Na/K-ATPase inhibition by ouabain induces CaMKII-dependent apoptosis in adult rat cardiac myocytes

The positive inotropic effect produced by Na⁺/K⁺-ATPase inhibition has been used for the treatment of heart failure for over 200 years. Recently, administration of toxic doses of ouabain has been shown to induce cardiac myocyte apoptosis. However, whether prolonged administration of non-toxic doses of ouabain can also promote cardiac myocyte cell death has never been explored. The aim of this study was to assess whether non-toxic doses of ouabain can induce myocyte apoptosis and if so, to examine the underlying mechanisms. For this purpose, cardiac myocytes from rat and cat, two species with different sensitivity to digitalis, were cultured for 24 h in the presence or absence of 2 µM (rat) and 25 nm-2 µM ouabain (cat). Cell viability and apoptosis assays showed that ouabain produced, in the rat, a 43 ± 5% decrease in cell viability due to apoptosis (enhanced caspase-3 activity, increased Bax/Bcl-2 and TUNEL-positive nuclei) and necrosis (LDH release and trypan blue staining). Similar results were obtained with 25 nM ouabain in the cat. Ouabain-induced reduction in cell viability was prevented by the NCX inhibitor KB-R7943 and by the CaMKII inhibitors, KN93 and AIP. Furthermore, CaMKII overexpression exacerbated ouabain-induced cell mortality which in contrast was reduced in transgenic mice with chronic CaMKII inhibition. However, KN93 failed to affect ouabain-induced inotropy. In addition, whereas ERK½ inhibition with PD-98059 had no effect on cell mortality, PI3K inhibition with wortmannin, exacerbated myocyte death. We conclude that ouabain triggers an apoptotic cascade that involves NCX and CaMKII as a downstream effector. Ouabain simultaneously activates an antiapoptotic cascade involving PI3K/AKT which is however, insufficient to completely repress apoptosis. The finding that KN93 prevents ouabain-induced apoptosis without affecting inotropy suggests the potential use of CaMKII inhibitors as an adjunct to digitalis treatment for cardiovascular disease.     

Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: involvement of Na+/H+ and Na+/Ca2+ exchangers

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.     

Activation of the cardiac Na-Ca exchanger by sorcin via the interaction of the respective Ca-binding domains

Sorcin is a penta-EF-hand protein that interacts with intracellular target proteins after Ca²⁺ binding. The sarcolemmal Na⁺/Ca²⁺ exchanger (NCX1) may be an important sorcin target in cardiac muscle. In this study, RNAi knockdown of sorcin, purified sorcin or sorcin variants was employed in parallel measurements of: (i) NCX activity in isolated rabbit cardiomyocytes using electrophysiological techniques and (ii) sorcin binding to the NCX1 calcium binding domains (CBD1 and (iii) using surface plasmon resonance and gel overlay techniques. Sorcin is activated by Ca²⁺ binding to the EF3 and EF2 regions, which are connected by the D helix. To investigate the importance of this region in the interaction with NCX1, three variants were examined: W105G and W99G, mutated respectively near EF3 and EF2, and E124A that does not bind Ca²⁺ due to a mutation at EF3. Downregulation of sorcin decreased and supplementation with wt sorcin (3 μM) increased NCX activity in isolated cardiomyocytes. The relative stimulatory effects of the sorcin variants were: W105G > wt sorcin > Sorcin Calcium Binding Domain (SCBD) > W99G > E124A. Sorcin binding to both CBD1 and 2 was observed. In the presence of 50 µM Ca²⁺, the interaction with CBD1 followed the order W105G > SCBD > wt sorcin > W99G > E124A. In sorcin, the interacting surface can be mapped on the C-terminal Ca²⁺-binding domain in the D helix region comprising W99. The fast association/dissociation rates that characterize the interaction of sorcin with CBD1 and 2 may permit complex formation/dissociation during an excitation/contraction cycle.     

Preparation and characterization of plasma membrane of cardiac tissue

A method of preparing highly purified plasma membrane from cardiac muscle is described using continuous ficoll-sucrose density gradient centrifugation in zonal rotors. 5′-Nucleotidase and ATPase (Mg²⁺ dependent, Na⁺ + K⁺ stimulated and ouabain inhibited) were found to be concentrated in the plasma membrane fraction. At 11% ficollsucrose Ca²⁺ and (Ca²⁺ + K⁺) stimulation of the ATPase was very small (13%) and very low level of succinic dehydrogenase activity was present. Electron microscopic studies of the fraction revealed the presence of membrane vesicles and sheets. Composition of these membranes was analyzed by 7% polyacrylamide gel electrophoresis in the presence of 1% SDS. One protein band of 68 000 Daltons accounted for 18% of protein on the gel. Lipid and carbohydrate stains revealed six and two bands respectively. Studies on the conformation of proteins within membranes were achieved using circular dichroism (CD) and optical rotatory dispersion (ORD) techniques. Corrections were applied using the pseudo reference state approach.     

Acute effects of glucose and insulin on vascular endothelium

Aims/hypothesis Chronic exposure to high concentrations of glucose has consistently been demonstrated to impair endothelium-dependent, nitric oxide (NO)-mediated vasodilation. In contrast, several clinical investigations have reported that acute exposure to high glucose, alone or in combination with insulin, triggers vasodilation. The aim of this study was to examine whether elevated glucose itself stimulates endothelial NO formation or enhances insulin-mediated endothelial NO release.Methods We measured NO release and vessel tone ex vivo in porcine coronary conduit arteries (PCAs). Intracellular Ca²⁺ was monitored in porcine aortic endothelial cells (PAECs) by fura-2 fluorescence. Expression of the Na⁺/glucose cotransporter-1 (SGLT-1) was assayed in PAECs and PCA endothelium by RT-PCR.Results Stimulation of PCAs with d-glucose, but not the osmotic control l-glucose, induced a transient increase in NO release (EC₅₀10 mmol/l), mediated by a rise in intracellular Ca²⁺ levels due to an influx from the extracellular space. This effect was abolished by inhibitors of the plasmalemmal Na⁺/Ca²⁺ exchanger (dichlorobenzamil) and the SGLT-1 (phlorizin), which was found to be expressed in aortic and coronary endothelium. Alone, d-glucose did not relax PCA, but did augment the effect of insulin on NO release and vasodilation.Conclusions/interpretation An increased supply of extracellular d-glucose appears to enhance the activity of the endothelial isoform of nitric oxide synthase by increasing intracellular Na⁺ concentrations via SGLT-1, which in turn stimulates an extracellular Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger. This mechanism may be responsible for glucose-enhanced, insulin-dependent increases in tissue perfusion (including coronary blood-flow), thus accelerating glucose extraction from the blood circulation to limit the adverse vascular effects of prolonged hyperglycaemia.     

Paradoxical resistance to myocardial ischemia and age-related cardiomyopathy in NHE1 transgenic mice: A role for ER stress?

Sarcolemmal Na⁺/H⁺ exchanger (NHE) activity, which is provided by the NHE isoform 1 (NHE1), has been implicated in ischemia/reperfusion-induced myocardial injury in animal models and humans, on the basis of studies with pharmacological NHE1 inhibitors. We generated a transgenic (TG) mouse model with cardiac-specific over-expression of NHE1 to determine whether this would be sufficient to increase myocardial susceptibility to ischemia/reperfusion-induced injury. TG mouse hearts exhibited increased sarcolemmal NHE activity and normal morphology and function. Surprisingly, they also showed reduced susceptibility to ischemia/reperfusion-induced injury, as reflected by improved functional recovery and smaller infarcts. Such protection was sustained in the presence of NHE1 inhibition with zoniporide, indicating a mechanism that is independent of sarcolemmal NHE activity. Immunoblot analysis revealed accumulation of immature NHE1 protein as well as marked upregulation of both cytoprotective (78/94 kDa glucose-regulated proteins, calreticulin, protein disulfide isomerase) and pro-apoptotic (C/EBP homologous protein) components of the endoplasmic reticulum (ER) stress response in TG myocardium. With increasing age, NHE1 TG mice exhibited increased myocyte apoptosis, developed left ventricular contractile dysfunction, underwent cardiac remodelling and died prematurely. Our findings indicate that: (1) Cardiac-specific NHE1 over-expression induces the ER stress response in mouse myocardium, which may afford protection against ischemia/reperfusion-induced injury despite increased NHE activity; (2) Ageing NHE1 TG mice exhibit myocyte apoptosis, cardiac remodelling and failure, likely as a result of sustained ER stress; (3) The pluripotent effects of the ER stress response may confound studies that are based on the chronic over-expression of complex proteins in myocardium.     

Remodeled cardiac calcium channels

Cardiac calcium channels play a pivotal role in the proper functioning of cardiac cells. In response to various pathologic stimuli, they become remodeled, changing how they function, as they adapt to their new environment. Specific features of remodeled channels depend upon the particular disease state. This review will summarize what is known about remodeled cardiac calcium channels in three disease states: hypertrophy, heart failure and atrial fibrillation. In addition, it will review the recent advances made in our understanding of the function of the various molecular building blocks that contribute to the proper functioning of the cardiac calcium channel.     

Role of protein phosphatase-1 inhibitor-1 in cardiac physiology and pathophysiology

The type 1 protein phosphatase (PP1) is a critical negative regulator of Ca²⁺ cycling and contractility in the cardiomyocyte. In particular, it mediates restoration of cardiac function to basal levels, after β-adrenergic stimulation, by dephosphorylating key phospho-proteins. PP1 is a holoenzyme comprised of its catalytic and auxiliary subunits. These regulatory proteins dictate PP1's subcellular localization, substrate specificity and activity. Amongst them, inhibitor-1 is of particular importance since it has been implicated as an integrator of multiple neurohormonal pathways, which finely regulate PP1 activity, at the level of the sarcoplasmic reticulum (SR). In fact, perturbations in the regulation of PP1 by inhibitor-1 have been implicated in the pathogenesis of heart failure, suggesting that inhibitor-1-based therapeutic interventions may ameliorate cardiac dysfunction and remodeling in the failing heart. This review will discuss the current views on the role of inhibitor-1 in cardiac physiology, its possible contribution to cardiac disease and its potential as a novel therapeutic strategy.     

Action potential prolongation in cardiac myocytes of old rats is an adaptation to sustain youthful intracellular Ca2+ regulation

Advanced age in rats is accompanied by reduced expression of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA-2). The amplitudes of intracellular Ca2+ (Ca2+(i)) transients and contractions in ventricular myocytes isolated from old (23-24-months) rats (OR), however, are similar to those of young (4-6-months) rat myocytes (YR). OR myocytes also manifest slowed inactivation of L-type Ca2+ current (I(CaL)) and marked prolongation of action potential (AP) duration. To determine whether and how age-associated AP prolongation preserves the Ca2+(i) transient amplitude in OR myocytes, we employed an AP-clamp technique with simultaneous measurements of I(CaL) (with Na+ current, K+ currents and Ca2+ influx via sarcolemmal Na+-Ca2+ exchanger blocked) and Ca2+(i) transients in OR rat ventricular myocytes dialyzed with the fluorescent Ca2+ probe, indo-1. Myocytes were stimulated with AP-shaped voltage clamp waveforms approximating the configuration of prolonged, i.e. the native, AP of OR cells (AP-L), or with short AP waveforms (AP-S), typical of YR myocytes. Changes in SR Ca2+ load were assessed by rapid, complete SR Ca2+ depletions with caffeine. As expected, during stimulation with AP-S vs AP-L, peak I(CaL) increased, by 21+/-4%, while the I(CaL) integral decreased, by 19+/-3% (P<0.01 for each). Compared to AP-L, stimulation of OR myocytes with AP-S reduced the amplitudes of the Ca2+(i) transient by 31+/-6%, its maximal rate of rise (+dCa2+(i)/dt(max); a sensitive index of SR Ca2+ release flux) by 37+/-4%, and decreased the SR Ca2+ load by 29+/-4% (P<0.01 for each). Intriguingly, AP-S also reduced the maximal rate of the Ca2+(i) transient relaxation and prolonged its time to 50% decline, by 35+/-5% and 33+/-7%, respectively (P<0.01 for each). During stimulation with AP-S, the gain of Ca2+-induced Ca2+ release (CICR), indexed by +dCa2+(i)/dt(max)/I(CaL), was reduced by 46+/-4% vs AP-L (P<0.01). We conclude that the effects of an application of a shorter AP to OR myocytes to reduce +dCa2+(i)/dt(max) and the Ca2+ transient amplitude are attributable to a reduction in SR Ca2+ load, presumably due to a reduced I(CaL) integral and likely also to an increased Ca2+ extrusion via sarcolemmal Na+-Ca2+ exchanger. The decrease in the Ca2+(i) transient relaxation rate in OR cells stimulated with shorter APs may reflect a reduction of Ca2+/calmodulin-kinase II-regulated modulation of Ca2+ uptake via SERCA-2, consequent to a reduced local Ca2+ release in the vicinity of SERCA-2, also attributable to reduced SR Ca2+ load. Thus, the reduction of CICR gain during stimulation with AP-S is the net result of both a diminished SR Ca2+ release and an increased peak I(CaL). These results suggest that ventricular myocytes of old rats utilize AP prolongation to preserve an optimal SR Ca2+ loading, CICR gain and relaxation of Ca2+(i) transients.     

Early reperfusion levels of Na(+) and Ca(2+) are strongly associated with postischemic functional recovery but are disassociated from K(ATP) channel-induced cardioprotection

We previously demonstrated that pinacidil does not affect Na(+)(i) accumulation, cellular energy depletion, or acidosis during myocardial ischemia, but dramatically improves the cationic/energetic status during reperfusion. We investigated the role of this latter effect in K(ATP) channel-induced cardioprotection. Employing (23)Na and (31)P nuclear magnetic resonance spectroscopy with perfused rat hearts, reperfusion Na(+)(i) was altered with brief infusions of ouabain and/or RbCl to transiently decrease or increase Na(+)/K(+) ATPase activity. The increases and decreases in functional recovery (%LVDP-R) with pinacidil or ouabain, respectively, were largely unaltered by each other's presence. Early reperfusion Na(+)(i) and cellular energy were greatly altered by ouabain and indicated linear relationships with %LVDP-R. Pinacidil shifted these relationships to higher %LVDP-R. Increasing early reperfusion Na(+)(i) decreased %LVDP-R but did not diminish pinacidil's capacity to improve %LVDP-R. Approximately 75% and 45% of the pinacidil-induced improvements in %LVDP-R, could be disassociated from early reperfusion Na(+)(i) and cellular energy, respectively. Both pinacidil and RbCl infusion blunted ouabain's elevation of reperfusion Na(+)(i), but RbCl did not improve %LVDP-R. Atomic absorption tissue Ca(2+) measurements indicated that pinacidil reduced late reperfusion Ca(2+) uptake, but did not reduce early reperfusion Ca(2+), and its beneficial effects were resistant to ouabain-induced early reperfusion Ca(2+) increases. In conclusion, K(ATP) channel-induced cardioprotection does not require moderation of Na(+)(i) accumulation, cellular energy depletion, or acidosis during ischemia. K(ATP) channel-induced cardioprotection is largely independent of the accelerated reperfusion Na(+)(i) recovery it induces and does not require early reperfusion reductions of tissue Ca(2+). A larger role for early reperfusion cellular energy cannot be excluded.     

Inhibition of the reverse mode of the Na+/Ca2+ exchange by KB-R7943 augments arrhythmogenicity in the canine heart during rapid heart rates

To test the hypothesis that the reverse mode of the Na+/Ca2+ exchange augmented by a rapid heart rate has an antiarrhythmic effect by shortening the action potential duration, we examined the effects of KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulfonate), a selective inhibitor of the reverse mode of the Na+/Ca2+ exchange, to attenuate this effect. We recorded the electrocardiogram, monophasic action potential (MAP), and left ventricular pressure in canine beating hearts. In comparison to the control, KB-R7943 significantly increased the QTc value and MAP duration. MAP alternans and left ventricular pressure alternans were observed after changing the cycle length to 300 milliseconds in the control studies. KB-R7943 magnified both types of alternans and produced spatially discordant alternans between right and left ventricles. Early after-depolarizations and nonsustained ventricular tachycardia occurred in the presence of KB-R7943. Our data suggest that the reverse mode of the Na+/Ca2+ exchange may contribute to suppression of arrhythmias by abbreviating action potential duration under pathophysiological conditions. This conclusion is based on further confirmation by future studies of the specificity of KB-R7943 for block of the reverse mode of the Na+/Ca2+ exchange.     

Reduced sarcoplasmic reticulum Ca2+ uptake and increased Na+–Ca2+ exchanger expression in left ventricle myocardium of dogs with progression of heart failure

Alteration of intracellular Ca²⁺ homeostasis in failing cardiomyocytes is associated with changes in regulatory proteins located in the sarcoplasmic reticulum (SR) and sarcolemma, which participate in Ca²⁺ fluxes across the membrane during the cardiac cycle. These regulatory proteins include Ca²⁺-ATPase (SERCA 2A), phospholamban (PLB), ryanodine-sensitive Ca²⁺ release channels (RR), and the sarcolemmal Na⁺–Ca²⁺ exchanger (NCX). Although their status is known in failed myocardium, it is poorly understood during the progression of heart failure (HF), particularly in large animals. We studied the left ventricular (LV) myocardium of six dogs with moderate HF and six with severe HF produced by multiple intracoronary microembolizations, compared with six normal dogs (NL). Oxalate-dependent SR Ca²⁺ uptake and expression of SERCA 2A, PLB, phosphorylated PLB at serine 16 (PLB-Ser) and threonine 17 (PLB-Thr), RR, and NCX were determined. Percent LV ejection fraction declined by 47% compared with NL (34.1% ± 1% vs 64% ± 2%) in dogs with moderate HF (HF-2W) 2 weeks after the last embolization and by 42% (20.5% ± 1% vs 34.1% ± 1%) in dogs with severe HF (HF-4M) at 4 months compared with HF-2W. Left ventricular pressure during isovolumic contraction (+dP/dt, mmHg/s) and relaxation (–dP/dt, mmHg/s) was significantly reduced in severe compared with moderate HF. Oxalate-dependent SR Ca²⁺ uptake (nmol ⁴⁵Ca²⁺ accumulated/min per milligram noncollagen protein) declined by 25% (21.3 ± 1 vs 28.5 ± 2) in HF-2W and 49% in HF-4M. Protein expression of SERCA 2A and PLB decreased by 67% and 35%, respectively, in HF-2W compared with NL, whereas SERCA 2A expression increased by 167% and PLB decreased by 40% in HF-4M compared with HF-2W. However, SERCA 2A protein was still significantly lower in HF-4M compared with NL. PLB-Ser and PLB-Thr increased significantly in HF-2W but decreased in HF-4M compared with NL. Similar changes in mRNA encoding PLB and SERCA 2A were observed in dogs with moderate and severe HF. The RR protein level declined in dogs with moderate and severe HF, whereas NCX protein did not change with moderate HF but increased with sever HF. These results suggest that the regulatory proteins responsible for Ca²⁺ uptake, Ca²⁺ release, and Na⁺–Ca²⁺ exchange are critically associated with the deterioration of LV function during the progression of HF.     

A Non-Ouabain Na/K Atpase Inhibitor Isolated from Bovine Hypothalamus. Its Relation to Hypothalamic Ouabain

We have isolated from bovine hypothalamic and pituitary tissue the sodium pump inhibitor HHIF that is structurally different from ouabain. By mass spectrometric analysis this purified factor revealed a single unique molecular ion with an accurate mass of 412.277 and a mass spectra different from ouabain. It has been previously shown that HHIF inhibits the Ca²⁺-ATPase of the plasma membrane of synaptosomes. HHIF increases free calcium levels in cultured rat mesangial cells as well as mesangial cell contraction and proliferation. With the same purification procedure we have isolated in parallel HHIF and Ouabain from central nervous tissue. Ouabain elutes prior to HHIF in the final purification HPLC systems. This endogenous Ouabain has, in all the systems tested, the same chromatographic behavior that synthetic cold or [³H] Ouabain     

Anti-hypertrophic effect of NHE-1 inhibition involves GSK-3β-dependent attenuation of mitochondrial dysfunction

Although Na⁺–H⁺ exchanger 1 (NHE-1) inhibition has been demonstrated to have anti-hypertrophic effect indirectly through mitochondria, the detailed cellular mechanisms mediating this effect remain elusive. In this study we sought to determine whether NHE-1 inhibition exerts an anti-hypertrophic effect by modulating the mitochondrial permeability transition pore (mPTP) opening through the AMP-activated protein kinase (AMPK)/glycogen synthase kinase 3β (GSK-3β) pathway during hypertrophy in cardiomyocytes. An in vivo model of hypertrophy was induced in male Sprague–Dawley rats by subjecting them to 3, 7 or 28 days of coronary artery ligation (CAL). To induce hypertrophy in vitro, cardiomyocytes isolated from hearts of neonatal (1–3 days) Sprague–Dawley rats were exposed to endothelin-1 (ET-1, 10 nM) in the presence or absence of various treatments. The results demonstrate that CAL affected both AMPKα and GSK-3β phosphorylation in a time-dependent manner. In cultured cardiomyocytes, ET-1 increased phosphorylation of AMPKα₁/α₂^{Ser485/Ser491} and GSK-3β^Ser9 by 80% (P < 0.05) and 225% (P < 0.05) respectively, both of which were significantly blunted by the NHE-1 inhibitor AVE-4890 (5 μM). ET-1-induced phosphorylation of GSK-3β^Ser9 was attenuated by inhibitors of phosphatidylinositol 3-kinase (LY294002), Akt (Akt inhibitor VIII), ERK1/2 (PD98059) and by the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). Prevention of GSK-3β^Ser9 phosphorylation was also accompanied by suppression of ET-1-induced increases in cell surface area, ANP and α-skeletal actin gene expression. Co-immunoprecipitation studies revealed that GSK-3β interacts with components of the mPTP, voltage-dependent anion channel (VDAC) and adenine nucleotide translocase. Furthermore, ET-1 reduced phosphorylation of VDAC, which was associated with both mPTP opening and mitochondrial membrane depolarization. These effects were mimicked by the GSK-3β inhibitor SB216763, thus showing that modulation of mPTP formation is GSK-3β-dependent. In conclusion, anti-hypertrophic effect of NHE-1 inhibition can be mediated through activation of GSK-3β which in turn induces inhibition of mPTP opening due to VDAC phosphorylation.     

The sting of salt on an old,but open,wound--is Na(+) the cause of mitochondrial and myocardial injury during ischemia/reperfusion?

D. B. Sawyer, T. M. Suter and C. S. Apstein. The Sting of Salt on an Old, but Open, Wound—is Na⁺ the Cause of Mitochondrial and Myocardial Injury During Ischemia/Reperfusion? Journal of Molecular and Cellular Cardiology (2002) 34, 699-702.     

Sequential dissection of multiple ionic currents in single cardiac myocytes under action potential-clamp

The cardiac action potential (AP) is shaped by myriad ionic currents. In this study, we develop an innovative AP-clamp Sequential Dissection technique to enable the recording of multiple ionic currents in the single cell under AP-clamp. This new technique presents a significant step beyond the traditional way of recording only one current in any one cell. The ability to measure many currents in a single cell has revealed two hitherto unknown characteristics of the ionic currents in cardiac cells: coordination of currents within a cell and large variation of currents between cells. Hence, the AP-clamp Sequential Dissection method provides a unique and powerful tool for studying individual cell electrophysiology.