Chronic SR Ca2+-ATPase inhibition causes adaptive changes in cellular Ca2+ transport

Phospholamban, the critical regulator of the cardiac SERCA2a Ca2+ affinity, is phosphorylated at Ser16 and Thr17 during beta-adrenergic stimulation (eg, isoproterenol). To assess the impact of nonphosphorylatable phospholamban, a S16A, T17A double-mutant (DM) was introduced into phospholamban knockout mouse hearts. Transgenic lines expressing DM phospholamban at levels similar to wild types (WT) were identified. In vitro phosphorylation confirmed that DM phospholamban could not be phosphorylated, but produced the same shift in EC50 of SERCA2a for Ca2+ as unphosphorylated WT phospholamban. Rates of basal twitch [Ca2+]i decline were not different in DM versus WT cardiomyocytes. Isoproterenol increased the rates of twitch [Ca2+]i decline in WT, but not DM myocytes, confirming the prominent role of phospholamban phosphorylation in this response. Increased L-type Ca2+ current (ICa) density, with unaltered characteristics, was the major compensation in DM myocytes. Consequently, the normal beta-adrenergic-induced increase in ICa caused larger dynamic changes in absolute ICa density. Isoproterenol increased Ca2+ transients to a comparable amplitude in DM and WT. There were no changes in myofilament Ca2+ sensitivity, or the expression levels and Ca2+ removal activities of other Ca2+-handling proteins. Nor was there evidence of cardiac remodeling up to 10 months of age. Thus, chronic inhibition of SERCA2a by ablation of phospholamban phosphorylation (abolishing its adrenergic regulation) results in a unique cellular adaptation involving greater dynamic ICa modulation. This ICa modulation may partly compensate for the loss in SERCA2a responsiveness and thereby partially normalize beta-adrenergic inotropy in DM phospholamban mice.     

Net transsarcolemmal Ca2+ shifts versus Ca/Ca exchange in guinea pig ventricular muscle

Summary We investigated the net transsarcolemmal Ca²⁺ shifts and Ca/Ca exchange by means of⁴⁵Ca in isolated, perfused ventricles of guinea pig heart treated with vanadate to inhibit ATP-driven sarcolemmal Ca²⁺ pump. The heart was stimulated (at the rate of 60/min) and perfused with a solution containing⁴⁵Ca for 60 min. Thereafter stimulation was stopped and either perfusion with radioactive solution was continued or the solution was exchanged for a non-radioactive one. In the first case, tissue⁴⁵Ca content (equivalent to the exchangeable Ca²⁺ content) dropped from 1.960±0.120 mmol/kg of wet weight (w.w.) to 0.715±0.049 mmol/kg w.w. and stabilized at this level between 5th and 10th min. In the second case, decrease in⁴⁵Ca content continued and within 40 min attained 0.047±0.004 mmol/kg w.w., despite stabilizing of the total exchangeable Ca²⁺ content. Drop of⁴⁵Ca content in the rested heart perfused (until the end of experiments) with radioactive solution resulted from the net transsarcolemmal Ca²⁺ shift and it was strongly inhibited by removal of extracellular Na⁺. The continuing drop in⁴⁵Ca content in the heart perfused with non-radioactive solution while total Ca²⁺ content stabilized must have resulted from Ca/Ca exchange; it was stimulated by removal of extracellular Na⁺. These experiments separate two modes of⁴⁵Ca fluxes and suggest that a common route of these fluxes is the Na/Ca exchanger.This work was supported by a grant: C.P.B.R.11.6 No 58.     

Effects of active oxygen generated by DTT/Fe2+ on cardiac Na+/Ca2+ exchange and membrane permeability to Ca2+

Sarcolemmal vesicles isolated from bovine heart were preincubated at 37 degrees C with an oxygen radical generating system consisting of 1 mM dithiothreitol (DTT) and 50 microM FeSO4. Exposure of the vesicles for 1 to 40 mins stimulated Na+/Ca2+ exchange about 2.5-fold. The DTT/Fe2+ treatment decreased the apparent Km for Ca2+ of Nai+-dependent Ca2+ uptake by 80% (from 63 to 13 microM). The effect on Vmax was much smaller however. The resulting stimulation of exchange activity was diminished by the presence of desferrioxamine (95%) or catalase (60%). In contrast, superoxide dismutase and sodium formate did not prevent the effects of DTT/Fe2+ on the exchanger. Neither Zn2+ nor Ga3+ could replace Fe2+ in the stimulation of Na+/Ca2+ exchange. Passive Ca2+ efflux was determined by first allowing Na+/Ca2+ exchange to continue to plateau values and then diluting the loaded vesicles in the presence of EGTA. Ca2+ leakage from the vesicles was slightly but significantly (P less than 0.05) increased by the action of DTT/Fe2+, the rate constants for the passive Ca2+ efflux being 0.22 and 0.26/min in control and treated groups, respectively. The calcium loading observed in myocytes in ischemia/reperfusion injury suggests that the stimulation of Na+/Ca2+ exchange by active oxygen may moderate the myocardial response to oxygen mediated injuries including ischemia/reperfusion injury. However, the clinical relevance of these phenomena is far from clear as the stimulation depends in part on the Km for Ca2+ prior to treatment.     

Ca2+-induced inhibition of the cardiac Ca2+ channel depends on calmodulin

Ca2+-induced inhibition of alpha1C voltage-gated Ca2+ channels is a physiologically important regulatory mechanism that shortens the mean open time of these otherwise long-lasting high-voltage-activated channels. The mechanism of action of Ca2+ has been a matter of some controversy, as previous studies have proposed the involvement of a putative Ca2+-binding EF hand in the C terminus of alpha1C and/or a sequence downstream from this EF-hand motif containing a putative calmodulin (CaM)-binding IQ motif. Previously, using site directed mutagenesis, we have shown that disruption of the EF-hand motif does not remove Ca2+ inhibition. We now show that the IQ motif binds CaM and that disruption of this binding activity prevents Ca2+ inhibition. We propose that Ca2+ entering through the voltage-gated pore binds to CaM and that the Ca/CaM complex is the mediator of Ca2+ inhibition.     

Arrhythmogenic effects of beta2-adrenergic stimulation in the failing heart are attributable to enhanced sarcoplasmic reticulum Ca load

Ventricular tachycardia in heart failure (HF) can initiate by nonreentrant mechanisms such as delayed afterdepolarizations. In an arrhythmogenic rabbit model of HF, we have shown that isoproterenol induces ventricular tachycardia in vivo and aftercontractions and transient inward currents in HF myocytes. To determine whether beta(2)-adrenergic receptor (beta(2)-AR) stimulation contributes, we performed in vivo drug infusion, in vitro myocyte and biochemical studies. Intravenous zinterol (2.5 microg/kg) led to ventricular arrhythmias, including ventricular tachycardia up to 13 beats long in 4 of 6 HF rabbits (versus 0 of 5 controls, P<0.01), an effect blocked by beta(2)-AR antagonist ICI-118,551 (0.2 mg/kg). In field-stimulated myocytes (0.5 to 4 Hz, 37 degrees C), beta(2)-AR stimulation (1 micromol/L zinterol+300 nmol/L beta(1)-AR antagonist CGP-29712A) induced aftercontractions and Ca aftertransients in 88% of HF versus 0% of control myocytes (P<0.01). beta(2)-AR stimulation in HF (but not control) myocytes increased Ca transient amplitude (by 29%), sarcoplasmic reticulum (SR) Ca load (by 28%), the rate of [Ca](i) decline (by 28%; n=12, all P<0.05), and phospholamban phosphorylation at Ser16, but Ca current was unchanged. All of these effects in HF myocytes were blocked by ICI-118,551 (100 nmol/L). Although total beta-AR expression was reduced by 47% in HF rabbit left ventricle, beta(2)-AR number was unchanged, indicating more potent beta(2)-AR-dependent SR Ca uptake and arrhythmogenesis in HF. Human HF myocytes showed similar beta(2)-AR-induced aftercontractions, aftertransients, and enhanced Ca transient amplitude, SR Ca load and twitch [Ca](i) decline rate. Thus, beta(2)-AR stimulation is arrhythmogenic in HF, mediated by SR Ca overload-induced spontaneous SR Ca release and aftercontractions.     

代谢抑制处理后大鼠心室肌细胞反向Na+/Ca2+交换体转运功能的变化

目的:利用荧光标记法观察代谢抑制处理后,大鼠心肌细胞反向Na+/Ca2+交换体（NCX）转运功能的变化。方法:酶解法分离制备钙耐受心肌细胞用Fura-2/AM负载,采用双激发荧光光电倍增系统（IonOptix Photom etry Sys-tem）检测钙信号。结果:细胞置于无Na+液后,可见[Ca2+]i逐渐升高,L-型Ca2+通道阻断剂n ifed ip ine在浓度为1μmol/L时,不影响此现象;而NCX的抑制剂N i2+,在浓度为1 mmol/L时,则完全阻断[Ca2+]i的升高。采用20mmol/L乳酸加10 mmol/L脱氧葡萄糖作为代谢抑制物处理心肌细胞不同时间,正常Tyrode液灌流10 m in,之后检测无Na+液引发[Ca2+]i升高效应的变化,发现5 m in处理与对照组无显著性差异,10和30 m in处理后此效应逐渐减弱。结论:首次发现,代谢抑制处理后心肌NCX的反向转运功能被抑制,阐明其调节机制,将为心肌缺血/再灌注损伤的治疗提供新思路。     

Na+/Ca2+ exchange current (I(Na/Ca)) and sarcoplasmic reticulum Ca2+ release in catecholamine-induced cardiac hypertrophy

OBJECTIVE: Catecholamines that accompany acute physiological stress are also involved in mediating the development of hypertrophy and failure. However, the cellular mechanisms involved in catecholamine-induced cardiac hypertrophy, particularly Ca2+ handling, are largely unknown. We therefore investigated the effects of cardiac hypertrophy, produced by isoprenaline, on I(Na/Ca) and sarcoplasmic reticulum (SR) function in isolated myocytes. METHODS: I(Na/Ca) was studied in myocytes from Wistar rats, using descending (+80 to -110 mV) voltage ramps under steady state conditions. Myocytes were also loaded with fura-2 and either field stimulated or voltage clamped to assess [Ca2+]i and SR Ca2+ content. RESULTS: Ca2+-dependent, steady state I(Na/Ca) density was increased in hypertrophied myocytes (P<0.05). Ca2+ release from the SR was also increased, whereas resting [Ca2+]i and the rate of decline of [Ca2+]i to control levels were unchanged. SR Ca2+ content, estimated by using 10.0 mmol/l caffeine, was also significantly increased in hypertrophied myocytes, but only when myocytes were held and stimulated from their normal resting potential (-80 mV) but not from -40 mV. However, the rate of decline of caffeine-induced Ca2+ transients or I(Na/Ca) was not significantly different between control and hypertrophied myocytes. Ca2+-dependence of I(Na/Ca), examined by comparing the slope of the descending phase of the hysteresis plots of I(Na/Ca) vs. [Ca2+]i, was also similar in the two groups of cells. CONCLUSION: Data show that SR Ca2+ release and SR Ca2+ content were increased in hypertrophied myocytes, despite an increase in the steady state I(Na/Ca) density. The observation that increased SR function occurred only when myocytes were stimulated from -80 mV suggests that Na+ influx may play a role in altering Ca2+ homeostasis in hypertrophied cardiac muscle, possibly through increased reverse Na+/Ca2+ exchange, particularly at low stimulation frequencies.     

Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments

Quantitative analysis of Ca(2+) fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca(2+)-dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca(2+) binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca(2+) release kinetics with an off-rate estimated at around 700 s(-1) and appropriate Ca(2+) binding affinity, likely attributable to local Ca(2+)-induced conformational changes around the designed Ca(2+) binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca(2+) dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca(2+) in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca(2+) release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca(2+) homeostasis.     

Heterogeneity in cellular response and intracellular distribution of Ca2+ concentration during and after metabolic inhibition

STUDY OBJECTIVE--To characterise cell injury during myocardial hypoxia and reoxygenation, [Ca2+]i was measured in guinea pig ventricular myocytes using digital images of fura-2 fluorescence. DESIGN--[Ca2+]i and cell morphology were measured during and after the perfusion of 2 mmol.litre-1 sodium cyanide (NaCN). EXPERIMENTAL MATERIAL--28 ventricular myocytes isolated from guinea pig hearts. MEASUREMENTS AND MAIN RESULTS--Eight out of 28 cells became rounded during the perfusion with NaCN, and [Ca2+]i increased from 48 (SEM 6) nmol-litre-1 to 163(24) (p less than 0.01) before cell rounding, and to 311(46) (p less than 0.01) after cell rounding. Ten cells were rounded after the washout of NaCN, and [Ca2+]i increased to 252(41) nmol.litre-1 before cell rounding (p less than 0.01), and to 314(48) after cell rounding (p less than 0.01). Intracellular distribution of [Ca2+]i was heterogeneous in some cells with high [Ca2+]i. The values of [Ca2+]i before and after cell rounding, during and after the perfusion of NaCN, were significantly lower than those during the perfusion of strophanthidin (0.1 mmol.litre-1). CONCLUSIONS--There appeared to be a mixed population of myocytes, some showing normal [Ca2+]i and shape, and others showing high [Ca2+]i and contracture, during the perfusion or the washout of NaCN. Ca2+ overload alone is unlikely to be the main and the only mechanism of cell injury during myocardial hypoxia/reoxygenation. Other mechanisms such as membrane disturbance could be involved.     

Sarcoplasmic reticulum Ca 2+ load in human heart failure

Excitation-contraction coupling and intracellular Ca ²⁺ homeostasis are altered in heart failure. We tested the hypothesis that these changes are related to disturbed Ca ²⁺ handling of the sarcoplasmic reticulum (SR). Isolated, electrically stimulated trabeculae were obtained from end-stage failing (NYHA IV) and nonfailing human hearts. Isometric twitch tension, intracellular Ca ²⁺ transients (aequorin method) and SR Ca ²⁺ content (rapid cooling contractures) were assessed under basal conditions (1 Hz, 37 °C) as well as after stepwise increasing rest intervals from 2 – 240 s (post-rest contractions). Protein expression of SERCA2a and phospholamban (Western blot) was assessed in a subset of failing trabeculae. In addition, the effects of SERCA1 overexpression on contractile function of isolated myocytes was tested. On average, post-rest twitch tension continuously increased with increasing rest intervals in nonfailing, but declined with rest intervals longer than 15s in failing myocardium. The rest-dependent contractile changes were accompanied by parallel changes in intracellular Ca ²⁺ transients. Failing trabeculae (n = 40) were grouped (group A: post-rest potentiation (force of contraction > pre-rest twitch force) after 120s rest interval; group B: post-rest decay (force of contraction < pre-rest twitch force) after 120 s rest interval), and post-rest contractile function was related to SERCA2a and PLB expression. While PLB protein expression was not different, SERCA2a protein expression as well as SERCA2a/PLB ratio was significantly higher in group A vs. group B. Transfection of SERCA1 increased shortening amplitude and enhanced relaxation kinetics in failing human myocytes. In conclusion, SR Ca ²⁺ handling is severely altered in human heart failure. Reduced SR Ca ²⁺ release is due to diminished SR Ca ²⁺ content directly related to a depressed expression of SERCA2a protein. Enhancing SERCA function or expression may improve SR Ca ²⁺ handling in failing human myocardium.     

Age and hypertrophy alter the contribution of sarcoplasmic reticulum and Na+/Ca2+ exchange to Ca2+ removal in rat left ventricular myocytes

Age and hypertension contribute significantly to cardiac morbidity and mortality, however the importance of each during the progression of hypertrophy is unclear. This investigation examined the effect of age and hypertension on Ca(2+) handling in rat ventricular myocytes by comparing a genetic model of hypertension and cardiac hypertrophy (spontaneously hypertensive rat, SHR) with its normotensive control (Wistar-Kyoto rat, WKY) at 5 and 8 months of age. Experiments were performed on single left ventricular myocytes isolated from SHR or WKY hearts. Intracellular Ca(2+) was measured optically using fura-2 or fluo-3. SHR myocytes had a significantly larger cell width and volume and a significantly decreased cell length/width ratio at 5 and 8 months compared to normotensive controls. Age had no effect on cell length, width, volume or the length/width ratio. Ca(2+) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) content and contraction amplitude were unaffected by age or hypertrophy. However at 8 months the contribution of the SR to Ca(2+) uptake during relaxation decreased, with a concomitant increase in the contribution of Na(+)/Ca(2+) exchanger (NCX) function to relaxation, in SHR and WKY myocytes. The incidence of non-synchronous SR Ca(2+) release decreased with age but not hypertrophy in SHR and WKY myocytes. These results show that the changes in Ca(2+) handling observed during progression of mild hypertrophy in SHR are the same as those that occur during ageing in normotensive control animals and can, therefore, be ascribed to maturation rather than hypertrophy.     

Ca2+ transport capacity of sarcolemmal Na+-Ca2+ exchange. Extrapolation of vesicle data to in vivo conditions

Na+-Ca2+ exchange activity is high in cardiac sarcolemmal vesicles suggesting an important physiologic role. Vesicular Na+-Ca2+ exchange, however, is usually measured under conditions which are far from physiologic. Using sarcolemmal vesicles, we have estimated the possible significance of both Ca2+ influx and efflux mediated by Na+-Ca2+ exchange under approximate in vivo ionic conditions. In this situation, Na+-Ca2+ exchange activity is far from maximal with intracellular Mg2+ causing significant inhibition. The capacity of the Na+-Ca2+ exchange system to extrude intracellular Ca2+ (at [Ca2+] = 6.0 microM) is about 1.2 mumol Ca2+/kg wet weight/s and approximately equals the capacity of the sarcolemmal ATP-dependent Ca2+ pump. The capacity of the sarcoplasmic reticular Ca2+ pump to remove cytoplasmic Ca2+ is much larger. Significant Ca2+ influx through the exchanger is unlikely to occur in normal mammalian myocardium and would require reduced extracellular Na+ or elevated intracellular Na+.     

The Na+/Ca2+ exchange blocker SEA0400 fails to enhance cytosolic Ca2+ transient and contractility in canine ventricular cardiomyocytes

AIMS: This study was designed to evaluate the effects of the Na(+)/Ca(2+) exchange (NCX) inhibitor SEA0400 on Ca(2+) handling in isolated canine ventricular myocytes. METHODS AND RESULTS: Intracellular Ca(2+) ([Ca(2+)](i)) transients, induced by either field stimulation or caffeine flush, were monitored using Ca(2+) indicator dyes. [Ca(2+)](i)-dependent modulation of the inhibitory effect of SEA0400 on NCX was characterized by the changes in Ni(2+)-sensitive current in voltage-clamped myocytes. Sarcoplasmic reticulum (SR) Ca(2+) release and uptake were studied in SR membrane vesicles. Gating properties of single-ryanodine receptors were analysed in lipid bilayers. Ca(2+) sensitivity of the contractile machinery was evaluated in chemically skinned myocytes. In myocytes paced at 1 Hz, neither diastolic [Ca(2+)](i) nor the amplitude of [Ca(2+)](i) transients was significantly altered by SEA0400 up to the concentration of 1 microM, which was shown to inhibit the exchange current. The blocking effect of SEA0400 on NCX decreased with increasing [Ca(2+)](i), and it was more pronounced in reverse than in forward mode operation at every [Ca(2+)](i) examined. The rate of decay of the caffeine-induced [Ca(2+)](i) transients was decreased significantly by 1 microM SEA0400; however, this effect was only a fraction of that observed with 10 mM NiCl(2). Neither SR Ca(2+) release and uptake nor cell shortening and Ca(2+) sensitivity of the contractile proteins were influenced by SEA0400. CONCLUSION: The lack of any major SEA0400-induced shift in Ca(2+) transients or contractility of myocytes can well be explained by its limited inhibitory effect on NCX (further attenuated by elevated [Ca(2+)](i) levels) and a concomitant reduction in Ca(2+) influx due to the predominantly reverse mode blockade of NCX and suppression of L-type Ca(2+) current.     

Abnormal Ca2+ handling and increased Mg2+ permeability in platelets of hypertensive rats.

In order to test the hypothesis that intracellular Na+ accumulation and cellular Mg2+ deficiency may be involved in the abnormalities in Ca2+ handling and reactivity in spontaneously hypertensive rats (SHR) platelets, the metabolism of Na+, Ca2+ and Mg2+ was determined in fluorescent dye loaded platelets from 15 SHR and 15 Wistar-Kyoto rats (WKY) at 12 weeks of age. Mg2+ leak was estimated as the Mg2+ influx induced by an increase in extracellular [Mg2+] (from 1 to 5 mmol/l) and Mg2+/Na+ exchange activity was estimated as the Mg2+ influx induced by a decrease in extracellular [Na+] (from 140 to 50 mmol/l). Cellular metabolism of the fluorescent dye was similar in the two groups. Mean platelet [Ca2+]i was significantly increased under basal and thrombin (0.1 U/ml)-stimulated conditions in SHR compared to WKY, both in the presence and absence of extracellular Ca2+. Mean Ca2+ discharge capacity was similar between the two groups. There was no difference in mean [Na+]i between the two groups. Basal [Mg2+]i was also increased in SHR platelets. Mg2+ leak was higher in SHR than in WKY, while Mg2+/Na+ exchange activity was similar in the two groups. There was no difference in serum Mg2+ concentration between SHR and WKY. These data suggest that abnormal Ca2+ handling is accompanied by elevation in [Mg2+]i via increased permeability of platelet cell membranes to Mg2+ in SHR without any alteration in [Na+]i, and do not support the Mg2+ deficiency hypothesis in genetically hypertensive rats.     

A comparison of adenovirally delivered molecular methods to inhibit Na+/Ca2+ exchange

The ability to use molecular biology tools to down-regulate Na+/Ca2+ exchanger (NCX) expression will allow us to better understand the regulation of Ca(i)2+ and contractility in heart. Three different techniques to deplete NCX expression were compared: short hairpin RNA (shRNA), antisense RNA and exchanger inhibitory peptide expression via adenoviral transfection. Our results demonstrate that the most efficient method to deplete NCX expression and activity from cardiomyocytes is shRNA. It is also possible to replace the endogenous NCX with alternative isoforms or mutant forms of the NCX. Adenovirally delivered shRNA is an efficient tool for the study of the NCX and could be adapted for many other cardiac proteins.     

Inhibition by KB-r7943 of the reverse mode of the Na+/Ca2+ exchanger reduces Ca2+ overload in ischemic-reperfused rat hearts.

Ca2+ influx via the Na+/Ca2+ exchanger (NCX) may lead to Ca2+ overload and myocardial injury in ischemia-reperfusion. Direct evidence that increased cytoplasmic Ca2+ concentration ([Ca2+]i) is mediated by the reverse mode of the NCX is limited, so in the present study the [Ca2+]i dynamics and left ventricular pressure were monitored in perfused beating hearts. The effects of KB-R7943 (KBR), a selective inhibitor of the NCX in the reverse mode, were analyzed during low-Na+ exposure and ischemia-reperfusion. Hearts from Sprague-Dawley rats were retrogradely perfused and loaded with 4 micromol/L fura-2 to measure the fluorescence ratio as an index of [Ca2+]i. To evaluate KBR effects on the reverse mode exchanger, the increase in [Ca2+]i induced by low-Na+ exposure (Na+: 30 mmol/L, 10 mmol/L caffeine pre-treatment) was measured with and without 10 micromol/L KBR (n=5). In another series, the hearts were subjected to 10 min of low-flow ischemia with pacing, followed by reperfusion in the absence (n=6) or in the presence of 10 micromol/L KBR (n=6). Background autofluorescence was subtracted to estimate the ratio in the ischemia-reperfusion protocol. KBR significantly suppressed the increase in [Ca2+]i induced by low-Na+ (40.2 +/- 11.2% of control condition, p=0.014), as well as on increase in diastolic [Ca2+]i during ischemia (% increase from pre-ischemia in [Ca2+]i at 10 min: KBR, 17.9 +/- 6.4%; no KBR, 44.4 +/- 7.7%; p=0.024). After reperfusion, diastolic [Ca2+]i normalized more rapidly in KBR-treated hearts (% increase at 1 min: KBR, 4.5 +/- 7.0%; no KBR, 39.8 +/- 12.2%; p=0.03). Treatment with KBR also accelerated recovery of the rate-pressure product on reperfusion (1 min: KBR, 8,944 +/- 1,554 min(-1) mmHg; no KBR, 4,970 +/- 1,325; p<0.05). Thus, inhibition of the reverse mode exchanger by KBR reduced ischemic Ca2+ overload and possibly improved functional myocardial recovery during reperfusion in a whole heart model.     

Erythrocyte (Ca+2 + Mg+2)-ATPase activity: increased sensitivity to oxidative stress in glucose-6-phosphate dehydrogenase deficiency

The effect of the thiol-oxidizing agent diamide on erythrocyte (Ca+2 + Mg+2)-ATPase activity was measured in normal and glucose-6-phosphate-dehydrogenase-deficient (G6PD-) cells. Although the enzyme activity before the oxidative stress was similar in both groups, diamide induced a markedly greater inhibition in the enzyme activity in the G6PD- cells than in the normal controls. These data indicate dependence of erythrocyte (Ca+2 + Mg+2)-ATPase, in part, on the redox status of the cell. The increased vulnerability of (Ca+2 + Mg+2)-ATPase to oxidative stress in G6PD- may be of pathophysiological relevance to their premature destruction in oxidant-induced hemolysis.