Protection of ischemic myocardium in diabetics by inhibition of electroneutral Na⁺-K⁺-2Cl cotransporter

¹ Division of Cardiology, Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032; and ² Department of Human Physiology and ³ Division of Cardiovascular Medicine, Department of Internal Medicine, University of California, Davis, California 95616

	ABSTRACT

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Diabetes increases both the incidence of cardiovascular disease and complications of myocardial infarction and heart failure. Studies using diabetic animals have shown that changes in myocardial sodium transporters result in alterations in intracellular sodium (Na_i) homeostasis. Because the changes in sodium homeostasis can be due to increased entry of Na⁺ via the electroneutral Na⁺-K⁺-2Cl cotransporter (NKCC), we conducted experiments in acute diabetic hearts to determine if 1) net inward cation flux via NKCC is increased, 2) this cotransporter contributes to a greater increase in Na_i during ischemia, and 3) inhibition of NKCC limits injury and improves function after ischemia-reperfusion. These issues were investigated in perfused type I diabetic and nondiabetic rat hearts subjected to ischemia and 60 min of reperfusion. A group of diabetic and nondiabetic hearts was perfused with 5 µM of bumetanide, an inhibitor of NKCC. Flux via NKCC, Na_i, and ATP was measured in each group with the use of radiotracer ⁸⁶Rb, ²³Na, and ³¹P nuclear magnetic resonance spectroscopy, respectively, whereas ischemic injury was assessed by measuring creatine kinase release on reperfusion. Cation flux via NKCC, as measured by ⁸⁶Rb uptake, was significantly increased in diabetic hearts. Inhibition of NKCC significantly reduced ischemic injury in diabetic hearts, improved functional recovery on reperfusion, attenuated the ischemic rise in Na_i, and conserved ATP during ischemia-reperfusion. Parallel studies in nondiabetic hearts showed that NKCC inhibition was not cardioprotective. These findings demonstrate that flux via NKCC is increased in type I diabetic hearts and that inhibition with bumetanide attenuates changes in Na_i and ATP during ischemia and protects against ischemic injury. The data suggest a therapeutic role for pharmacological agents that inhibit flux via NKCC in diabetic patients with myocardial ischemia.

diabetes; ischemic injury; sodium transporters

	INTRODUCTION

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DIABETIC PATIENTS WITH coronary artery disease have high morbidity and mortality due to cardiovascular complications, including a greater incidence of left ventricular (LV) dysfunction following myocardial infarction (13, 20, 47). Studies using animal models of diabetes, although providing conflicting data on the extent of ischemic injury and infarction in diabetes (10), have consistently shown detrimental alterations in myocardial metabolism and myocyte ion homeostasis (22, 36, 38, 43, 50).

In nondiabetic animals, there are data showing (4, 9, 27, 30, 36, 41, 42, 44, 45) that functional recovery on reperfusion of ischemic myocardium can be enhanced by interventions that maintain tissue ATP availability, and limit derangements in ion homeostasis. The critical role for ion regulation in protecting ischemic myocardium is supported by experiments (2, 14, 28) in which reduced ischemic injury due to global ischemia-reperfusion has been associated with reduced intracellular acidification and lower intracellular sodium and calcium accumulation. Therapeutic interventions that protect ischemic myocardium by limiting ionic derangements have not been completely tested in diabetics, in part due to lack of complete understanding of the alterations in ion transport pathways in diabetics. Of the data available, studies have shown that the Na⁺-K⁺ ATPase (32, 35, 43), Na⁺-H⁺ exchanger (16, 17, 37), and Na⁺-Ca²⁺ exchanger are impaired in diabetic hearts (8, 43). The result of these changes in transporter activities is an increase in intracellular sodium under baseline conditions in diabetic hearts (12, 35, 37). A component of this increase may be an alteration in other sodium entry pathways, such as the electroneutral Na⁺-K⁺-2Cl cotransporter in diabetic hearts. Our goal in this study was to address the following questions. First, is the net inward flux via the electroneutral Na⁺-K⁺-2Cl cotransporter increased in a genetically Type I acute diabetic rat heart? Second, does this cotransporter contribute to increases in intracellular sodium during ischemia in diabetic hearts? Third, does inhibition of the electroneutral Na⁺-K⁺-2Cl cotransporter limit injury and functional impairment in diabetic hearts during ischemia-reperfusion? We examined these questions in diabetic and nondiabetic rat hearts by inhibiting the Na⁺-K⁺-2Cl cotransporter with bumetanide and measuring radioactive ⁸⁶Rb uptake, intracellular sodium (by using ²³Na spectroscopy), intracellular pH, and high-energy phosphates (by using ³¹P NMR spectroscopy) during global ischemia-reperfusion. The data indicate that the flux via Na⁺-K⁺-2Cl cotransporter is increased in diabetic hearts and that inhibition with bumetanide attenuates the rise in intracellular sodium during ischemia and reduces ischemic injury in diabetic hearts.

	MATERIALS AND METHODS

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All experiments were performed with the approval of the Research committee for Animal Care and Use at Columbia University (New York, NY) and the University of California (Davis, CA).

Rats

We used spontaneously acute diabetic Bio-Bred (BB/W) rats from a colony maintained at the University of Massachusetts Medical Center (Worcester, MA). The 3- to 4-mo-old BB/W rats weighed between 300 and 350 g, with the duration of diabetes being 12 ± 3 days. The rats were receiving daily insulin therapy, which was discontinued 24 h before the isolated heart perfusion studies were performed. The blood glucose levels in these rats were 486 ± 81 mg/dl. The age-matched nondiabetic littermates, also from the colony maintained at the University of Massachusetts Medical Center, were used in this study. The mean blood glucose levels in the littermate controls were 112 ± 12 mg/dl.

Isolated Perfused Heart Model

Experiments were performed with the use of an isovolumic isolated rat heart preparation as previously described (34, 36). Acutely diabetic male BB/W rats and nondiabetic age-matched littermates were pretreated with heparin (100 U ip), followed by pentobarbital sodium (65 mg/kg ip). After deep anesthesia was achieved (determined by the absence of a foot reflex), the hearts were rapidly excised and placed into ice-cold saline. The arrested hearts were retrogradely perfused (in a nonrecirculating mode) through the aorta within 2 min. LV developed pressure (LVDP) was determined with the use of a latex balloon in the left ventricle with high-pressure tubing connected to a pressure transducer. Perfusion pressure was monitored with the use of high-pressure tubing off the perfusion line. Hemodynamic measurements were recorded on a four-channel Gould recorder. This design allowed us to rapidly change perfusion media. The hearts were perfused with the use of an accurate roller pump. The perfusate consisted of (in mM) 118 NaCl, 4.7 KCl, 1.2 CaCl₂, 1.2 MgCl₂, and 25 NaHCO₃ (the substrate was 11 mM glucose unless otherwise noted). The perfusion apparatus was tightly temperature controlled, with heated baths used for the perfusate and for the water jacket around the perfusion tubing to maintain heart temperature at 37°C under all conditions. Coronary venous effluent was collected via a cannula that was placed into the pulmonary artery. PO₂ was measured in the effluent with the use of a pH-blood gas analyzer (model IL 1306, Instrumentation Laboratories). Myocardial oxygen consumption was calculated as (0.003 × arterial PO₂  0.003 × effluent PO₂) × total flow/LV weight.

Choice of Bumetanide to Inhibit Na+-K⁺-2Cl Cotransporter

Several loop diuretics (such as bumetanide, furosemide, piretanide, and benzmetanide) are available to inhibit Na⁺-K⁺-2Cl cotransporter. Among the diuretics, bumetanide has been shown to be specific and well characterized for its ability to inhibit the Na⁺-K⁺-2Cl cotransporter from several organs and cells [see the recent review by Russell (40)]. At concentrations of 10⁶-10⁷ M, bumetanide (inhibitory constant ~ 1 × 10⁶ M) has been shown to inhibit Na⁺-K⁺-2Cl cotransporter in several organs, including the heart (19, 40). In this study, we used 5 µM bumetanide to inhibit the myocardial Na⁺-K⁺-2Cl cotransporter.

Measurements of ⁸⁶RbCl Uptake via Na+-K⁺-2Cl Cotransporter

Bumetanide-sensitive ⁸⁶Rb (an analog of K⁺) uptake was measured by perfusing hearts with radioactive ⁸⁶RbCl (0.1 mCi/ml) and cold RbCl (1 mM) under recirculating conditions for 1 h. Hearts from diabetic (n = 6) and nondiabetic rats were perfused with ⁸⁶RbCl buffer for 60 min to measure the uptake of ⁸⁶Rb by all K⁺ pathways. To measure the ⁸⁶Rb uptake via the bumetanide-sensitive Na⁺-K⁺-2Cl cotransporter, diabetic (n = 6) and nondiabetic (n = 6) hearts were perfused with the buffer containing ⁸⁶RbCl and bumetanide for 60 min. The uptake of ⁸⁶Rb in the absence and presence of bumetanide was measured in the hearts at the end of the perfusion period in a gamma-well counter. Hearts were perfused with the cold Krebs buffer (without any RbCl) for 2 min before being counted to remove ⁸⁶Rb from the vasculature. Myocardial ⁸⁶Rb uptake was quantified by normalizing for perfusate radioactivity and heart weight.

Na+-K⁺-2Cl Protein

Hearts (n = 6 in each group) were homogenized in the presence of protease inhibitors, and membranes were prepared by differential centrifugation as previously described (49). Membranes were analyzed by Western blot after separating samples on 7.5% Tricine gels and electrophoretic transfer to polyvinylidene difluoride membrane (Immobilon-P, Milipore). Equal amounts of protein (100 mg of protein) were loaded from each sample (diabetic or nondiabetic hearts). The anti-Na-K-Cl cotransporter monoclonal antibody (T4 antibody) (23) and enhanced chemiluminescence assay (ECL, Amersham) was used to detect the Na⁺-K⁺-2Cl protein. The intensity of the bands in the Western blots was compared among the groups to get a qualitative estimate of changes in protein expression.

Creatine Kinase

Creatine kinase (CK) was measured from timed 5-min collections of the effluents for 60 min of reperfusion after the ischemic period. Each 5-min collection was analyzed with the use of established spectrophotometric methods (34, 41). Total integrated CK activity over the reperfusion was calculated for each heart and corrected for the dry weight of the heart. CK release was expressed in units per gram of dry weight. Total CK activity was calculated by measuring tissue CK activity and integrated CK release on reperfusion. The percentage of CK released on reperfusion was calculated as
This normalization of CK release enabled us to correct for lower total myocardial CK content in the diabetic hearts.

NMR Spectroscopy

³¹P NMR spectroscopy. All spectroscopy were performed on an AMX 400 (Bruker) or Omega-300 (General Electric) vertical-bore spectrometer using a dual-tuned probe. ³¹P NMR spectroscopy was performed by using 248 acquisitions of a 45° pulse and 1.21-s interpulse delay, with spectra processed by using an exponential multiplication of 20 Hz and manual phasing. Intracellular pH was determined from the chemical shift of the P_i resonance by using a titration curve established in this laboratory. All metabolites were referenced to their baseline value determined in duplicate at the start of the experiment and are expressed as a fraction of baseline.

²³Na NMR spectroscopy. Intracellular sodium concentration ([Na]_i) was determined by using the shift reagent 1,4,7,10-tetrazacyclododecane-N,N',N'',N''',tetra(methylene-phosphonate) (TmDOTP⁵) (5, 24), supplied by Magnetic Resonance Solutions (Dallas, TX). Sodium spectra were acquired on a Bruker AMX 400-MHz spectrometer by using a broad-band probe tuned to 105.85 MHz. One thousand free-induction decays were signal averaged over 5 min using 90° pulses with a ±4,000-Hz sweep width. [Na]_i was calculated by using the formula
where A_Nai and A_Nao are the intracellular and extracellular areas of the sodium resonances, respectively, f_o and f_i are the fractional visibilities of extracellular and intracellular sodium (assumed as 1.0 and 0.4, respectively), [Na]_o is the extracellular sodium concentration, and V_o/V_i is the ratio of intracellular and extracellular volume, respectively (assumed to be 1) (46). Because the changes in intracellular sodium visibilities and relative volumes were not determined during ischemia, the changes in intracellular sodium during ischemia-reperfusion are reported as a percentage of baseline values.

Statistical Methods

Data were analyzed with the use of INSTAT software (GraphPad; San Diego, CA) operating on an IBM-compatible personal computer. The differences among different groups were assessed with the use of analysis of variance (ANOVA) for repeated measures, with subsequent Student-Newman-Keuls multiple comparisons post tests if the P value for ANOVA was significant. P < 0.05 was used to reject the null hypothesis. All data are expressed as means ± SD.

Ischemia Protocol

Four groups of hearts, diabetic and nondiabetic, without and with bumetanide, were studied continuously with the use of ³¹P or ²³Na NMR spectroscopy. NMR spectra were obtained every 5 min during baseline, ischemia, and reperfusion along with simultaneous measurements of heart rate, LV end-diastolic pressure (LVEDP), and LVDP. All hearts were perfused at 12.5 ml/min before and after ischemia, without recirculating the buffer; reperfusion was performed for 60 min by using standard perfusate without bumetanide. The ischemic period of 20 min was initiated after the equilibration period (20 min) with the use of standard perfusate or perfusate also containing the Na⁺-K⁺-2Cl cotransport inhibitor bumetanide (5 µM) for 10 min before ischemia.

	RESULTS

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Cation Flux Via Na+-K⁺-2Cl Cotransporter in Diabetic Hearts

⁸⁶Rb uptake studies. Because Rb⁺ is an analog of K⁺, measurements of radiotracer Rb⁺ uptake was undertaken to estimate the flux via the Na⁺-K⁺-2Cl cotransporter in perfused hearts. Figure 1 illustrates the baseline measurements of ⁸⁶Rb uptake in diabetic and nondiabetic hearts. In diabetic hearts, bumetanide reduced the uptake of ⁸⁶Rb by ~50%, consistent with an increase in flux via the Na⁺-K⁺-2Cl cotransporter in diabetic hearts. In comparison, bumetanide did not significantly reduce ⁸⁶Rb uptake in nondiabetic hearts.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   86RbCl uptake in nondiabetic (CON), diabetic (DM), bumetanide-perfused nondiabetic (CON-BUM), and bumetanide-perfused diabetic (DM-BUM) hearts (n = 6 in each group). Normalized Rb uptake is determined by dividing the tissue Rb radioactive counts by weight of heart and the mean count. *P < 0.05, DM-BUM hearts had significantly reduced 86RbCl uptake than DM hearts.

Western blot analysis of the Na+-K⁺-2Cl cotransporter levels. Because one potential explanation for the greater inhibition of Na⁺-K⁺-2Cl cotransporter flux in diabetic hearts is greater levels of the protein, we measured the cotransporter by using Western blot techniques with a polyclonal antibody specific for the Na⁺-K⁺-2Cl cotransporter. Western blot analysis using a mouse monoclonal anti-Na⁺-K⁺-2Cl cotransporter antibody (T4) was performed on membranes prepared from control and diabetic hearts (Fig. 2). Similar to the observations by Lytle et al. (23) on other cell types, two major bands were observed prominently in the control hearts. These two bands represent mature glycosylated protein present at the plasma membrane (upper band; ~170 kDa) and immature unprocessed protein (lower band; ~135 kDa). The intensity of the mature protein, in arbitrary units, was 176 ± 36 in diabetic and 162 ± 55 in control hearts. These data demonstrate no significant differences in the mature Na⁺-K⁺-2Cl cotransporter expression in the diabetics and controls. Thus the elevated Na⁺-K⁺-2Cl cotransporter activity observed in the diabetic hearts cannot be explained by an increased level of Na⁺-K⁺-2Cl cotransporter protein expression.

View larger version (107K): [in this window] [in a new window]   Fig. 2.   Western blot analysis of the Na+-K+-2Cl cotransporter protein in rat heart membranes. Membranes were loaded onto a 7.5% Tricine-sodium dodecyl sulfate gel at 100 mg per lane as noted. Separated proteins were transferred to polyvinylidene difluoride membranes and probed with anti-Na+-K+-2Cl cotransporter monoclonal antibody (used at a 1:1,000 dilution). Densitometric analysis of the bands did not show any differences in the total intensity of the bands for diabetic and nondiabetic hearts.

Impact of Na+-K⁺-2Cl Cotransporter Inhibition on Cardiac Function and Ischemic Injury

Functional changes during ischemia-reperfusion. LVDP and EDP were similar in all groups under baseline conditions (Table 1). Ischemia resulted in significant reductions in LVDP in all groups. The rise in EDP during ischemia was attenuated in the bumetanide-treated diabetics compared with other groups of hearts. During reperfusion, the bumetanide-treated diabetic hearts exhibited greater LVDP recovery than the untreated diabetic, nondiabetic control, and bumetanide-treated nondiabetic hearts. Na⁺-K⁺-2Cl cotransporter inhibition did not affect myocardial oxygen consumption.

View this table: [in this window] [in a new window]   Table 1.   Hemodynamic values from isolated rat hearts

Ischemia-Reperfusion Injury

CK release, a measure of ischemia-reperfusion injury, was measured during reperfusion in all the groups of hearts studied. Consistent with previous data, diabetic hearts had a lower CK release than nondiabetic control hearts (Table 2). Since it was earlier demonstrated (26, 33) that the total amount of myocardial CK and its activity is lower in streptozotocin-induced diabetic animals, we measured the total myocardial CK activity in these hearts and normalized the release of CK during reperfusion. As demonstrated (26, 33) with the use of chemically induced diabetic animal models, we also show that the total myocardial CK activity is significantly lower in the genetically diabetic BB/W rat hearts. However, when expressed as a percentage of total CK activity, it can be seen that the both diabetic and nondiabetic hearts released ~60% of total CK on reperfusion (Table 2). Bumetanide markedly reduced the fraction of CK released in the diabetic hearts but did not limit CK release in the nondiabetic hearts. These data demonstrate that inhibition of the bumetanide-sensitive Na⁺-K⁺-2Cl cotransporter protected the diabetic hearts from ischemia-reperfusion injury.

View this table: [in this window] [in a new window]   Table 2.   Creatine kinase data

Contribution of Flux via Na+-K⁺-2Cl Cotransporter Toward Changes in Intracellular Sodium During Ischemia-Reperfusion

²³Na NMR studies were conducted to investigate the contribution of the Na⁺-K⁺-2Cl cotransporter pathway to the changes in intracellular sodium during ischemia in diabetic and nondiabetic hearts. Relative changes in intracellular sodium in each group of hearts during baseline, ischemia, and reperfusion are displayed in Fig. 3. In each group, baseline intracellular sodium was set to 100%, and the changes in sodium during ischemia-reperfusion are reported relative to the baseline values. The intracellular sodium increased to 229.8 ± 18.1% in diabetic hearts compared with a greater increase of 309.1 ± 14.2% in nondiabetic hearts (P < 0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Changes in intracellular sodium concentration ([Na]i), expressed as a percentage of baseline during 20 min of global ischemia and 40 min of reperfusion in diabetic (A) and nondiabetic (B) heart groups. In the untreated diabetic and nondiabetic group, 9 hearts were studied, whereas in the bumetanide-treated group 6 hearts were studied. Each arrow represents the data obtained every 5 min. *P < 0.05, significantly greater than the bumetanide-treated group. Baseline intracellular sodium was significantly lower than those obtained for the entire ischemia period and during the first 20 min of reperfusion within the respective groups.

Inhibition of the Na⁺-K⁺2Cl cotransporter with bumetanide significantly attenuated the rise in intracellular sodium during ischemia in both diabetic and nondiabetic hearts, with the end-ischemic [Na]_i being 130.2 ± 12.2% in diabetes-bumetanide and 258.2 ± 13.5% in the control-bumetanide hearts. Comparing changes in intracellular sodium in the presence and in the absence of bumetanide, it is evident that the Na⁺-K⁺-2Cl cotransporter contributed ~18% of the rise in sodium during ischemia in control hearts, whereas the Na⁺-K⁺-2Cl cotransporter was responsible for ~44% of the increase in sodium during ischemia in diabetics. These results clearly indicate that the flux via Na⁺-K⁺-2Cl cotransporter was increased in diabetic hearts.

Metabolic Consequences of Na+-K⁺-2Cl Cotransporter Inhibition

Phosphocreatine. All hearts had rapid loss of phosphocreatine (PCr) at the onset of global ischemia and partial return of PCr levels on reperfusion. Treatment with bumetanide increased the levels of PCr more in diabetic than in nondiabetic hearts (Fig. 4). Bumetanide did not affect the loss or return of PCr in nondiabetic hearts.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Phosphocreatine (PCr), expressed as fraction of baseline, in DM, CON, DM-BUM, and CON-BUM hearts during baseline and at the end of reperfusion. These data were obtained from 31P nuclear magnetic resonance (NMR) measurements. At the end of ischemia, all groups of hearts had no NMR-measurable PCr levels. *P < 0.05, bumetanide-treated diabetic hearts had significantly higher levels of PCr than other groups. Baseline levels of PCr measured with the use of HPLC and, expressed in micromoles per gram dry weight, were 34.89 ± 5.07 in CON and 34.55 ± 4.66 in DM hearts (n = 6 in each group). #, @, , P < 0.05 vs. baseline values in its group.

ATP. ATP, expressed as a fraction of baseline (Fig. 5), was similar in diabetic and bumetanide perfused diabetic hearts before ischemia. During ischemia, ATP content was preserved in bumetanide-treated diabetic hearts, whereas in all other groups, ATP loss was quite severe. At the midpoint of the reperfusion period, bumetanide-perfused diabetic hearts had significantly higher levels of ATP than untreated diabetic hearts (fraction of baseline ATP was 0.52 ± 0.07 in diabetes-bumetanide vs. 0.16 ± 0.04 in diabetic hearts, P < 0.05). In nondiabetic hearts, inhibition of the Na⁺-K⁺-2Cl cotransporter with bumetanide did not conserve ATP levels during ischemia or reperfusion.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   ATP, expressed as a fraction of baseline, in DM, CON, DM-BUM, and CON-BUM hearts during baseline, end of ischemia, and end of reperfusion. These data were obtained from 31P NMR measurements. *P < 0.05, BUM-treated diabetic hearts had significantly greater levels of ATP than other groups. #P < 0.05 vs. baseline values in its group. @P < 0.05 vs. baseline values in its group.  P < 0.05 vs. baseline values in its group. P < 0.05 vs. baseline values in its group. Baseline concentration of ATP was measured by HPLC and, expressed as micromoles per gram dry weight, was significantly lower in DM compared with CON hearts (15.51 ± 2.05 in DM vs. 23.11 ± 3.52 in CON, P < 0.05, n = 6 in each group).

Intracellular pH during ischemia-reperfusion. Bumetanide perfusion did not alter intracellular pH in diabetic hearts before ischemia (Table 3). However, during ischemia, bumetanide-perfused diabetic hearts became less acidotic than untreated diabetic hearts, resulting in an end-ischemic pH of 6.02 ± 0.03 in diabetes versus 6.38 ± 0.02 in diabetes-bumetanide hearts (P < 0.05). Reperfusion resulted in pH recovery to baseline values in both hearts within 10 min.

View this table: [in this window] [in a new window]   Table 3.   Intracellular pH during ischemia-reperfusion

Intracellular pH was not affected by bumetanide perfusion under any condition in nondiabetic hearts. The end-ischemic pH was 5.98 ± 0.02 in control versus 6.01 ± 0.04 in bumetanide-treated nondiabetic hearts. Reperfusion resulted in pH recovery to baseline values with no differences in the rate of recovery.

	DISCUSSION

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The results of the present study show that the flux via Na⁺-K⁺-2Cl cotransporter is higher in hearts isolated from diabetic animals under baseline and ischemic conditions. As a consequence, inhibition of the cotransporter with bumetanide limits the rise in intracellular sodium, conserves ATP and limits acidosis during ischemia, and reduces ischemic injury. The mechanisms of these responses can be postulated in light of known effectors of sodium regulation in the heart.

Increased Na+-K⁺-2Cl Cotransporter Flux and Intracellular Sodium in Diabetic Hearts

Steady-state [Na]_i is maintained by a balance between sodium influx and efflux. Under baseline conditions, sodium influx occurs via the sodium channels, the Na⁺-H⁺ exchanger, the Na⁺-K⁺-2Cl cotransporter, and the Na⁺/Ca²⁺ exchanger, whereas sodium efflux occurs mainly through the Na⁺-K⁺- ATPase (19). In hearts from diabetic animals, inhibition of the myocardial Na⁺-K⁺-ATPase in the presence of active sodium influx via other transporters results in an increased baseline [Na]_i (18, 35, 43). In the current experiments, the baseline ⁸⁶RbCl uptake measurements demonstrated an increased flux via the bumetanide-sensitive Na⁺-K⁺-2Cl cotransporter in diabetic hearts, findings that mirror a report (25) that demonstrated a 70% increase in flux via the Na⁺-K⁺-2Cl cotransporter in aortas from diabetic rats. The flux studies using ⁸⁶Rb⁺ (K⁺ analog) and bumetanide indicate that the net flux via the bidirectional Na⁺-K⁺-2Cl cotransporter is directed toward increasing intracellular ion concentration (Na, K, and Cl).

An increase in the Na⁺-K⁺-2Cl cotransporter activity could be due to an increase in the levels of the protein or to activation by endogenous factors, such as changes in cell volume, and cAMP-dependent-, and non-cAMP-dependent protein phosphorylation (3, 6, 29, 31). The Na⁺-K⁺-2Cl cotransporter protein levels measured by western blots were similar in diabetic and nondiabetic hearts, indicating that the observed increases in the Na⁺-K⁺-2Cl cotransporter activity were likely due to activation by endogenous factors.

Increased Flux Via Na+-K⁺-2Cl Cotransporter and Intracellular Sodium During Ischemia

The changes in intracellular sodium during ischemia result from the balance of changes in Na⁺ influx and efflux. Figure 3 shows that bumetanide decreased peak intracellular sodium in diabetic and nondiabetic hearts. The magnitude of the decrease in intracellular sodium, due to bumetanide treatment, is greater in diabetics than in nondiabetics. These findings are consistent with a greater contribution of the Na⁺-K⁺-2Cl cotransporter to the increase in intracellular sodium during ischemia in diabetic hearts. Two studies support our observations that the Na⁺-K⁺-2Cl cotransporter may play a greater role in the influx of sodium during ischemia when Na⁺-K⁺-ATPase activity is reduced. Anderson et al. (1) have shown that inhibition of the Na⁺-K⁺-2Cl cotransporter with bumetanide decreased the intracellular Na⁺ uptake and accumulation during ischemia in ouabain-treated neonatal hearts. Similarly, Rubin and Navon (39) demonstrated that the rise in intracellular sodium, due either to inhibition of Na⁺-K⁺ ATPase or to hypothermic ischemia, could be attenuated by treatment with furosemide (an inhibitor of the Na⁺-K⁺-2Cl cotransporter). That study also showed that inhibition of the Na⁺-K⁺-2Cl cotransporter protected the hearts during hypothermic preservation (39). These studies support our findings that in diabetic hearts with reduced Na⁺-K⁺-ATPase activity, inhibition of the Na⁺-K⁺-2Cl cotransporter reduces the rise in intracellular sodium during ischemia.

Inhibition of the Na+-K⁺-2Cl Cotransporter on Acidosis, ATP, and Ischemic Injury

In this study, bumetanide perfusion resulted in both attenuation of intracellular sodium and acidosis, as well as higher levels of ATP, in diabetic hearts during ischemia. Potential reasons for reduced acidosis during ischemia include reduced proton production from anaerobic glycolysis, reduced ATP hydrolysis, or greater proton efflux from ion transport mechanisms (7, 11, 48). While it is difficult to elucidate the precise sequence of events that resulted in less acidosis, greater ATP preservation, and a lower rise in intracellular sodium during ischemia, it is possible that the requirements of ATP for transport pathways to maintain intracellular sodium homeostasis may have been attenuated in bumetanide-treated diabetic hearts, thus impacting net changes in acidosis and ATP levels. One possible scenario is that the reduction in overall intracellular sodium load during ischemia, as well as during reperfusion, may facilitate proton exchange and alter sodium gradient thereby reducing intracellular calcium load. Attenuation of intracellular calcium accumulation has been associated with greater ATP production by the mitochondria (45).

Inhibition of the Na⁺-K⁺-2Cl cotransporter with bumetanide reduced ischemia-reperfusion injury by ~50% in diabetic hearts, while the reduction was <20% in nondiabetic hearts treated with bumetanide. These data suggest that inhibition of the Na⁺-K⁺-2Cl cotransporter with the use of bumetanide was protective only in diabetic hearts. The likely explanation can be postulated in light of higher activity of the Na⁺-K⁺-2Cl cotransporter and its contribution to Na⁺ uptake in diabetic hearts. The data presented here is consistent with earlier studies (2, 21, 35, 45), which demonstrated protection of ischemic myocardium by interventions that reduce intracellular acidification, and lower intracellular sodium and calcium accumulation.

Limitations

The findings from our isolated perfused heart experiments, demonstrating protection of ischemic myocardium in diabetics by bumetanide, must be interpreted within the limitations of the experimental design. The use of glucose as a sole substrate limited potential effects of exogenous fatty acids on the actions of bumetanide. Furthermore, insulin was not used in our preparations to mimic conditions of insulin deficiency. The presence of insulin may influence or alter the effects of bumetanide observed in this study. However, clinical studies suggest that the presence of insulin does not impact the efficacy of bumetanide or other loop diuretics. Because hyperglycemia has been shown to influence the severity of ischemic injury in a dog diabetes model (15), as well as in nondiabetic rat hearts (42), it is likely that we may have underestimated the severity of ischemic injury by maintaining a lower glucose concentration during perfusion.

In conclusion, the data from our experiments show that net inward flux though the Na⁺-K⁺-2Cl cotransporter activity is increased in diabetic hearts under normoxic conditions. Furthermore, Na⁺-K⁺-2Cl cotransporter inhibition using bumetanide attenuates the ischemic rise in intracellular sodium, maintains higher levels of high-energy phosphates, reduces CK release, and significantly improves functional recovery after ischemia in diabetic, but not in nondiabetic hearts. These findings suggest a potential role for Na⁺-K⁺-2Cl cotransporter inhibitors as novel therapeutic agents in the treatment of myocardial ischemia, specifically in diabetic patients.

	ACKNOWLEDGEMENTS

R. Ramasamy was supported by an American Diabetes Association Career Development Award, an Established Investigator Award from the American Heart Association, by Juvenile Diabetes Foundation International Grant 196098, and by National Heart, Lung, and Blood Institute Grants HL-58408 and HL-61783.

	FOOTNOTES

Address for reprint requests and other correspondence: R. Ramasamy, Div. of Cardiology, PH 3-342, College of Physicians and Surgeons, Columbia Univ., 630 W. 168th St., New York, NY 10032 (E-mail: rr260{at}columbia.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 July 2000; accepted in final form 13 April 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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