a faster beating rate (108 to 152%) than the control cells. AVP (1 μM) treated PV cardiomyocytes had higher late sodium (Na+) and Na+/Ca2+ exchanger (NCX) currents than control PV cardiomyocytes. AVP (1 μM) treated PV cardiomyocytes had smaller Ca2+i transients, and sarcoplasmic reticulum (SR) Ca2+ content as well as higher Ca2+ leak. However, combined AVP (1 μM) and OPC 21268 (0.1 μM) treated PV cardiomyocytes had a slower PV beating rate, larger Ca2+i transients and SR Ca2+ content, smaller late Na+ and NCX currents than AVP (1 μM)-treated PV cardiomyocytes. Western blot experiments showed that AVP (1 μM) treated PV cardiomyocytes had higher expression of NCX and p-CaMKII, and a higher ratio of p-CaMKII/CaMKII.ConclusionsAVP increases PV arrhythmogenesis with dysregulated Ca2+ homeostasis through vasopressin V1 signaling.

Coupling of the Na+/K+-ATPase to Ankyrin B controls Na+/Ca2+ exchanger activity in cardiomyocytes.

Ankyrin B (AnkB) is an adaptor protein that assembles Na+/K+-ATPase (NKA) and Na+/Ca2+ exchanger (NCX) in the AnkB macromolecular complex. Loss-of-function mutations in AnkB cause the AnkB syndrome in humans, characterized by ventricular arrhythmias and sudden cardiac death. It is unclear to what extent NKA binding to AnkB allows regulation of local Na+ and Ca2+ domains and hence NCX activity. To investigate the role of NKA binding to AnkB in cardiomyocytes, we synthesized a disruptor peptide (MAB peptide) and its AnkB binding ability was verified by pulldown experiments. As opposed to control, the correlation between NKA and NCX currents was abolished in adult rat ventricular myocytes dialyzed with MAB peptide, as well as in cardiomyocytes from AnkB+/- mice. Disruption of NKA from AnkB (with MAB peptide) increased NCX-sensed cytosolic Na+ concentration, reduced Ca2+ extrusion through NCX, and increased frequency of Ca2+ sparks and Ca2+ waves without concomitant increase in Ca2+ transient amplitude or SR Ca2+ load, suggesting an effect in local Ca2+ domains. Selective inhibition of the NKAα2 isoform abolished both the correlation between NKA and NCX currents and the increased rate of Ca2+ sparks and waves following NKA/AnkB disruption, suggesting that an AnkB/NKAα2/NCX domain controls Ca2+ fluxes in cardiomyocytes. NKA binding to AnkB allows ion regulation in a local domain, and acute disruption of the NKA/AnkB interaction using disruptor peptides lead to increased rate of Ca2+ sparks and waves. The functional effects were mediated through the NKAα2 isoform. Disruption of the AnkB/NKA/NCX domain could be an important pathophysiological mechanism in the AnkB syndrome.

Neurokinin-1 receptor antagonist decreases [Ca2+]i in airway smooth muscle cells by reducing the reverse-mode Na+/Ca2+ exchanger current

Airway smooth muscle (ASM) is involved in asthma airway inflammation. The aim of this study was to evaluate the effect of substance P and neurokinin-1 receptor (NK-1R) antagonist on intracellular calcium concentration ([Ca2+]i) in airway smooth muscle cells (ASMCs), ASMC contraction, and the effect on reverse-mode Na+–Ca2+ exchanger (NCX) currents in ASMCs. In our study, primary rat ASMCs were cultured. ASMCs were identified by immunofluorescence. [Ca2+]i variations were measured by fluorescence microscopy. Cell shortening (%) and relaxation (%) were analyzed with phase-contrast microscopy. Patch clamp techniques were used to assess NCX currents in ASMCs. We found that substance P increased, and NK-1R antagonist decreased [Ca2+]i in ASMCs. Substance P induced ASMCs contraction, and NK-1R antagonist can make ASMC relax. Patch clamp techniques were implemented to analyze NCX currents in ASMCs. Substance P increased reverse-mode NCX currents in ASMCs but the current density was lower than the one treated with acetylcholine (Ach). NK-1R antagonist reduced reverse-mode NCX current activity in ASMCs, and the current density was similar to the one treated with the reversed NCX inhibitor. So, we concluded that substance P increased [Ca2+]i in ASMCs by promoting the reverse-mode NCX current and stimulating ASMCs, whereas NK-1R antagonist decreased [Ca2+]i in ASMCs by decreasing the reverse-mode NCX current to make ASMCs relax.

Acute Genetic Ablation of Sodium-Calcium Exchange: Adaptations of Excitation-Contraction Coupling and Calcium Regulation

Previously described murine models of sodium-calcium exchange (NCX) ablation exhibit cardiac remodeling that complicates interpretation, likely a result of embryonic excision of the NCX gene and chronic adaptation over time. To investigate the effect of acute ablation of NCX, we created a cardiac specific NCX KO in adult mice using the tamoxifen-inducible cardiac specific (αMHC-MerCreMer) promoter. We injected tamoxifen (40mg/kg/day IP) for 5 consecutive days. One week post tamoxifen, NCX protein expression was reduced by 96% in ventricular tissue and NCX current was completely eliminated in 4/9 cells (85% average reduction). LV function by echocardiography was preserved and, unlike ventricular-specific NCX KO mice produced by the MLC2v promoter, we observed no fibrosis or hypertrophy. We observed a 12% increase of resting calcium and normal Ca transient amplitudes in isolated cardiomyocytes loaded with fura-2 AM (30 min, 2.5 μM), despite a 37% reduction in ICa (P < 0.001), a two-fold increase of SERCA2 (P<0.01) and a two-fold increase of PMCA (P<0.05). Reduced ICa and preserved Ca release are indicative of increased EC coupling gain, which we have observed in MLC2v NCX KO mice. However, changes in SERCA and PMCA are distinct from the MLC2v NCX KO at 8-12 weeks, where resting Ca was similar to wildtype and SERCA and PMCA protein expression were unchanged. 4 weeks after tamoxifen, NCX expression and current were virtually eliminated and ICa was reduced by 47%. SERCA and PMCA continued to increase but fibrosis and hypertrophy developed despite normalization of diastolic Ca. In conclusion, mice survive after acute ablation of NCX by reducing Ca influx, increasing EC coupling gain, and increasing alternative Ca efflux and SR uptake mechanisms. However, prolonged absence of NCX results in cardiac remodeling, possibly by activating Ca-dependent gene programming.

NCX-Mediated Subcellular Ca2+ Dynamics Underlying Early Afterdepolarizations in LQT2 Cardiomyocytes

Long QT syndrome type 2 (LQT2) is a congenital disease characterized by loss of function mutations in hERG potassium channels (IKr). LQT2 is associated with fatal ventricular arrhythmias promoted by triggered activity in the form of early afterdepolarizations (EADs). We previously demonstrated that intracellular Ca2+ handling is remodeled in LQT2 myocytes. Remodeling leads to aberrant late RyR-mediated Ca2+ releases that drive forward-mode Na+-Ca2+ exchanger (NCX) current and slow repolarization to promote reopening of L-type calcium channels and EADs. Forward-mode NCX was found to be enhanced despite the fact that these late releases do not significantly alter the whole-cell cytosolic calcium concentration during a vulnerable period of phase 2 of the action potential corresponding to the onset of EADs. Here, we use a multiscale ventricular myocyte model to explain this finding. We show that because the local NCX current is a saturating nonlinear function of the local submembrane calcium concentration, a larger number of smaller-amplitude discrete Ca2+ release events can produce a large increase in whole-cell forward-mode NCX current without increasing significantly the whole-cell cytosolic calcium concentration. Furthermore, we develop novel insights, to our knowledge, into how alterations of stochastic RyR activity at the single-channel level cause late aberrant Ca2+ release events. Experimental measurements in transgenic LTQ2 rabbits confirm the critical arrhythmogenic role of NCX and identify this current as a potential target for antiarrhythmic therapies in LQT2.

Heparin Oligosaccharides Have Antiarrhythmic Effect by Accelerating the Sodium-Calcium Exchanger.

Background: Blockage of the Na+/Ca2+ exchanger (NCX) is used to determine the role of NCX in arrhythmogenesis. Trisulfated heparin disaccharide (TD) and Low Molecular Weight Heparins (LMWHs) can directly interact with the NCX and accelerate its activity.Objective: In this work, we investigated the antiarrhythmic effect of heparin oligosaccharides related to the NCX activity.Methods: The effects of heparin oligosaccharides were tested on the NCX current (patch clamping) and intracellular calcium transient in rat cardiomyocytes. The effects of heparin oligosaccharides were further investigated in arrhythmia induced in isolated rat atria and rats in vivo.Results: The intracellular Ca2+ concentration decreases upon treatment with either enoxaparin or ardeparin. These drugs abolished arrhythmia induction in isolated atria. The NCX antagonist KB-R7943 abolished the enoxaparin or ardeparin antiarrhythmic effects in isolated atria. In the in vivo measurements, injection of TD 15 min both before coronary occlusion or immediately after reperfusion, significantly prevented the occurrence of reperfusion-induced arrhythmias (ventricular arrhythmia and total AV block) and reduced the lethality rate. The patch clamping experiments showed that, mechanistically, TD increases the forward mode NCX current.Conclusion: Together, the data shows that heparin oligosaccharides may constitute a new class of antiarrhythmic drug that acts by accelerating the forward mode NCX under calcium overload.

Swimming exercise reverses aging-related contractile abnormalities of female heart by improving structural alterations.

The objective of this study was to examine the effect of swimming exercise on aging-related Ca2+ handling alterations and structural abnormalities of female rat heart. For this purpose, 4-month and 24-month old female rats were used and divided into three following groups: sedentary young (SY), sedentary old (SO), and exercised old (Ex-O). Swimming exercise was performed for 8 weeks (60 min/day, 5 days/week). Myocyte shortening, L-type Ca2+ currents and associated Ca2+ transients were measured from ventricular myocytes at 36 ± 1°C. NOX-4 levels, aconitase activity, glutathione measurements and ultrastructural examination by electron microscopy were conducted in heart tissue. Swimming exercise reversed the reduced shortening and slowed kinetics of aged cardiomyocytes. Although the current density was similar for all groups, Ca2+ transients were higher in SO and Ex-O myocytes with respect to the SY group. Caffeine-induced Ca2+ transients and the integrated NCX current were lower in cardiomyocytes of SY rats compared with other groups, suggesting an increased sarcoplasmic reticulum Ca2+ content in an aged heart. Aging led to upregulated cardiac NOX-4 along with declined aconitase activity. Although it did not reverse these oxidative parameters, swimming exercise achieved a significant increase in glutathione levels and improved structural alterations of old rats' hearts. We conclude that swimming exercise upregulates antioxidant defense capacity and improves structural abnormalities of senescent female rat heart, although it does not change Ca2+ handling alterations further. Thereby, it improves contractile function of aged myocardium by mitigating detrimental effects of oxidative stress.

Abstract 12646: Cellular Mechanisms Underlying Post-operative Atrial Fibrillation in Patients

Background: Post-operative atrial fibrillation (poAF) after cardiac surgery is a common condition. Although usually self-limiting, poAF increases length of hospital stay and can cause debilitating strokes. A multifactorial pathophysiology involving triggered activity has been hypothesized, but the underlying cellular and molecular mechanisms are unknown and were the subject of this study. Methods: Multicellular action potentials (APs) were recorded with sharp microelectrodes in right atrial trabeculae obtained from sinus-rhythm patients during open-heart surgery and correlated with subsequent non-poAF (Ctl) or poAF status. Membrane currents (whole-cell voltage clamp) and [Ca 2+ ] i (Fluo-3) were also measured in right atrial cardiomyocytes isolated from Ctl and poAF patient samples. Protein expression was quantified by immunoblot. Results: In poAF, AP duration at 90% repolarization (APD 90 ) was slightly prolonged (Fig A), whereas AP amplitude, upstroke velocity, APD 20 , APD 50, and resting membrane potential (RMP) were unchanged. I Ca,L -triggered [Ca 2+ ] i transient (CaT) amplitude was 37% lower in poAF despite greater Ca 2+ influx via L-type Ca 2+ current (integral I Ca,L ), pointing to sarcoplasmic reticulum (SR) dysfunction (Fig B). Na + -Ca 2+ exchange (NCX) activity was similar in both groups (Fig C). SR Ca 2+ content calculated by integrating NCX current (I NCX ) during caffeine-induced SR Ca 2+ release was decreased by 50% in poAF (Fig C), likely because of smaller expression of SR Ca 2+ -ATPase-2a (SERCA2a) and decreased Ser16-phosphorylation (stronger SERCA2a inhibition) of the SERCA2a regulator phospholamban (PLB, Fig D). Expression of cardiac SR ryanodine receptor (RyR2) was reduced by 56% (Fig D), consistent with the reduced CaT amplitude. Conclusions: PoAF is associated with prolonged APD and impaired SR function. These abnormalities likely result in the Ca 2+ mishandling that predisposes patients to triggered activity underlying poAF.