INTRODUCTION

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REPERFUSION of ischemic myocardium leads to myocardial stunning, which is characterized by changes in the myocardial metabolic and contractile states. Alterations in Ca²⁺ homeostasis and cardiac myofilament responsiveness to Ca²⁺ are thought to be responsible for the contractile dysfunction in the stunned myocardium. Cardiomyocyte cytosolic Ca²⁺ overload causes numerous potentially degenerative states, including alterations in Ca²⁺ transport processes, altered contraction, arrhythmogenesis, and cell death. It has been suggested that the burst in reactive oxygen species (ROS) on reperfusion may contribute to Ca²⁺ overload (14).

ROS are by-products of oxygen consumption that are easily managed under normal conditions with reactive oxygen scavengers (16, 26). Several forms of ROS are generated during I/R, including superoxide (O), H₂O₂, and the highly reactive hydroxyl radical (·OH). ROS have been demonstrated to cause lipid peroxidation and myocardial injury, and are thought to trigger the contractile dysfunction observed during reperfusion (9). Increased levels of ROS production have been shown in the isolated perfused rabbit heart peaking 10-30 s after reperfusion after an ischemic episode (41, 42). Scavengers of free radicals such as catalase and superoxide dismutase can reduce myocardial stunning and reperfusion arrhythmias (3). Finally, exposure of nonischemic myocardium or myocytes to ROS can produce cell injury similar to that seen in ischemia-reperfusion (I/R) (14).

Indirect measures of oxidant stress have also been studied in humans after myocardial I/R. For example, electron spin resonance has been used to show peak production of ROS at 5 and 25 min after reperfusion in patients undergoing coronary artery bypass graft surgery (10) and an increase in oxidative stress during transient ischemia in patients undergoing elective coronary angioplasty (6).

The cellular mechanisms of oxidant injury and its relationship to Ca²⁺ overload in the cardiomyocyte have not been elucidated. High concentrations of H₂O₂ (1-10 mM) have been shown to produce Ca²⁺ overload via regulation of L-type Ca²⁺ channels, Na⁺/Ca²⁺ exchanger (NCX), and sarcoplasmic reticulum (SR) Ca²⁺ release (13, 17, 39). However, these concentrations of H₂O₂ are ~10- to 100-fold higher than those observed for the burst in ROS production during I/R.

There has been considerable interest in determining whether the effects of ROS at pathophysiological doses similar to those observed during I/R could lead to myocardial tissue damage. Utilizing cultured neonatal rat ventricular myocytes (NRVM) as an in vitro model, we (31) previously found that low doses of H₂O₂ (similar to those generated during I/R) cause contractile dysfunction, which was associated with activation of the Na⁺/H⁺ exchanger (NHE). Activity of NHE-1, the only NHE isoform in the myocardium (8), is low during normal physiological conditions. Increased NHE-1 activity during reperfusion after an ischemic episode, although protective against acidosis, paradoxically contributes to the subsequent myocardial injury. NHE-1 inhibition has been shown to protect the I/R myocardium (18).

The relationship among ROS, Ca²⁺ overload, and enhanced NHE-1 activity during I/R injury remains to be determined. One possibility is that the increased NHE-1 activity results in excess intracellular Na⁺, leading to an alteration in the activity of the NCX, favoring Ca²⁺ accumulation within the cell (19). Thus NHE-1 could have deleterious effects on myocardial tissue during I/R by contributing to the Ca²⁺ overload in the cardiomyocyte.

The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinases (MAPK) represent attractive candidate kinases for regulating NHE-1 in response to I/R and or ROS. ERK1/2 are activated during I/R in conscious rabbits (29), and H₂O₂ and hypoxia activate members of the MAPK family, including p38, c-jun NH₂-terminal kinase, and ERK1/2 (1, 23, 31). We have also shown that ERK1/2 are responsible for H₂O₂-stimulated NHE-1 activation in NRVM (31) and for NHE-1 phosphorylation in the I/R rat myocardium (26).

The present study investigates the role of ERK1/2 MAPK and NHE-1 in H₂O₂-induced Ca²⁺ overload in spontaneously beating NRVM. Short-term exposure to low levels of H₂O₂ (50 µM) caused a dramatic diastolic Ca²⁺ overload that was reduced by ERK1/2 inhibition and NHE-1 blockade. Our results are consistent with the hypothesis that H₂O₂ may cause Ca²⁺ overload in the cardiomyocyte partially through ERK1/2-mediated phosphorylation of NHE-1.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Cell preparation and culture. Primary cultures of rat ventricular myocytes were obtained from 1- to 2-day-old Sprague-Dawley rats by enzymatic dissociation of ventricular tissue (32). Myocytes were further purified with differential preplatings for 5 min on collagen-coated plates. The remaining cells were then plated onto collagen-coated glass coverslips (Warner) in 12-well plates at a density of 1,600 per mm² in complete serum-free PC-1 medium (BioWhittaker) supplemented with antibiotic-antimycotic solution (GIBCO) and 10 µM arabinosidase C and cultured for 24 h at 37°C. These dense cultures beat spontaneously within 24 h and exhibit <10% contamination by nonmuscle cells. NVRM media was changed after 24 h and then maintained for 2-24 h in a 2:1 mixture of serum-free Dulbecco's modified Eagle's-Ham's F-12-PC-1 media supplemented with an antibiotic-antimycotic solution.

Measurement of intracellular Ca²+. NVRM were loaded with 3 µM fura 2-acetoxymethyl ester in Tyrode basic salt solution (Sigma), supplemented with 0.1% bovine serum albumin for 20 min at 37°C, followed by a 30-min unloading period in Tyrode basic salt solution at room temperature, minimizing dye compartmentalization. The coverslip was placed on an inverted microscope (model IX50, Olympus) secured in an imaging chamber (Warner) and perfused with Tyrode basic salt solution for 5 min (with or without pharmacological inhibitors). This equilibration period was followed by 5 min of perfusion of Tyrode basic salt solution with 50 µM H₂O₂ and a subsequent 5- to 10-min washout period in Tyrode basic salt solution (with or without pharmacological inhibitors). For all inhibitor studies, the drug was used at the same concentration during the unloading, equilibration, H₂O₂ perfusion, and washout periods.

Measurement of intracellular pH. NRVMs were plated at a density of 3 × 10⁶ cells per 35-mm dish containing two 9 × 22-mm collagen-coated glass coverslips. Forty-eight hours after being plated, the cells were loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) by incubation with BCECF-acetoxymethyl ester (2 µM), 1 mg/ml bovine serum albumin, and 0.02% Pluronic F127 in Tyrode basic salt solution (Sigma) for 15 min in the dark at room temperature. The cells were then washed three times with Krebs solution and incubated in Krebs solution and 5 µM HOE-642 for ~1 h in the dark before BCECF fluorescence was recorded. BCECF fluorescence was recorded using a Perkin-Elmer LS50B fluorescence spectrophotometer, as previously described (31). A ratio of fluorescence emitted at 515 nm from excitation at 490 nm to that at 440 nM was converted to intracellular pH using the nigericin high-K⁺ protocol of Thomas et al. (36).

Preparation of cell lysates for MAPK experiments with Western blot analysis. Cell lysates (25 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to nitrocellulose (Hybond, Amersham). The blots were incubated for 1 h with primary antibody and 1 h with secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG). Immunoreactive bands were visualized with the use of enhanced chemiluminescence (Amersham). Autoradiograms were scanned with an AFGA scanner and densitometric analysis performed with NIH Image version 1.6.

In-gel kinase assay. NHE-1 kinase activity and MAPK activity were analyzed by the in-gel kinase assay as described previously (37). Cell lysates (30 µg) were fractionated by SDS-PAGE in a gel in which 0.15 mg/ml of NHE-1 fusion protein [amino acids 639-815 of NHE-1 coupled to glutathione S-transferase (GST)] had been copolymerized. The phosphorylation assay was performed by placing the gel in 10 ml of buffer containing 50 µM ATP with 100 µCi [-³²P]ATP and incubating for 1 h at 30°C. The reaction was terminated by immersion of the gel in a fixative solution (5% trichloroacetic acid and 10 mM sodium pyrophosphate). The radioactivity was quantified by densitometric analysis of scanned images.

Data analysis. Data were analyzed using InStat statistical software (GraphPad). All results are expressed as means ± SE. One-way analysis of variance with Dunnett's posttest was used to compare control versus treated groups. Differences between groups were considered statistically significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

H₂O₂ causes diastolic [Ca²+]_i overload in NRVM. We (31) reported that exposure of spontaneously beating NRVM to low concentrations of H₂O₂ resulted in a sustained decrease in contractility. To determine whether this contractile dysfunction was associated with intracellular Ca²⁺ overload, we examined the effects of H₂O₂ on diastolic [Ca²⁺]_i. Acute exposure to low levels of H₂O₂ caused a profound increase in diastolic [Ca²⁺]_i during the washout period, similar to the Ca²⁺ overload observed during I/R. Compared with baseline, exposure to 50 µM H₂O₂ for 5 min led to a significant 1,210 ± 325 nM (from 74.8 ± 43.7 to 1,280 ± 314 nM) increase in diastolic [Ca²⁺]_i during the subsequent 10-min washout period (Fig. 1). NRVM responded to H₂O₂ with both a time- and dose-dependent increase in resting [Ca²⁺]_i during washout. A significant alteration in Ca²⁺ overload was detected during washout after a 2.5-min exposure to 50 µM H₂O₂ (273 ± 97 nM, P < 0.05) and was maximum at 5 min (Fig. 1B). As shown in Fig. 1C, H₂O₂ elicited a concentration-dependent diastolic Ca²⁺ overload on washout that was observed at 10 µM, significant at 25 µM (584 ± 179 nM, P < 0.05) and maximum at 50 µM H₂O₂.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   H2O2 induced increases in diastolic intracelluar Ca2+ concentration ([Ca2+]i) during washout. Fura 2-loaded, spontaneously beating neonatal rat ventricular myocytes (NRVM) were perfused with H2O2 in Tyrode basic salt solution for 5 min, followed by a 10-min washout period in Tyrode basic salt solution alone. Change in diastolic [Ca2+]i was calculated as the difference between the beginning and the end of washout with Tyrode basic salt solution (10 min) after H2O2 perfusion. A: representative trace with 50 µM H2O2 for n = 10 experiments. B: time-dependent changes in [Ca2+]i. Cumulative results from n = 4 experiments. C: concentration-dependent changes in diastolic [Ca2+]i. Cumulative results from n = 4 experiments. *P < 0.05 compared with control.

Involvement of NHE-1 and ERK1/2 MAPK in H₂O₂-induced diastolic Ca²+ overload. We have previously demonstrated that H₂O₂ activates NHE-1 in an ERK1/2 MAPK-dependent manner. To link these events to H₂O₂-induced diastolic Ca²⁺ overload, we examined the effects of the NHE-1 blocker HOE-642 and the ERK kinase (MEK) inhibitor U-0126 on H₂O₂-induced increases in resting [Ca²⁺]_i (Fig. 2A). The concentrations of the inhibitors used were based on preliminary experiments that determined the minimal effective concentration that blocked H₂O₂-induced NHE-1 activity and ERK1/2 activation, respectively (data not shown). Pretreatment with HOE-642 (5 µM) led to a 50% reduction (from 1,208 ± 132 to 639 ± 92 nM, P < 0.05) in H₂O₂-induced diastolic Ca²⁺ overload (Fig. 2B). To confirm that this concentration of HOE-642 blocked NHE-1 activity, we examined its effects on H₂O₂-induced, Na⁺-dependent recovery from an acid load in spontaneously beating NRVM. As shown in Fig. 3, NRVM treated with 50 µM H₂O₂ in the absence of pharmacological inhibitors exhibited complete recovery from an acid load. Treatment with HOE-642 completely abolished NHE-1 activity. These results are consistent with HOE-642 blunting H₂O₂-induced diastolic Ca²⁺ overload secondary to NHE-1 inhibition.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Effects of inhibitors of the extracellular signal-related kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) and Na+/H+ exchanger-1 (NHE-1) on H2O2-induced increase in diastolic [Ca2+]i. Fura 2 loaded NRVM were pretreated with no inhibitor (H2O2 alone), ERK kinase (MEK) inhibitor (5 µM U-0126), or NHE-1 inhibitor (5 µM HOE-642) for 30 min. Inhibitors were used to pretreat the cells during the 30-min unloading of fura 2 and were present throughout the duration of the experiment. Change in diastolic [Ca2+]i was calculated as the difference between the beginning and the end of washout with Tyrode basic salt solution (10 min) following H2O2 perfusion. A: representative traces during a 10-min washout in Tyrode basic salt solution. B: cumulative data from n = 5 experiments. *P < 0.05 compared with control; P < 0.05 compared with H2O2 alone.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   HOE-642 blocks Na+-dependent pH recovery from an acid load. Intracellular pH (pHi) was measured with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in NRVM during exposure to NH4Cl (25 mM, light gray box), followed by removal of external Na+ (to induce an acid load) and reintroduction of Na+ (Tyrode basic salt solution). The latter two solutions also contained 50 µM H2O2. Experiments were conducted in the absence (Control) or presence of 5 µM HOE-642 (cells were pretreated with 5 µM HOE-642 for 48 min before recording began and the drug was present in all solutions). Recovery from the acid load in the presence of Na+ is an indication of NHE activity. Results are representative of 3 independent experiments.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   H2O2-induced ERK1/2 activation and NHE-1 phosphorylation are blocked by MEK inhibition. Cultured NRVM were treated with H2O2 for 20 min ± pretreatment for 45 min with U-0126. A: Western blot analysis was performed with anti-phospho-ERK1/2 antibodies (1:2,000, top) or total ERK1/2 antibodies (1:3,000, bottom). Representative blots of n = 3 experiments. B: in-gel kinase assay using 0.15 mg/ml of NHE-1 fusion protein [amino acids 501-815 of NHE-1 coupled to glutathione S-transferase (GST)] as a substrate. Top: representative blot; bottom: cumulative data from n = 3 experiments. *P < 0.01 vs. control; P < 0.01 vs. H2O2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

It is widely accepted that reperfusion of ischemic myocardium leads to contractile dysfunction and injury. Although the pathogenesis of this injury is complex, Ca²⁺ overload, ROS, and the NHE-1 have been implicated as key mechanisms that are responsible for the deleterious effects of I/R. The goal of the present study was to further elucidate the mechanisms that link ROS to altered Ca²⁺ homeostasis in cardiac myocytes. Previously, our laboratory (31, 37) showed H₂O₂-induced activation of MAPKs, increased NHE-1 activity, and decreased contractility in NRVM and adult RVM. The results from the current study provide direct evidence that low concentrations of H₂O₂ caused a significant diastolic Ca²⁺ overload that was reduced by NHE-1 blockade and abolished by MEK inhibition. This is the first evidence that directly links low levels of H₂O₂ to increases in cardiomyocyte resting [Ca²⁺]_i through NHE-1 activation.

There are several functional alterations in Ca²⁺ homeostatic proteins that could contribute to the H₂O₂-induced Ca²⁺ overload. These mechanisms include increased Ca²⁺ influx through voltage-sensitive Ca²⁺ channels (either direct modulation of channel activity or indirect through inhibition of K⁺ channels and subsequent membrane depolarization), enhanced SR Ca²⁺ release, inhibition of Ca²⁺ uptake by the SR Ca²⁺ ATPase, or enhanced Na⁺/Ca²⁺ exchange through direct modulation of the NCX or indirect activation secondary to increased NHE-1 activity. Our data are most consistent with the indirect modulation of Na⁺/Ca²⁺ exchange secondary to NHE-1 activation. The following lines of evidence support this contention. First, we have previously shown that low concentrations of H₂O₂ increase NHE-1 activity in NRVM. Second, this study demonstrates that H₂O₂-induced Ca²⁺ overload was significantly inhibited by NHE-1 blockade with HOE-642 at a concentration that completely blocks NHE-1 activity (Figs. 2 and 3). The results indicating that NHE-1 contributes to the Ca²⁺ overload that occurs in response to oxidative stress are in close agreement with previous studies (19, 38) that have demonstrated the crucial relationship between NHE activation, NCX modulation, and the development of cardiomyocyte Ca²⁺ overload. However, we cannot rule out a direct modulation of NCX by H₂O₂ because HOE-642 did not completely block H₂O₂-induced Ca²⁺ overload at concentrations that completely inhibited H₂O₂-induced, Na⁺-dependent recovery from an acid load. This possibility is supported by a recent report (33) demonstrating that direct activation of the reverse mode of NCX contributes to reoxygenation-induced cardiomyocyte injury and altered Ca²⁺ flux. In addition, a recent report (13) shows that high concentrations of H₂O₂ activate the reverse mode of NCX in guinea pig ventricular myocytes. Taken together, these results suggest that H₂O₂ causes diastolic Ca²⁺ overload by modulation of both NHE-1 and NCX.

Our results are in close agreement with the concept that specific NHE-1 inhibitors exert beneficial effects on recovery from I/R injuries and diminish post-I/R Ca²⁺ overload. Early studies (18, 27) demonstrated that amiloride or its derivatives reduce Na⁺/Ca²⁺ loading and enhance postischemic myocardial recovery. These inhibitors, however, are known to affect other sarcolemmal proteins, including NCX (11). Newer, more specific NHE-1 inhibitors have been shown to reduce myocardial damage after I/R injury that is characterized by an improvement in the postischemic recovery of left ventricular developed pressure, decreases in creatine phosphate release, and a reduction in tissue Na⁺ and Ca²⁺ content (5, 16, 24, 40).

To elucidate the mechanisms by which H₂O₂ regulates NHE-1 activity, we examined the effects of MEK inhibition with U-0126. We previously showed that activation of NHE-1 by H₂O₂ was dependent on ERK1/2 MAPKs (31). Using in-gel kinase assays, we showed that U-0126 inhibited at least three kinases that were able to phosphorylate the NHE-1 COOH tail: ERK1, ERK2, and p90^rsk (Fig. 4B). Moor et al. (25, 26), Snabaitis et al. (34), and our laboratory (37) have shown an important role of ERK MAPKs in the regulation of NHE-1 activity in cardiovascular tissue in response to neurohormones, serum, and I/R. In addition to directly phosphorylating NHE-1, ERK1/2 MAPKs also participate in NHE-1 regulation indirectly through p90^rsk because U-0126 also blocked p90^rsk-dependent NHE-1 phosphorylation. The identification of p90^rsk as a NHE-1 kinase is similar to a report in vascular smooth muscle (28) and is in close agreement with recent data (26) that demonstrated NHE-1 phosphorylation by p90^rsk in response to myocardial I/R injury. In fact, a recent study (22) indicated that although recombinant NHE-1 was a substrate for ERK1/2 and p90^rsk, the stoichiometry of phosphorylation observed for p90^rsk was greater than that for ERK1/2. p90^rsk has been shown to phosphorylate NHE-1 at Ser⁷⁰³ but the ERK1/2 site has not been identified (35). Therefore, it is likely that H₂O₂-stimulated NHE-1 activation involves phosphorylation by p90^rsk and/or ERK1/2 MAPK pathway.

Although H₂O₂ activates p38 MAPK in NRVM, it is not a major regulator of NHE-1, because the p38 inhibitor SB-203580 had no effect on H₂O₂-induced NHE-1 phosphorylation. A recent report (12) ruled out the involvement of p38 MAPK in NHE-1 activation in a human fibroblast cell line in response to osmotic stress. In agreement, also found was a diminished or insignificant role of p38 in NRVM (26). In contrast, p38 was shown to be a negative regulator of NHE-1 in VSMC treated with angiotensin II (22). Another recent report (21) has shown that p38 can phosphorylate and activate the NHE-1 and induce alkalinization in some tissues, but these effects of p38 may be tissue specific or may vary with the isoform of p38 involved.

Our results also suggest that the ERK1/2 pathway makes a significant contribution to H₂O₂-induced diastolic Ca²⁺ overload independent of NHE-1 phosphorylation, because U-0126, but not HOE-642, completely inhibited H₂O₂-induced increases in resting Ca²⁺ levels (Fig. 2). This suggests that ERK1/2 can regulate other ion transporters independently of NHE-1. The possible targets for ERK1/2 that could contribute to the cytosolic Na⁺ and Ca²⁺ load include the Na⁺-K⁺-2Cl cotransporter, which is activated by ₁-adrenergic agonist in an ERK1/2-dependent mechanism in cardiac myocytes (2), or the Na⁺-HCO₃ cotransporter, which is coupled to muscarinic receptor activation by ERK1/2 in renal epithelial cells (30). We cannot rule out the possibility that ERK1/2 can directly modulate NCX or Na⁺/K⁺ pump activity that could also contribute to alterations in Ca²⁺ or Na⁺ homeostasis. However, there is no evidence that ERK1/2 MAPKs actually regulate NCX through phosphorylation. Moreover, H₂O₂, albeit at high concentrations, actually increased NCX activity (13). On the basis of this study, one would predict that the MEK inhibitor U-0126 would actually increase intracellular Ca²⁺ levels by eliminating the increase in forward mode of NCX.

On the basis of our results, we propose that H₂O₂-induced Ca²⁺ overload is primarily mediated by ERK1/2 MAPKs and partially through the phosphorylation and activation of NHE-1 (Fig. 5). The subsequent rise in intracellular Na⁺ results in an inhibition of the forward mode and/or activation of reverse mode of NCX to ultimately cause a rise in intracellular Ca²⁺. The diastolic Ca²⁺ overload may then contribute to the contractile dysfunction that is a hallmark of I/R injury. However, it is likely that other cellular processes participate in oxidative stress-induced myocardial contractile dysfunction. These mechanisms include alterations in excitation-contraction coupling, decreased myofilament sensitivity and/or responsiveness to Ca²⁺, diminished mitochondrial function, and apoptosis. Further studies are needed to precisely define the signal transduction pathways that contribute to the altered cardiomyocyte function in response to ROS.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Proposed link between H2O2 and diastolic Ca2+ overload in spontaneously beating NRVM, shown by the circled numbers. Exposure to H2O2 (similar to the increase in ROS during ischemia-reperfusion) results in the alteration of signaling proteins involved in the MAPK pathway, ultimately leading to MEK activation (1). MEK phosphorylates and activates ERK1/2 (2). ERK1/2 MAPK phosphorylate the COOH tail of NHE-1 increasing exchanger activity (3). The resulting rise in intracellular Na+ alters NCX activity (4) leading to an increase in diastolic Ca2+ levels (5).

In conclusion, our results indicate that low levels of ROS cause a decrease in contractility and Ca²⁺ overload in NRVM. Therapies targeting ERK1/2 kinases and/or NHE-1 activation may ameliorate the alterations in Ca²⁺ homeostasis that contribute to myocardial tissue injury following I/R.