Introduction
Morbidity and mortality due to cardiac arrest (CA) remains unacceptably high, yet effective treatments for CA have proven to be elusive [
1]. In patients who initially achieve return of spontaneous circulation (ROSC) after CA, the significant subsequent morbidity and mortality are largely due to the myocardial dysfunction that accompanies prolonged whole-body ischemia. Furthermore, CA contributes to hemodynamic disorders that cause the systemic release of massive oxygen free radicals, lactic acid and metabolites of arachidonic acid, which could reach the different tissues via blood circulation and could cause ischemia/reperfusion (I/R) injury [
2]. Postresuscitation myocardial dysfunction, an important component of the “post–cardiac arrest syndrome,” is caused by I/R injury and includes primary manifestations such as arrhythmias, myocyte apoptosis and contractile dysfunction [
3]. In addition, myocardial dysfunction aggravates persistent precipitating pathologies, such as microcirculatory dysfunction, requiring lifelong medication and clinical follow-up.
Nitric oxide (NO) has been identified as an important mediator of the physiological and pathological processes in I/R injury [
4]. Sildenafil is a selective inhibitor of the isoform 5 of the enzyme phosphodiesterase (PDE5), which is responsible for the breakdown of 39,59-cyclic guanosine monophosphate (cGMP) in smooth muscle cells [
5]. As the intracellular level of cGMP is controlled by the activity of PDE5, it is expected that pharmacological inhibition of PDE5 by sildenafil could improve cardioprotection in the myocardium. As a pharmacological stimulator of ischemic preconditioning, sildenafil now represents a powerful therapeutic tool for treating several cardiovascular disorders and provides direct cardioprotection against ischemia through NO-dependent pathways [
5],[
6]. Well-tolerated for long-term treatment with few side effects, sildenafil reduces pulmonary vascular resistance, improves arterial oxygenation in patients with pulmonary artery hypertension and prevents altitude-induced hypoxemia [
7],[
8]; however, the potential role of sildenafil as a novel pharmacologic adjunct to resuscitation from CA for the purpose of attenuating the myocardial dysfunction caused by I/R injury remains unexplored.
The microdialysis technique is capable of detecting real-time metabolic changes
in vivo, which makes it a sensitive and site-specific method for monitoring metabolism in the myocardial interstitial fluid (ISF) during CA [
9]. With this background in mind, we designed this study to investigate changes of intracardial metabolism measured by microdialysis under different management methods in an established porcine model of CA, and we also sought to determine whether administration of sildenafil is optimal for attenuating postresuscitation myocardial dysfunction by obtaining extracellular metabolic evidence.
Discussion
We found that pretreatment with sildenafil reduced the severity of postresuscitation myocardial dysfunction during the no-flow or low-flow state of CA in this porcine model of CA. In our analysis of the changes of ISF metabolites (glucose, lactate, glutamate, pyruvate and glycerol) during VF and reperfusion, we observed that sildenafil inhibited abrupt increases in ISF lactate and glutamate levels, which indicates that sildenafil could improve myocardial energy metabolism during ischemia and reperfusion.
Although several studies have focused on the role of PDE5 inhibition on I/R injury in the myocardium [
5]-[
7], this study is, to our knowledge, the first to demonstrate that pretreatment with sildenafil prior to VF after CA is cardioprotective. Myocardial I/R injury occurs in a wide spectrum of patients, ranging from survivors of out-of-hospital CA to patients who have acute myocardial infarction and patients undergoing cardiac surgery. Ockaili
et al. conducted the first study showing a powerful preconditioning-like effect of sildenafil against myocardial I/R injury in an
in vivo rabbit model [
20]. The anti-ischemic effects of sildenafil against I/R-triggered ventricular arrhythmias were also observed, as well as improvement of postischemic ventricular contractile function [
21]. In another previous study, researchers demonstrated that, in an atherosclerotic mice model, sildenafil reduced oxidative stress and increased NO bioavailability, which culminated in protection against DNA damage [
7]. All of these results are in accordance with our findings that the postresuscitation hemodynamic profile in the sildenafil group seemed to correspond with a less severe myocardial injury (higher ATP and Na
+-K
+-ATPase levels) and less dysfunction. This may be explained by an enhanced or better-preserved oxygen supply/demand ratio after successful ROSC in the sildenafil group (higher mean aortic pressure (MAP) and a lower HR/pressure radio), whereas the 24-hour survival rate showed a promising trend in the sildenafil group.
The magnitude of cardiac damage observed in piglets in the present study is similar to the damage observed in clinical practice (after CA and/or trauma). CA represents the most severe shock state, during which the delivery of oxygen and metabolic substrates is abruptly halted and metabolites are no longer removed [
22]. Several studies have conclusively demonstrated that opening mitochondrial ATP-dependent potassium (mito-K
ATP) channels plays an important role in ischemic as well as pharmacological preconditioning in the heart [
23]. Opening of the mito-K
ATP channel partially compensates for the loss in membrane potential, which enables additional protons to be pumped out to form a H
+ electrochemical gradient for both ATP synthesis and Ca
2+ transport. In our present study, we found that the activities of Na
+-K
+-ATPase and Ca
2+-ATPase were significantly increased in the sildenafil group compared with the SA group at 24 hours after ROSC, and we found that the contents of ATP, ADP and phosphodiesterase in the SA group were much lower than in the sildenafil group, indicating greater energy exhaustion in the SA group, which indicated that the myocardium energy metabolism system was damaged much more severely in the SA group than in the sildenafil group. We therefore suggest that sildenafil may lead to cardioprotection through beneficial effects on energy metabolism by opening mito-K
ATP channels. Our results are consistent with some previous experiments in which administration of sildenafil was shown to inhibit the breakdown of cGMP and subsequent high cGMP levels led to acute and delayed cardioprotection via mito-K
ATP channel opening and mediated preconditioning in rat hearts. These features would in principle lead to cardioprotection [
20].
In vivo microdialysis techniques have made it possible to directly determine myocardial ISF metabolite changes. Therefore, in contrast to many previous studies focused only on serum markers, the findings of the present study provide comprehensive insight regarding metabolic changes and cellular energy preservation in ISF during CA by microdialysis.
The microdialysis results demonstrated real-time changes of several metabolic markers of basic metabolism in the ISF. It has been reported that the glucose level in the dialysate is closely correlated to the oxygen supply to the mycardium [
24]. In the present study, after CA, glucose concentrations were significantly lower in the SA and sildenafil groups compared to the sham group (
P < 0.05), whereas the glucose level remained higher in the sildenafil group than in the SA group during CA and in the early reperfusion phase. The reason for these effects is the shift of cellular metabolism to anaerobic conditions, which causes formation of lactate and an accelerated uptake of extracellular glucose during ischemia to sustain the energetically inefficient anaerobic glycolysis [
25].
Lactate is produced by anaerobic glycolysis when the metabolic demand for oxygen exceeds the available oxygen supply [
26]. Furthermore, lactate is the end product of anaerobic metabolism, and pyruvate is the intermediate product between aerobic and anaerobic metabolism. In aerobic conditions, pyruvate is produced via glycolysis and then enters the tricarboxylic acid cycle, largely bypassing the production of lactate [
27]. In our study, the SA group had the highest lactate level and the lowest pyruvate level among the groups. Interestingly, compared with the SA group, sildenafil had almost a lower level of lactate and a higher level of pyruvate, suggesting that the sildenafil group had a potent capacity to clear metabolic waste accumulated in a relatively short CA duration.
The L/P ratio is a more reliable variable than lactate that characterizes the relationship between aerobic and anaerobic metabolism [
28]. The animals in the SA group showed rapid deterioration of energy supply with an immediate increase of the L/P ratio as a sign of nutritional disorder. The L/P ratio in the sildenafil group was lower than that in the SA group at most time points, which demonstrated that animals treated with sildenafil had a better energy reservoir. Nevertheless, the curve of the L/P ratio for the sildenafil group suggested that sildenafil could minimize energy depletion to the utmost extent.
Increasing levels of glycerol indicated the degree of destruction in the cell membrane [
29]. Sildenafil attenuated the production of glycerol at most time points, which indicates that it had a fair capability to protect myocardial cells.
There are obvious limitations to the design of this study. First, we acknowledge that our study may lack the power to detect some additional differences because of a relatively small sample size. Second, a major limitation is that the animals were administered sildenafil 30 minutes prior to VF, which cannot reflect sildenafil’s clinical applicability truly. Third, it also should be noted that the endpoints of this study were focused on the acute changes in postresuscitation myocardial dysfunction. The animals were killed immediately at the end of the study, resulting in a lack of observation of survival rate changes over a longer time course. An additional disadvantage of the study is the use of heparin to avoid hypercoagulability. Heparin may have impaired thrombus formation and vasoconstriction, which are physiologic responses in unanesthetized animals. Also, in the interpretation of our findings, repetitive electrical shocks themselves may increase the severity of postresuscitation myocardial dysfunction in settings of myocardial ischemia [
30]. Further, because our studies were performed in an animal model in the absence of underlying cardiovascular disease, direct applicability to humans cannot be assured. Finally, optimal doses and methods of administration of sildenafil also deserve additional investigation. This information would be helpful in applying the use of sildenafil in CPR.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
QZ contributed to the study design, method development, data interpretation and writing of the manuscript. WY conducted experiments and contributed to the writing of the manuscript. GXW contributed to the experimental design of the studies and participated in the writing of the manuscript. JYW conducted experiments. MMW conducted experiments. CSL conceived of the study design, was responsible for the coordination of the experiments, helped to draft the manuscript and finalized the manuscript. All authors read and approved the final manuscript.