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Ann Thorac Surg 1998;65:1303-1309
© 1998 The Society of Thoracic Surgeons

Cardioprotection by Activation of NO/cGMP Pathway After Cardioplegic Arrest and 8-Hour Storage

^a Division of Cardiothoracic Surgery, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada
^b Department of Pharmacology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada
^c Department of Anaesthesia, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada

Accepted for publication December 16, 1997.

Address reprint requests to Dr Clanachan, Department of Pharmacology, University of Alberta, 9-43 Medical Sciences Building, Edmonton, AB, Canada, T6G 2H7
e-mail: (sandy.clanachan{at}ualberta.ca)

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. We determined whether activation of the nitric oxide/cyclic guanosine monophosphate pathway by sodium nitroprusside (SNP) protects hearts subjected to cardioplegic arrest and prolonged hypothermic storage.

Methods. Isolated rat hearts arrested with St. Thomas’ II cardioplegia and stored at 3° ± 1°C for 8 hours were reperfused at 37°C in Langendorff (10 minutes) and working (60 minutes) modes.

Results. During reperfusion, left ventricular work was depressed in stored hearts relative to fresh hearts. When present during arrest, storage, and both reperfusion phases, SNP (200 µmol/L) improved work to values close to those in fresh hearts. When added only during the 10-minute period of Langendorff reperfusion, SNP also improved the subsequent recovery of work. This effect was antagonized by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Poststorage coronary perfusion was not increased by SNP.

Conclusions. The ability of SNP to enhance recovery independent of changes in coronary perfusion and in an ODQ–sensitive manner suggests that SNP–induced protection is due to activation of the myocardial nitric oxide/cyclic guanisine monophosphate pathway. These results suggest that supplementing cardioplegic solutions with SNP, administering SNP during early reperfusion, or both may offer additional means to improve donor heart preservation.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Heart transplantation is an accepted therapeutic modality for select patients with end-stage heart disease. Unlike liver and kidney transplantation in which graft preservation times of 12 to 24 hours are clinically feasible, the "safe" ischemic time for cardiac allografts remains 4 to 6 hours. This limitation greatly exacerbates the critical shortage of donor organs and precludes long-distance organ procurement and sharing. Furthermore, inadequate myocardial preservation continues to be a significant cause of early graft failure and recipient mortality [1].

The key to extending the safe ischemic time of cardiac allografts is the elucidation of the mechanisms underlying the etiology of myocardial ischemia-reperfusion injury. Recent studies have suggested that this injury is worsened by a diminished bioavailability of nitric oxide (NO) [2] and that an enhancement of NO production is cardioprotective in models of normothermic, short-term, regional ischemia [3]. In a heterotopic rat heart transplant model, augmentation of the NO/cyclic guanosine monophosphate (cGMP) pathway enhanced survival of the donor organ after 12 hours of storage [4]. One mechanism for the salutary effect of NO that has emerged from in vivo models is improved coronary perfusion mediated either by coronary vasodilation or by inhibition of endothelial-leukocyte interactions [5]. Other potential mechanisms of NO–induced cardioprotection may be independent of coronary vasodilation. These include a direct myocardial relaxant effect such as that mediated by the NO donors sodium nitroprusside (SNP) and nitroglycerin [6]. Also, NO donors may be cardioprotective during ischemia plus reperfusion by favorably altering myocardial metabolism during ischemia [7]. Although the NO donor SNP has cardioprotective properties, little is known about the role of the NO/cGMP pathway in the preservation of mechanical function after prolonged hypothermic storage and reperfusion of working hearts, a scenario analogous to clinical donor-heart preservation.

This study was designed to examine four possibilities: test if SNP would enhance the recovery of mechanical function of hearts reperfused after cardioplegic arrest and prolonged hypothermic storage; determine whether the timing of SNP administration affected the recovery of mechanical function; investigate whether any observed protective effect was related to coronary vasodilation; and clarify the role of the NO/cGMP pathway in the cardioprotective action of SNP. Experiments were performed in isolated, fatty acid–perfused working rat hearts in the absence of bloodborne cellular elements, thereby eliminating any major contribution of neutrophils to ischemia-reperfusion injury.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Heart extraction
Male Sprague-Dawley rats 7 to 8 weeks old and weighing 300 to 350 g that had been fed ad libitum were used. All animals received care according to the Canadian Council on Animal Care and the University of Alberta Health Sciences Animal Welfare Committee.

Hearts were excised after the induction of anesthesia with sodium pentobarbital. The aortae were cannulated, and retrograde perfusion using Krebs-Henseleit solution (37°C, pH 7.4, and gassed with a 95% oxygen and 5% carbon dioxide mixture) containing 11 mmol/L glucose and 2.5 mmol/L Ca²⁺ was initiated at a constant perfusion pressure of 60 mm Hg.

Perfusion protocol and experimental groups
Hearts were subjected to a perfusion protocol comprising several phases that were performed in the following sequence:

Initial Langendorff perfusion that comprised a 10- minute period of stabilization in the nonworking, nonrecirculating Langendorff mode (L);

Storage phase in which hearts were arrested with 25 mL of ice-cold St. Thomas’ II cardioplegic solution (110 mmol/L NaCl, 10 mmol/L NaHCO₃, 16 mmol/L KCl, 16 mmol/L MgCl₂ · 6H₂O, and 1.2 mmol/L CaCl₂, pH 7.8) administered at a constant perfusion pressure of 60 mm Hg and thereafter removed from the perfusion rig and immersion stored in St. Thomas’ II solution at 3° ± 1°C for 8 hours (S);

Reperfusion after storage in the nonworking, nonrecirculating Langendorff mode for 10 minutes with Krebs-Henseleit solution at 37°C (R);

Working-mode reperfusion for 60 minutes in which hearts were paced at a rate of 300 beats/min (Grass SD9 stimulator) (W). The reperfusion fluid (recirculating volume of 100 mL) consisted of a modified Krebs-Henseleit solution at 37°C containing 2.5 mmol/L Ca²⁺, 11 mmol/L glucose, 100 µU/mL insulin, and 1.2 mmol/L palmitate prebound to 3% bovine serum albumin (fraction V). Reperfusion in the working mode was performed at a constant left atrial preload (11.5 mm Hg) and a constant aortic afterload (80 mm Hg).

Six freshly excised hearts that were not subjected to cardioplegic arrest and storage were perfused in the Langendorff mode for 10 minutes and then in the working mode for 60 minutes to assess coronary flow and mechanical function under normal (nonstored) aerobic conditions. Hearts that were subjected to cardioplegic arrest, hypothermic storage, and reperfusion were randomly assigned to an untreated group (CPL, n = 8) or drug treatment groups as follows:

Sodium nitroprusside present throughout perfusion: Separate groups of hearts were exposed throughout all phases (L + S + R + W) of the protocol to graded concentrations of SNP—10 µmol/L (n = 4), 50 µmol/L (n = 4), or 200 µmol/L (n = 8)—to investigate the concentration-dependent effects of SNP on the recovery of mechanical function.

Timing of SNP treatment: Groups of hearts (n = 8) were exposed to SNP (200 µmol/L) during only cardioplegic arrest and storage (S), during only Langendorff reperfusion after storage (R), or during initial Langendorff perfusion, cardioplegic arrest and storage, and poststorage Langendorff reperfusion (L + S + R).

Inhibition of NO/cGMP pathway: Two further groups of hearts were exposed during only the poststorage Langendorff reperfusion period (R) to either the specific guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (3 µmol/L, n = 8) or a combination of SNP (200 µmol/L) and ODQ (3 µmol/L) (n = 8). The recovery of mechanical function in these groups was compared with that in hearts treated with SNP (200 µmol/L) alone during only the poststorage Langendorff reperfusion period (R).

Measurements of coronary perfusion and mechanical function
Measurements of baseline coronary flow (milliliters per minute) were made in all hearts during initial Langendorff perfusion prior to the initiation of arrest and in select groups, after 1 minute of perfusion with cardioplegic solution. Coronary flow was also measured during the poststorage Langendorff perfusion phase (R). In the working-mode reperfusion phase (W), heart rate and systolic and diastolic aortic pressures (millimeters of mercury) were measured using a Gould P21 pressure transducer attached to the aortic outflow line. Cardiac output (milliliters per minute) and aortic flow (milliliters per minute) were measured using ultrasonic flow probes (Transonic T206) placed in the left atrial inflow line and the aortic outflow line, respectively. Coronary flow during the working mode (milliliters per minute) was calculated from the difference between cardiac output and aortic flow. Left ventricular minute work (LV work), calculated as cardiac output x (systolic pressure - left atrial preload), served as a continuous index of mechanical function. Coronary vascular conductance (milliliters per minute per millimeters of mercury) was calculated as the ratio of coronary flow to mean aortic pressure.

Sources of drugs
Sodium nitroprusside was prepared by adding precalculated amounts of commercially available substance (SNP, Hoffmann-La Roche) to either the Krebs-Henseleit solution or the St. Thomas’ II cardioplegia to prepare final concentrations of 10, 50, or 200 µmol/L as required by the experimental protocol. In this way, isolated hearts could be exposed to a known and constant concentration of SNP. The perfusion apparatus and storage bottles were covered with aluminum foil to protect SNP from light-induced decomposition. The ODQ was purchased from Tocris Cookson Inc, and was dissolved in 100 µL of dimethyl sulfoxide and diluted in Krebs-Henseleit solution to achieve a final concentration of 3 µmol/L (final dimethyl sulfoxide, 0.005%).

Statistical analysis
Data are expressed as the mean ± the standard error of the mean. The significance of differences between groups was estimated by analysis of variance. In addition, the significance of differences in LV work between treatment groups at the individual time points during the 60-minute perfusion period was estimated by repeated-measures analysis of variance. If significant, selected data sets were compared by Dunnett’s multiple comparison test. Differences were considered significant when the value of p was less than 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Concentration-dependent effects of SNP
Left ventricular minute work in hearts subjected to cardioplegic arrest, hypothermic storage for 8 hours, and reperfusion (CPL group) recovered to only 24% of the work level measured in fresh hearts (Fig 1). Cardiac output and aortic flow were also significantly reduced (Table 1). Treatment with low concentrations of SNP (10 µmol/L or 50 µmol/L) during all phases of the perfusion protocol had no significant effects on the recovery of LV work. However, treatment with 200 µmol/L SNP throughout perfusion significantly improved the recovery of LV work throughout the working reperfusion period (see Fig 1). Other indices of mechanical function were also improved by this concentration of SNP (see Table 1).

View larger version (25K): [in this window] [in a new window]   Fig 1. Left ventricular function in working mode. Data are shown for freshly excised hearts (Fresh, black circles,n = 6) and stored hearts that were either untreated (CPL, white circles, n = 8) or treated with sodium nitroprusside (SNP), 10 µmol/L (downward triangles, n = 4), 50 µmol/L (squares, n = 4), or 200 µmol/L (upward triangles, n = 8), which was present throughout all phases of the perfusion protocol. Data are shown as the mean ± the standard error of the mean. (* =p < 0.05 versus CPL group by repeated-measures analysis of variance.)

View larger version (21K): [in this window] [in a new window]   Fig 2. Effects of sodium nitroprusside (SNP) on coronary flow. Coronary flow (milliliters per minute) was measured either during the initial phase of Langendorff perfusion before arrest and storage (Pre-storage, white circles) or during Langendorff reperfusion (Post-storage, black circles). Data are shown as the mean ± the standard error of the mean for hearts that were either untreated (0 µmol/L, n = 8) or treated with sodium nitroprusside, 10 µmol/L (n = 4), 50 µmol/L (n = 4), or 200 µmol/L (n = 8). (* = p < 0.05 by analysis of variance between values in absence and presence of SNP.)

Timing of SNP treatment
When hearts were exposed to SNP (200 µmol/L) during only the period of cardioplegic arrest and storage (S), recovery of LV work was not significantly improved relative to untreated hearts (Table 2). However, treatment with SNP during only poststorage Langendorff reperfusion (R) or during all phases of the protocol prior to working reperfusion (L + S + R) significantly improved recovery of LV work, and these improvements were accompanied by a normalization of coronary perfusion as indicated by coronary flow and coronary vascular conductance (see Table 2). Interestingly, exposure of the hearts to SNP (200 µmol/L) for only the 10-minute period of poststorage Langendorff reperfusion (R) increased the subsequent recovery of LV work from 24% to 65% of values measured in fresh hearts. Furthermore, this short treatment period accounted for 78% of the benefit derived from exposure of hearts to SNP throughout all phases of the perfusion protocol.

View larger version (22K): [in this window] [in a new window]   Fig 3. Effect of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on sodium nitroprusside (SNP)–induced improvement in left ventricular function. Data are shown for freshly excised hearts (Fresh, black circles, n = 6) and stored hearts either untreated (CPL, white circles, n = 8) or treated with SNP, 200 µmol/L, during the 10-minute period of Langendorff reperfusion in absence (SNP, squares, n = 8) or presence of ODQ (SNP + ODQ, triangles, n = 8). Data are shown as the mean ± the standard error of the mean. (* = p < 0.05 by repeated-measures analysis of variance between SNP–treated and CPL groups; # = p < 0.05, by repeated-measures analysis of variance between groups exposed to SNP in absence and presence of ODQ.)

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

The experimental protocol to assess the effects of SNP on mechanical function was designed to mimic the harvest and storage phases and the nonworking and working reperfusion phases associated with clinical cardiac transplantation. An 8-hour hypothermic storage period was selected, as preliminary pilot experiments indicated that this interval was associated with a marked impairment of functional recovery.

The isolated, buffer-perfused rat heart model offered a number of advantages for this study. Assessment of mechanical function after cardioplegic arrest and prolonged storage was made during working mode when the left ventricle was required to perform external work to eject fluid against a hydrostatic afterload equivalent to aortic pressure. This simulates an appropriate energy demand and is unlike nonworking isolated (Langendorff) or heterotopic transplant models [4] in which hearts do not perform external work. Energy substrates were provided in the form of both fatty acid and glucose, and hearts were perfused with a high concentration of fatty acid (1.2 mmol/L palmitate) that simulates levels that occur during cardiac surgical procedures in humans [8]. Perfusion with this level of fatty acid is critical not only because postischemic hearts derive up to 90% of their adenosine triphosphate requirements from fatty acid oxidation, but also because high rates of fatty acid oxidation during reperfusion contribute to postischemic dysfunction [9].

Under conditions of appropriate energy supply and demand, the SNP–induced improvement in the recovery of mechanical function during working-mode reperfusion was greatest when SNP was present during all phases of the perfusion protocol. Although previous studies have investigated the cardioprotective potential of NO, NO donor, or both, beneficial effects were reported only in models of short periods (20 to 60 minutes) of normothermic, global ischemia [7, 10, 11].

The relationship between SNP–induced protection and timing of SNP administration was also studied. Although greatest benefit was obtained when SNP was present during all phases of the perfusion protocol (L + S + R + W), significant beneficial effects were also observed when SNP was present only during the initial poststorage phase (R) when hearts were subjected to nonworking Langendorff reperfusion. This indicates that mechanisms operative during early reperfusion are critical determinants of the subsequent recovery of mechanical function. Studies [12] in working rat hearts have recently found convincing protection with L-arginine given only at the time of reperfusion after shorter 4-hour cardioplegic arrest. Treatment with L-arginine during cardioplegic arrest and reperfusion, however, elicited no further improvement over that seen during reperfusion alone [13].

The lack of cardioprotective efficacy of SNP that was observed in our study when SNP was present only during the storage phase [5] indicates that this phase may be a relatively less important period for NO–related strategies of cardioprotection. Similar conclusions regarding the importance of the "early reperfusion" phase may be drawn from a brief communication [14] reporting clinical transplantation of 14 hearts after 10 to 13 hours of hypothermic storage. The study employed a low reperfusion pressure and low cardiopulmonary bypass output for the first 10 minutes of reperfusion, and this resulted in survivals comparable to those with hearts stored for less than 4 hours. Pharmacologic interventions during the early reperfusion phase have the advantage that they are clinically feasible and avoid the potential of adverse hemodynamic effects or other effects arising from systemic administration. As recovery of mechanical function was greatest when SNP was present during all phases of the perfusion protocol, it appears that beneficial effects of SNP continue to be operative during working-mode reperfusion.

Sodium nitroprusside–induced protection may be due to coronary vasodilation, a direct effect on the myocardium, or both. During Langendorff perfusion before cardioplegic arrest and storage, SNP at the lowest concentration studied (10 µmol/L) elicited maximal increases in coronary flow. Sodium nitroprusside–induced increases in coronary flow, and associated positive inotropic effects, have been reported previously [15], and these may have contributed to the enhanced recovery of mechanical function. However, during poststorage Langendorff reperfusion, we found that SNP was unable to increase coronary flow, even at the highest concentration examined (200 µmol/L). These results indicate that SNP–induced cardioprotection occurred by a mechanism independent of improved coronary perfusion. Improved delivery of cardioplegic solution may have been an alternative flow-related mechanism to explain SNP–induced protection. However, because the delivery rate of cardioplegic solution was similar in SNP–treated hearts and untreated hearts, this possibility can also be excluded.

In the present study, cardioplegic arrest and prolonged hypothermic storage did not reduce baseline coronary flow in nonworking reperfusion but did induce a type of vascular stunning manifested by an inhibition on rewarming of the vasodilator response to normally effective concentrations of SNP. During working-mode reperfusion, the higher coronary flow in SNP–treated hearts than in untreated hearts was likely a consequence of the higher workload and oxygen demand rather than a cause of the improved workload. The coronary flows of SNP–treated hearts were never higher than those of freshly excised, nonstored hearts. Thus, it is unlikely that the enhanced recovery of mechanical function was due to any SNP–induced coronary hyperemia. This indicates that the beneficial effects of SNP are due to a direct action on the myocardium.

Although other studies have also found evidence of a direct protective effect of another nitrovasodilator (ie, nitroglycerin) on the myocardium [16], excessive NO concentrations can cause adverse effects. These occur either by direct myocardial depression [17, 18] or through the formation of damaging peroxynitrite ions (ONOO^-) from the interaction between NO and oxygen free radicals released during reperfusion [19].

The role of the NO/cGMP pathway in the mechanism of SNP–induced cardioprotection was assessed using the specific inhibitor of guanylyl cyclase ODQ. Studies using a range of NO donors provide indirect evidence that these agents replenish NO that potentially enhances cGMP production [4] through stimulation of guanylyl cyclase. The ability of the specific guanylyl cyclase inhibitor ODQ [20] to antagonize SNP–induced protection in our study provides support for the involvement of a cGMP mechanism in the cardioprotective actions of SNP. Activation of soluble guanylyl cyclase appears beneficial, but it remains to be established whether it is NO production, guanylyl cyclase activity, or mechanisms downstream of cGMP that are impaired after cardioplegic arrest and storage.

The precise mechanism by which SNP and the NO/cGMP pathway lead to cardioprotection is not defined in the present study. A role for inhibition of neutrophil-mediated reperfusion injury by the NO donor SNP has been excluded in this model because the crystalloid perfusate was devoid of bloodborne cellular elements. Rather, the data support a direct myocardial action of SNP mediated by activation of the NO/cGMP pathway. This may involve enhanced ventricular relaxation [17] mediated by a reduction in L-type calcium current [21] that would improve ventricular filling and the ability of the heart to perform LV work on reperfusion. Elevation of cGMP content has been shown to favorably influence myocardial energy substrate metabolism by reducing the rate of myocardial glycolysis [22], which, in turn, improves the coupling between glycolysis and glucose oxidation and attenuates proton production and acidosis [23]. This attenuation of proton production then improves recovery of LV work and cardiac efficiency of the postischemic heart [24], possibly by limiting Na⁺-H⁺ exchange and Ca²⁺ overload.

In summary, this study demonstrates that SNP markedly improves the recovery of LV work in hearts subjected to cardioplegic arrest and prolonged hypothermic storage. The ability of ODQ to inhibit the beneficial actions of SNP provides strong evidence that cardioprotection arises as a consequence of activation of the NO/cGMP pathway. Although strict reperfusion techniques have been recommended for the preservation of function of transplanted hearts [14], our results suggest that agents that enhance the NO/cGMP pathway during the critical early period of reperfusion will provide further improvements in the recovery of mechanical function of transplanted hearts.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
This work was supported in part by a grant from the Medical Research Council of Canada.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Imtiaz S. Ali Arvind Koshal Alexander S. Clanachan Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ali, I. S. Articles by Clanachan, A. S. Search for Related Content PubMed PubMed Citation Articles by Ali, I. S. Articles by Clanachan, A. S.