Nucleoside transport inhibitor, dipyridamole, induced myocardial protection following hemorrhagic shock in ex vivo perfused rat hearts

Abstract

Introduction

Successful protection against post-resuscitation myocardial injury is not available for trauma patients. Whereas intensive improvement in resuscitation strategies reduce myocardial injury, death among trauma patients are among the highest in the world due to myocardial dysfunction and multiple organ failure. Dipyridamole is a nucleoside transport inhibitor. Recent studies have shown that elevation of serum adenosine caused by dipyridamole improve cardiac function. The purpose of the present study was to examine the myocardial protective effects of dipyridamole therapy following 1 h of hemorrhagic shock.

Methods

Sprague–Dawley rats were used. The study consisted of three phases: Phase I to examine the direct effects of dipyridamole on myocardial function by perfusion of the isolated hearts with Krebs Henseleit buffer (KHB) + dipyridamole on the Langendorff apparatus. Phase II examined the protective effects of dipyridamole following 60 min of hemorrhagic shock (HS) by ex vivo treatment with dipyridimole 20 μg/L for 5 min followed by resuscitation with KHB for 55 min. Phase III: 60 min HS followed by in vivo treatment by injecting 1 ml of (20 μg/L) dipyridamole intra-arterially, and resuscitation for 30 min. Myocardial protection was assessed by measuring left ventricular generated pressure (LVGP) and end diastolic pressures (LVEDP).

Results

During ex vivo resuscitation, hearts from dipyridamole treated animals had significantly higher LVGP, and significantly lower LVEDP versus controls.

Conclusion

Dipyridamole therapy produces protection against post-resuscitation myocardial injury in rats.

1. Introduction

Emergent resuscitation therapies have improved survival after trauma (Crookes et al., 2004). Unfortunately, these resuscitation therapies are rarely carried out before considerable myocardial dysfunction and injury has occurred. This has led researchers for identifying therapies that protect against post-resuscitation myocardial injury.

Several lines of evidence suggest that nucleoside transport inhibitors, which increase adenosine levels by inhibiting uptake into myocyte, might offer protection against ischemia–reperfusion injury (Gokgoz et al., 1992). Adenosine is rapidly taken up by cells via nucleoside transporter in the cell membrane. Adenosine has cardioprotective effects: it activates adenosine receptors, resulting in attenuation of catecholamine release, β adrenoceptor-mediated myocardial hypercontraction, and Ca²⁺ overload via A1 receptors, and increases coronary blood flow and inhibits platelet and leukocyte activation via A2 receptors (Gresele et al., 1986).

Nucleoside transport inhibitors, including dipyridamole (Auchampach and Gross, 1993; Amrani et al., 1992), reduce ischemia–reperfusion injury when infused immediately before experimental myocardial infarction. In recent studies, elevation of serum adenosine by administration of dipyridamole improved cardiac function in patients with heart failure (Strauer et al., 1996; Bedetti et al., 2005). However, the cardioprotective effects of nucleoside transport inhibitors following resuscitation of hemorrhagic shock are unknown.

The present study examined the myocardial protective effects of adenosine transport inhibitor, dipyridamole, on myocardial contractile function and structure following hemorrhagic shock and resuscitation.

2. Materials and methods

This study was approved by the Continuous Medical Research Center at the College of Medicine, King Saud University. Male Sprague–Dawley rats (450–500 GWT) were used. The study consisted of three phases to which rats were randomly assigned: Phase I was designed to examine the direct effects of dipyridamole on myocardial function by perfusion of the isolated hearts with Krebs Henseleit buffer (KHB) + dipyridamole for 5 min then KHB for 55 min on the Langendorff apparatus. Phase II examined the protective effects of dipyridamole following 60 min of hemorrhagic shock (HS) by ex vivo treatment with dipyridimole 20 μg/L for 5 min then KHB for 55 min. Phase III: examined the protective effects of in vivo treatment with dipyridamole following 60 min HS by injecting 1 ml of (20 μg/L) dipyridamole intra-arterially, and resuscitation for 30 min.

2.1. Phase I: effects of dipyridamole on myocardial function in the isolated perfused hearts

Rats were injected intra-peritonealy (i.p.) with heparin sodium 2000 I.U. 15 min prior to anesthesia. The rats were then anaesthetized using urethane 125 mg/kg intra-peritonealy. Rats were randomly assigned to either: (1) control group: of 60 min perfusion with Krebs solution in the Langendorff system, or (2) dipyridamole treated group: perfusion of the isolated hearts with 20 μg/L Dipyridamole + Krebs for 5 min, then shifted to perfusion with Krebs solution for 55 min (n = 6 per group).

2.1.1. 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 two solutions: (1) Krebs solution; (2) Krebs + dipyridamole 20 μg/L. 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.

2.2. Phase II: ex vivo treatment with dipyridamole following hemorrhagic shock

Rats were randomly assigned to: (1) hemorrhage, (2) hemorrhage + dipyridamole, (3) sham hemorrhage and (4) sham hemorrhage + dipyridamole groups (n = 6 per group). Rats were hemorrhaged using a reservoir model. 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. Arterial blood pressure was monitored for 1 h. In the hemorrhage group, blood was withdrawn to reach mean arterial blood pressure of 40 mmHg, via an intra-arterial catheter inserted into the left carotid artery. In the sham hemorrhage group, the same surgical procedure was performed without hemorrhage. Isolated hearts were resuscitated ex vivo either with KHB solution for 60 min, or 20 μg/L dipyridamole + KHB for the first 5 min then KHB for 55 min.

2.3. Phase III: in vivo resuscitation following 1 h of hemorrhagic shock

Rats were randomly assigned to: (1) hemorrhage, (2) hemorrhage + dipyridamole, (3) sham hemorrhage and (4) sham hemorrhage + dipyridamole groups (n = 6 per group). Rats were hemorrhaged as described earlier. Rats were resuscitated in vivo by reinjecting the shed blood to restore normotension. In the treated group, rats were injected intra-arterially with dipyridamole then resuscitated. After 60 min, hearts were isolated and perfused with KHB for measurement of myocardial function.

2.4. Hemodynamic and cardio dynamic measurements

Left ventricular end diastolic pressure (LVEDP) and the left ventricular peak systolic pressure (LVPSP) were measured continuously. Left ventricular generated pressure (LVGP) was calculated.

2.5. 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 <0.05.

3. Results

In isolated perfused hearts, treatment with dipyridamole has been shown to induce positive ionotropic effect in the isolated perfused hearts. Fig. 1A and B show the changes in the left ventricular generated pressure and max + dP/dt during ex vivo perfusion of the hearts in the Langendorff system for 1 h with KHB only or dipyridamole treated. Left ventricular generated pressure and max + dP/dt were significantly higher in the dipyridamole perfused group as compared to the hearts perfused with only KHB (p < 0.05).

Figure 1.

(A) Left ventricular generated pressure (LVGP) and (B) positive change in pressure over time (+dP/dt) in the isolated perfused hearts in the dipyridamole + KHB and the KHB perfused hearts (n = 6 per group). All values are means ± SD. ^∗Represents p < 0.05 versus perfusion with KHB only (n = 6 per group).

As shown in Fig. 2A and B, left ventricular generated pressure and max + dP/dt were significantly improved after 60 min of ex vivo perfusion in hearts treated with dipyridamole compared to hearts perfused with normal physiological KHB buffer as well as to sham control group (p < 0.05). These data suggest that dipyridamole therapy before resuscitation following hemorrhagic shock improved contractile function in the ex vivo perfused hearts in the Langendorff system.

Figure 2.

(A) Left ventricular generated pressure (LVGP) in the sham, hemorrhagic shock and hemorrhagic shock + dipyridamole and (B) positive change in pressure over time (+dP/dt) in ex vivo resuscitated hearts in the Langendorff system with KHB only or KHB + dipyridamole following 1 h of experimental protocol (n = 6 per group). All values are means ± SD. ^∗Represents p < 0.05 versus hemorrhagic shock group, ^•represents p < 0.05 versus hemorrhagic shock group (n = 6 per group).

As shown in Fig. 2A and B, left ventricular generated pressure and max + dP/dt were significantly lower in hearts from hemorrhagic shock animals perfused with KHB only compared to hearts from sham group (p < 0.05). This shows that myocardial contractile function is decreased following resuscitation of hemorrhagic shock (see Fig. 3).

Figure 3.

(A) Left ventricular generated pressure (LVGP) in the sham, hemorrhagic shock and hemorrhagic shock + dipyridamole and (B) positive change in pressure over time (+dP/dt) in the in vivo dipyridamole treated rats and resuscitated isolated hearts in the Langendorff system with KHB only following 1 h of experimental protocol (n = 6 per group). All values are means ± SD. ^∗Represents p < 0.05 versus hemorrhagic shock group, ^•represents p < 0.05 versus hemorrhagic shock group (n = 6 per group).

4. Discussion

The purpose of this study was to determine the myocardial protective effects of dipyridamole following hemorrhagic shock in isolated perfused hearts as well as in the in vivo treated animals. Dipyridamole increased the myocardial contractility in the isolated perfused hearts. Hemorrhagic shock results in myocardial dysfunction. Successful resuscitation following hemorrhagic shock requires complete recovery of myocardial function. This can be achieved by increase in contractility mediated by pharmacological vasodilatation of coronary bed (Stahl et al., 1986).

Dipyridamole inhibits the transport of extracellular adenosine into the cells resulting in increased extracellular adenosine, which provides protection against post-resuscitation myocardial injury (Gresele et al., 1986).

The mechanisms by which dipyridamole can improve myocardial contractility following hemorrhagic shock and resuscitation are not completely understood. The most likely mechanism is the dipyridamole induced increase in blood flow and vascular wall within the ventricular wall, which in turn leads to a stretching of myocardial fibers and to an increase in contractility through the Frank–Starling effect.

Experimental studies support the concept that vasodilators may reduce ischemia by increasing collateral blood flow to ischemic myocardium (Cohen et al., 1973).

Vasodilator-induced hypotension is generally beneficial because of the associated decrease in an LV wall tension, but an excessive reduction in perfusion pressure may be harmful (Kattus and Gregg, 1959).

Other possible mechanisms are sympathetic activation secondary to the systemic vasodilatation or a decrease of ventricular afterload (Picano et al., 1992; Lucarini et al., 1992).

In addition, dipyridamole has documented anti-inflammatory properties, including antioxidant effects (Weyrich et al., 2005). Dipyridamole potentiates synthesis of nitric oxide, a vasodilating antioxidant molecule that prevents abnormal platelet and leukocyte adherence to vessel wall. Increased extracellular adenosine levels resulting from extended-release dipyridamole treatment also affect the expression of inflammatory factors. Healthy vascular endothelium produces adenosine that minimizes the inflammatory response. Elevated adenosine levels inhibit neutrophil activation and associated superanion production. Reduced expression of adhesion molecules (CD11/ CD18, MAC-1, β₂-integrins) on neutrophils and polymorphonuclear cells has also been shown to occur (Wollner et al., 1993).

Dipyridamole has been reported to attenuate nuclear factor κB transcription in monocytes. Dipyridamole treatment also reduced expression of monocyte chemotactic protein-1 and matrix metalloproteinase-9 (Weyrich et al., 2005). Monocyte chemotactic protein-1 attracts monocytes to the atherosclerotic region and promotes atherosclerosis (Charo and Taubman, 2004). Reduction of matrix metalloproteinase-9 is of particular interest because matrix metalloproteinase-9 is associated with unstable plaques and has been found to be elevated in carotid plaques from patients with TIA and stroke (Verhoeven et al., 2005).

Another way of evaluating the effects of dipyridamole on circulating levels of inflammatory markers, which may reflect abnormal responses occurring at the vessel wall (Libby and Theroux, 2005).

5. Summary

Based on the results of previous experimental and clinical studies, the present study showed that dipyridamole protects the myocardium against post-resuscitation myocardial dysfunction and failure.