Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+ exchanger

Although the Na(+)/H(+) exchanger (NHE) is considered to be involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the Na(+)/Ca(2+) exchanger, the exact mechanisms of its participation in Ca(2+) handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify Ca(2+) movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-N-isobutyl)amiloride (MIA). [Ca(2+)](i) in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [Ca(2+)](i) and augmented the KCl-induced increase in [Ca(2+)](i) in a concentration-dependent manner. The MIA-induced increase in basal [Ca(2+)](i) was unaffected by extracellular Ca(2+), antagonists of the sarcolemmal (SL) L-type Ca(2+) channel, and inhibitors of the SL Na(+)/Ca(2+) exchanger, SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. However, the MIA-induced increase in basal [Ca(2+)](i) was attenuated by inhibitors of SL Na(+)-K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+) transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular Ca(2+) concentration and attenuated by agents that inhibit SL L-type Ca(2+) channels, the SL Na(+)/Ca(2+) exchanger, SL Na(+)-K(+)-ATPase, and SR Ca(2+) release channels and the SR Ca(2+) pump. However, the effect of MIA on the KCl-induced increase in [Ca(2+)](i) remained unaffected by treatment with inhibitors of SL Ca(2+) pump ATPase and mitochondrial Ca(2+) uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [Ca(2+)](i), whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [Ca(2+)](i) in cardiomyocytes. These results suggest that NHE may be involved in regulation of [Ca(2+)](i) and that MIA-induced increases in basal [Ca(2+)](i), as well as augmentation of the KCl-induced increase in [Ca(2+)](i), in cardiomyocytes are regulated differentially.     

ROS are required for rapid reactivation of Na+/Ca2+ exchanger in hypoxic reoxygenated guinea pig ventricular myocytes

The cardiac Na(+)/Ca(2+) exchanger (NCX) contributes to cellular injury during hypoxia, as its altered function is largely responsible for a rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)). In addition, the NCX in guinea pig ventricular myocytes undergoes profound inhibition during hypoxia and rapid reactivation during reoxygenation. The mechanisms underlying these changes in NCX activity are likely complex due to the participation of multiple inhibitory factors including altered cytosolic Na(+) concentration, pH, and ATP. Our main hypothesis is that oxidative stress is an essential trigger for rapid NCX reactivation in guinea pig ventricular myocytes and is thus a critical factor in determining the timing and magnitude of Ca(2+) overload. This hypothesis was evaluated in cardiac myocytes using fluorescent indicators to measure [Ca(2+)](i) and oxidative stress. An NCX antisense oligonucleotide was used to decrease NCX protein expression in some experiments. Our results indicate that NCX activity is profoundly inhibited in hypoxic guinea pig ventricular myocytes but is reactivated within 1-2 min of reoxygenation at a time of rising oxidative stress. We also found that several interventions to decrease oxidative stress including antioxidants and diazoxide prevented NCX reactivation and Ca(2+) overload during reoxygenation. Furthermore, application of exogenous H(2)O(2) was sufficient by itself to reactivate the NCX during sustained hypoxia and could reverse the suppression of reoxygenation-mediated NCX reactivation by diazoxide. These data suggest that elevated oxidative stress in reoxygenated guinea pig ventricular myocytes is required for rapid NCX reactivation, and thus reactivation should be viewed as an active process rather than being due to the simple decline of NCX inhibition.     

Hypoxia inhibits the Na(+)/Ca(2+) exchanger in pulmonary artery smooth muscle cells.

The cellular mechanisms underlying hypoxic pulmonary vasoconstriction are not fully understood. We examined the effect of hypoxia on Ca(2+) efflux from the cytosol in single Fura-2-loaded pulmonary artery myocytes. During mild hypoxia (pO(2)=50-60 Torr), peak [Ca(2+)](i) was increased and the rate of Ca(2+) removal from the cytosol was markedly slowed after stimuli that elevated [Ca(2+)](i). Removal of extracellular Na(+) potentiated the peak [Ca(2+)](i) rise and slowed the Ca(2+) decay rate in cells recorded under normoxic conditions; it did not further slow the Ca(2+) decay rate or potentiate the [Ca(2+)](i) increase in hypoxic cells. An Na(+)/Ca(2+) exchange current was recorded in isolated pulmonary artery myocytes. Switching from Li(+) to Na(+) (130 mM) revealed an inward current with reversal potential consistent with the Na(+)/Ca(2+) exchange current in cells in which [Ca(2+)](i) was clamped at 1 microM similar currents, although smaller, were observed with normal resting [Ca(2+)](i) using the perforated patch clamp technique. The Na(+)/Ca(2+) exchange current was markedly inhibited in myocytes exposed to mild hypoxia. RT-PCR revealed the expression of specific alternatively spliced RNAs of NCX1 in rat pulmonary arteries. These findings provide an enhanced understanding of the molecular mechanisms underlying hypoxic sensing in pulmonary arteries.     

ATP steal between cation pumps: a mechanism linking Na+ influx to the onset of necrotic Ca2+ overload

We set out to identify molecular mechanisms underlying the onset of necrotic Ca(2+) overload, triggered in two epithelial cell lines by oxidative stress or metabolic depletion. As reported earlier, the overload was inhibited by extracellular Ca(2+) chelation and the cation channel blocker gadolinium. However, the surface permeability to Ca(2+) was reduced by 60%, thus discarding a role for Ca(2+) channel/carrier activation. Instead, we registered a collapse of the plasma membrane Ca(2+) ATPase (PMCA). Remarkably, inhibition of the Na(+)/K(+) ATPase rescued the PMCA and reverted the Ca(2+) rise. Thermodynamic considerations suggest that the Ca(2+) overload develops when the Na(+)/K(+) ATPase, by virtue of the Na(+) overload, clamps the ATP phosphorylation potential below the minimum required by the PMCA. In addition to providing the mechanism for the onset of Ca(2+) overload, the crosstalk between cation pumps offers a novel explanation for the role of Na(+) in cell death.     

N-n-Butyl haloperidol iodide inhibits the augmented Na+/Ca2+ exchanger currents and L-type Ca2+ current induced by hypoxia/reoxygenation or H2O2 in cardiomyocytes

N-n-butyl haloperidol iodide (F(2)), a novel quaternary ammonium salt derivative of haloperidol, was reported to antagonize myocardial ischemia/reperfusion injuries. To investigate its mechanisms, we characterized the effects of F(2) on Na(+)/Ca(2+) exchanger currents (I(NCX)) and the L-type Ca(2+) channel current (I(Ca,L)) of cardiomyocytes during either hypoxia/reoxygenation or exposure to H(2)O(2). Using whole-cell patch-clamp techniques, the I(NCX) and I(Ca,L) were recorded from isolated rat ventricular myocytes. Exposure of cardiomyocytes to hypoxia/reoxygenation or H(2)O(2) enhanced the amplitude of the inward and outward of I(NCX) and I(Ca,L). F(2) especially inhibited the outward current of Na(+)/Ca(2+) exchanger, as well as the I(Ca,L), in a concentration-dependent manner. F(2) inhibits cardiomyocyte I(NCX) and I(Ca,L) after exposure to hypoxia/reoxygenation or H(2)O(2) to antagonize myocardial ischemia/reperfusion injury by inhibiting Ca(2+) overload.     

Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca(2+) overload and acidosis

We investigated the question of whether inhibition of the Na(+)/H(+) exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca(2+) accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na(+)-HCO(3)(-) symporter (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca(2+) (fura 2), Na(+) [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 micromol/l HOE 642 or 1 micromol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Na(i) and Ca(i), respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 +/- 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pH(i)), Na(i), and Ca(i), but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 +/- 1.4%, EIPA: 68.2 +/- 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Ca(i) overload.     

Exogenous nitric oxide triggers classic ischemic preconditioning by preventing intracellular Ca2+ overload in cardiomyocytes

The involvement of nitric oxide (NO) in the late phase of ischemic preconditioning is well established. However, the role of NO as a trigger or mediator of "classic preconditioning" remains to be determined. The present study was designed to investigate the effects of NO on calcium homeostasis in cultured newborn rat cardiomyocytes in normoxia and hypoxia. We found that treatment with the NO donor, sodium nitroprusside (SNP) induced a sustained elevation of intracellular calcium level ([Ca(2+)](i)) followed by a decrease to control levels. Elevation of extracellular calcium, which generally occurs during ischemia, caused an immediate increase in [Ca(2+)](i) and arrhythmia in cultures of newborn cardiomyocytes. Treatment with SNP decreased [Ca(2+)](i) to control levels and re-established synchronized beating of cardiomyocytes. A decrease in extracellular [Na(+)], which inhibits the Na(+)/Ca(2+) exchanger, did not prevent [Ca(2+)](i) reduction by SNP. In contrast, application of thapsigargin, an inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), increased [Ca(2+)](i), and in its presence, SNP did not reduce [Ca(2+)](i), indicating that Ca(2+) reduction is achieved via activation of SERCA2a. The results obtained suggest that activation of SERCA2a by SNP increases Ca(2+) uptake into the sarcoplasmic reticulum (SR) and prevents cytosolic Ca(2+) overload, which might explain the protective effect of SNP from hypoxic damage.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.     

Conformational changes of the Ca(2+) regulatory site of the Na(+)-Ca(2+) exchanger detected by FRET

The Na(+)-Ca(2+) exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca(2+) for 3 Na(+) ions entering the cell, regulating intracellular Ca(2+) levels and thereby contractility. Na(+)-Ca(2+) exchanger activity is regulated by intracellular Ca(2+), which binds to a region (amino acids 371-508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca(2+) activates the exchanger and removes Na(+)-dependent inactivation. The physiological role of intracellular Ca(2+) regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH(2)- and CO(2)H-termini of the exchanger Ca(2+) binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca(2+) concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca(2+) concentrations. Titrations of Ca(2+) concentration versus FRET efficiency indicate a K(D) for Ca(2+) of approximately 140 nM, which increased to approximately 400 nM in the presence of 1 mM Mg(2+). Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca(2+) on a beat-to-beat basis during excitation-contraction coupling.     

Regulation of in vivo cardiac contractility by phospholemman: role of Na+/Ca2+ exchange

Phospholemman (PLM), when phosphorylated at serine 68, relieves its inhibition on Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger 1 (NCX1) in cardiac myocytes. Under stress when catecholamine levels are high, enhanced Na(+)-K(+)-ATPase activity by phosphorylated PLM attenuates intracellular Na(+) concentration ([Na(+)](i)) overload. To evaluate the effects of PLM on NCX1 on in vivo cardiac contractility, we injected recombinant adeno-associated virus (serotype 9) expressing either the phosphomimetic PLM S68E mutant or green fluorescent protein (GFP) directly into left ventricles (LVs) of PLM-knockout (KO) mice. Five weeks after virus injection, ～40% of isolated LV myocytes exhibited GFP fluorescence. Expression of S68E mutant was confirmed with PLM antibody. There were no differences in protein levels of α(1)- and α(2)-subunits of Na(+)-K(+)-ATPase, NCX1, and sarco(endo)plasmic reticulum Ca(2+)-ATPase between KO-GFP and KO-S68E LV homogenates. Compared with KO-GFP myocytes, Na(+)/Ca(2+) exchange current was suppressed, but resting [Na(+)](i), Na(+)-K(+)-ATPase current, and action potential amplitudes were similar in KO-S68E myocytes. Resting membrane potential was slightly lower and action potential duration at 90% repolarization (APD(90)) was shortened in KO-S68E myocytes. Isoproterenol (Iso; 1 μM) increased APD(90) in both groups of myocytes. After Iso, [Na(+)](i) increased monotonically in paced (2 Hz) KO-GFP but reached a plateau in KO-S68E myocytes. Both systolic and diastolic [Ca(2+)](i) were higher in Iso-stimulated KO-S68E myocytes paced at 2 Hz. Echocardiography demonstrated similar resting heart rate, ejection fraction, and LV mass between KO-GFP and KO-S68E mice. In vivo closed-chest catheterization demonstrated enhanced contractility in KO-S68E compared with KO-GFP hearts stimulated with Iso. We conclude that under catecholamine stress when [Na(+)](i) is high, PLM minimizes [Na(+)](i) overload by relieving its inhibition of Na(+)-K(+)-ATPase and preserves inotropy by simultaneously inhibiting Na(+)/Ca(2+) exchanger.     

Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes

In this study, we examined the acute effects of thyroid hormones (TH) T(3) and T(4), leading to improvement of myocardial function through activation of Ca(2+) extrusion mechanisms and, consequently, prevention of intracellular calcium overload. Extracellular calcium elevation from 1.8 to 3.8 mM caused immediate increase in intracellular calcium level ([Ca(2+)](i)) in newborn cardiomyocyte cultures. Administration of 10 or 100 nM T(3) or T(4) rapidly (within 10 sec) decreased [Ca(2+)](i) to its control level. Similar results were obtained when [Ca(2+)](i) was elevated by decreasing extracellular Na(+) concentration, causing backward influx of Ca(2+) through Na(+)/Ca(2+) exchanger, or by administration of caffeine, releasing Ca(2+) from the sarcoplasmic reticulum (SR). Under these conditions, T(3) or T(4) decreased [Ca(2+)](i). T(3) and T(4) also exhibited protective effects during ischemia. T(3) or T(4) presence during hypoxia for 120 min in culture medium restricted the increase of [Ca(2+)](i) and prevented the pathological effects of its overload. An inhibitor of SR Ca(2+)-ATPase (SERCA2a), thapsigargin, increases [Ca(2+)](i) and in its presence neither T(3) nor T(4) had any effect on the [Ca(2+)](i) level. The reduction of [Ca(2+)](i) level by T(3) and T(4) was also blocked in the presence of H-89 (a PKA inhibitor), and by calmodulin inhibitors. The effect of TH on the reduction of [Ca(2+)](i) was prevented by propranolol, indicating that the hormones exert their effect through interaction with adrenergic receptors. These results support our hypothesis that TH prevent calcium overload in newborn rat cardiomyocytes, most likely by a direct, acute, and nongenomic effect on Ca(2+) transport into the SR.     

Calcium transport mechanisms in muskrat and rat hearts

Mammalian hearts experience calcium overload during extreme and prolonged hypoxia and the calcium overload may lead to enzyme activation and cell death. Several calcium transport systems were examined in muskrat hearts and compared to those found in rat hearts to determine if there is a species difference that might be related to the muskrats' superior ability to survive hypoxia. Radiolabeled nitredendipine binding was determined in rat and muskrat hearts to estimate the density of voltage gated calcium channels in surface membranes. There were no species differences. Calcium release channel density in the sarcoplasmic reticulum was estimated by the determination of radiolabeled ryanodine binding in muskrat and rat heart SR membranes. No differences were revealed between species. The SR uptake of calcium was measured in SR membranes from the hearts of the two species. No differences were found in the B(max) values, however, the muskrat SR membranes did have a slightly lower K(m) value. There were large species differences in Na(+)/Ca(2+) exchange in SL membranes with the muskrat heart having approximately 3.5 times the transport capacity of rat SL membranes. During hypoxic conditions in which there is extensive ATP depletion leading to [Na(+)](i) accumulation and discharge of cellular membrane potential, the Na(+)/Ca(2+) exchanger may operate in the reverse mode and import calcium into the cell and accelerate hypoxic damage. Prior to reaching this state a robust Na(+)/Ca(2+) exchange would facilitate the maintenance of normal diastolic calcium levels and calcium cycling. Muskrats hearts are hypoxia tolerant by virtue of their ability to reduce metabolic demand and generate ATP anaerobically thus, maintaining a favorable ATP balance. Therefore, the relative overexpression of Na(+)/Ca(2+) exchangers in muskrat hearts may be beneficial in the preservation of contractile function and calcium homeostasis in this freshwater diving mammal.     

Enhanced gene expression of Na(+)/Ca(2+) exchanger attenuates ischemic and hypoxic contractile dysfunction

Enhanced gene expression of the Na(+)/Ca(2+) exchanger in failing hearts may be a compensatory mechanism to promote influx and efflux of Ca(2+), despite impairment of the sarcoplasmic reticulum (SR). To explore this, we monitored intracellular calcium (Ca(i)(2+)) and cardiac function in mouse hearts engineered to overexpress the Na(+)/Ca(2+) exchanger and subjected to ischemia and hypoxia, conditions known to impair SR Ca(i)(2+) transport and contractility. Although baseline Ca(i)(2+) and function were similar between transgenic and wild-type hearts, significant differences were observed during ischemia and hypoxia. During early ischemia, Ca(i)(2+) was preserved in transgenic hearts but significantly altered in wild-type hearts. Transgenic hearts maintained 40% of pressure-generating capacity during early ischemia, whereas wild-type hearts maintained only 25% (P < 0.01). During hypoxia, neither peak nor diastolic Ca(i)(2+) decreased in transgenic hearts. In contrast, both peak and diastolic Ca(i)(2+) decreased significantly in wild-type hearts. The decline of Ca(i)(2+) was abbreviated in hypoxic transgenic hearts but prolonged in wild-type hearts. Peak systolic pressure decreased by nearly 10% in hypoxic transgenic hearts and >25% in wild-type hearts (P < 0.001). These data demonstrate that enhanced gene expression of the Na(+)/Ca(2+) exchanger preserves Ca(i)(2+) homeostasis during ischemia and hypoxia, thereby preserving cardiac function in the acutely failing heart.     

Hypoxic pulmonary vasoconstriction in intact rat intrapulmonary arteries is not initiated by inhibition of Na+-Ca2+ exchange

It has been proposed that a hypoxia-induced inhibition of the Na(+)-Ca(2+) exchanger (NCX) contributes to hypoxic pulmonary vasoconstriction (HPV). By recording isometric tension development in rat intrapulmonary arteries (IPA), we examined the effect on HPV of maneuvers that reduce the ability of NCX to regulate intracellular Ca(2+) concentration ([Ca(2+)](i)). In some experiments, fura pentakis(acetoxymethyl) ester-3 (fura PE-3) was also used to monitor [Ca(2+)](i). HPV was elicited in IPA that were pretreated with 10 microM diltiazem and slightly preconstricted with PGF(2alpha), which enhances the hypoxic response. Substitution of Na(+) with Li(+) increased HPV and the associated rise in [Ca(2+)](i). Pretreatment with ouabain (100 microM) to diminish the Na(+) gradient or with the reverse-mode NCX inhibitor KB-R7943 (3 or 10 microM) had no significant effect on HPV. Combined treatment with ouabain and low-[Na(+)] (24 mM) solution enhanced HPV strongly. The role of NCX in Ca(2+) extrusion was examined by assessing the decrease in [Ca(2+)](i) in Ca(2+)-free physiological saline solution either containing or lacking Na(+) following a high K(+)-induced loading of cellular [Ca(2+)]. Although the large initial rapid fall in [Ca(2+)] was Na(+) independent, final recovery of [Ca(2+)] to its basal level was delayed in the absence of Na(+). Therefore, HPV persisted or was increased under conditions in which forward-mode NCX was already attenuated or prevented, demonstrating that inhibition of NCX by hypoxia is unlikely to initiate HPV. Instead, NCX appears to act to inhibit HPV as would be expected if it is functioning to extrude Ca(2+).     

H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes

The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular Ca(2+) handling in hydrogen sulfide (H(2)S)-preconditioned cardiomyocytes and its consequent effects on ischemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an H(2)S donor, 10(-4) M) preconditioning (SP, 30 min). Intracellular Ca(2+) was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKCalpha, PKCepsilon, and PKCdelta to membrane fraction but only translocation of PKCepsilon and PKCdelta was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca(2+)] transients, which were reversed by a selective PKC inhibitor chelerythrine. These data suggest that SP facilitated Ca(2+) removal via both accelerating uptake of Ca(2+) into sarcoplasmic reticulum and enhancing Ca(2+) extrusion through Na(+)/Ca(2+) exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca(2+) overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKCalpha, PKCepsilon, and PKCdelta in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular Ca(2+) handling.     

Hypoxic postconditioning reduces cardiomyocyte loss by inhibiting ROS generation and intracellular Ca2+ overload

We have shown that intermittent interruption of immediate reflow at reperfusion (i.e., postconditioning) reduces infarct size in in vivo models after ischemia. Cardioprotection of postconditioning has been associated with attenuation of neutrophil-related events. However, it is unknown whether postconditioning before reoxygenation after hypoxia in cultured cardiomyocytes in the absence of neutrophils confers protection. This study tested the hypothesis that prevention of cardiomyocyte damage by hypoxic postconditioning (Postcon) is associated with a reduction in the generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h of hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned after the 3-h index hypoxia by three cycles of 5 min of reoxygenation and 5 min of rehypoxia applied before 6 h of reoxygenation. Relative to sham control and hypoxia alone, the generation of ROS (increased lucigenin-enhanced chemiluminescence, SOD-inhibitable cytochrome c reduction, and generation of hydrogen peroxide) was significantly augmented after immediate reoxygenation as was the production of malondialdehyde, a product of lipid peroxidation. Concomitant with these changes, intracellular and mitochondrial Ca(2+) concentrations, which were detected by fluorescent fluo-4 AM and X-rhod-1 AM staining, respectively, were elevated. Cell viability assessed by propidium iodide staining was decreased consistent with increased levels of lactate dehydrogenase after reoxygenation. Postcon treatment at the onset of reoxygenation reduced ROS generation and malondialdehyde concentration in media and attenuated cardiomyocyte death assessed by propidium iodide and lactate dehydrogenase. Postcon treatment was associated with a decrease in intracellular and mitochondrial Ca(2+) concentrations. These data suggest that Postcon treatment reduces reoxygenation-induced injury in cardiomyocytes and is potentially mediated by attenuation of ROS generation, lipid peroxidation, and intracellular and mitochondrial Ca(2+) overload.     

Intracellular Ca2+ oscillations, a potential pacemaking mechanism in early embryonic heart cells

Early (E9.5-E11.5) embryonic heart cells beat spontaneously, even though the adult pacemaking mechanisms are not yet fully established. Here we show that in isolated murine early embryonic cardiomyocytes periodic oscillations of cytosolic Ca(2+) occur and that these induce contractions. The Ca(2+) oscillations originate from the sarcoplasmic reticulum and are dependent on the IP(3) and the ryanodine receptor. The Ca(2+) oscillations activate the Na(+)-Ca(2+) exchanger, giving rise to subthreshold depolarizations of the membrane potential and/or action potentials. Although early embryonic heart cells are voltage-independent Ca(2+) oscillators, the generation of action potentials provides synchronization of the electrical and mechanical signals. Thus, Ca(2+) oscillations pace early embryonic heart cells and the ensuing activation of the Na(+)-Ca(2+) exchanger evokes small membrane depolarizations or action potentials.     

Na(+)] in the subsarcolemmal 'fuzzy' space and modulation of [Ca(2+)](i) and contraction in cardiac myocytes

The strength of the heart beat depends on the amplitude and time course of the transient increase in [Ca(2+)] in the myocytes with each cycle. [Na(+)](i) modulates cardiac contraction through its effect on the Ca(2+) flux through the Na/Ca exchanger. Cardiac excitation-contraction coupling has been postulated to occur in a microdomain or 'fuzzy' space at the junction of the T-tubules and the sarcoplasmic reticulum. This 'fuzzy' space is well described for the Ca(2+) fluxes and the interaction between the L-type Ca(2+) channel, the Ca(2+) release channel of the sarcoplasmic reticulum and the Na/Ca exchanger. Co-localization of the Na(+) transporters, in particular the Na/K pump and the Na(+) channel, within this 'fuzzy' space is not as well established. The functional and morphological characteristics of the 'fuzzy' space for Na(+) and its interaction with the Ca(2+) handling suggest that this space is not strictly co-inciding with the Ca(2+) microdomain. In this space [Na(+)] can be several-fold higher or lower than [Na(+)] in the bulk cytosol. This has implications for modulation of [Ca(2+)](i) during a single beat as well as during alterations in Na(+) fluxes seen in pathological conditions.