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1.
It has been suggested that the sodium/calcium exchanger NCX1 may have a more important physiological role in embryonic and neonatal hearts than in adult hearts. However, in chick heart sarcolemmal vesicles, sodium-dependent calcium transport is reported to be small and, moreover, to be 3–12 times smaller in hearts at embryonic day (ED) 4–5 than at ED18, the opposite of what would be expected of a transporter that is more important in early development. To better assess the role of NCX1 in calcium regulation in the chick embryonic heart, we measured the activity of NCX1 in chick embryonic hearts as extracellular calcium-activated exchanger current (INCX) under controlled ionic conditions. With intracellular calcium concentration ([Ca2+]i) = 47 nM, INCX density increased from 1.34 ± 0.28 pA/pF at ED2 to 3.22 ± 0.55 pA/pF at ED11 (P = 0.006); however, with [Ca2+]i = 481 nM, the increase was small and statistically insignificant, from 4.54 ± 0.77 to 5.88 ± 0.73 pA/pF (P = 0.20, membrane potential = 0 mV, extracellular calcium concentration = 2 mM). Plots of INCX density against [Ca2+]i were well fitted by the Michaelis-Menton equation and extrapolated to identical maximal currents for ED2 and ED11 cells (extracellular calcium concentration = 1, 2, or 4 mM). Thus the increase in INCX at low [Ca2+]i appeared to reflect a developmental change in allosteric regulation of the exchanger by intracellular calcium rather than an increase in the membrane density of NCX1. Supporting this conclusion, RT-PCR demonstrated little change in the amount of mRNA encoding NCX1 expression from ED2 through ED18. NCX1; chick embryo; allosteric regulation; sodium/calcium exchange current  相似文献   

2.
In cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca2+ current (ICa), provides a critical mechanism for the maintenance of Ca2+ homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na+-Ca2+ exchanger knockout mice: reduced transsarcolemmal Ca2+ flux. Circ Res 97: 1288–1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (Ito) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak Ito was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased Ito, ECGs from KO mice exhibited shortened QT intervals. Expression of the Ito-generating K+ channel subunit Kv4.2 and the K+ channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378–1403, 2004) to determine the relative contributions of increased Ito, reduced ICa, and reduced NCX current (INCX) on the shape and kinetics of the AP. Reduction of ICa and elimination of INCX had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when Ito was increased in the computer model. Thus, the increase in Ito, and not the reduction of ICa or INCX, is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of Ito may comprise an important regulatory mechanism to limit Ca2+ influx via a reduction of AP duration, thus preventing Ca2+ overload in situations of reduced myocyte Ca2+ extrusion capacity. genetically altered mice; cardiac myocytes; short QT interval; transient outward current  相似文献   

3.
The cardiacNa+/Ca2+ exchanger (NCX), an importantregulator of cytosolic Ca2+ concentration in contractionand relaxation, has been shown in trout heart sarcolemmal vesicles tohave high activity at 7°C relative to its mammalian isoform. Thisunique property is likely due to differences in protein structure. Inthis study, outward NCX currents (INCX) of thewild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressedin oocytes were measured to explore the potential contributions ofregulatory vs. transport mechanisms to this observation. cRNA wastranscribed in vitro from both wild-type cDNA and was injected intoXenopus oocytes. INCX of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30°C using the giant excised patch technique. TheINCX for both isoforms exhibitedNa+-dependent inactivation and Ca2+-dependentpositive regulation. The INCX of NCX1.1exhibited typical mammalian temperature sensitivities withQ10 values of 2.4 and 2.6 for peak and steady-statecurrents, respectively. However, the INCX ofNCX-TR1.0 was relatively temperature insensitive with Q10values of 1.2 and 1.1 for peak and steady-state currents, respectively.INCX current decay was fit with a singleexponential, and the resultant rate constant of inactivation () wasdetermined as a function of temperature. As expected,  decreasedmonotonically with temperature for both isoforms. Although  wassignificantly greater in NCX1.1 compared with NCX-TR1.0 at alltemperatures, the effect of temperature on  was not differentbetween the two isoforms. These data suggest that thedisparities in INCX temperature dependencebetween these two exchanger isoforms are unlikely due to differences intheir inactivation kinetics. In addition, similar differences intemperature dependence were observed in both isoforms after-chymotrypsin treatment that renders the exchanger in a deregulatedstate. These data suggest that the differences in INCX temperature dependence between the twoisoforms are not due to potential disparities in either theINCX regulatory mechanisms or structuraldifferences in the cytoplasmic loop but are likely predicated ondifferences within the transmembrane segments.

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4.
In mammalian cardiac myocytes, calcium released into the dyadic space rapidly inactivates calcium current (ICa). We used this Ca2+ release-dependent inactivation (RDI) of ICa as a local probe of sarcoplasmic reticulum Ca2+ release activation. In whole cell patch-clamped rat ventricular myocytes, Ca2+ entry induced by short prepulses from —50 mV to positive voltages caused suppression of peak ICa during a test pulse. The negative correlation between peak ICa suppression and ICa inactivation during the test pulse indicated that RDI evoked by the prepulse affected only calcium channels in those dyads in which calcium release was activated. Ca2+ ions injected during the prepulse and during the subsequent tail current suppressed peak ICa in the test pulse to a different extent. Quantitative analysis indicated that equal Ca2+ charge was 3.5 times less effective in inducing release when entering during the prepulse than when entering during the tail. Tail Ca2+ charge injected by the first voltage-dependent calcium channel (DHPR) openings was three times less effective than that injected by DHPR reopenings. These findings suggest that calcium release activation can be profoundly influenced by the recent history of L-type Ca2+ channel activity due to potentiation of ryanodine receptors (RyRs) by previous calcium influx. This conclusion was confirmed at the level of single RyRs in planar lipid bilayers: using flash photolysis of the calcium cage NP-EGTA to generate two sequential calcium stimuli, we showed that RyR activation in response to the second stimulus was four times higher than that in response to the first stimulus. excitation-contraction coupling  相似文献   

5.
Antisense oligodeoxynucleotides (AS-oligos) targeted to theNa+/Ca2+exchanger (NCX) inhibit NCX-mediatedCa2+ influx in mesenteric artery(MA) myocytes [Am. J. Physiol.269 (Cell Physiol. 38):C1340-C1345, 1995]. Here, we show AS-oligo knockdown ofNCX-mediated Ca2+ efflux. Ininitial experiments, the cytosolic freeCa2+ concentration([Ca2+]cyt)was raised, and sarcoplasmic reticulum (SR)Ca2+ sequestration was blockedwith caffeine and cyclopiazonic acid; the extracellularNa+-dependent (NCX) component ofCa2+ efflux was then selectivelyinhibited in AS-oligo-treated cells but not in controls (no oligos ornonsense oligos). In contrast, theLa3+-sensitive (plasmalemmaCa2+ pump) component ofCa2+ efflux was unaffected inAS-oligo-treated cells. Knockdown of NCX activity was reversed byincubating AS-oligo-treated cells in normal media for 5 days. Transient[Ca2+]cytelevations evoked by serotonin (5-HT) at 15-min intervals inAS-oligo-treated cells were indistinguishable from those in controls.When cells were stimulated every 3 min, however, the peak amplitudes ofthe second and third responses were larger, and[Ca2+]cytreturned to baseline more slowly, in AS-oligo-treated cells than incontrols. Peak 5-HT-evoked responses in the controls, but notAS-oligo-treated cells, were augmented more than twofold inNa+-free media. This implies thatNCX is involved in Na+ gradientmodulation of SR Ca2+ stores andcell responsiveness. The repetitive stimulation data suggest that theNCX may be important during tonic activation of arterial myocytes.

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6.
The effect of -adrenergic stimulation on cardiac Na+/Ca2+ exchange has been controversial. To clarify the effect, we measured Na+/Ca2+ exchange current (INCX) in voltage-clamped guinea pig, mouse, and rat ventricular cells. When INCX was defined as a 5 mM Ni2+-sensitive current in guinea pig ventricular myocytes, 1 µM isoproterenol apparently augmented INCX by 32%. However, this increase was probably due to contamination of the cAMP-dependent Cl current (CFTR-Cl current, ICFTR-Cl), because Ni2+ inhibited the activation of ICFTR-Cl by 1 µM isoproterenol with a half-maximum concentration of 0.5 mM under conditions where INCX was suppressed. Five or ten millimolar Ni2+ did not inhibit ICFTR-Cl activated by 10 µM forskolin, an activator of adenylate cyclase, suggesting that Ni2+ acted upstream of adenylate cyclase in the -adrenergic signaling pathway. Furthermore, in a low-extracellular Cl bath solution, 1 µM isoproterenol did not significantly alter the amplitude of Ni2+-sensitive INCX at +50 mV, which is close to the reversal potential of ICFTR-Cl. No change in INCX amplitude was induced by 10 µM forskolin. When INCX was activated by extracellular Ca2+, it was not significantly affected by 1 µM isoproterenol in guinea pig, mouse, or rat ventricular cells. We concluded that -adrenergic stimulation does not have significant effects on INCX in guinea pig, mouse, or rat ventricular myocytes. cystic fibrosis transmembrane conductance regulator; nickel ion  相似文献   

7.
Phospholamban(PLB) ablation is associated with enhanced sarcoplasmic reticulum (SR)Ca2+ uptake and attenuation of thecardiac contractile responses to -adrenergic agonists. In thepresent study, we compared the effects of isoproterenol (Iso) on theCa2+ currents(ICa) ofventricular myocytes isolated from wild-type (WT) and PLB knockout(PLB-KO) mice. Current density and voltage dependence ofICa were similarbetween WT and PLB-KO cells. However, ICa recorded fromPLB-KO myocytes had significantly faster decay kinetics. Iso increasedICa amplitude inboth groups in a dose-dependent manner (50% effective concentration,57.1 nM). Iso did not alter the rate ofICa inactivationin WT cells but significantly prolonged the rate of inactivation inPLB-KO cells. When Ba2+ was usedas the charge carrier, Iso slowed the decay of the current in both WTand PLB-KO cells. Depletion of SRCa2+ by ryanodine also slowed therate of inactivation ofICa, and subsequent application of Iso further reduced the inactivation rate ofboth groups. These results suggest that enhancedCa2+ release from the SR offsetsthe slowing effects of -adrenergic receptor stimulation on the rateof inactivation ofICa.

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8.
Store-operated Ca2+ entry (SOCE), which is Ca2+ entry triggered by the depletion of intracellular Ca2+ stores, has been observed in many cell types, but only recently has it been suggested to occur in cardiomyocytes. In the present study, we have demonstrated SOCE-dependent sarcoplasmic reticulum (SR) Ca2+ loading (loadSR) that was not altered by inhibition of L-type Ca2+ channels, reverse mode Na+/Ca2+ exchange (NCX), or nonselective cation channels. In contrast, lowering the extracellular [Ca2+] to 0 mM or adding either 0.5 mM Zn2+ or the putative store-operated channel (SOC) inhibitor SKF-96365 (100 µM) inhibited loadSR at rest. Interestingly, inhibition of forward mode NCX with 30 µM KB-R7943 stimulated SOCE significantly and resulted in enhanced loadSR. In addition, manipulation of the extracellular and intracellular Na+ concentrations further demonstrated the modulatory role of NCX in SOCE-mediated SR Ca2+ loading. Although there is little knowledge of SOCE in cardiomyocytes, the present results suggest that this mechanism, together with NCX, may play an important role in SR Ca2+ homeostasis. The data reported herein also imply the presence of microdomains unique to the neonatal cardiomyocyte. These findings may be of particular importance during open heart surgery in neonates, in which uncontrolled SOCE could lead to SR Ca2+ overload and arrhythmogenesis. cardiac ontogeny; cardiac excitation-contraction coupling; calcium homeostasis  相似文献   

9.
Xu L  Chen J  Li XY  Ren S  Huang CX  Wu G  Li XY  Jiang XJ 《Molecular biology reports》2012,39(4):3847-3852
Na+/Ca2+ exchanger (NCX) plays important roles in cardiac electrical activity and calcium homeostasis. NCX current (INCX) shows transmural gradient across left ventricle in many species. Previous studies demonstrated that NCX expression was increased and transmural gradient of INCX was disrupted in failing heart, but the mechanisms underlying INCX remodeling still remain unknown. In present study, we used patch clamp technique to record INCX from subepicardial (EPI) myocytes and subendocardial (ENDO) myocytes isolated from sham operation (SO) mice and heart failure (HF) mice. Our results showed that INCX was higher in normal EPI cells compared with that in ENDO, whatever for forward mode or reverse mode. In HF group, INCX was significantly up-regulated, but EPI-ENDO difference was disrupted because of a more increase of INCX in ENDO myocytes. In order to explore the molecular mechanism underlying remodeling of INCX in failing heart, we detected the protein expression of NCX1 and Ca2+/calmodulin-dependent protein kinase II (CaMKII) by Western blot. We found that CaMKII activity was dramatically enhanced and parallel with the expression of NCX1 in failing heart. Our study demonstrated that transmural gradient of INCX existed in murine left ventricle, and increased activity of CaMKII should account for INCX remodeling in failing heart.  相似文献   

10.
This study examines whether fluid pressure (FP) modulates the L-type Ca2+ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (16 dyn/cm2) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca2+ current (ICa) and cytosolic Ca2+ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed ICa (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of ICa. The level of ICa suppression by FP depended on the level and duration of pressure. The Ba2+ current through the Ca2+ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca2+ channel during FP stimulation. The cytosolic Ca2+ transients and the basal Ca2+ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the ICa and on the Ca2+ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca2+ buffer, eliminated the FP-induced acceleration of ICa inactivation and reduced the inhibitory effect of the FP on ICa by 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca2+ release, eliminated the accelerating effect of FP on the ICa inactivation, and they reduced the inhibitory effect of FP on the ICa. These results suggest that the fluid pressure indirectly suppresses the Ca2+ channel by enhancing the Ca2+-induced intracellular Ca2+ release in rat ventricular myocytes. L-type Ca2+ current; fluid pressure; ventricular myocytes; cytosolic Ca2+ transient  相似文献   

11.
The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (If)/neuronal (Ih) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and Ih channels in neurons. This raises the possibility of Ca2+ permeation in If, the Ih counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca2+ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by If in rat ventricular myocytes. We observed Ca2+ influx when HCN/If channels were activated. Ca2+ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an If channel blocker, inhibited If and Ca2+ influx at the same time. Quantitative analysis revealed that Ca2+ flux contributed to 0.5% of current produced by the HCN2 channel or If. The associated increase in Ca2+ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which If current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca2+ chelator), preactivation of If channels significantly reduced the action potential duration, and the effect was blocked by another selective If channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of If channels had no effects on action potential duration. Our data extend our previous discovery of Ca2+ influx in Ih channels in neurons to If channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels  相似文献   

12.
The cardiac Na+/Ca2+ exchanger (NCX1) is almost certainly the major Ca2+ extrusion mechanism in cardiac myocytes, although the driving force for Ca2+ extrusion is quite small. To explain multiple recent results, it is useful to think of the exchanger as a slow Ca2+ buffer that can reverse its function multiple times during the excitation-contraction cycle (ECC). An article by the group of John Reeves brings new insights to this function by analyzing the role of regulatory domains of NCX1 that mediate its activation by a rise of cytoplasmic Ca2+. It was demonstrated that the gating reactions are operative just in the physiological range of Ca2+ changes, a few fold above resting Ca2+ level, and that they prevent the exchanger from damping out the influence of mechanisms that transiently increase Ca2+ levels. Furthermore, exchangers with deleted regulatory domains are shown to reduce resting Ca2+ to lower levels than achieved by wild-type exchangers. A study by the group of Kenneth Philipson demonstrated that the NCX1 regulatory domain can bind and respond to Ca2+ changes on the time scale of the ECC in rat myocytes. At the same time, studies of transgenic mice and NCX1 knockout mice generated by the Philipson group revealed that large changes of NCX1 activity have rather modest effects on ECC. Simple simulations predict these results very well: murine cardiac ECC is very sensitive to small changes of the Na+ gradient, very sensitive to changes of the sarcoplasmic reticulum Ca2+ pump activity, and very insensitive to changes of NCX1 activity. It is speculated that the NCX1 gating reactions not only regulate coupled 3Na+:1Ca2+ exchange but also control the exchanger’s Na+ leak function that generates background Na+ influx and depolarizing current in cardiac myocytes. excitation-contraction cycle  相似文献   

13.
We previously reported thatlysoplasmenylcholine (LPlasC) altered the action potential (AP) andinduced afterdepolarizations in rabbit ventricular myocytes. In thisstudy, we investigated how LPlasC alters excitation-contractioncoupling using edge-motion detection, fura-PE3 fluorescent indicator,and perforated and whole cell patch-clamp techniques. LPlasC increasedcontraction, myofilament Ca2+ sensitivity, systolic anddiastolic free Ca2+ levels, and the magnitude ofCa2+ transients concomitant with increases in the maximumrates of shortening and relaxation of contraction and the rising anddeclining phases of Ca2+ transients. In some cells, LPlasCinduced arrhythmias in a pattern consistent with early and delayedaftercontractions. LPlasC also augmented the caffeine-inducedCa2+ transient with a reduction in the decay rate.Furthermore, LPlasC enhanced L-type Ca2+ channel current(ICa,L) and outward currents. LPlasC-induced alterations in contraction and ICa,L wereparalleled by its effect on the AP. Thus these results suggest thatLPlasC elicits distinct, potent positive inotropic, lusitropic, andarrhythmogenic effects, resulting from increases in Ca2+influx, Ca2+ sensitivity, sarcoplasmic reticular (SR)Ca2+ release and uptake, SR Ca2+ content, andprobably reduction in sarcolemmal Na+/Ca2+ exchange.

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14.
We examined the effectsof metabolic inhibition on intracellular Ca2+ release insingle pulmonary arterial smooth muscle cells (PASMCs). Severemetabolic inhibition with cyanide (CN, 10 mM) increased intracellularcalcium concentration ([Ca2+]i) and activatedCa2+-activated Cl currents[ICl(Ca)] in PASMCs, responses that were greatlyinhibited by BAPTA-AM or caffeine. Mild metabolic inhibition with CN (1 mM) increased spontaneous transient inward currents andCa2+ sparks in PASMCs. In Xenopus oocytes, CNalso induced Ca2+ release and activatedICl(Ca), and these responses were inhibited by thapsigarginand cyclopiazonic acid to deplete sarcoplasmic reticulum (SR)Ca2+, whereas neither heparin nor anti-inositol1,4,5-trisphosphate receptor (IP3R) antibodies affected CNresponses. In both PASMCs and oocytes, CN-evoked Ca2+release was inhibited by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and oligomycin or CCCP andthapsigargin. Whereas hypoxic stimuli resulted in Ca2+release in pulmonary but not mesenteric artery myocytes, CN induced release in both cell types. We conclude that metabolic inhibition withCN increases [Ca2+]i in both pulmonary andsystemic artery myocytes by stimulating Ca2+ release fromthe SR and mitochondria.

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15.
We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca2+ concentrations ([Ca2+]i). Because the sarcoplasmic reticulum (SR) and Na+/Ca2+ exchanger (NCX) play crucial roles in regulating [Ca2+]i and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca2+ overload by maintaining Ca2+ homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca2+ transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca2+ transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca2+]i levels and attenuated the depression of the Ca2+ transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca2+ release channels and/or ryanodine receptors (RyRs) and SR Ca2+ pump ATPase (SERCA2) and SR Ca2+ release and uptake. In addition, a delayed decay rate time constant of Ca2+ transients and cell shortening of Ca2+ transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca2+ homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R. intracellular Ca2+ concentration; Ca2+ transients; Ca2+ transporters; myofilament Ca2+ sensitivity  相似文献   

16.
In cardiac myocytes, excitation-contraction coupling depends upon sarcoplasmic reticular Ca2+ release triggered by Ca2+ influx through L-type Ca2+ channels. Although Na+-Ca2+ exchange (NCX) is essential for Ca2+ extrusion, its participation in the trigger process of excitation-contraction coupling is controversial. To investigate the role of NCX in triggering, we examined Ca2+ sparks in ventricular cardiomyocytes isolated from wild-type (WT) and cardiac-specific NCX knockout (KO) mice. Myocytes from young NCX KO mice are known to exhibit normal resting cytosolic Ca2+ and normal Ca2+ transients despite reduced L-type Ca2+ current. We loaded myocytes with fluo-3 to image Ca2+ sparks using confocal microscopy in line-scan mode. The frequency of spontaneous Ca2+ sparks was reduced in KO myocytes compared with WT. However, spark amplitude and width were increased in KO mice. Permeabilizing the myocytes with saponin eliminated differences between spontaneous sparks in WT and KO mice. These results suggest that sarcolemmal processes are responsible for the reduced spark frequency and increased spark width and amplitude in KO mice. When myocytes were loaded with 1 mM fluo-3 and 3 mM EGTA via the patch pipette to buffer diadic cleft Ca2+, the number of sparks triggered by action potentials was reduced by 60% in KO cells compared to WT cells, despite similar SR Ca2+ content in both cell types. When EGTA was omitted from the pipette solution, the number of sparks triggered in KO and WT myocytes was similar. Although the number of sparks was restored in KO cells, Ca2+ release was asynchronous. These results suggest that high subsarcolemmal Ca2+ is required to ensure synchronous triggering with short spark latency in the absence of NCX. In WT mice, high subsarcolemmal Ca2+ is not required for synchronous triggering, because NCX is capable of priming the diadic cleft with sufficient Ca2+ for normal triggering, even when subsarcolemmal Ca2+ is lowered by EGTA. Thus, reducing subsarcolemmal Ca2+ with EGTA in NCX KO mice reveals the dependence of Ca2+ release on NCX.  相似文献   

17.
Palytoxin is a coral toxin that seriously impairs heart function, but its effects on excitation-contraction (E-C) coupling have remained elusive. Therefore, we studied the effects of palytoxin on mechanisms involved in atrial E-C coupling. In field-stimulated cat atrial myocytes, palytoxin caused elevation of diastolic intracellular Ca2+ concentration ([Ca2+]i), a decrease in [Ca2+]i transient amplitude, Ca2+ alternans followed by [Ca2+]i waves, and failures of Ca2+ release. The decrease in [Ca2+]i transient amplitude occurred despite high sarcoplasmic reticulum (SR) Ca2+ load. In voltage-clamped myocytes, palytoxin induced a current with a linear current-voltage relationship (reversal potential 5 mV) that was blocked by ouabain. Whole cell Ca2+ current and ryanodine receptor Ca2+ release channel function remained unaffected by the toxin. However, palytoxin significantly reduced Ca2+ pumping of isolated SR vesicles. In current-clamped myocytes stimulated at 1 Hz, palytoxin induced a depolarization of the resting membrane potential that was accompanied by delayed afterdepolarizations. No major changes of action potential configuration were observed. The results demonstrate that palytoxin interferes with the function of the sarcolemmal Na+-K+ pump and the SR Ca2+ pump. The suggested mode of palytoxin toxicity in the atrium involves the conversion of Na+-K+ pumps into nonselective cation channels as a primary event followed by depolarization, Na+ accumulation, and Ca2+ overload, which, in turn, causes arrhythmogenic [Ca2+]i waves and delayed afterdepolarizations. atrial myocytes; intracellular calcium  相似文献   

18.
We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca2+ at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and Na+/Ca2+ exchanger (NCX) handled Ca2+ in rat ventricular myocytes preconditioned with a -opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca2+ transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca2+ homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca2+ fluxes via NCX by the use of 45Ca2+ in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca2+ from the sarcoplasmic reticulum via RyR and in the removal of Ca2+ from the cytoplasm by reuptake of Ca2+ to the SR via SERCA and by extrusion of Ca2+ out of the cell via NCX. UP also reduced mitochondrial Ca2+ accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca2+ homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca2+ overload, improved recovery in contractile function, and cardioprotection. intracellular Ca2+, -opioid receptor; Na+/Ca2+ exchanger; ryanodine receptor; sarco(endo)plasmic reticulum Ca2+-ATPase  相似文献   

19.
This study examined Ca2+ handling mechanisms involved in cardioprotection induced by chronic intermittent hypoxia (CIH) against ischemia-reperfusion (I/R) injury. Adult male Sprague-Dawley rats were exposed to 10% inspired O2 continuously for 6 h daily from 3, 7, and 14 days. In isolated perfused hearts subjected to I/R, CIH-induced cardioprotection was most significant in the 7-day group with less infarct size and lactate dehydrogenase release, compared with the normoxic group. The I/R-induced alterations in diastolic Ca2+ level, amplitude, time-to-peak, and the decay time of both electrically and caffeine-induced Ca2+ transients measured by spectrofluorometry in isolated ventricular myocytes of the 7-day CIH group were less than that of the normoxic group, suggesting an involvement of altered Ca2+ handling of the sarcoplasmic reticulum (SR) and sarcolemma. We further determined the protein expression and activity of 45Ca2+ flux of SR-Ca2+-ATPase, ryanodine receptor (RyR) and sarcolemmal Na+/Ca2+ exchange (NCX) in ventricular myocytes from the CIH and normoxic groups before and during I/R. There were no changes in expression levels of the Ca2+-handling proteins but significant increases in the RyR and NCX activities were remarkable during I/R in the CIH but not the normoxic group. The augmented RyR and NCX activities were abolished, respectively, by PKA inhibitor (0.5 µM KT5720 or 0.5 µM PKI14-22) and PKC inhibitor (5 µM chelerythrine chloride or 0.2 µM calphostin C) but not by Ca2+/calmodulin-dependent protein kinase II inhibitor KN-93 (1 µM). Thus, CIH confers cardioprotection against I/R injury in rat cardiomyocytes by altered Ca2+ handling with augmented RyR and NCX activities via protein kinase activation. cardioprotection; intracellular calcium  相似文献   

20.
Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (±dP/dtmax) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca2+ homeostasis that determines the contractile function. Particular emphasis was given to Ca2+ handling by ryanodine receptor (RyR) and Na+-Ca2+ exchange (NCX). 45Ca2+ fluxes via the RyR, NCX, and Ca2+-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca2+ by these Ca2+ handling proteins with the overall Ca2+ homeostasis by determining the Ca2+ transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca2+ concentration ([Ca2+]i) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca2+ handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca2+ fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca2+ through RyR and removal of Ca2+ by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx. ovariectomy  相似文献   

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