Our observation that sildenafil administered during the first 10 min of reperfusion efficiently reduced infarct size is consistent with a number of studies also showing that it limits injury in several models of myocardial ischemia and reperfusion injury. Sildenafil administered before ischemia has been found to reduce infarct size in an in vivo rabbit model (3
). Similar results have been observed in isolated rabbit (28
), rat (8
), and mouse hearts (33
). Sildenafil also protects against other forms of cardiac dysfunction, such as progression of hypertrophy (26
Despite these observations, the precise mechanism by which sildenafil is cardioprotective remains to be fully elucidated. Also, most studies investigating sildenafil-mediated protection have used healthy animals before the onset of ischemia (28
) or treatment 5 min before reperfusion, which may equate to partial early reperfusion (11
). Such interventions are not as clinically relevant as sildenafil treatment at reperfusion, which theoretically could be used during nonelective revascularization procedures. Consequently, we focused on sildenafil at reperfusion, trying to define the underlying cellular basis of this cardioprotective intervention. Sildenafil given for 10 min at reperfusion was protective at 0.1 μM but surprisingly was not protective at lower (0.01 μM) or higher (1 or 10 μM) concentrations. We cannot explain the loss of protection by sildenafil at higher concentrations, leading to a bell-shaped dose-response curve. Nevertheless, this finding is consistent with others (10
) who also found that sildenafil only reduced myocardial infarct size in a relatively narrow therapeutic concentration window. However, the loss of protection at higher concentrations of sildenafil may involve the nonspecific activation of additional molecular targets (other than PDE5) that mediate deleterious effects. We then tested whether longer reperfusion times (of 30, 60, or 120 min) with sildenafil improved protection, which they did not. This observation is consistent with the early minutes of reperfusion being a crucial period for establishing tissue survival and viability (49
), especially in the context of ion handling (9
We compared myocardial cGMP levels in hearts subjected to ischemia and reperfusion with or without sildenafil. We had anticipated that sildenafil would elevate cGMP, as it inhibits PDE5 to prevent the breakdown of this second messenger (2
). Perhaps surprisingly, sildenafil did not increase cGMP above controls, although it is clearly “bioactive” since it reduces infarction and, as outlined below, triggers phosphorylation of PLM. Our observations are consistent with those of Wilson and colleagues (44
) who also found sildenafil did not elevate global cGMP. The likely explanation of this is that sildenafil increases cGMP in defined compartments of cardiomyocytes, which has been observed by others (4
). In addition, Elrod and colleagues (11
) recently found that sildenafil protected through endothelial and inducible NO synthases but, as in our studies, this did not alter myocardial cGMP. These findings contradict those of Das and colleagues (6
) who found endothelial and inducible NO synthases were required for sildenafil-mediated protection. There is a recognized basis for the elevation of disparate, compartmentalized cGMP pools, as opposed to global cell-wide increases, namely pGC. sGC is a cytosolic enzyme that is activated by NO to produce cGMP (16
), whereas pGC enzymes are membrane-bound receptors that produce cGMP after natriuretic peptide (atrial, brain, and C-type) binding (24
). pGC can trigger discrete signal transduction events compared with the soluble enzyme (37
), elevating cGMP locally instead of globally throughout the cell (4
). Sildenafil can synergize with particulate-derived cGMP, for example potentiating the protective effects of brain natriuretic peptide against heart failure (12
). In addition, NPR-A KO mice, which cannot produce cGMP following atrial natriuretic peptide treatment, have a deficit in their response to sildenafil compared with WT mice. These KO mice have enhanced hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and vascular remodeling (48
). These observations highlight the prospect that sildenafil-mediated cardioprotection could involve stabilization of cGMP produced by receptor cyclases in the particulate fraction, as opposed to that derived from sGC. This would be consistent with the failure of sildenafil to globally elevate cGMP. To address this issue, we assessed the protective effect of sildenafil at reperfusion in NPR-A KO mice, comparing it with littermate WT controls. Sildenafil significantly protected both groups of mice, suggesting that cGMP derived from the particulate NPR-A receptor was not required for protection. Therefore, it remains possible that total myocardial cGMP does not necessarily reflect localized cGMP signaling, which may exist within compartmentalized subdomains within the cell.
While this study has focused on elevating cGMP using sildenafil-induced PDE5 inhibition, the scheme outlined in is consistent with other mechanisms of elevating cGMP being cardioprotective. One way in which this might be achieved would be to use GC activators (36
). Elevating cGMP by alternate mechanisms such as GC activators may provide protection from reperfusion injury without the potential limitations of using sildenafil, such as coronary “steal.”
Schematic diagram to illustrate the potential signaling cardioprotective mechanism for sildenafil at reperfusion. NO, nitric oxide; sGC, soluble guanylate cyclase; PDE5, phosphodiesterase enzyme type 5; 8-Br-cGMP, 8-bromo-cGMP.
Having established that sildenafil protects against reperfusion injury, we performed further experiments to test whether this was PKG mediated. A role for PKG might be expected, since this kinase is activated by cGMP and sildenafil prevents the hydrolysis of this cyclic nucleotide (2
). The PKG inhibitor, KT-5823, attenuated sildenafil-induced protection from reperfusion injury, confirming a role for this kinase as hypothesized. This is consistent with the general perception of PKG as a “good” kinase, integral to several forms of cardioprotection, including that by NO (32
) and ischemic preconditioning (15
In this study we used KT-5823, choosing this antagonist for a combination of reasons. This inhibitor is the most commonly used in isolated heart studies investigating PKG-dependent cardioprotection (1
), perhaps primarily because it is affordable in perfusion studies. cGMP-derivative inhibitors are generally too expensive for use in heart perfusion work and indeed can have their own issues due to actually activating PKG in some scenarios (40
). Other options for pharmacological inhibition are several DT peptides, some of which are cell permeable as well as being PKG isoform selective. While some studies have shown these to be useful in experiments investigating vasotone in cerebral arteries, they were ineffective in isolated heart preparations, being trapped in the endothelium and failing to reach the myocytes (21
). Routine genetic manipulation of PKG signaling is not possible in the isolated heart, and PKG null mice are not available to us and also have a complex phenotype and have health issues that shorten their life (17
). Following these considerations, we used KT-5823 for this study.
The downstream targets of PKG in the context of cardioprotection are less well established, although pathways involving PKCε and phosphoactivation of the mitochondrial ATP-sensitive K+
channel, inhibition of the mitochondrial transition pore, are routinely cited (5
). However, the precise order of these events is less clear, such as the molecular connections between PKG and the putative end-effector kinase PKCε. We therefore attempted to define whether there was a possible link between PKC, PKG, and PLM Ser/Thr69. Either sildenafil or the PKG activator 8-bromo-cGMP stimulated efficient PLM Thr69 phosphorylation, and in both cases this was blocked by PKC inhibition using bisindolylmaleimide. It is tempting to speculate that PKCε is the end-effector kinase that phosphorylates PLM, consistent with the known protective role of this protein. However, this idea is difficult to reconcile with the fact that Ser68, an established target for PKC, was not phosphorylated concomitantly with Ser69. One possibility is that the detection of Ser/Thr69 phosphorylation by antibody binding was compromised when the adjacent Ser68 site was modified. However, the precise relationship between PKG, PKC, and PLM phosphorylation remains to be defined. The involvement of PKG and PKC in sildenafil-induced cardioprotection is consistent with other studies that also implicate these kinases in cardioprotection during ischemic pre- or postconditioning. However, as in these other studies on protection, it remains unclear exactly how the PKG pathway and PKC pathways integrate with each other. Substrates of PKG, other than PLM, such as vasodilator-stimulated phosphoprotein or the L-type Ca2+
channel may also be phosphorylated in response to sildenafil (46
) and could contribute to the cardioprotection observed. cGMP can also directly modulate the activity of ion translocating membrane proteins and so could recruit their activity and signaling to protection.
The loss of ionic homeostasis is a key event in reperfusion injury (9
), and it is logical that protective interventions such as sildenafil treatment should somehow address this major mode of dysfunction, especially Na+
overload. NO donors stimulate the Na+
-ATPase in isolated ventricular myocytes (43
), with additional evidence that this enzyme has reduced function during ischemia and early reperfusion (14
). This NO-mediated activation highlights a potential role for PKG in stimulating the Na+
-ATPase. The Na+
-ATPase is regulated by PLM, the phosphorylation of which activates ion pump activity. Although there are no studies reporting that PKG phosphorylates PLM, it is a target for PKA that can target the same substrates as PKG (45
). We undertook several experiments that indicate that stimulating the PKG pathway culminates in PLM phosphorylation. This phosphorylation was not at the established Ser63 or Ser68 sites but at a newly confirmed phosphorylation site (13
), namely, Ser/Thr69. In isolated myocytes treated with 8-bromo-cGMP, a highly selective activator of PKG, we observed PLM Thr69 phosphorylation. Thr69 phosphorylation was also induced by sildenafil treatment of isolated myocytes, an event that stimulated the Na+
-ATPase. The prospect that sildenafil activates the Na+
-ATPase in isolated hearts when given at reperfusion was supported by our observation that PLM Ser/Thr69 was phosphorylated during this intervention in isolated hearts. Furthermore, sildenafil also induced PLM Ser/Thr69 phosphorylation in isolated ventricular myocytes, which patch-clamp analysis confirmed activated the Na+
-ATPase. Consequently, we examined whether sildenafil enhanced Na+
-ATPase activity during early reperfusion, using Rb uptake into the intact myocardium to index this. Consistent with the other supporting data, we found that sildenafil did indeed enhance Na+
-ATPase activity in the isolated perfused heart, which would serve to limit Na+
accumulation and attenuate injury. In addition, these Rb uptake experiments also confirmed a central tenet of these studies, namely, that the Na+
-ATPase activity is attenuated during ischemia and reperfusion. Na+
-ATPase inhibition during ischemia and reperfusion contributes to injury, and interventions that limits this deficit (here sildenafil) provide protection.
Although our studies are consistent with the phosphorylation of PLM being integral to the protection afforded by sildenafil against injury during postischemic reperfusion, this is only a correlative association. A role for PLM phosphorylation is supported by the fact that PLM is phosphorylated during this intervention, and that the Na+ loading at reperfusion is attenuated, this is only correlative. We cannot exclude the possibility that sildenafil triggers other cardioprotective mechanisms, and indeed there may be multiple, synergistic pathways that are recruited by PDE5 inhibition. Establishing the cause and effect in cardioprotection is notoriously difficult, but perhaps this could only be most definitive if a PLM T69A knock-in mouse was generated and this was not protected by sildenafil.
Our observations are consistent with a recent study that identified improved Ca2+
handling as a crucial mechanism in sildenafil-mediated cardioprotection from hypertrophy (26
). Sildenafil enhanced the expression of sarco(endo)plasmic reticulum Ca2+
-ATPase (SERCA2a) sarcoplasmic reticulum Ca2+
-pump expression, as well as the phosphorylation of its phosphoregulatory protein phospholamban (PLB). In our acute protection experiments involving 10 min of sildenafil treatment at reperfusion, an alteration of ion-translocating protein expression such as the Na+
-ATPase or SERCA2a is unlikely. However, their observations of PLB activation and enhanced Ca2+
handling have strong parallels with our studies of enhanced Na+
handling as a result of PLM Ser/Thr69 phosphorylation. Indeed, both PLM and PLB serve as a brake on the activities of their respective ATPase enzyme, which is relieved by the phosphorylation of these small accessory proteins.
In summary, we have shown that sildenafil protects against myocardial reperfusion injury and clarified in part the mechanism involved. outlines the cardioprotective pathway that our data suggest underlies sildenafil-mediated protection. Sildenafil at reperfusion activates PKG, leading to the phosphorylation of PLM Ser/Thr69, but not Ser63 or Ser68, in a PKC-dependent manner. The phosphorylation of PLM at position 69 leads to the activation of the Na+/K+-ATPase, which provides a mechanism for sildenafil-mediated cardioprotection against reperfusion injury, namely, by attenuating an otherwise damaging Na+ overload that occurs at this time.