Cardiac Sodium-calcium Exchanger Research Articles

SAR296968, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Ca2+ Handling and Contractile Function in Human Atrial Cardiomyocytes.

Background: In reverse-mode, cardiac sodium-calcium exchanger (NCX) can increase the cytoplasmic Ca2+ concentration in response to high intracellular Na+ levels, which may contribute to diastolic contractile dysfunction. Furthermore, increased spontaneous Ca2+ release from intracellular stores can activate forward mode NCX. The resulting transient inward current causes delayed afterdepolarization (DAD)-dependent arrhythmias. Moreover, recently, NCX has been associated with impaired relaxation and reduced cardiac function in heart failure with preserved ejection fraction (HFpEF). Since NCX is upregulated in human chronic atrial fibrillation (AF) as well as heart failure (HF), specific inhibition may have therapeutic potential. Objective: We tested the antiarrhythmic, lusitropic and inotropic effects of a novel selective NCX-inhibitor (SAR296968) in human atrial myocardium. Methods and Results: Right atrial appendage biopsies of 46 patients undergoing elective cardiac surgery in a predominant HFpEF cohort (n = 24/46) were investigated. In isolated human atrial cardiomyocytes, SAR296968 reduced the frequency of spontaneous SR Ca2+ release events and increased caffeine transient amplitude. In accordance, in isolated atrial trabeculae, SAR296968 enhanced the developed tension after a 30 s pause of electrical stimulation consistent with reduced diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Moreover, compared to vehicle, SAR296968 decreased steady-state diastolic tension (at 1 Hz) without impairing developed systolic tension. Importantly, SAR296968 did not affect the safety parameters, such as resting membrane potential or action potential duration as measured by patch clamp. Conclusion: The novel selective NCX-inhibitor SAR296968 inhibits atrial pro-arrhythmic activity and improves diastolic and contractile function in human atrial myocardium, which may have therapeutic implications, especially for treatment of HFpEF.

Cardioprotection in ischemia-reperfusion injury achieved by a single amino acid substitution of NCX1 (H165A)

We used genetically modified mice to test the hypothesis that pH regulation of the cardiac sodium calcium-exchanger (NCX1) contributes to ischemia/reperfusion (I/R) injury. NCX1 is the dominant calcium (Ca) efflux mechanism of cardiomyocytes and is strongly regulated by pH. During ischemia, when pH is lowered by anaerobic glycolysis, inhibition of NCX1 by acidosis may potentiate the Na overload imposed by Na/K pump failure and Na-H exchange. Upon reperfusion and restoration of normal pH, this high intracellular Na concentration drives the now functional NCX1 into reverse mode resulting in Ca overload.

Design of a Proteolytically Stable Sodium-Calcium Exchanger 1 Activator Peptide for In Vivo Studies.

The cardiac sodium–calcium exchanger (NCX1) is important for normal Na+- and Ca2+-homeostasis and cardiomyocyte relaxation and contraction. It has been suggested that NCX1 activity is reduced by phosphorylated phospholemman (pSer68-PLM); however its direct interaction with PLM is debated. Disruption of the potentially inhibitory pSer68-PLM-NCX1 interaction might be a therapeutic strategy to increase NCX1 activity in cardiac disease. In the present study, we aimed to analyze the binding affinities and kinetics of the PLM-NCX1 and pSer68-PLM-NCX1 interactions by surface plasmon resonance (SPR) and to develop a proteolytically stable NCX1 activator peptide for future in vivo studies. The cytoplasmic parts of PLM (PLMcyt) and pSer68-PLM (pSer68-PLMcyt) were found to bind strongly to the intracellular loop of NCX1 (NCX1cyt) with similar K D values of 4.1 ± 1.0 nM and 4.3 ± 1.9 nM, but the PLMcyt-NCX1cyt interaction showed higher on/off rates. To develop a proteolytically stable NCX1 activator, we took advantage of a previously designed, high-affinity PLM binding peptide (OPT) that was derived from the PLM binding region in NCX1 and that reverses the inhibitory PLM (S68D)-NCX1 interaction in HEK293. We performed N- and C-terminal truncations of OPT and identified PYKEIEQLIELANYQV as the minimum sequence required for pSer68-PLM binding. To increase peptide stability in human serum, we replaced the proline with an N-methyl-proline (NOPT) after identification of N-terminus as substitution tolerant by two-dimensional peptide array analysis. Mass spectrometry analysis revealed that the half-life of NOPT was increased 17-fold from that of OPT. NOPT pulled down endogenous PLM from rat left ventricle lysate and exhibited direct pSer68-PLM binding in an ELISA-based assay and bound to pSer68-PLMcyt with a K D of 129 nM. Excess NOPT also reduced the PLMcyt-NCX1cyt interaction in an ELISA-based competition assay, but in line with that NCX1 and PLM form oligomers, NOPT was not able to outcompete the physical interaction between endogenous full length proteins. Importantly, cell-permeable NOPT-TAT increased NCX1 activity in cardiomyocytes isolated from both SHAM-operated and aorta banded heart failure (HF) mice, indicating that NOPT disrupted the inhibitory pSer68-PLM-NCX1 interaction. In conclusion, we have developed a proteolytically stable NCX1-derived PLM binding peptide that upregulates NCX1 activity in SHAM and HF cardiomyocytes.

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.

Protein Phosphatase 1c Associated with the Cardiac Sodium Calcium Exchanger 1 Regulates Its Activity by Dephosphorylating Serine 68-phosphorylated Phospholemman

The sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis. Serine 68-phosphorylated phospholemman (pSer-68-PLM) inhibits NCX1 activity. In the context of Na(+)/K(+)-ATPase (NKA) regulation, pSer-68-PLM is dephosphorylated by protein phosphatase 1 (PP1). PP1 also associates with NCX1; however, the molecular basis of this association is unknown. In this study, we aimed to analyze the mechanisms of PP1 targeting to the NCX1-pSer-68-PLM complex and hypothesized that a direct and functional NCX1-PP1 interaction is a prerequisite for pSer-68-PLM dephosphorylation. Using a variety of molecular techniques, we show that PP1 catalytic subunit (PP1c) co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes, left ventricle lysates, and HEK293 cells. Bioinformatic analysis, immunoprecipitations, mutagenesis, pulldown experiments, and peptide arrays constrained PP1c anchoring to the K(I/V)FF motif in the first Ca(2+) binding domain (CBD) 1 in NCX1. This binding site is also partially in agreement with the extended PP1-binding motif K(V/I)FF-X5-8Φ1Φ2-X8-9-R. The cytosolic loop of NCX1, containing the K(I/V)FF motif, had no effect on PP1 activity in an in vitro assay. Dephosphorylation of pSer-68-PLM in HEK293 cells was not observed when NCX1 was absent, when the K(I/V)FF motif was mutated, or when the PLM- and PP1c-binding sites were separated (mimicking calpain cleavage of NCX1). Co-expression of PLM and NCX1 inhibited NCX1 current (both modes). Moreover, co-expression of PLM with NCX1(F407P) (mutated K(I/V)FF motif) resulted in the current being completely abolished. In conclusion, NCX1 is a substrate-specifying PP1c regulator protein, indirectly regulating NCX1 activity through pSer-68-PLM dephosphorylation.

GATA-Binding Factor 6 Contributes to Atrioventricular Node Development and Function.

Several transcription factors regulate cardiac conduction system (CCS) development and function but the role of each in specifying distinct CCS components remains unclear. GATA-binding factor 6 (GATA6) is a zinc-finger transcription factor that is critical for patterning the cardiovascular system. However, the role of GATA6 in the embryonic heart and CCS has never been shown. We report that Gata6 is expressed abundantly in the proximal CCS during midgestation in mice. Myocardial-specific deletion of the carboxyl zinc-finger of Gata6 induces loss of hyperpolarizing cyclic nucleotide-gated channel, subtype 4 staining in the compact atrioventricular node with some retention of hyperpolarizing cyclic nucleotide-gated channel, subtype 4 staining in the atrioventricular bundle, but has no significant effect on the connexin-40-positive bundle branches. Furthermore, myocardial-specific deletion of the carboxyl zinc-finger of Gata6 alters atrioventricular conduction in postnatal life as assessed by surface and invasive electrophysiological evaluation, as well as decreasing the number of ventricular myocytes and inducing compensatory myocyte hypertrophy. Myocardial-specific deletion of the carboxyl zinc-finger of Gata6 is also associated with downregulation of the transcriptional repressor ID2 and the cardiac sodium-calcium exchanger NCX1 in the proximal CCS, where GATA6 transactivates both of these factors. Finally, carboxyl zinc-finger deletion of Gata6 reduces cell-cycle exit of TBX3+ myocytes in the developing atrioventricular bundle during the period of atrioventricular node specification, which results in fewer TBX3+ cells in the proximal CCS of mature mutant mice. GATA6 contributes to the development and postnatal function of the murine atrioventricular node by promoting cell-cycle exit of specified cardiomyocytes toward a conduction system lineage.

Liberation of Pser68-Plm Inhibition of NCX1 by an Optimized Anchoring Disruptor Peptide

The cardiac sodium-calcium exchanger (NCX1) is an important regulator of intracellular Ca2+ ([Ca2+]i) and a potential therapeutic target in heart failure. Among potential interacting partners regulating NCX1 activity is the transmembrane protein phospholemman (PLM); a substrate for protein kinase A and protein kinase C. Several reports have demonstrated that binding of phosphorylated PLM (pSer68-PLM) to NCX1 leads to NCX1 inhibition, while other studies have failed to demonstrate a functional interaction of these proteins. In present study, we aimed to analyze the biological function of the pSer68-PLM-NCX1 interaction by developing anchoring disruptor peptides. We observed that PLM co-fractionated and co-immunoprecipitated with NCX1 in rat left ventricle (LV) and in co-transfected HEK293 cells. Strong binding of pSer68-PLM to one of the previously reported NCX1-QKHPD regions was demonstrated in pull-down and overlay assays. Single amino acid substitutions of native NCX1 peptide sequence 1-KHPDKEIEQLIELANYQVLS-20 revealed that single substitutions at position 1-11 (particularly with tryptophan or tyrosine) increased binding affinity of the peptide to pSer68-PLM. Interestingly, we found that double substitution at position 4 and 6 with tyrosine (D4Y and E6Y) in the native peptide sequence enhanced the binding affinity to pSer68-PLM 8-fold, compared to native sequence. The optimized peptide 1-KHPYKYIEQLIELANYQVLS-20 was further tested in a series of electrophysiological experiments using whole-cell voltage clamp technique, with voltage-ramp protocol employed to elicit NCX current. Constitutively phosphorylated (S68D) PLM was observed to inhibit NCX1 current in both forward and reverse mode. Inclusion of the disruptor peptide in the patch pipette reversed S68D-PLM inhibition of NCX1, while NCX current was not altered by a scrambled peptide control. Taken together these data strongly suggest that PLM interacts directly with NCX1, and that when PLM is phosphorylated at serine 68, the interaction inhibits NCX1 activity.

Burst Pacemaker Activity in NCX1 Knockout Mice: Is it Funny Current?

The cardiac Sodium-Calcium exchanger (NCX1) is the dominant Ca efflux protein in SinoAtrial Node (SAN) cells. NCX1 has also been implicated in the generation of pacemaker activity, through the “Ca clock”. To study SAN pacing in the absence of NCX1, we created an atrial-specific NCX1 KO mouse using a Cre/loxP system under the control of the endogenous sarcolipin promoter. NCX1 KO mice have complete AV block and no P waves. The latter is caused by conduction interference between SAN and atria. To test the hypothesis that SAN pacemaking can be maintained by funny current (If) in the absence of NCX1, we recorded Ca transients (using Cal 520 and high speed 2D confocal imaging) in an ex vivo tissuepreparation that included the SAN, right atrium and left atrium. At ∼36°C, the SAN pacemaker rate was 5.3±0.3Hz in WT (n=14) but only 2±0.2Hz (n=17) in KO. The pattern of pacing in KO was characterized by frequent bursts of Ca transients alternating with long pauses. During bursts in KO, the rate was comparable to WT (4.1±0.3Hz). WT SANs (n=5) showed a 70.5±6.1% increase in rate after exposure to the β-adrenergic agonist ISO (10μM), while KO showed no increase in average rate. However, when considering only rate during burst activity in KO, 6 out of 10 KO SANs responded significantly to ISO 10μM (52±16% increase). The specific If inhibitor ivabradine (IVA, 9μM) reduced the spontaneous pacemaker rate in both WT (n=6) and KO (n=8) SANs (44±9% and 58.9±6.8% decrease, respectively), and even during the bursts in the KO (36±17.9% decrease). Thus, NCX1 KO SANs can generate bursts of pacemaker activity similar to WT. These bursts are responsive to both ISO and IVA, consistent with If-mediated pacemaker activity despite the absence of NCX.

The cardiac sodium–calcium exchanger NCX1 is a key player in the initiation and maintenance of a stable heart rhythm

The complex molecular mechanisms underlying spontaneous cardiac pacemaking are not fully understood. Recent findings point to a co-ordinated interplay between intracellular Ca(2+) cycling and plasma membrane-localized cation transport determining the origin and periodicity of pacemaker potentials. The sodium-calcium exchanger (NCX1) is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. Here, we investigated the contribution of NCX1 to cardiac pacemaking. We used an inducible and sinoatrial node-specific Cre transgene to create micelacking NCX1 selectively in cells of the cardiac pacemaking and conduction system (cpNCX1KO). RT-PCR and immunolabeling experiments confirmed the precise tissue-specific and temporally controlled deletion. Ablation of NCX1 resulted in a progressive slowing of heart rate accompanied by severe arrhythmias. Isolated sinoatrial tissue strips displayed a significantly decreased and irregular contraction rate underpinning a disturbed intrinsic pacemaker activity. Mutant animals displayed a gradual increase in the heart-to-body weight ratio and developed ventricular dilatation; however, their ventricular contractile performance was not significantly affected. Pacemaker cells from cpNCX1KO showed no NCX1 activity in response to caffeine-induced Ca(2+) release, determined by Ca(2+) imaging. Regular spontaneous Ca(2+) discharges were frequently seen in control, but only sporadically in knockout (KO) cells. The majority of NCX1KO cells displayed an irregular and a significantly reduced frequency of spontaneous Ca(2+) signals. Furthermore, Ca(2+) transients measured during electrical field stimulation were of smaller magnitude and decelerated kinetics in KO cells. Our results establish NCX1 as a critical target for the proper function of cardiac pacemaking.

Atrial-Specific NCX KO Mice Reveal Dependence of Sinoatrial Node Pacemaker Activity on Sodium-Calcium Exchange

The cardiac sodium-calcium exchanger (NCX1) is hypothesized to play a major role in sinoatrial node (SAN) pacemaker activity. To test this hypothesis directly, we used cre/loxP technology to generate an atrial-specific knockout (KO) of NCX1 in mice using the sarcolipin promoter. At 12 weeks, there is no evidence of NCX1 in the atrium or SAN by immunostaining or immunoblot. KO mice exhibit atrial dilation and ventricular hypertrophy with mild ventricular dysfunction. On electrocardiography, KO mice have no P waves and a relatively slow junctional escape rhythm (231 ± 16 bpm, n = 4) compared to normal sinus rhythm in WT (422 ± 63 bpm, n = 2; p = 0.01). Furthermore, recordings of cardiac electrograms in Langendorff-perfused hearts show no evidence of atrial activity in KO. In patch clamped SAN cells isolated from KO mice, there is no NCX activity in response to caffeine-induced SR Ca2+ release. L-type Ca2+ current is decreased in KO by ∼50% but there is no significant difference in funny current (If) amplitude between WT and KO. Spontaneous action potentials (APs) are absent in KO, even after application of isoproterenol (ISO, 1μM). However, we were still able to evoke APs in patch clamped KO cells under current clamp conditions, indicating that KO cells are capable of electrically stimulated depolarization. The maximum diastolic potential (MDP) was slightly more depolarized in KO (- 57 ± 2.0 mV) compared to WT (- 70 ± 2.5 mV, p < 0.001), which could theoretically reduce spontaneous activity. However, reducing extracellular K+ to lower the MDP in KO to WT values failed to restore rhythmic pacemaker activity. Thus, we conclude that NCX1 is required for normal pacemaker activity in the murine SAN.

Regional genomic regulation of cardiac sodium–calcium exchanger by oestrogen

Female rabbit hearts are more susceptible to torsade de pointes (TdP) in acquired long QT type 2 than males, in-part due to higher L-type Ca2+ current (ICa,L) at the base of the heart. In principle, higher Ca2+ influx via ICa,L should be balanced by higher efflux, perhaps mediated by parallel sex differences of sodium-calcium exchange (NCX) current (INCX). We now show that NCX1, like Cav1.2α, is greater at the base of female than male left ventricular epicardium and greater at the base than at the apex in both sexes. In voltage-clamp studies, inward (0, +20 mV, P < 0.04) and outward (-80, -60, -40, -20 mV, P < 0.01) INCX densities were significantly higher (1.5-2 fold) in female base compared to apex and male (base and apex) myocytes. Myocytes were incubated ±17β-oestradiol (E2 = 1 nm) and INCX was measured on days 0, 1, 2 and 3. Inward and outward INCX decreased over 2 days in female base myocytes becoming similar to INCX at the apex. E2 incubation (24 h) increased NCX1 (50%) and INCX (∼3-fold at 60 mV) in female base but not endocardium, apex or in male base myocytes. INCX upregulation by E2 was blunted by an oestrogen receptor (ER) antagonist (fulvestrant, 1 μm), and inhibition of transcription (actinomycin D, 5 μg ml-1) or translation (cycloheximide, 20 μg ml-1). Dofetilide (an IKr blocker) induced early afterdepolarizations (EADs) in female base myocytes cultured for 1 day if incubated with E2, but not without E2 or with E2+KB-R4973 (an INCX inhibitor), E2+fulvestrant or E2 with apex myocytes. Thus, E2 upregulates NCX1 by a genomic mechanism mediated by ERs, and de novo mRNA and protein biosynthesis, in a sex- and region-dependent manner which contributes to the enhanced propensity to EADs and TdP in female hearts.

Ec Coupling is Preserved in Ncx Knockout Mice Exposed to Metabolic Inhibitors

To determine whether ablating the cardiac sodium-calcium exchanger (NCX) prevents couplon loss during metabolic inhibition (MI), we recorded Ca transients or resting Ca spark frequency (CaSpF) in ventricular myocytes isolated from WT and ventricular-specific NCX knockout (KO) mice. In field stimulated myocytes loaded with 10 µM Fluo-3 AM, application of the mitochondrial and glycolytic inhibitors FCCP (50 nM) and 2-DG (10 mM) reduced Ca transients by 54.2 ± 3.3% in WT but only by 19.0 ± 6.0% in KO (p <0.05). Similarly, MI suppressed action potential evoked Ca sparks in patch clamped WT myocytes loaded with 1 mM fluo-3 salt by 60.0 ± 8.2% (p <0.05), but in KO by only 33.0 ± 3.4%. The reduction in ICa during MI was similar for both WT and KO and SR Ca stores remained at control levels in both cell types during MI. In patch clamped myocytes loaded with 1 mM K5Fluo-3 and held at −75 mV, MI reduced resting CaSpF in WT by 92.4 ± 6.7%, (p=0.008) but had no effect on CaSpF in KO Abolishing Na and Ca gradients by using the same “internal” solution for both the bath and pipette (current clamp mode, VRest = 0 mV), eliminated the KO's resistance to MI, causing CaSpF to decline to a similar extent in both WT and KO (59.0 ± 6.2% and 60.7 ± 10.9%, respectively). This finding suggests that elevated diadic cleft Ca, a major characteristic of KO myocytes (Pott et al, Biophys J, 92: 1431, 2007; Neco et al. Biophys J, 99: 755, 2010), maintains effective triggering of SR Ca release during MI despite reduced ICa.

Local Control of Cardiac Sodium-Calcium Exchanger by PMCA in Submembrane Microdomain in Mouse Heart Cells

OBJECT: This study aimed at unveiling functional interaction between the Na/Ca exchanger (NCX) and the plasma membrane Ca2+-ATPase (PMCA) on the sarcolemmal membrane of heart cells. METHOD: The Na/Ca exchange current (INCX) was recorded from whole-cell clamped mouse ventricular myocytes under physiological conditions at 37°C. Functions of the ryanodine receptor and SERCA Ca2+-pump on the sarcoplasmic reticulum were abolished by using ryanodine and thapsigargin. The INCX was isolated as Ni2+-sensitive current component, under the conditions that eliminate other major membrane current systems. RESULTS: With the [Ca2+]i strongly buffered with 10 mM-BAPTA, the INCX was recorded as a time-independent current. However, with the [Ca2+]i only weakly buffered with 0.1 mM-BAPTA, the INCX showed a small current amplitude and a slow activation time-course. This was not observed when the [Ca2+]i was strongly buffered (with 10 mM-BAPTA). Inhibition of PMCA by intracellular administration of orthovanadate (VO43-) dramatically increased the amplitude of INCX and accelerated its activation kinetics. At the same time, orthovanadate shifted the [Ca2+]i-dependence of the INCX amplitude (EC50 = 0.97 µM) to lower levels (EC50 = 0.40 µM). Moreover, similar effects were observed by intracellular application of a selective PMCA inhibitor 5(6)-carboxyeosin. CONCLUSION: The PMCA regulates the operation of the NCX by altering local [Ca2+]i level around the NCX molecule. Because PMCA is driven by ATP, this functional coupling might serve as a mechanism that the intracellular metabolic status to the [Ca2+]i regulation and Ca2+ signaling in heart cells.

Myocardial Function With Reduced Expression of the Sodium-Calcium Exchanger

Background The complete removal of the cardiac sodium-calcium exchanger (NCX1) is associated with embryonic lethality, whereas its overexpression is linked to heart failure. To determine whether or not a reduced expression of NCX1 is compatible with normal heart structure and function, we studied 2 knockout (KO) mouse models with reduced levels of NCX1: a heterozygous global KO (HG-KO) with a 50% level of NCX1 expression in all myocytes, and a ventricular-specific KO (V-KO) with NCX1 expression in only 10% to 20% of the myocytes. Methods and Results Both groups of mice were evaluated at baseline, after transaortic constriction (TAC), and after acute or chronic β-adrenergic stimulation. At baseline, the HG-KO mice had smaller hearts and the V-KO mice had larger hearts than their wild-type (WT) controls ( P < .05). The HG-KO and their control WT mice had normal responses to TAC and β-adrenergic stimulation. However, the V-KO group was intolerant to TAC and had a significantly ( P < .05) blunted response to β-adrenergic stimulation as compared with the HG-KO mice and WT controls. Unlike the HG-KO mice, the V-KO mice did not tolerate chronic isoproterenol infusion. Telemetric analysis of the electrocardiogram, body temperature, and activity revealed a normal diurnal rhythm in all groups of mice, but confirmed shorter QT intervals along with increased arrhythmias and reduced R wave to P wave amplitude ratios in the V-KO mice. Conclusions Though NCX1 can be reduced by half in all myocytes without significant functional alterations, it must be expressed in more than 20% of the myocytes to prevent severe remodeling and heart failure in mouse heart.

The Cardiac Sodium‐Calcium Exchanger Gene (NCX‐1) is a Potential Canine Cardiac Biomarker of Chronic Mitral Valvular Insufficiency

The cardiac sodium-calcium exchanger gene (NCX-1) is upregulated in humans and mice with congestive heart failure (CHF). NCX-1 expression is upregulated in dogs with heart failure from chronic mitral valvular insufficiency (CMVI). Client-owned 14 healthy control dogs and 30 dogs with CMVI. Prospective, controlled, observational study. We investigated the levels of NCX-1 expression in dogs at different stages of CMVI with real-time reverse transcription polymerase chain reaction. The mRNA expression levels of NCX-1 were determined in peripheral blood samples obtained from the animals used in this study. Dogs were graded by the severity of disease. The fold differences in the levels of mRNA expression compared with controls were 1.39 +/- 0.88 (group I), 1.32 +/- 0.65 (group II), 4.86 +/- 1.25 (group III), and 5.96 +/- 1.69 (group IV). NCX-1 expression was significantly higher in groups III and IV (P < .05) compared with the healthy controls, whereas groups I and II were not. The level of NCX-1 expression was significantly higher in groups of dogs with moderate to severe CMVI (groups III and IV) compared with the controls. Our findings indicate that NCX-1 can be a biomarker for chronic valvular disease in dogs and is a potential biomarker for severity of heart disease.

Lentiviral Vectors Bearing the Cardiac Promoter of the Na+-Ca2+ Exchanger Report Cardiogenic Differentiation in Stem Cells

Cardiosphere-derived resident cardiac stem cells (CDCs) are readily isolated from adult hearts and confer functional benefit in animal models of heart failure. To study cardiogenic differentiation in CDCs, we developed a method to genetically label and selectively enrich for cells that have acquired a cardiac phenotype. Lentiviral vectors achieved significantly higher transduction efficiencies in CDCs than any of the nine adeno-associated viral (AAV) serotypes tested. To define the most suitable vector system for reporting cardiogenic differentiation, we compared the cell specificity of five commonly-used cardiac-specific promoters in the context of lentiviral vectors. The promoter of the cardiac sodium-calcium exchanger (NCX1) conveyed the highest degree of cardiac specificity, as assessed by transducing seven cell types with each vector and measuring fluorescence intensity by flow cytometry. NCX1-GFP-positive CDC subpopulations, demonstrating prolonged expression of a variety of cardiac markers, could be isolated and expanded in vitro. Finally, we used chemical biology to validate that lentiviral vectors bearing the cardiac NCX1-promoter can serve as a highly accurate biosensor of cardiogenic small molecules in stem cells. The ability to accurately report cardiac fate and selectively enrich for cardiomyocytes and their precursors has important implications for drug discovery and the development of cell-based therapies.

Hypertrophy and Heart Failure in Mice Overexpressing the Cardiac Sodium-Calcium Exchanger

The cardiac sodium-calcium exchanger (NCX1) is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. Because heart failure is associated with increased expression of NCX1, heterozygous (HET) and homozygous (HOM) transgenic mice overexpressing NCX1 were developed and evaluated. The NCX1 transgenic mice display 2.3-fold (HET) and 3.1-fold (HOM) increases in exchanger activity from wild-type (WT) mice. Functional information was obtained by echocardiography and catheterizations before and after hemodynamic stress from pregnancy, treadmill exercise or transaortic constriction (TAC). HET and HOM mice exhibited hypertrophy and blunted responses with beta-adrenergic stimulation. Postpartum mice from all groups were hypertrophied, but only the HOM mice exhibited premature death from heart failure. HOM mice became exercise intolerant after 6 weeks of daily treadmill running. After 21 days TAC, HET, and HOM mice exhibited significant contractile dysfunction and 15% to 40% mortality with clinical evidence of heart failure. Hemodynamic stress results in a compensated hypertrophy in WT mice, but NCX1 transgenic mice exhibit decreased contractile function and heart failure in proportion to their level of NCX1 expression. Thus exchanger overexpression in mice leads to abnormal calcium handling and a decompensatory transition to heart failure with stress.

Cardiac sodium–calcium exchanger regulation by Nkx2–5 and three human Nkx2–5 mutations

Cardiac sodium–calcium exchanger regulation by Nkx2–5 and three human Nkx2–5 mutations

Functional alterations after cardiac sodium-calcium exchanger overexpression in heart failure

The sodium-calcium exchanger (NCX) is discussed as one of the key proteins involved in heart failure. However, the causal role and the extent to which NCX contributes to contractile dysfunction during heart failure are poorly understood. NCX overexpression was induced by infection with an adenovirus coding for NCX, which coexpressed green fluorescence protein (GFP) (AdNCX) by ex vivo gene transfer to nonfailing and failing rabbit cardiomyocytes. Myocardial gene transfer in rabbits in vivo was achieved by adenoviral delivery via aortic cross-clamping. Peak cell shortening of cardiomyocytes was determined photo-optically. Hemodynamic parameters in vivo were determined by echocardiography (fractional shortening) and tip catheter [maximal first derivative of left ventricular (LV) pressure (dP/dt(max)); maximal negative derivative of LV pressure (-dP/dt(max))]. Peak cell shortening was depressed after NCX gene delivery in isolated nonfailing and in failing cardiomyocytes. In nonfailing rabbits in vivo, basal systolic contractility (fractional shortening and dP/dt(max)) and maximum rate of LV relaxation (-dP/dt(max)) in vivo were largely unaffected after NCX overexpression. However, during heart failure, long-term NCX overexpression over 2 wk significantly improved fractional shortening and dP/dt(max) compared with AdGFP-infected rabbits, both without inotropic stimulation and after beta-adrenergic stimulation with isoproterenol. -dP/dt(max) was also improved after NCX overexpression in the failing rabbits group. These results indicate that short-term effects of NCX overexpression impair contractility of isolated failing and nonfailing rabbit cardiomyocytes. NCX overexpression over 2 wk in vivo does not seem to affect myocardial contractility in nonfailing rabbits. Interestingly, in vivo overexpression of NCX decreased the progression of systolic and diastolic contractile dysfunction and improved beta-adrenoceptor-mediated contractile reserve in heart failure in rabbits in vivo.