Inhibition of Na⁺–H⁺ exchange before resuscitation following hemorrhagic shock is cardioprotective in rats

Abstract

Background

Stimulation of the Na⁺–H⁺ exchanger during resuscitation following hemorrhagic shock results in myocardial injury and dysfunction. Inhibition of the Na⁺–H⁺ exchanger appears to be a new pharmacological tool for myocardial protection following ischemia–reperfusion. Our lab showed that inhibition of the Na⁺–H⁺ exchanger, using amiloride and dimethyl amiloride, before ex vivo resuscitation of isolated perfused hearts protected the myocardium and improved the post-resuscitation myocardial function. The purpose of the present study was to examine the myocardial protective effects of treating the hemorrhagic shocked rats by intra-arterial injection of 20 μM dimethyl amiloride (DMA), a specific Na⁺–H⁺ exchanger blocker, before in vivo resuscitation.

Methods

Sprague–Dawley rats were assigned to hemorrhagic treated or untreated groups (n = 4 per group). After 60 min of hemorrhagic shock, rats were treated or not by injection of 20 μM 5-(N,N-dimethyl)-amiloride (DMA) intra-arterially. Rats were then resuscitated in vivo and monitored for 30 min. Then hearts were harvested and perfused in the Langendorff system for 60 min for measurements of hemodynamic function.

Results

Administration of DMA before in vivo resuscitation following 60 min of hemorrhagic shock and 30 min of in vivo resuscitation, 20 μM DMA intra-arterially significantly improved post-resuscitation myocardial function.

Conclusion

Our results suggest that DMA protects the heart against post-resuscitation myocardial injury.

1. Introduction

Myocardial dysfunction and failure are among the major causes of mortality following hemorrhagic shock (Gutierrez et al., 2004). Activation of the Na⁺–H⁺ exchanger at the time of resuscitation is a major component of the hemorrhagic shock associated myocardial dysfunction and injury (Lazdunski et al., 1985). The Na⁺–H⁺ exchanger is activated at reperfusion due to the intracellular acidosis caused by anaerobic metabolism. The exchange of intracellular H⁺ for extracellular Na⁺ causes Na⁺overload and subsequent activation of the Na⁺–Ca²⁺exchanger that leads to an increase in intracellular Ca²⁺, with its deleterious effects on myocardial contractility. This has been supported by data from different research groups in case of ischemia–reperfusion (Eng et al., 1998; Meng et al., 1993; Pierce et al., 1993; Meng and Pierce, 1991, 1990). Thus, inhibition of Na⁺–H⁺ exchanger, which would lead to prevention of Ca²⁺ overload, is a promising therapeutic approach against myocardial ischemia and reperfusion injury (Piper et al., 1996; Frohlich and Karmazyn, 1997). However, the myocardial protective effects of blocking the Na⁺–H⁺ exchanger before resuscitation following hemorrhagic shock is unclear.

Our laboratory studied the effects of blocking the Na⁺–H⁺ exchanger using amiloride before ex vivo as well as in vivo resuscitation of hemorrhagic shocked rats. The results were very promising showing improved myocardial contractility in the treated animals. Previous work from our lab also showed myocardial protective effects of blocking the Na⁺–H⁺ exchanger using 20 μM dimethyl amiloride (DMA), a specific Na⁺–H⁺ exchanger blocker, before ex vivo resuscitation of isolated hearts from hemorrhagic shock model. The objective of this study was to determine the myocardial protective effects of treating the hemorrhagic shock rats using DMA, i.e. before in vivo resuscitation of rats.

2. Materials and methods

This study was approved by the Continuous Medical Research Center at the College of Medicine, King Saud University. Sixteen male Sprague–Dawley rats were used.

2.1. Surgical procedure

Male Sprague–Dawley rats were injected intra-peritonealy (i.p.) with heparin sodium 2000 I.U. 15 min prior to anesthesia. The rats were then anaesthetized used urethane 125 mg/kg intra-peritonealy. The left carotid artery was cannulated using polyethylene tubing size 60, and was connected to an in-line pressure transducer for continuous blood pressure monitoring. Animals were allowed to stabilize for a period of 30 min. Animals were assigned randomly to either hemorrhagic shock or time matched control group (n = 4 per group).

2.2. Hemorrhage and resuscitation

After 15 min stabilization, rats were hemorrhaged using a reservoir (a 10 ml syringe). Blood was withdrawn from the carotid artery until MAP reached 35–40 mmHg. Where in vivo resuscitation was required, shed blood was reinfused to achieve normotension (80–110 mmHg). If the blood pressure drops below 30 mmHg or failed to be restored to normal, experiments were excluded. The same surgical procedures were performed for the sham hemorrhage groups except that rats were not hemorrhaged.

2.3. Experimental groups

• 1.Hemorrhagic shock group (HS)
• 2.DMA treated hemorrhagic shock group (DMA-HS)
• 3.Sham hemorrhage group (Sham)
• 4.DMA treated sham hemorrhage group (DMA-Sham)

2.4. Experimental design

Rats were randomly assigned to either: (1) hemorrhagic shock of 60 min followed by in vivo resuscitation by reinfusion of the shed blood to restore normotension, (2) DMA treated hemorrhagic shock group where animals hemorrhaged for 60 min followed by treatment by injecting 1 ml of (20 μM) DMA dissolved in KHB intra-arterially, and resuscitation for 30 min, (3) sham hemorrhage group where rats underwent same surgical procedure and blood pressure monitored for 90 min and (4) DMA treated sham hemorrhage group where rats underwent same surgical procedure and blood pressure monitored for 60 min, then rats where treated by injecting 1 ml of (20 μM) DMA dissolved in KHB intra-arterially, and monitored for another 30 min.

2.5. Isolated heart perfusion

Hearts were excised quickly and rapidly placed onto a Langendorff system for perfusion in retrograde mode via the aorta; at a rate of 10 ml/min. Hearts were perfused with KHB of the following composition (in mM): sodium chloride, 118; calcium chloride, 1.25; potassium chloride, 4.7; sodium bicarbonate, 21; magnesium sulphate, 1.2; glucose, 11; potassium biphosphate, 1.2; and EDTA, 0.5. An apical stab incision was made in the left ventricle (LV) using a #15 scalpel blade. A saline-filled cellophane balloon-tipped catheter was placed into the LV via the mitral valve and was used to measure LV pressure and balloon volume. LV and perfusion pressures were measured using transducers placed at the levels of the heart and aorta. Hearts were stimulated electrically at 5 Hz using electrical stimulator (6020 Stimulator from Harvard Apparatus). Perfusion pressure was maintained at 50 mmHg. LVEDP was maintained at 5 mmHg. Perfusate temperature was maintained at 37 °C. The perfusate was gassed with a mixture of 95% O₂ + 5% CO₂ at a pH of 7.4 for the duration of the experiment.

2.6. Hemodynamic and cardio dynamic measurements

Left ventricular end diastolic pressure (LVEDP) and the left ventricular peak systolic pressure (LVPSP) were measured continuously. Left ventricular max ±dP/dt were calculated.

2.7. Statistical analysis

Data were initially analyzed with Bartlett’s test for homogeneity. Data found not to be homogeneous were transformed and reanalyzed. Data are presented as means and standard deviations or standard errors. Data were analyzed with multivariate analysis of variance (ANOVA). Means were analyzed using Duncan’s test. Data were considered significant when yielding a p-value less than 0.05.

3. Results

The myocardial contractile function was measured in the isolated hearts after treatment and resuscitation of the animals. Exposure to hemorrhagic shock resulted in myocardial contractile dysfunction as compared to controls. Treatment with the Na⁺–H⁺ exchanger blocker improved the myocardial contractile function. LVEDP was elevated in the hemorrhage group compared to the sham hemorrhage group (Fig. 1). Left ventricular peak systolic pressure was significantly higher in the hemorrhage treated group as compared to the untreated hemorrhage group (p < 0.05) (Fig. 2). Left ventricular generated pressure was significantly low in the hemorrhage group compared to the sham hemorrhage group and the hemorrhage treated (Fig. 3). Left ventricular maximum +d P/dt (Fig. 4) was lower in the hemorrhage group as compared to the sham hemorrhage group and the hemorrhage treated. Maximum −dP/dt was significantly higher in the hemorrhage group than the sham hemorrhage and the hemorrhage treated group (p < 0.05) (Fig. 5), while −dP/dt was significantly lower in the hemorrhage treated compared to the hemorrhage group (p < 0.05). In the hemorrhagic shock group, there was a significant drop in blood pressure as compared to controls (Fig. 6). There was no significant drop in blood pressure in the sham hemorrhage treated as compared to the sham untreated group (Fig. 6).

Figure 1.

Left ventricular end-diastolic pressure of the sham-hemorrhage treated and untreated, hemorrhage treated and untreated group over 1 h of ex vivo perfusion (n = 4).

Figure 2.

Left ventricular peak systolic pressure of the sham-hemorrhage treated and untreated, hemorrhage treated and untreated group over 1 h ex vivo perfusion (n = 4). a = p < 0.05 compared to hemorrhage + DMA; b = p < 0.05 compared to sham hemorrhage; c = p < 0.05 compared to sham hemorrhage + DMA.

Figure 3.

Left ventricular generated pressure of the sham-hemorrhage treated and untreated, hemorrhage treated and untreated group over 1 h ex vivo perfusion (n = 4). a = p < 0.05 compared to hemorrhage + DMA; b = p < 0.05 compared to sham hemorrhage; c = p < 0.05 compared to sham hemorrhage + DMA.

Figure 4.

Left ventricular +dP/dt of the sham-hemorrhage treated and untreated, hemorrhage treated and untreated group over 1 h ex vivo perfusion (n = 4).

Figure 5.

Left ventricular −dP/dt of the sham-hemorrhage treated and untreated, hemorrhage treated and untreated group over 1 h ex vivo perfusion (n = 4). a = p < 0.05 compared to hemorrhage + DMA; b = p < 0.05 compared to sham hemorrhage; c = p < 0.05 compared to sham hemorrhage + DMA.

Figure 6.

Effects of adenosine on MAP at 90 min after resuscitation of 1 h of hemorrhagic shock. Data are means ± SE (n = 4). ^∗p < 0.05 as compared to the sham hemorrhage group.

4. Discussion

The potential for successful resuscitation from traumatic hemorrhagic shock remains a challenge for management of trauma patients. Hemorrhagic shock involves a complex interaction of many pathophysiological pathways, metabolic acidosis, release of ischemic metabolites and the stimulation of a cascade of inflammatory process (Wu et al., 2008). The Na⁺–H⁺ exchanger has been known to participate in the ischemia–reperfusion injury and myocardial dysfunction by activating a series of events that leads to cellular dysfunction and failure (Pierce and Czubryt, 1995). Hemorrhagic shock results in the stimulation of the Na⁺–H⁺ exchanger due to the metabolic acidosis. This will cause Na⁺ overload which will eventually cause subsequent activation of the Na⁺−Ca²⁺ exchanger and an increase in the intracellular Ca²⁺, with its deleterious effects on myocardial contractility (Eng et al., 1998; Meng et al., 1993; Pierce et al., 1993; Meng and Pierce, 1990, 1991). Inhibition of the Na⁺–H⁺ exchanger will prevent the rapid wash out of the intracellular H⁺ by the Na⁺–H⁺ exchanger, and will allow the cell to recover from acidosis by other pathways. This is for a short period, then the exchanger will be stimulated gradually. In this way, the Ca²⁺ overload is prevented and its consequences on myocardial contractile function and structure would be prevented.

Inhibition of the Na⁺–H⁺ exchanger has been shown to protect the cells and preserve myocardial function in ischemia-reperfusion injury (Kaba et al., 2008). However, there is lack of information on the role of the Na⁺–H⁺ exchange inhibitors in protecting the myocardial structure and function following resuscitation of hemorrhagic shock. Our laboratory has previously shown that blocking the Na⁺–H⁺ exchanger using dimethyl amiloride for short time before ex vivo resuscitation of the isolated hearts in the Langendorff system, protected the myocardium against post-resuscitation contractile dysfunction (Soliman, in press). In the present study we investigated the myocardial protective effects of blocking the Na⁺–H⁺ exchanger using DMA, a specific inhibitor, by treating the animals before in vivo resuscitation following 1 h of hemorrhagic shock.

The present study showed that blocking the exchanger for a short period of time before resuscitation improved the myocardial contractile function with comparison to the non-treated hemorrhagic shock group and prevent the post-resuscitation dysfunction.

In summary our results demonstrate that inhibition of the exchanger before resuscitation of hemorrhagic shock has a strong cardioprotective effect. Our result, therefore, suggests that the exchanger is active during hemorrhagic shock and this activity is an important contributory factor in myocardial dysfunction and injury. This work strengthens the previous data that showed the cardioprotective effects of blocking the exchanger in an isolated resuscitated heart model (Soliman, in press) and opens a new window for therapeutic strategies for the treatment of trauma patients to prevent ischemia–reperfusion injury.