Na+/H+ exchange and its inhibition in cardiac ischemia and reperfusion

Summary The characterization of various ion transport systems has led to a better understanding of the effects, which seem to take part in the impairment of ischemic and reperfused cardiac tissue. This review discusses the role of the Na⁺/H⁺ exchange system in the pathophysiology of ischemia and reperfusion and the beneficial effects of its inhibition.At the onset of ischemia intracellular pH (pH_i) decreases due to anaerobic metabolism and ATP hydrolysis, leading to an activation of Na⁺/H⁺ exchange. This in turn increases intracellular Na⁺ (Na⁺ _i) and activates Na⁺/K⁺ ATPase, with a consecutive increase of energy consumption. Since cellular Na⁺ and Ca⁺⁺ transport are coupled by the Na⁺/Ca⁺⁺ exchange system, which depends on the Na⁺ gradient, the high Na⁺ _i leads to increased intracellular Ca⁺⁺ (Ca⁺⁺ _i). After a certain period, Na⁺/H⁺ exchange is inactivated by a decrease of extracellular pH.In case of reperfusion the acid extracellular fluid is washed out, which reactivates Na⁺/H⁺ exchange, leading to an unfavourably fast restoration of pH_i and a second time to Na⁺ and Ca⁺⁺ _i overflow.High Ca⁺⁺ _i is assumed to be one of the main reasons for ischemic and reperfusion injury, like arrhythmias, myocardial contracture, stunning and necrosis.It seems that the inhibition of Na⁺/H⁺ exchange can interrupt this process at an early phase and prevent or delay the consequences of ischemia and reperfusion as demonstrated by numerous investigators.     

Role of the cardiac Na+/H+ exchanger during ischemia and reperfusion

The coupled exchanger theory describes one of the central mechanisms of damage in the ischemic heart. The theory proposes that anaerobic glycolysis produces lactate and protons and that the protons can leave the cardiac cell on the cardiac Na+/H+ exchanger (NHE1). The subsequent rise in [Na+]i stimulates the cardiac Na+/Ca2+ exchanger (NCX) and results in an increase in [Ca2+]i which promotes myocardial cell damage. Although the general features of this theory are widely accepted, there is dispute about some aspects, specifically whether the NHE1 remains active during ischemia or not. We review the evidence on this issue and conclude that NHE1 is substantially inhibited during ischemia. This issue is central to the design of a clinical trial of NHE1 inhibitors in the treatment of human cardiac ischemia and the existing clinical trials are considered in this light.     

The role of Na+/H+ exchange in ischemia-reperfusion

In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pH_i-regulating transport systems involved is the Na⁺/H⁺ exchanger. Inhibitors of the Na⁺/H⁺ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na⁺ and secondary Ca²⁺ influx. Under reperfusion conditions Na⁺/H⁺ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na⁺/H⁺ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.     

Estrogen receptor regulation of the Na+/H+ exchange regulatory factor.

To better understand the actions of estrogens and antiestrogens in estrogen target cells, we have searched for estrogen-regulated genes in human breast cancer cells, in which the number of genes known to be directly activated by estrogen is quite small. Using differential display RNA methods, we have identified the human homolog of the Na+ -H+ exchanger regulatory factor (NHE-RF), an approximately 50-kDa protein that is also an ezrin-radixin-moesin-binding phosphoprotein, as being under rapid and direct regulation by estrogen in estrogen receptor (ER)-containing breast cancer cells. Stimulation by estrogen of NHE-RF RNA is rapid, being near maximal (approximately 6-fold) by 1 h, and is not blocked by cycloheximide, indicating that it is a primary response. Stimulation is selective for estrogen ligands, with no stimulation by other classes of steroid hormones, and stimulation by estrogen is suppressed by the antiestrogens tamoxifen and ICI 182,780. Induction is shown to require an active ER through several approaches, including the use of ER-negative breast cancer cells containing a stably integrated ER. NHE-RF protein levels, monitored using antibodies specific for this protein, increase after estrogen and reach maximal levels at 24-48 h. Interestingly, NHE-RF is a PDZ domain-containing protein that is enriched in polarized epithelia, where it is known to be localized in microvilli. Among various human tissues we have examined, we found that NHE-RF is expressed at a fairly high level in mammary tissue. NHE-RF regulates protein kinase A inhibition of the Na+ -H+ exchanger and may serve as a scaffold adaptor protein that contributes to the specificity of signal transduction events. Our findings suggest that the early, known effects of estrogen on cell cytoarchitecture (e.g. increasing microvilli on breast cancer cells) and on some cell signaling pathways (e.g. those involving cAMP) may involve rapid estrogen-mediated changes in the production of NHE-RF.     

Prevention of reperfusion-induced arrhythmia by Na+/H+ exchange block

Using the isolated papillary muscle and rat hearts, perfused by Langendorf, the effects of the Na+/H+ exchange blocker, ethylisopropylamiloride (EIPA), on electrical activity and contractility, and induction of ischemic and reperfusion arrhythmias were studied. In the experiments with regional ischemia and reperfusion of an isolated heart (the ligation of the left anterior descending coronary artery for 10 minutes), EIPA (5 microM) effectively abolished reperfusion fibrillations, reducing the incidence of the long fibrillations from 60% (in the controls) to 8%, and increased nearly five-fold the time interval prior to their onset. Antiarrhythmic action of EIPA seems to be unconnected with the direct block of ionic channels, because 5 microM of this compound did not significantly change the action potential parameters, first derivative Vmax and the contractile response of the papillary muscle in normal conditions. The results obtained show a significant role of the postischemic activation of the Na+/H+ exchange in the initiation of reperfusion-induced arrhythmias and possible use of amiloride derivatives for their prevention.     

Pre-treatment with the Na+/H+ exchange inhibitor cariporide delays cell-to-cell electrical uncoupling during myocardial ischemia

OBJECTIVE: Inhibition of Na(+)-H(+) exchange (NHE) delays the onset of myocardial rigor contracture during ischemia. The aim of this study was to analyse the effects of NHE inhibition on cell-to-cell electrical uncoupling during myocardial ischemia/reperfusion. METHODS: Twenty-six isolated rat hearts and 23 in situ porcine hearts were submitted to no-flow ischemia followed by reperfusion, with or without pre-treatment with cariporide (7 microM in rats and 3 mg/kg in pigs). Ischemic rigor and hypercontracture, conduction velocity and myocardial electrical impedance were monitored. RESULTS: Pre-treatment with cariporide delayed ATP depletion (luminescence assay in rat myocardium) and onset of rigor contracture (tension recordings or ultrasonic crystals) during ischemia both in rat and pig hearts (P<0.05). In addition, cariporide delayed the onset of sharp changes in tissue resistivity and phase angle in impedance recordings (four-electrode probes) from 10+/-1 to 13+/-1 min (P<0.001) in rat hearts, and from 22+/-1 to 38+/-2 min (P<0.001) in pigs. Blockade of impulse propagation (transmembrane action potentials in rat hearts) was also markedly delayed by cariporide (from 14+/-1 to 20+/-1 min, P<0.001). Reperfusion-induced LDH release in rat hearts and infarct size in pigs were markedly reduced by pre-treatment with cariporide. CONCLUSIONS: Inhibition of NHE with cariporide slows the progression of ischemic injury during myocardial ischemia, and delays the onset of cell-to-cell electrical uncoupling.     

Angiotensin II and Na+/H+ exchange in human blood platelets

Human blood platelets have a high potency for Na+/H+ exchange activity; they also bear receptors for angiotensin II (Ang II). To explore the effect of Ang II on the exchange, we have used a fluorimetric analysis capable of detecting cytoplasmic changes of 0.01 pH units in platelets, together with an Ang II preparation which has been proven to be effective as a vasoconstrictor. Thrombin and adenosine diphosphate (ADP) affected Na+/H+ exchange, but Ang II did not affect Na+/H+ exchange for a wide range of concentrations and experimental conditions. It is concluded that in contrast to its function in vascular smooth muscle cells, Ang II apparently does not play a role in the regulation of Na+/H+ exchange in human blood platelets.     

Coupling of histamine H3 receptors to neuronal Na+/H+ exchange: a novel protective mechanism in myocardial ischemia

In myocardial ischemia, adrenergic nerves release excessive amounts of norepinephrine (NE), causing dysfunction and arrhythmias. With anoxia and the concomitant ATP depletion, vesicular storage of NE is impaired, resulting in accumulation of free NE in the axoplasm of sympathetic nerves. Intraneuronal acidosis activates the Na(+)/H(+) exchanger (NHE), leading to increased Na(+) entry in the nerve terminals. These conditions favor availability of the NE transporter to the axoplasmic side of the membrane, causing massive carrier-mediated efflux of free NE. Neuronal NHE activation is pivotal in this process; NHE inhibitors attenuate carrier-mediated NE release. We previously reported that activation of histamine H(3) receptors (H(3)R) on cardiac sympathetic nerves also reduces carrier-mediated NE release and alleviates arrhythmias. Thus, H(3)R activation may be negatively coupled to NHE. We tested this hypothesis in individual human SKNMC neuroblastoma cells stably transfected with H(3)R cDNA, loaded with the intracellular pH (pH(i)) indicator BCECF. These cells possess amiloride-sensitive NHE. NHE activity was measured as the rate of Na(+)-dependent pH(i) recovery in response to an acute acid pulse (NH(4)Cl). We found that the selective H(3)R-agonist imetit markedly diminished NHE activity, and so did the amiloride derivative EIPA. The selective H(3)R antagonist thioperamide abolished the imetit-induced NHE attenuation. Thus, our results provide a link between H(3)R and NHE, which may limit the excessive release of NE during protracted myocardial ischemia. Our previous and present findings uncover a novel mechanism of cardioprotection: NHE inhibition in cardiac adrenergic neurons as a means to prevent ischemic arrhythmias associated with carrier-mediated NE release.     

Activation of electrogenic Na+/K+ exchange by extracellular K+ in canine cardiac Purkinje fibers.

Transient increments in sodium pump current were elicited in small voltage-clamped Purkinje fibers suspended in a fast flow system by briefly exposing them to K+-free fluid, to temporarily inhibit the pump, and then suddenly returning them to K+-containing fluid. The exponential time course of decay of the current increment provides a measure of the pump rate constant for Na+ extrusion. The dependence of that rate constant, and of the peak amplitude of the increment in pump current, on the extracellular K+ concentration was determined. The results indicate: that in cardiac Purkinje cells, as in many other cells, the pump is half-maximally activated by about 1 mM K+; that the coupling ratio for Na+/K+ exchange is independent of either intracellular Na+ concentration or external K+ concentration; and that a simple model in which intracellular Na+ concentration is determined by a passive "leak," and an active extrusion of Na+, seems sufficient to account for moderate changes in cellular Na+ concentration.     

Role of brush border Na+/H+ exchange in canine ileal absorption

Na⁺/H⁺ exchanger isoforms have been identified in mammalian intestinal enterocytes and cloned: NHE1 on the basolateral membrane regulating intracellular pH; and NHE2 and NHE3 on the brush border serving transcellular absorption of Na⁺, NHE1 and NHE2 are much more sensitive to inhibition by amiloride than NHE3, theirin vitro IC₅₀s for amiloride being 1 μM, 1 μM and 39 μM, respectively. This study tested the hypothesis that the brush border NHE3 isoform plays the predominant role in basal and meal-stimulated ileal absorption. Absorption studies (N=72) were performed in dogs with 25-cm ileal Thiry-Vella fistulae. Six groups were studied over 4 hr. Perfusion with [¹⁴C]PEG and 140 mM Na⁺ was used to calculate absorption of water, ions, and glucose. Luminal amiloride was administered from the second to the fourth hours at doses of 20 μM in groups 3 and 4 to inhibit NHE1 and NHE2, and 1 mM in groups 5 and 6 to also inhibit NHE3. A 480-kcal canine meal was ingested after the second hour in groups 2, 4, and 6. Meal ingestion was followed by significant increases in water and electrolyte absorption. Amiloride (1 mM) caused significant reductions in basal and meal-stimulated ileal absorption, while the 20 μM dose had no effect on either. These data are consistent with the hypothesis that NHE3, but not NHE2, is involved in basal and meal-stimulated ileal water and Na⁺ absorption.     

Contribution of Na+/H+ exchange to Na+ overload in the ischemic hypertrophied hyperthyroid rat heart

OBJECTIVE: The mechanisms responsible for intracellular ion homeostasis in ischemic hypertrophied myocardium are not fully known. Moderately hypertrophied hyperthyroid hearts (T3) are characterized by the bioenergetic changes and increased Na+/H+ exchange (NHE) activity comparable with those observed in humans and experimental models of hypertrophy. Here we test the hypothesis whether NHE inhibition in T3 heart improves ion homeostasis during ischemia and contractile function during recovery. METHODS: We compared intracellular H+ (H+i) and Na+ (Na+i) accumulations during 28 min global ischemia in isolated perfused T3 and euthyroid (EUT) rat hearts with and without NHE inhibition by using 31P and 23Na NMR. Heart function was measured during control perfusion and 30 min following ischemic insult. RESULTS: In T3 hearts ischemia caused: (1) faster and greater Na+i accumulation (534+/-25% of preischemic level versus 316+/-22% in EUT, P<0.001); (2) lower acidification (pH(i) 6.66+/-0.66 versus 6.12+/-0.12 in EUT, P<0.001); and (3) faster hydrolysis of ATP. NHE inhibition (amiloride 1 mM) in T3 hearts lead to: (1) delayed and lower Na+i accumulation by 35+/-5%; (2) faster and greater acidification (pH(i) 6.45+/-0.15, P<0.05); (3) delayed ATP degradation; and (4) improved heart function during recovery. When NHE was inhibited, all T3 hearts (n=11) recovered 68+/-10% of their preischemic rate pressure product (RPP), while only two untreated T3 hearts (from 11) recovered approximately 40% of preischemic RPP. CONCLUSIONS: These data suggest that NHE inhibition could be useful intervention for the prevention of ischemic/reperfusion cell injury and could improve the function of the hypertrophied heart after acute ischemia.     

Na+/H+ exchange is not operative under low-flow ischemic conditions

Using 31P-NMR the existence of Na+/H+ exchange system and its contribution to intracellular pH (pHi) regulation were examined in the isolated isovolumic rat heart under physiological and pathophysiological conditions. Ethylisopropylamiloride (EIPA) was used as a tool to search into the role of Na+/H+ exchange system. In the normal well-oxygenated heart dose-dependent negative chronotropic effects were observed with 10(-6) to 10(-5) M EIPA. After 10(-4) M the heart ceased to beat and a progressive fall of high energy phosphates compounds occurred. However, contrary to expectation pHi did not fall but rose after EIPA. In NH4Cl-loaded hearts removal of NH4Cl resulted in a fall of the pHi followed by a rapid recovery to the normal pHi. After 10(-5) M EIPA the fall of pHi became greater and there was no recovery within 35 min of observation period. This dose of EIPA, however, did not affect the time course of changes in the pHi during 60 min of low-flow ischemia (0.2 ml/min). It is concluded that pHi regulation following an acute acid loading is dependent on amiloride-sensitive Na+/H+ exchange. However, Na+/H+ exchange system does not play an important role in maintenance of the pHi under normoxic or ischemic condition. In the normoxic heart EIPA produced a decrease in heart rate without producing any change either in myocardial energy metabolism or in pHi. Thus, the compound could be categorized as a bradycardic agent.     

Na+/H+ exchange mediates postprandial ileal water and electrolyte transport

Feeding stimulates fluid and electrolyte absorption in the small intestine. Previous studies have suggested that Na⁺/glucose cotransport is important in initiating this response in the jejunum. The purpose of this study was to determine whether Na⁺/H⁺ exchange plays a role in meal-induced absorption. Exteriorized, neurovascularly intact jejunal and ileal loops (25 cm) were constructed in dogs. Following a two-week period of postoperative recovery, the loops of awake dogs were perfused with standard buffer alone or with increasing concentrations of amiloride, a Na⁺/H⁺ exchange inhibitor. Water, sodium, and chloride fluxes were calculated following a meal using [¹⁴C]PEG as a volume marker. The meal significantly increased absorption in both the jejunum (P<0.001) and ileum (P<0.01) in those animals perfused with buffer alone. More significantly, amiloride suppressed the increased absorption seen following a meal in the ileum (P<0.001) but not the jejunum. The response in the ileum was dose dependent. These findings suggest that a major mediator of postprandial sodium and water absorption in the ileum is the Na⁺/H⁺ exchanger.Supported by NIH R29-DK-47326 (S.W.A.), R01-DK-39879 (M.J.Z.), and a VA Merit Review (D.W.M.).Portions of this work were presented at the Annual Meeting of the American Gastroenterological Association, May 1993, Boston, Massachusetts.     

Regression of isoproterenol-induced cardiac hypertrophy by Na+/H+ exchanger inhibition

Cardiac hypertrophy is often associated with an increased sympathetic drive, and both in vitro and in vivo studies have demonstrated the development of cardiomyocyte hypertrophy in response to either alpha- or beta-adrenergic stimulation. Because an association between the Na+/H+ exchanger and cellular growth has been proposed, this study aimed to analyze the possible role of the antiporter in isoproterenol-induced cardiac hypertrophy. Isoproterenol alone (5 mg/kg IP once daily) or combined with a selective inhibitor of the Na+/H+ exchanger activity (3 mg x kg(-1) x d(-1) BIIB723) was given to male Wistar rats for 30 days. Sex- and age-matched rats that received 0.9% saline IP daily served as controls. Echocardiographic follow-up showed a 33% increase in left ventricular mass in the isoproterenol-treated group, whereas it did not increase in the isoproterenol+BIIB723-treated group. Heart weight-to-body weight ratio at necropsy was 2.44+/-0.11 in controls and increased to 3.35+/-0.10 (P<0.05) with isoproterenol, an effect that was markedly attenuated by BIIB723 (2.82+/-0.07). Intense cardiomyocyte enlargement and severe subendocardial fibrosis were found in isoproterenol-treated rats, and both effects were attenuated by BIIB723. Myocardial Na+/H+ exchanger activity and protein expression significantly increased in isoproterenol-treated rats compared with the control group (1.45+/-0.11 vs 0.91+/-0.05 arbitrary units, P<0.05). This effect was significantly reduced by BIIB723 (1.17+/-0.02, P<0.05). In conclusion, our results show that Na+/H+ exchanger inhibition prevented the development of isoproterenol-induced hypertrophy and fibrosis, providing strong evidence in favor of a key role played by the antiporter in this model of cardiac hypertrophy.     

Regulation of the cardiac Na+/H+ exchanger in health and disease

The Na⁺ gradient produced across the cardiac sarcolemma by the ATP-dependent Na⁺-pump is a constant source of energy for Na⁺-dependent transporters. The plasma membrane Na⁺/H⁺ exchanger (NHE) is one such secondary active transporter, regulating intracellular pH, Na⁺ concentration, and cell volume. NHE1, the major isoform found in the heart, is activated in response to a variety of stimuli such as hormones and mechanical stress. This important characteristic of NHE1 is intimately linked to heart diseases, including maladaptive cardiac hypertrophy and subsequent heart failure, as well as acute ischemic-reperfusion injury. NHE1 activation results in elevation of pH and intracellular Na⁺ concentration, which potentially enhance downstream signaling cascades in the myocardium. Therefore, in addition to determining the mechanism underlying regulation of NHE1 activity, it is important to understand how the ionic signal produced by NHE1 is transmitted to the downstream targets. Extensive studies have identified many accessory factors that interact with NHE1. Here, we have summarized the recent progress on understanding the molecular mechanism underlying NHE1 regulation and have shown a possible signaling pathway leading to cardiac remodeling, which is initiated from NHE1. This article is part of a Special Issue entitled “Na⁺ Regulation in Cardiac Myocytes”.