In this study, the protective effect of pentoxifylline against hypoxia-reoxygenation injury and the possible involvement of nitric oxide (NO)-mediated pathways in this protection were investigated in isolated rat papillary muscles. Papillary muscles were excised and isolated in Krebs–Henseleit solution aerated with 95% O2 and 5% CO2. Hypoxia was simulated by substituting O2 with argon. Three sets of experiments, testing 30, 60, and 90 min of hypoxia, were performed. The effects of different pentoxifylline concentrations on papillary muscle contractile parameters and responsiveness to isoproterenol were assessed. To investigate the role of NO, N(ω)-nitro-L-arginine methyl ester was added before pentoxifylline treatment. Pentoxifylline did not show any inotropic effect on papillary muscles. Hypoxia caused a profound depression of contractile parameters, which was not affected by pentoxifylline treatment. Reoxygenation resulted in significant partial recovery of contractile parameters after 30 and 60 but not 90 min of hypoxia. In experiments with 30 and 60 min of hypoxia, reoxygenation-induced contractile recovery and responsiveness to isoproterenol were improved by pentoxifylline in a concentration-dependent fashion. These functional improvements were completely blocked by N(ω)-nitro-L-arginine methyl ester pretreatment. No improvement was observed in 90-min hypoxia experiment. In conclusion, pentoxifylline improved contractile recovery during reoxygenation and postreoxygenation responsiveness to β-adrenergic stimulation through the NO-dependent mechanism.
*Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
†Department of Cardiothoracic Surgery, Tehran Heart Center, Tehran
Reprints: Dr. Ahmad Reza Dehpour (e-mail: firstname.lastname@example.org).
Received for publication February 4, 2006; accepted March 21, 2006
Cardiovascular effects of pentoxifylline have been studied extensively.1–3 However, its actions in this system are still controversial. Some studies have shown that pentoxifylline results in vasodilation and tachycardia besides its positive chronotropic and inotropic effects,3 whereas other studies have failed to show these effects.2,4 Modulations of vagal and vasomotor centers in the brainstem, peripheral cardiovascular system, and either direct or reflex release of catecholamines have also been suggested as mediating pentoxifylline's diverse cardiovascular effects.3
Pentoxifylline exerts anti-inflammatory and antioxidant effects through inhibition of neutrophil activation and production of inflammatory mediators, as well as scavenging free oxygen radicals under pathological circumstances.5,6 It increases nitric oxide (NO) production and intracellular cGMP level by the stimulation of endothelial NO synthase and inhibition of phosphodiesterase activity, respectively.7–9 The involvement of NO-mediated pathways in the protective effect of pentoxifylline against ischemia-reperfusion injury in gastrointestinal tissue has been shown.10 Its anti-inflammatory, antioxidant, and phosphodiesterase inhibitory effects (increasing cellular levels of cAMP)11 have caused it to be suggested as a potential cardioprotective agent in ischemia-reperfusion injury,12–15 but there are also disagreements on this aspect of its action. Some previous in vivo studies have shown that pentoxifylline reduces leukocyte sequestration,16 preserves endothelial function,16 limits myocardial injury (assessed by loss of enzymes and ST segment elevation),15,17 and improves functional recovery during the postischemic period.15 No reduction in infarct size has been reported in other studies.4 Although the mechanisms of these discrepancies are not clear, methodological differences, which are inherent in in vivo studies, may be involved.
For many years, the model of isolated papillary muscle, which enables strict control of experimental conditions, has been used to examine the role of specific substances on myocardial function in different physiological or pathological conditions. Therefore, we investigated the effect of pentoxifylline on contractility, protection against hypoxia-reoxygenation injury, and postreoxygenation responsiveness to β-adrenergic stimulation in isolated rat papillary muscles. Moreover, considering the cardioprotective effect of NO against ischemia-reperfusion injury,18,19 its role as a mediatory agent in inducing pentoxifylline effect was also assessed.
MATERIALS AND METHODS
The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by US National Institute of Health (NIH Publication No. 85-23, revised 1985). Male Sprague-Dawley rats weighing 200 to 250 g were used in the experiments. Animals were housed in groups of 3 to 4 in a room controlled at 22°±1°C and maintained in an alternating 12-h light/12-h dark cycles; they were given free access to food and water.
Papillary Muscle Contractility Study
Animals were anesthetized by an intraperitoneal injection of ketamine HCl (50 mg/kg; Gedoon Richter, Budapest, Hungary) and chlorpromazine HCl (10 mg/kg; Daroupakhsh, Tehran, Iran). Then hearts were removed and left ventricular papillary muscles were excised and isolated in Krebs–Henseleit solution aerated with 95% O2 and 5% CO2. The composition of Krebs–Henseleit (in millimoles) was as follows: NaCl, 118.0; KCl, 4.70; CaCl2, 2.52; MgSO4, 1.64; NaHCO3, 24.88; KH2PO4, 1.18; glucose, 5.55; sodium pyruvate, 2.0 (pH 7.4). Papillary muscles were suspended vertically to isometric force transducer (MLT 1030/D, ADInstruments, PowerLab, Spain) in the 25-mL glass chambers of organ bath (ADInstruments). The temperature of the bathing buffer was 33°C. The papillary muscles were gradually stretched to search for the optimal preload according to the Frank–Starling relationship. Once the maximal contraction force was achieved, the preparation was relaxed to 90% of optimal tension and left for 90 min of stabilization. After stabilization, the muscles were subjected to electrical-field stimulation at 0.5 Hz, 3 ms duration, and 30 V, which is about 20% higher than the threshold.
In the first set of experiments (n=20), the influence of pentoxifylline (Aventis) on papillary muscle contractile parameters (contractile force and velocity of contractility) was assessed. Pentoxifylline was applied in increasing concentrations, starting with 10−7 mmol/L and increased in negative logarithm half-molar cumulative steps up to 10−3 mmol/L to establish the concentration–contractility relationship.
Papillary Muscle Hypoxia-Reoxygenation Study
During subsequent experiments, hypoxia was simulated by substituting O2 in carbogen with argon (95% argon, 5% CO2).6 This resulted in the drop of tissue bath O2 partial pressure from 453±42 to 49±2.1 mm Hg (P<0.001) and caused significant and rapid impairment of muscle contractility. During reoxygenation, carbogen (95% O2, 5% CO2) was added to the organ bath again.
We evaluated the effects of different concentrations of pentoxifylline (10−7–10−3 mmol/L), added 30 min before 30, 60, and 90 min of hypoxia, on contractile parameters (Fig. 1). We did not observe any significant effect of concentrations lower than 10−4 mmol/L, so the effects of higher concentrations (10−4, 10−3.5, and 10−3 mmol/L) were shown in the results section. An additional group of time-related nonhypoxic control (TRNHC) rats was also studied to evaluate contractility changes during this time. Each group consisted of 6 animals. After 120 min of reoxygenation, isoproterenol (a nonselective β-adrenoceptor agonist, 10−5 mmol/L) was added to test post-reoxygenation responsiveness to β-adrenergic stimulation.20–24
To evaluate the role of NO in the cardioprotective effect of pentoxifylline against hypoxia-reoxygenation injury, N(ω)-nitro-L-arginine methyl ester (L-NAME, a nonselective NO synthase inhibitor, 10−4 mmol/L), was added to the organ bath 30 min before pentoxifylline treatment (Fig. 1). L-NAME concentration was selected based on the previous studies and shown to effectively reduce NO production and inhibit the cardioprotective effect of NO on the cardiovascular system.25,26
Contractile parameters were evaluated during hypoxia and reoxygenation periods and expressed as the percentage of baseline (before induction of hypoxia). For each preparation, recorded contractile parameters were compared with baseline and expressed in percentage, the initial contraction force and velocity being 100%. Contractile response to isoproterenol was also expressed as the percentage of the postreoxygenation contractility.
Data are expressed as mean±SEM. Statistical evaluation of the data was performed with the analysis of variance followed by the Holm–Sidak method for the post hoc multiple comparison procedure. A value of P<0.05 was considered statistically significant. SPSS for Windows Version 10 (SPSS, Chicago, IL) was used for statistical analysis.
Pentoxifylline Concentration–Contractility Relationship
Pentoxifylline (10−7–10−3 mmol/L) did not show any inotropic effect on rat papillary muscles in our study (data not shown).
Basal Papillary Muscle Contractility
Basal contractile parameters are shown in Table 1. Basal contractile force and velocity of pentoxifylline-treated rats were not significantly different from control group.
Papillary Muscle Contractility During Hypoxia and Reoxygenation Period
Hypoxia resulted in a profound depression of contractile parameters (contractile force and velocity), which partially recovered during reoxygenation, depending on the hypoxia duration (Fig. 2; graph related to velocity not shown). Pentoxifylline treatment had no significant effect on the posthypoxic contractile impairment (Figs. 3–5; graphs related to velocity not shown). Although reoxygenation resulted in significant but partial recovery of contractile parameters following 30 and 60 min of hypoxia (P<0.05, for each variable), no recovery was found after 90 min of hypoxia (Fig. 2). This recovery achieved the plateau within 60 min, and no significant change was seen thereafter (Figs. 3–5). In experiments with 30 min of hypoxia (Fig. 3), pentoxifylline, at concentrations of 10−4, 10−3.5, and 10−3 mmol/L, was able to improve reoxygenation-induced contractile recovery (P<0.05 for 10−4 mmol/L and P<0.01 for 10−3.5 and 10−3 mmol/L), which was significantly higher in 10−3.5 and 10−3 compared to 10−4 mmol/L (P<0.05, for each variable). In 60-min hypoxia experiments (Fig. 4), a low concentration of pentoxifylline (10−4 mmol/L) failed to induce statistically significant improvement during the reoxygenation period, but higher concentrations (10−3.5 and 10−3 mmol/L) significantly improved contractile recovery compared with both control (P<0.05, for each variable) and low concentration pentoxifylline (P<0.05, for each variable) groups. Better reoxygenation-induced contractile recovery was achieved with neither low nor high concentrations of pentoxifylline, in 90-min hypoxia experiments (Fig. 5).
Isoproterenol Stimulation Study
Although 30 and 60 min of hypoxia resulted in an attenuation of isoproterenol responsiveness (P<0.001), a longer period of hypoxia almost abolished the isoproterenol response (Fig. 6; graph related to velocity not shown). In a 30-min hypoxia experiment, we showed a higher response to isoproterenol in papillary muscles treated with 10−3.5 and 10−3 mmol/L pentoxifylline, not only in comparison with control (P<0.01, for each variable) but also in comparison with the lower pentoxifylline concentration (P<0.05, for each variable). After 60 min of hypoxia, the lower pentoxifylline concentration (10−4 mmol/L), unlike the higher concentrations (10−3.5 and 10−3 mmol/L; P<0.05, for each variable), did not improve contractile response to isoproterenol. At the end of the 90-min hypoxia experiment, no concentration of pentoxifylline was able to increase papillary muscle contractile responsiveness to isoproterenol.
According to our results, reoxygenation-induced contractile recovery and responsiveness to isoproterenol stimulation were most effectively improved by the highest concentration of pentoxifylline (10−3 mmol/L) in the shortest period of hypoxia (30 min). Therefore, we evaluated the involvement of NO in the cardioprotective effect of pentoxifylline against hypoxia-reoxygenation injury at these concentration and hypoxic period. L-NAME treatment had no significant effect on contractile parameters of control rats at baseline (Table 1) during the hypoxia-reoxygenation period (Fig. 7; graph related to velocity not shown) and their responses to isoproterenol stimulation (Fig. 8; graph related to velocity not shown). However, it could block the pentoxifylline-induced improvement in both contractile recovery during the reoxygenation period and the responsiveness to isoproterenol stimulation (Figs. 7 and 8).
In this study, pentoxifylline did not show any inotropic effect on papillary muscles. This finding is in agreement with that of the Vittone et al in vitro study,2 which showed that despite increasing cAMP level, pentoxifylline had no effect on contractile force of isolated rat heart. However, it has been reported that intravenous administration of pentoxifylline may increase myocardial contractility, thought to be mediated by catecholamine release.3
Hypoxia resulted in a profound depression of contractile force, which was significantly related to the hypoxia duration. This depression was partially recovered during the reoxygenation periods of 30 and 60 but not 90 min of hypoxia experiments. Similar to the Speechly-Dick et al study,24 this contractile recovery achieved the plateau about 60 min after reoxygenation and no further contractile change was observed thereafter. However, Deja et al20 have demonstrated the decline of contractility during the reperfusion period in their study. They attributed it to the use of the Krebs–Henseleit solution with low pyruvate concentration and no replacement of bath solution during the reperfusion period. Because we have used the same method, it seems that other reasons may underlie these discrepancies.
With respect to diverse actions of pentoxifylline, as an anti-inflammatory, antioxidant, and phosphodiesterase inhibitor agent, many studies have been focused on the cardioprotective effects of pentoxifylline against hypoxia-reoxygenation damage, but the findings are contradictory. Some previous in vivo studies have shown that pentoxifylline reduces leukocyte sequestration by preserving endothelial function,16 limits myocardial injury (assessed by loss of enzymes and ST segment elevation)15,17 and also improves functional recovery during the postischemic period.15 Nevertheless, no cardioprotection has been reported in studies evaluating infarct size as a marker of cardiac damage.4 Although the complexity of in vivo experiments may be postulated to underlie these discrepancies, it is interesting that pentoxifylline has been shown to produce both protective12 and detrimental27 effects on cardiomyocyte cultures exposed to anoxia. Although, in our in vitro model, pentoxifylline conferred no significant protection against posthypoxic contractile depression, it did result in concentration-dependent improvement of reoxygenation-induced contractile recovery. A clear advantage of using higher pentoxifylline concentration was visible when assessing the functional recovery after 30 and 60 min of hypoxia. A longer hypoxic period of 90 min resulted in severe and irreversible decline of contractile force, which does not significantly recover even with a high concentration of pentoxifylline.
To investigate whether pentoxifylline could also improve postreoxygenation responsiveness to β-adrenergic stimulation, we added isoproterenol to the tissue bath at the end of the reoxygenation period. Although 30 and 60 min of hypoxia resulted in a significant reduction in isoproterenol responsiveness, a longer period of hypoxia almost abolished this response in control groups. We observed that a high pentoxifylline concentration was able to improve papillary muscle responsiveness to isoproterenol following 30 and 60 min of hypoxia, whereas its low concentration was only effective in the 30-min hypoxia experiment. These findings would suggest that, alongside better contractile recovery during reoxygenation period, pentoxifylline could improve postreoxygenation responsiveness to catecholamines in a concentration-dependent fashion.
Previous studies have shown that stimulation of endothelial NO production by pentoxifylline contributes to its effects on cardiovascular function.7–9 It has been demonstrated that NO-dependent mechanisms are also involved in protective effects of pentoxifylline against ischemia-reperfusion injury in gastrointestinal tissue.10 Although NO has been widely known as a cardioprotective agent against ischemia-reperfusion injury,18,19 its mediatory role in pentoxifylline-induced cardioprotection has not yet been investigated. According to our results, L-NAME pretreatment completely blocked the improvement in contractile recovery during reoxygenation period as well as contractile response to isoproterenol, induced by pentoxifylline. These findings suggest that NO production may contribute to the cardioprotective effect of pentoxifylline against hypoxia-reoxygenation injury.
This study demonstrates that in spite of no significant effect on hypoxia-induced contractile suppression, pentoxifylline can improve reoxygenation-induced contractile recovery and postreoxygenation responsiveness to β-adrenergic stimulation. In addition, this study provides the first evidence for the contribution of NO-mediated pathways in pentoxifylline-induced cardioprotection.
1. Argel MI, Vittone L, Grassi AO, et al. Effect of phosphodiesterase inhibitors on heart contractile behavior, protein kinase activity and cyclic nucleotide levels. J Mol Cell Cardiol. 1980;12:939–954.
2. Vittone L, Chiappe LE, Argel MI, et al. The mechanical and biochemical effects of pentoxifylline on the perfused rat heart. Experientia. 1980;36:1088–1090.
3. Watanabe H, Furukawa Y, Chiba S. Cardiovascular effects of aminophylline and pentoxifylline on intact dogs and isolated dog atria. Jpn Heart J. 1982;23:235–243.
4. Campbell CA, Clavenna CF, Wynne J, et al. Pentoxifylline does not reduce infarct size in a canine model of acute myocardial infarction. Br J Pharmacol. 1988;93:587–590.
5. Bhat VB, Madyastha KM. Antioxidant and radical scavenging properties of 8-oxo derivatives of xanthine drugs pentoxifylline and lisofylline. Biochem Biophys Res Commun. 2001;288:1212–1217.
6. Horvath B, Marton Z, Halmosi R, et al. In vitro antioxidant properties of pentoxifylline, piracetam, and vinpocetine. Clin Neuropharmacol. 2002;25:37–42.
7. Berkenboom G, Fang ZY, Unger P, et al. Endothelium-dependent effects of pentoxifylline in rat aorta. Eur J Pharmacol. 1991;193:81–86.
8. Hansen PR. In vitro studies on responses to pentoxifylline and aminophylline of rat mesenteric resistance vessels. Eur J Pharmacol. 1994;261:105–110.
9. Wang P, Ba ZF, Stepp KJ, et al. Pentoxifylline attenuates the depressed endothelial cell function and vascular muscle contractility following trauma and hemorrhagic shock. J Trauma. 1995;39:121–126.
10. Kwiecien S, Brzozowski T, Konturek PC, et al. Gastroprotection by pentoxyfilline against stress-induced gastric damage. Role of lipid peroxidation, antioxidizing enzymes and proinflammatory cytokines. J Physiol Pharmacol. 2004;55:337–355.
11. Nandi JS, Nair KG, Deo S. Inhibition cAMP-phosphodiesterase in the rat heart by pentoxifylline: a new xanthine derivative. Adv Myocardiol. 1980;1:359–365.
12. Hansen PR, Stawski G. Neutrophil mediated damage to isolated myocytes after anoxia and reoxygenation. Cardiovasc Res 1994;28:565–569.
13. Horton JW, White DJ. Free radical scavengers prevent intestinal ischemia-reperfusion-mediated cardiac dysfunction. J Surg Res. 1993;55:282–289.
14. Pridjian AK, Bove EL, Bolling SF, et al. Developmental differences in myocardial protection in response to 5′-nucleotidase inhibition. J Thorac Cardiovasc Surg. 1994;107:520–526.
15. Ulus AT, Aksoyek A, Katircioglu SF, et al. Preservation of myocardial functions by pentoxyphylline cardioplegia during and after cardiopulmonary bypass. Panminerva Med. 2000;42:253–256.
16. Gale SC, Hokama JY, Ritter LS, et al. Pentoxifylline reduces coronary leukocyte accumulation early in reperfusion after cold ischemia. Ann Thorac Surg. 2001;71:1305–1311.
17. Gallenkamper W, Rucker W, Schror K. Cardioprotective actions of pentoxifylline in an animal model of acute myocardial ischaemia. Br J Pharmacol. 1984;81:575–581.
18. Lochner A, Marais E, Genade S, et al. Nitric oxide: a trigger for classic preconditioning? Am J Physiol Heart Circ Physiol. 2000;279:H2752–H2765.
19. Novalija E, Hogg N, Kevin LG, et al. Ischemic preconditioning: triggering role of nitric oxide-derived oxidants in isolated hearts. J Cardiovasc Pharmacol. 2003;42:593–600.
20. Deja MA, Golba KS, Kolowca M, et al. Diazoxide provides protection to human myocardium in vitro that is concentration dependent. Ann Thorac Surg. 2004;77:226–232.
21. Iwashiro K, Criniti A, Sinatra R, et al. Felodipine protects human atrial muscle from hypoxia reoxygenation dysfunction: a force-frequency relationship study in an in vitro model of stunning. Int J Cardiol. 1997;62:107–132.
22. Monti F, Iwashiro K, Picard S, et al. Adenosine triphosphate-dependent potassium channel modulation and cardioplegia induced protection of human atrial muscle in an in vitro model of myocardial stunning. J Thorac Cardiovasc Surg. 2000;119:842–848.
23. Picard S, Criniti A, Iwashiro K, et al. Protection of human myocardium in vitro by KATP activation with low concentration of bimakalim. J Cardiovasc Pharmacol. 1999;34:162–172.
24. Speechly-Dick ME, Grover GJ, Yellon DM, et al. Does ischemic preconditioning in the human involves protein kinase C and the ATP-dependent K channel? Studies of contractile function after simulated ischemia in an atrial in vitro model. Circ Res. 1995;77:1030–1035.
25. Bell RM, Smith CC, Yellon DM, et al. Nitric oxide as a mediator of delayed pharmacological (A(1) receptor triggered) preconditioning; is eNOS masquerading as iNOS? Cardiovasc Res. 2002;53:405–413.
26. Hattori R, Otani H, Maulik N, et al. Pharmacological preconditioning with resveratrol: role of nitric oxide. Am J Physiol Heart Circ Physiol. 2002;282:H1988–H1995.
27. Geisbuhler TP, Schwager TL, Ervin HD. 3-Isobutyl-1-methylxanthine (IBMX) sensitizes cardiac myocytes to anoxia. Biochem Pharmacol. 2002;63:2055–2062.
Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
contractility; hypoxia/reoxygenation; nitric oxide; pentoxifylline