Marcel M. G. J. van Borren

Intracellular calcium modulation of voltage-gated sodium channels in ventricular myocytes

AIMS Cardiac voltage-gated sodium channels control action potential (AP) upstroke and cell excitability. Intracellular calcium (Ca(i)(2+)) regulates AP properties by modulating various ion channels. Whether Ca(i)(2+) modulates sodium channels in ventricular myocytes is unresolved. We studied whether Ca(i)(2+) modulates sodium channels in ventricular myocytes at Ca(i)(2+) concentrations ([Ca(i)(2+)]) present during the cardiac AP (0-500 nM), and how this modulation affects sodium channel properties in heart failure (HF), a condition in which Ca(i)(2+) homeostasis is disturbed. METHODS AND RESULTS Sodium current (I(Na)) and maximal AP upstroke velocity (dV/dt(max)), a measure of I(Na), were studied at 20 and 37 degrees C, respectively, in freshly isolated left ventricular myocytes of control and HF rabbits, using whole-cell patch-clamp methodology. [Ca(i)(2+)] was varied using different pipette solutions, the Ca(i)(2+) buffer BAPTA, and caffeine administration. Elevated [Ca(i)(2+)] reduced I(Na) density and dV/dt(max), but caused no I(Na) gating changes. Reductions in I(Na) density occurred simultaneously with increase in [Ca(i)(2+)], suggesting that these effects were due to permeation block. Accordingly, unitary sodium current amplitudes were reduced at higher [Ca(i)(2+)]. While I(Na) density and gating at fixed [Ca(i)(2+)] were not different between HF and control, reductions in dV/dt(max) upon increases in stimulation rate were larger in HF than in control; these differences were abolished by BAPTA. CONCLUSION Ca(i)(2+) exerts acute modulation of I(Na) density in ventricular myocytes, but does not modify I(Na) gating. These effects, occurring rapidly and in the [Ca(i)(2+)] range observed physiologically, may contribute to beat-to-beat regulation of cardiac excitability in health and disease.

Ae2(a,b)-Deficient mice exhibit osteopetrosis of long bones but not of calvaria

Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pHi) is thought to be kept at a near‐neutral level by chloride/bicarbonate exchange. Here we show that the Na+‐independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the longbonesof Ae2a,b–/– mice (deficient in the main isoforms Ae2a, Ae2b1, and Ae2b2). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long‐bone marrow cells from Ae2a,b–/– mice suggested a role for Ae2 in osteoclast formation, as fusion of preosteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2a,b–/– mice showed no alterations, indicating that calvaria osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvaria osteoclasts—but not long‐bone osteoclasts—possess a sodium‐dependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvaria osteoclasts of Ae2a,b–/– mice.—Jansen, I. D. C., Mardones, P., Lecanda, F., de Vries, T. J., Recalde, S., Hoeben, K. A., Schoenmaker, T., Ravesloot, J.‐H., van Borren, M. M. G. J., van Eijden, T. M., Bronckers, A. L. J. J., Kellokumpu, S., Medina, J. F., Everts, V., Oude Elferink, R. P. J. Ae2a,b‐Deficient mice exhibit osteopetrosis of long bones but not of calvaria. FASEB J. 23, 3470–3481 (2009). www.fasebj.org

Ca2+‐activated Cl− current in rabbit sinoatrial node cells

The Ca2+‐activated Cl− current (ICl(Ca)) has been identified in atrial, Purkinje and ventricular cells, where it plays a substantial role in phase‐1 repolarization and delayed after‐depolarizations. In sinoatrial (SA) node cells, however, the presence and functional role of ICl(Ca) is unknown. In the present study we address this issue using perforated patch‐clamp methodology and computer simulations. Single SA node cells were enzymatically isolated from rabbit hearts. ICl(Ca) was measured, using the perforated patch‐clamp technique, as the current sensitive to the anion blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS). Voltage clamp experiments demonstrate the presence of ICl(Ca) in one third of the spontaneously active SA node cells. The current was transient outward with a bell‐shaped current‐voltage relationship. Adrenoceptor stimulation with 1 μm noradrenaline doubled the ICl(Ca) density. Action potential clamp measurements demonstrate that ICl(Ca) is activate late during the action potential upstroke. Current clamp experiments show, both in the absence and presence of 1 μm noradrenaline, that blockade of ICl(Ca) increases the action potential overshoot and duration, measured at 20 % repolarization. However, intrinsic interbeat interval, upstroke velocity, diastolic depolarization rate and the action potential duration measured at 50 and 90 % repolarization were not affected. Our experimental data are supported by computer simulations, which additionally demonstrate that ICl(Ca) has a limited role in pacemaker synchronization or action potential conduction. In conclusion, ICl(Ca) is present in one third of SA node cells and is activated during the pacemaker cycle. However, ICl(Ca) does not modulate intrinsic interbeat interval, pacemaker synchronization or action potential conduction.

Reduced swelling-activated Cl− current densities in hypertrophied ventricular myocytes of rabbits with heart failure

OBJECTIVE Hypertrophied myocytes of failing hearts have prolonged action potential durations. It is unknown how the swelling-activated Cl(-) current (I(Cl,swell)) affects the abnormal AP configuration. METHODS We studied I(Cl,swell) in ventricular myocytes isolated from failing and age-matched normal rabbit hearts. We applied whole-cell patch-clamp methodology and activated I(Cl,swell) by lowering tonicity of the superfusate. RESULTS Neither with ruptured-patch nor with amphotericin B perforated-patch, whole-cell clamp we found I(Cl,swell) active under isotonic conditions in either the normal or the hypertrophied failing heart (HFH) myocytes. I(Cl,swell) caused AP shortening and resting membrane potential (V(m)) depolarization in an osmotic gradient-dependent fashion. However, in the HFH myocytes swelling-induced AP changes were significantly smaller, even though the cells underwent the same relative change in planar cell surface area. Voltage-clamp experiments revealed that in HFH myocytes I(Cl,swell) current density was approximately 50% reduced. CONCLUSION Reduced I(Cl,swell) densities in HFH myocytes cause limited AP shortening and V(m) depolarization upon swelling of the cells.

Sphingosine‐1‐Phosphate Receptor 1 Regulates Cardiac Function by Modulating Ca2+ Sensitivity and Na+/H+ Exchange and Mediates Protection by Ischemic Preconditioning

Background Sphingosine‐1‐phosphate plays vital roles in cardiomyocyte physiology, myocardial ischemia–reperfusion injury, and ischemic preconditioning. The function of the cardiomyocyte sphingosine‐1‐phosphate receptor 1 (S1P1) in vivo is unknown. Methods and Results Cardiomyocyte‐restricted deletion of S1P1 in mice (S1P1 α MHCC re) resulted in progressive cardiomyopathy, compromised response to dobutamine, and premature death. Isolated cardiomyocytes from S1P1 α MHCC re mice revealed reduced diastolic and systolic Ca2+ concentrations that were secondary to reduced intracellular Na+ and caused by suppressed activity of the sarcolemmal Na+/H+ exchanger NHE‐1 in the absence of S1P1. This scenario was successfully reproduced in wild‐type cardiomyocytes by pharmacological inhibition of S1P1 or sphingosine kinases. Furthermore, Sarcomere shortening of S1P1 α MHCC re cardiomyocytes was intact, but sarcomere relaxation was attenuated and Ca2+ sensitivity increased, respectively. This went along with reduced phosphorylation of regulatory myofilament proteins such as myosin light chain 2, myosin‐binding protein C, and troponin I. In addition, S1P1 mediated the inhibitory effect of exogenous sphingosine‐1‐phosphate on β‐adrenergic–induced cardiomyocyte contractility by inhibiting the adenylate cyclase. Furthermore, ischemic precondtioning was abolished in S1P1 α MHCC re mice and was accompanied by defective Akt activation during preconditioning. Conclusions Tonic S1P1 signaling by endogenous sphingosine‐1‐phosphate contributes to intracellular Ca2+ homeostasis by maintaining basal NHE‐1 activity and controls simultaneously myofibril Ca2+ sensitivity through its inhibitory effect on adenylate cyclase. Cardioprotection by ischemic precondtioning depends on intact S1P1 signaling. These key findings on S1P1 functions in cardiac physiology may offer novel therapeutic approaches to cardiac diseases.

Ca2+ cycling properties are conserved despite bradycardic effects of heart failure in sinoatrial node cells

Background: In animal models of heart failure (HF), heart rate decreases due to an increase in intrinsic cycle length (CL) of the sinoatrial node (SAN). Pacemaker activity of SAN cells is complex and modulated by the membrane clock, i.e., the ensemble of voltage gated ion channels and electrogenic pumps and exchangers, and the Ca2+ clock, i.e., the ensemble of intracellular Ca2+ ([Ca2+]i) dependent processes. HF in SAN cells results in remodeling of the membrane clock, but few studies have examined its effects on [Ca2+]i homeostasis. Methods: SAN cells were isolated from control rabbits and rabbits with volume and pressure overload-induced HF. [Ca2+]i concentrations, and action potentials (APs) and Na+–Ca2+ exchange current (INCX) were measured using indo-1 and patch-clamp methodology, respectively. Results: The frequency of spontaneous [Ca2+]i transients was significantly lower in HF SAN cells (3.0 ± 0.1 (n = 40) vs. 3.4 ± 0.1 Hz (n = 45); mean ± SEM), indicating that intrinsic CL was prolonged. HF slowed the [Ca2+]i transient decay, which could be explained by the slower frequency and reduced sarcoplasmic reticulum (SR) dependent rate of Ca2+ uptake. Other [Ca2+]i transient parameters, SR Ca2+ content, INCX density, and INCX-[Ca2+]i relationship were all unaffected by HF. Combined AP and [Ca2+]i recordings demonstrated that the slower [Ca2+]i transient decay in HF SAN cells may result in increased INCX during the diastolic depolarization, but that this effect is likely counteracted by the HF-induced increase in intracellular Na+. β-adrenergic and muscarinic stimulation were not changed in HF SAN cells, except that late diastolic [Ca2+]i rise, a prominent feature of the Ca2+ clock, is lower during β-adrenergic stimulation. Conclusions: HF SAN cells have a slower [Ca2+]i transient decay with limited effects on pacemaker activity. Reduced late diastolic [Ca2+]i rise during β-adrenergic stimulation may contribute to an impaired increase in intrinsic frequency in HF SAN cells.

Increased sarcolemmal Na+/H+ exchange activity in hypertrophied myocytes from dogs with chronic atrioventricular block

Dogs with compensated biventricular hypertrophy due to chronic atrioventricular block (cAVB), are more susceptible to develop drug-induced Torsade-de-Pointes arrhythmias and sudden cardiac death. It has been suggested that the increased Na+ influx in hypertrophied cAVB ventricular myocytes contribute to these lethal arrhythmias. The increased Na+ influx was not mediated by Na+ channels, in fact the Na+ current proved reduced in cAVB myocytes. Here we tested the hypothesis that increased activity of the Na+/H+ exchanger type 1 (NHE-1), commonly observed in hypertrophic hearts, causes the elevated Na+ influx. Cardiac acid-base transport was studied with a pH-sensitive fluorescent dye in ventricular myocytes isolated from control and hypertrophied cAVB hearts; the H+ equivalent flux through NHE-1, Na+-HCO−3 cotransport (NBC), Cl−/OH− exchange (CHE), and Cl−/HCO−3 exchange (AE) were determined and normalized per liter cell water and corrected for surface-to-volume ratio. In cAVB, sarcolemmal NHE-1 flux was increased by 65 ± 6.3% in the pHi interval 6.3–7.2 and NBC, AE, and CHE fluxes remained unchanged. Accordingly, at steady-state intracellular pH the total sarcolemmal Na+ influx by NHE-1 + NBC increased from 8.5 ± 1.5 amol/μm2/min in normal myocytes to 15 ± 2.4 amol/μm2/min in hypertrophied cAVB myocytes. We conclude that compensated cardiac hypertrophy in cAVB dogs is accompanied with an increased sarcolemmal NHE-1 activity. This in conjunction with unchanged activity of the other acid-base transporters will raise the intracellular Na+ in hypertrophied cAVB myocytes.