The role of Na+/H+ exchanger in Ca2+ overload and ischemic myocardial damage in hearts from type 2 diabetic db/db mice.

BACKGROUND: A higher increase in intracellular Na(+) via Na(+)/H(+) exchanger (NHE) during ischemia has been reported in type 2 diabetic mouse hearts. We investigated the role of NHE in inducing changes in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and alterations in ventricular function during ischemia-reperfusion in type 2 diabetic mouse hearts. METHODS: Hearts from male type 2 diabetic db/db (12-15 weeks old) and age-matched control db/+ mice were subjected to Langendorff perfusion and loaded with 4 µM of the Ca(2+) indicator fura-2. The hearts were exposed to no-flow ischemia for 15 minutes and then reperfused. [Ca(2+)](i) was measured by monitoring fura-2 fluorescence at 500 nm (excitation wavelengths of 340 and 380 nm), while left ventricular (LV) pressure was simultaneously measured. RESULTS: db/db hearts exhibited a lower recovery of LV developed pressure than db/+ hearts during reperfusion following ischemia. Diastolic [Ca(2+)](i) was increased to a greater level in diabetic hearts than in the control hearts during ischemia and reperfusion. Such an increase in cytoplasmic Ca(2+) overload during ischemia-reperfusion in diabetic hearts was markedly reduced in the presence of the NHE inhibitor cariporide. This was accompanied by a significantly improved recovery of ventricular function on reperfusion, as shown by a lower increase in diastolic pressure and increased recovery of developed pressure. CONCLUSION: NHE plays a key role in enhancing cytoplasmic Ca(2+) overload during ischemia-reperfusion and severely impairing post-ischemic cardiac function in hearts from type 2 diabetic db/db mice.

3-(R)-[3-(2-methoxyphenylthio-2-(S)-methylpropyl]amino-3,4-dihydro-2H-1,5-benzoxathiepine bromhydrate (F 15845) prevents ischemia-induced heart remodeling by reduction of the intracellular Na+ overload.

The present study investigates whether 3-(R)-[3-(2-methoxyphenylthio-2-(S)-methylpropyl]amino-3,4-dihydro-2H-1,5-benzoxathiepine bromhydrate (F 15845), a new, persistent sodium current blocker, can reduce the ischemic Na(+) accumulation and exert short- and long-term cardioprotection after myocardial infarction. First, F 15845 concentration-dependently reduced veratrine-induced diastolic contracture (IC(50) = 0.14 microM) in isolated atria. Second, F 15845 from 1 microM preserved viability in 54.2 +/- 12.5% of isolated cardiomyocytes exposed to lysophosphatidylcholine. Third, the effect of F 15845 on intracellular Na(+) of isolated hearts from control and diabetic db/db mice was monitored using (23)Na-nuclear magnetic resonance spectroscopy. F 15845 (0.3 microM) significantly counteracted [Na(+)](i) increase during no-flow ischemia in control mouse hearts. In diabetic db/db mouse hearts, the reduction in [Na(+)](i) was delayed relative to control. However, it was more marked and maintained upon reperfusion. The cardioprotective properties after myocardial infarction associated with short- (24-h) and long-term (14-day) reperfusion were measured in anesthetized rats. After 24-h reperfusion, F 15845 (5 mg/kg) significantly reduced infarct size (32.4 +/- 1.7% with vehicle and 24.2 +/- 3.4% with F 15845; P < 0.05) and decrease of troponin I levels (524 +/- 93 microg/l with vehicle versus 271 +/- 63 microg/l with F 15845; P < 0.05). It is important that F 15845 limits the long-term expansion of infarct size (35.2 +/- 2.6%, n = 19 versus 46.7 +/- 1.6%, n = 27 in the vehicle group; P < 0.001). Overall, F 15845 attenuates [Na(+)](i) and prevents (or reverses) contractile and biochemical dysfunction in ischemic and remodeling heart. F 15845 constitutes a new generation of cardioprotective agent.

ERM proteins mediate the effects of Na+/H+ exchanger (NHE1) activation in cardiac myocytes.

AIMS: Ezrin, radixin, and moesin (ERM) proteins have been implicated in regulating signalling molecules. The aim of the present study was to investigate the activity and subcellular distribution of ERM proteins in cardiac myocytes from both Wistar and diabetic Goto-Kakizaki (GK) rats, and the role of these proteins in mediating the downstream effects of the cardiac sarcolemmal Na+/H+ exchanger (NHE1) activation in response to cell acidification. METHODS AND RESULTS: Immunofluorescence microscopy revealed that activated ERM proteins were localized predominantly at the intercalated disc regions in left ventricular (LV) myocytes of both Wistar and GK rats under basal conditions. After acid loading, profound changes in activated ERM distribution were observed in both groups of myocytes, with immunolabelling detected in regions corresponding to the transverse tubules. This correlated with a marked increase in phospho-ERM levels in both groups, which was higher in GK myocytes and blocked by NHE1 inhibitor treatment. Levels of phospho-Akt paralleled those of phospho-ERM under the various experimental conditions used; in particular, the marked acid-induced increase in both phospho-ERM and phospho-Akt in GK myocytes was abolished by an NHE1 inhibitor treatment. Moreover, the pattern of glycogen synthase kinase-3beta (GSK-3beta) phosphorylation in these myocytes was strikingly similar to that observed for Akt activity under the conditions used. CONCLUSION: Activated ERM proteins mediate the effects of acid-induced NHE1 activation in LV myocytes. Akt is a downstream effector in the cascade activated by NHE1-ERM interaction. In addition, GSK-3beta phosphorylation is required for downstream effects of NHE1/ERM-Akt signalling.

High glucose-induced apoptosis through store-operated calcium entry and calcineurin in human umbilical vein endothelial cells.

Diabetes mellitus causes multiple cardiovascular complications. Previous studies have shown that prolonged exposure (96 h) of human umbilical vein endothelial cells (HUVECs) to hyperglycemia causes a significant increase in apoptosis. We report here that this increase in apoptosis is associated with an increase in Ca(2+) current (whole cell patch-clamp recorded) resulting from Ca(2+) entry mediated by store-operated channels (SOCs). The number of apoptotic cells after prolonged high glucose (HG, 30 mmol/L) exposure was significantly reduced in the presence of the SOC inhibitor 2-APB or of La(3+). A marked increase (approximately 80%) in Ca(2+)-dependent calcineurin (CN-A) phosphatase activity also occurred after prolonged HG exposure. Prolonged HG exposure-induced increase in CN-A activity was prevented by 2-APB, and selective CN-A phosphatase inhibition by FK506 or calmodulin inhibition by calmidazolium decreased HG-induced apoptosis. Blocking hydrogen peroxide production using catalase or inhibiting the tyrosine kinase pp60(src) during prolonged exposure to HG, resulted in a marked decrease in apoptosis and was further associated with a significant reduction in CN-A phosphatase activity. The results demonstrate a significant role for Ca(2+) entry in HG-induced apoptosis in HUVECs, and suggest that this role is mediated via H(2)O(2) generation and the action of the Ca(2+)-activated protein phosphatase calcineurin.

Different pathways for sodium entry in cardiac cells during ischemia and early reperfusion.

A number of data are consistent with the hypothesis that increases in intracellular Na+ concentration (Na+i) during ischemia and early reperfusion lead to calcium overload and exacerbation of myocardial injury. However, the mechanisms underlying the increased Na+i remain unclear. 23Na nuclear magnetic resonance spectroscopy was used to monitor Na+i in isolated rat hearts perfused with a high concentration of fatty acid as can occur under some pathological conditions. Whole-cell patch-clamp experiments were also performed on isolated cardiomyocytes in order to investigate the role of voltage-gated sodium channels. Na+i increased to substantially above control levels during no-flow ischemia. The results show that a pharmacological reduction of Na+i increase by cariporide (1 micromol/L, a Na+/H+ exchange blocker) is not the only protection against ischemia-reperfusion damage, but that such protection may also be brought about by metabolic action aimed at reducing fatty acid utilization by myocardial cells. This action was obtained in the presence of etomoxir (0.1 micromol/L), an inhibitor of carnitine palmitoyltransferase-1 (the key enzyme involved in fatty acid uptake by the mitochondria) which also decreases long-chain acyl carnitine accumulation. The possibility of Na+ channels participating in Na+i increase as a consequence of alterations in cardiac metabolism was studied in isolated cells. Sustained I(Na) was stimulated by the presence of lysophosphatidylcholine (LPC, 10 micromol/L) whose accumulation during ischemia is, at least partly, dependent on increased long-chain acyl carnitine. Current activation was particularly significant in the range of potentials between -60 and -20 mV. This may have particular relevance in ischemia. The quantity of charge carried by sustained I(Na) was reduced by 24% in the presence of 1 micromol/L cariporide. Therefore, limitation of long-chain fatty acid metabolism, and consequent limitation of ischemia-induced long-chain acyl carnitine accumulation, may contribute to reducing intracellular Na+ increase during ischemia-reperfusion.

The ERK pathway regulates Na(+)-HCO(3)(-) cotransport activity in adult rat cardiomyocytes.

The sarcolemmal Na(+)-HCO cotransporter (NBC) is stimulated by intracellular acidification and acts as an acid extruder. We examined the role of the ERK pathway of the MAPK cascade as a potential mediator of NBC activation by intracellular acidification in the presence and absence of angiotensin II (ANG II) in adult rat ventricular myocytes. Intracellular pH (pH(i)) was recorded with the use of seminaphthorhodafluor-1. The NH method was used to induce an intracellular acid load. NBC activation was significantly decreased with the ERK inhibitors PD-98059 and U-0126. NBC activity after acidification was increased in the presence of ANG II (pH(i) range of 6.75-7.00). ANG II plus PD-123319 (AT(2) antagonist) still increased NBC activity, whereas ANG II plus losartan (AT(1) antagonist) did not affect it. ERK phosphorylation (measured by immunoblot analysis) during intracellular acidification was increased by ANG II, an effect that was abolished by losartan and U-0126. In conclusion, the MAPK(ERK)-dependent pathway facilitates the rate of pH(i) recovery from acid load through NBC activity and is involved in the AT(1) receptor-mediated stimulation of such activity by ANG II.

Anti-ischemic compound KC 12291 prevents diastolic contracture in isolated atria by blockade of voltage-gated sodium channels.

Several lines of evidence support a fundamental role for voltage-gated sodium channels in mediating ischemic Na rise. We examined the effect of the novel anti-ischemic compound KC 12291 on veratridine-stimulated and lysophosphatidylcholine (LPC)-induced sustained sodium current (I(NAL)) mediated by sodium channels in isolated myocytes obtained from guinea-pig atria, by using the whole-cell patch-clamp technique. We also analyzed the effect of KC 12291 on veratridine- and LPC-induced contractures in isolated guinea-pig atria. Veratridine as well as LPC increased I(NAL) measured at 20 ms of a 2 s pulse evoked from -100 to -30 mV (47.5 and 12 pA/pF in the presence of 40 microM veratridine and 10 microM LPC, respectively, vs. 6.7 pA/pF under control conditions). A significant reduction by KC 12291 in the quantity of charge carried by veratridine-stimulated I(NAL) in the range of test potentials between -50 mV and +10 mV was observed and similar effects were obtained on LPC-induced I(NAL). Thus, the quantity of charge carried by LPC-induced I(NAL) over a 2 s pulse to -30 mV was reduced by 48% in the presence of 10 microM KC 12291 vs. a reduction by 50% of veratridine-stimulated I(NAL) at the same test potential. Veratridine- and LPC-induced submaximal contractures in isolated atria were significantly inhibited by KC 12291 in a concentration-dependent manner, with an IC of 0.55 microM and 0.79 microM, respectively. The data indicate that veratridine- and LPC-induced increases in diastolic tension are inhibited by KC 12291 by a mechanism that involves blockade of voltage-gated sodium channels mediating sustained sodium current.