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Temperature preconditioning (TP) provides very powerful protection against ischaemia/reperfusion. Understanding the signalling pathways involved may enable the development of effective pharmacological cardioprotection. We investigated the interrelationship between activation of protein kinase A (PKA) and protein kinase C (PKC) in the signalling mechanisms of TP and developed a potent pharmacological intervention based on this mechanism.
Isolated rat hearts were subjected to TP, 30 min global ischaemia, and 60 min reperfusion. Other control and TP hearts were perfused with either sotalol (β-adrenergic blocker) or H-89 (PKA inhibitor). Some hearts were pre-treated with either isoproterenol (β-adrenergic agonist) or adenosine (PKC activator) that were given alone, simultaneously, or sequentially. Pre-treatment with isoproterenol, adenosine, and the consecutive isoproterenol/adenosine treatment was also combined with the PKC inhibitor chelerythrine. Cardioprotection was evaluated by haemodynamic function recovery, lactate dehydrogenase release, measurement of mitochondrial permeability transition pore opening, and protein carbonylation during reperfusion. Cyclic AMP and PKA activity were increased in TP hearts. H-89 and sotalol blocked the cardioprotective effect of TP and TP-induced PKC activation. Isoproterenol, adenosine, and the consecutive treatment increased PKC activity during pre-ischaemia. Isoproterenol significantly reduced myocardial glycogen content. Isoproterenol and adenosine, alone or simultaneously, protected hearts but the consecutive treatment gave the highest protection. Cardioprotective effects of adenosine were completely blocked by chelerythrine but those of the consecutive treatment only attenuated.
The signal transduction pathway of TP involves PKA activation that precedes PKC activation. Pharmacologically induced consecutive PKA/PKC activation mimics TP and induces extremely potent cardioprotection.
Reperfusion following a prolonged period of ischaemia induces myocardial dysfunction and necrotic damage.1 We have recently described a novel cardioprotective protocol in which hearts are subject to a few brief, transient hypothermic (26°C) episodes interspersed with normothermic perfusion prior to index ischaemia. Such temperature preconditioning (TP) is as good, if not better than ischaemic preconditioning (IP) in restoring haemodynamic function and reducing arrhythmias, oxidative stress, and lactate dehydrogenase (LDH) release.2 We showed that TP involves a modest increase in reactive oxygen species (ROS) that activates protein kinase Cε (PKCε), although AMP-activated protein kinase may play some role. The cardioprotective effects of TP, like IP,3 were associated with decreased oxidative stress at the end of ischaemia and during reperfusion. We proposed that this prevents opening of the mitochondria permeability transition pore (MPTP) leading to both improved contractile function and decreased necrotic damage.2
The switch from hypothermic to normothermic perfusion during the TP protocol is accompanied by a rapid augmentation of haemodynamic function that subsequently returns to the initial value.2 These changes could reflect β-adrenergic stimulation following a TP cycle with activation of cyclic AMP (cAMP)-dependent protein kinase A (PKA) that others have shown to be cardioprotective.4 In this paper, we test this hypothesis and also study the temporal relationship between PKA and PKC activation in TP hearts. We demonstrate that PKA stimulation prior to PKC stimulation provides optimal cardioprotection. Understanding the signalling pathways and molecular targets through which TP exerts its effects may lead to the development of more effective pharmacological interventions.5
Unless otherwise stated, all biochemicals were from Sigma and general chemicals from Fischer Scientific or VWR-Jencons.
All procedures conform to the UK Animals (Scientific Procedures) Act 1986 and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Ethical approval was granted by the University of Bristol, UK (Investigator number ub/09/012). Male Wistar rats (250–260 g) were killed by stunning and cervical dislocation. Hearts (~0.75 g) were rapidly removed into ice-cold Krebs–Henseleit buffer (KH) and perfused in Langendorff mode with haemodynamic measurements of left ventricular developed pressure (LVDP), LV systolic pressure, LV end-diastolic pressure, work index (RPP), heart rate (HR), and time derivatives of pressure during contraction (+dP/dt) and relaxation (−dP/dt) as described previously6 and detailed in Supplementary Methods.
Four series of experiments were performed as shown schematically in Figure 1. Further details are provided in Supplementary Methods. In brief, after pre-ischaemia, global normothermic ischaemia (37°C) was induced for 30 min and then normothermic perfusion reinstated for 60 min. In Series 1, hearts were divided into two groups: control (15 hearts) and TP (12 hearts). TP hearts experienced three cycles of 2 min hypothermic perfusion at 26°C interspersed with 6 min normothermic perfusion prior to ischaemia. Samples of perfusate were collected for determination of LDH activity. Eight and six additional hearts of each group were freeze-clamped following 44 min pre-ischaemia and 15 min reperfusion, respectively, ground under liquid nitrogen, and stored at −80°C for later analysis of cAMP, PKA activity, and Akt and GSK3 phosphorylation.
In Series 2 (Figure 1B), six groups of six to eight hearts were employed; three control groups (C, CS, and CH) and three TP groups (TP, TPS, and TPH) in the presence or absence of 10 µM of the non-selective β-adrenergic blocker sotalol (CS and TPS) or 10 µM H-89 (PKA inhibitor) (CH and TPH). Sotalol and H-89 were washed out for 5 min before index ischaemia. Eight additional hearts of C, CH, TP, and TPH groups were freeze-clamped following 51 min pre-ischaemia for further analysis of PKC activity.
In Series 3 (Figure 1C), hearts were perfused with either adenosine, a PKC activator well known for its cardioprotective effects,7 or isoproterenol, a non-selective β-adrenergic agonist widely used on isolated perfused heart. Hearts were divided into four groups (7–9 hearts in each): control, hearts perfused with 0.2 µM isoproterenol for 2 min followed by 10 min washout, hearts perfused with 30 µM adenosine for 5 min followed by 5 min washout, and hearts perfused with isoproterenol followed by perfusion with adenosine and 5 min washout. Eight additional non-ischaemic hearts of each group were freeze-clamped following 27 min KH perfusion (control group) or immediately after isoproterenol or adenosine treatment for later analysis of PKC activity. Eight more frozen pre-ischaemic hearts were used for measuring myocardial glycogen content in each group, whereas another 7–10 hearts of each group were used to prepare mitochondria after 30 min global ischaemia for the measurement of MPTP opening and analysis of protein carbonylation.
In Series 4 (Figure 1C), hearts were divided into eight groups (5–8 heats each): Group 1 control; Groups 2–4, hearts subjected to either isoproterenol, adenosine, or consecutive isoproterenol and adenosine treatment (see Series 3); in Groups 5–7, the PKC inhibitor chelerythrine (10 µM) was added 5 min before isoproterenol (Groups 5 and 7) or adenosine (Group 6) perfusion and removed prior to ischaemia. Chelerythrine at this concentration has no effect on heart recovery during reperfusion.2 Hearts of Group 8 were perfused with 30 µM adenosine for 5 min with 0.2 µM isoproterenol also added after 1.5 min for 2 min.
PKA and PKC activities and cAMP concentration were determined in freeze-clamped heart powders using kits supplied by Sigma (cAMP) and Promega (PKA and PKC) according to the manufacturer's instructions. The assays of PKA and PKC activity rely on a change in charge of the fluorescent PepTag® A1 and PepTag® C1 peptides from +1 to −1 following phosphorylation. Bands were visualized under UV light and the ratio of fluorescence intensity of phosphorylated to non-phosphorylated peptide was quantified using AlphaInotech ChemiImager 4400 with AlphaEase v5.5 software. The phosphorylation of Akt and GSK3α/β was determined in freeze-clamped, powdered hearts by a method based on that of Hausenloy et al.8 using western blotting with antibodies (Cell Signaling Technology) against phosphorylated and total Akt and GSK3α/β. The ratio of the band intensity for phosphoprotein to total protein was used as a measure of phosphorylation state.
The assay of glycogen in freeze-clamped pre-ischaemic hearts was performed using α-glycosidase hydrolysis with measurement of the released glucose performed spetrophotometrically (340 nm) as described previously.9
Mitochondria were isolated as described in Supplementary Methods and measurement of MPTP opening performed in de-energized conditions at 30°C as described previously3 using the decrease in light scattering (A520) that accompanies mitochondrial swelling following addition of 100 µM Ca2+.
Protein carbonyls were determined in frozen mitochondria following derivatization with dinitrophenylhydrazine (Chemicon International, Chandlers Ford, UK) and western blotting with anti-dinitrophenyl antibodies (Sigma) exactly as described previously.10
Further details may be found in Supplementary Methods.
Data are presented as mean ± SEM. Statistical significances of the differences between groups were evaluated using Student's t-test (Series 1) or one-way ANOVA followed by Tukey's multiple comparison post hoc test (Series 2–4) using GraphPad Prism v5.0 software. Differences were considered significant where P < 0.05.
In Table 1, we show that during reperfusion, recovery of LVDP and RPP in TP hearts was two-fold greater than for control hearts with a 60% increase in the time derivatives of LV pressure. Protection against necrotic damage (LDH release) during the first 15 min of reperfusion showed a similar pattern to the recovery of haemodynamic function. Figure 2 shows that after the TP protocol, the tissue concentration of cAMP was significantly increased (Figure 2B) as was PKA activity (Figure 2A). However, neither GSK3α/β (Figure 2C and E) nor Akt (Figure 2D and F) showed any change in phosphorylation following the TP protocol or after 15 min reperfusion.
The role of β-adrenergic stimulation and PKA activation in TP was investigated using the β-adrenergic blocker (±)-sotalol11 and the PKA inhibitor H-89.12 In preliminary experiments (data not shown), we found that both 10 µM sotalol and 10 µM H-89 completely and reversibly abolished the increase in haemodynamic function induced by isoproterenol (0.2 µM).
Prior to ischaemia, the RPP of sotalol-treated hearts (TPS group) was significantly lower than untreated hearts during the first and third hypothermic episodes, and sotalol also suppressed the rise of HR during the subsequent normothermia leading to a smaller increase in RPP (Figure 3A). H-89 (TPH group) also decreased LVDP, although HR of these hearts was higher than in the TPS hearts in all three normothermic episodes (Supplementary material online, Table S1). The combined effect was a lower RPP in TPH hearts relative to TP (Figure 3A), but less so than in TPS hearts. H-89 also blocked the increase in PKC activity seen in TP hearts without affecting PKC activity in control hearts (Figure 3B).
Neither sotalol (CS) nor H-89 (CH) affected recovery of LVDP or RPP in control hearts but they did attenuate (sotalol, TPS group) or prevent (H-89, TPH group) the increased haemodynamic recovery seen in TP hearts. The effects of sotalol and H-89 on haemodynamic function were matched by their ability to reduce or abolish the protection TP offers against necrosis (LDH release) (Table 2).
Adenosine reduced RPP by ~20% (P < 0.05) with subsequent gradual return of this parameter to the initial value, whereas perfusion with isoproterenol increased RPP 2.5-fold. When hearts perfused with isoproterenol were switched to adenosine, RPP was reduced to 60% of the initial value (P < 0.01) and was significantly lower than in adenosine-treated hearts (P < 0.01). At the end of pre-ischaemia, this parameter was still slightly decreased in hearts of the consecutive isoproterenol + adenosine group (P < 0.05 vs. initial value) (Figure 4A). Perfusion with isoproterenol but not adenosine also reduced the glycogen content of the hearts by >50%. There was no additive effect of adenosine on the response to isoproterenol (Figure 4B). PKC activity, measured immediately after perfusion with isoproterenol or adenosine, was significantly higher in all three groups of isoproterenol and adenosine-treated hearts (Figure 4C).
Treatment of hearts with either isoproterenol or adenosine alone improved haemodynamic function recovery after 30 min global ischaemia and 60 min reperfusion (Table 3); the recovered LVDP and RPP reached twice the values of control reperfused hearts. However, consecutive treatment of hearts with isoproterenol followed by adenosine resulted in a complete recovery of haemodynamic function accompanied by the lowest LDH release (Table 3).
Haemodynamic function recovery of hearts treated simultaneously (as opposed to consecutively) with the mixture of adenosine and isoproterenol (Table 4) was similar to hearts treated with isoproterenol or adenosine alone and significantly lower than for consecutive treatment. The PKC inhibitor chelerythrine completely abolished the improvement of haemodynamic function recovery in adenosine-treated hearts and considerably reduced the beneficial effect of the consecutive isoproterenol and adenosine treatment but had no significant effect on the recovery of isoproterenol-treated hearts. LDH release was the lowest in hearts with the consecutive treatment (Tables 3 and 44); in hearts with the simultaneous isoproterenol and adenosine treatment this parameter was not significantly less than controls (Table 4). In hearts treated with the combination of adenosine and chelerythrine, LDH release was the same as control.
Both isoproterenol and adenosine reduced Ca2+-induced mitochondria swelling (MPTP opening) after 30 min global ischaemia to 17 and 41% of control ischaemic values, respectively, whereas the sequential treatment with the two agents reduced swelling to 5% indicating almost total prevention of MPTP opening (Figure 5A). Parallel measurements of mitochondrial protein carbonylation showed that only the consecutive isoproterenol + adenosine group gave a significant reduction in this parameter (Figure 5B).
Here, we report that a β-adrenergic-mediated rise in cAMP and subsequent activation of PKA is important for TP-induced cardioprotection which is consistent with our earlier observation2 that the switch from hypothermic perfusion to normothermia during TP caused a fast and significant augmentation of haemodynamic function (see also Figure 3A; see Supplementary material online, Table S1). These changes of contractility might reflect the increased accumulation of intracellular calcium that accompanies hypothermic perfusion13 and our current data suggest that endogenous β-adrenergic agonists may cause this through their known effects on intracellular calcium homeostasis. Thus, we have shown that myocardial cAMP concentration and PKA activity were increased at the end of TP protocol (Figure 2A and B) and that the non-selective β-adrenergic blocker sotalol and the PKA inhibitor H-89 attenuate the cardioprotective effects of TP. Both agents reduced the augmentation of haemodynamic function during the TP protocol (see Supplementary material online, Table S1; Figure 3A), whereas H-89 completely abolished and sotalol partially abolished the TP-mediated improvement of haemodynamic function and reduction of LDH release during reperfusion (Table 2). These data suggest that β-adrenergic stimulation is only partially responsible for PKA activation during TP protocol and that another pathway, perhaps ROS-mediated,14 may also be involved. Indeed, we have shown previously that the free radical scavenger N-(2-mercaptopropionyl)-glycine (300 µM) applied during pre-ischaemia abolishes cardioprotection by TP as it does for IP.2 Furthermore, Stowe and colleagues15 have reported that hypothermia moderately enhances superoxide concentration in myocardium.
Interestingly, it has been shown by others that β-adrenergic stimulation can be considered as a trigger of IP4 and that repeated stimulation with norepinephrine or isoproterenol mimics IP.16 Proposed mechanisms for cardioprotection by β-adrenergic activation of PKA include β-adrenergic desensitization4 and attenuation of calpain-mediated degradation pathways.12
A major problem with using protein kinase inhibitors to dissect signal transduction pathways is their lack of specificity.17 Although H-89 is a potent PKA inhibitor, it can inhibit other kinases including Akt.17 The Akt–GSK3 pathway has been implicated in cardioprotection by IP,18 although our own data3 and that of other19 have questioned the central role of this pathway prior to ischaemia and we were unable to detect any change in phosphorylation of either protein following the TP protocol (Figure 2C and D). Hausenloy et al.8 showed IP-induced Akt phosphorylation at 15 min of reperfusion following prolonged ischaemia, but we were also unable to detect any changes in Akt or GSK3 phosphorylation by TP at 15 min reperfusion (Figure 2E and F). However, we cannot completely exclude involvement of this pathway in TP because phosphorylation could be transient; although in studies where Akt activation and GSK3 inhibition were found to be important for cardioprotection, phosphorylation of these kinases was very consistent and significant during preischaemia20 and reperfusion.8,21 Thus, our data do not support a major role of Akt and GSK3 phosphorylation in the TP signalling mechanism.
We have previously shown that PKC activation is critical for TP-mediated cardioprotection,2 and here, we demonstrate that the PKA inhibitor H-89, which itself has little effect on PKC activity,17 is able to prevent both this PKC activation (Figure 3B) and cardioprotection (Table 2), implying that PKA activation is upstream of PKC activation in the TP signalling pathway. Our data further support this finding. Thus, treatment of rat hearts consecutively with the β-adrenergic agonist isoproterenol and then adenosine, to activate PKC, resulted in extremely potent cardioprotection that considerably exceeded the protection afforded by either agent alone (Table 3) or added simultaneously (Table 4) and allowed hearts to recover completely after 30 min normothermic (37°C) global ischaemia. Thus, the consecutive activation of these enzymes (PKA followed by PKC activation) is of critical importance in terms of cardioprotection.
The decreased glycogen content found in isoproterenol-treated hearts (Figure 4B) could contribute to the observed cardioprotective effect. Thus, it has been reported that glycogen depletion of rat hearts by anoxic perfusion prior to global ischaemia significantly improved recovery of ventricular function during reperfusion, whereas lactate accumulation was harmful for the heart.22 Others showed that IP is associated with glycogen depletion leading to less anaerobic glycolysis during the subsequent prolonged ischaemia, and hence reduced accumulation of lactate and H+. This causes a smaller decrease in intracellular pH during ischaemia and thus less compensatory increases in intracellular Na+ and Ca2+.23 The decreased calcium loading will reduce the likelihood that the MPTP opens which might partially explain the observed cardioprotection.24 However, reduced glycogen content alone cannot entirely account for the cardioprotection in our experiments since this parameter was similar in hearts treated with isoproterenol alone or with adenosine; yet, the combined treatment gave much better protection. Our data suggest that the additional factor is activation of PKC.
It has been shown that isoproterenol can augment the negative inotropic effect of adenosine25 which we also observed in the pre-ischaemic phase of the combined treatment (Figure 4A). This would be consistent with PKA activation of PKC, and in support of this, our data show that perfusion with the β-adrenergic agonist isoproterenol does increase PKC activity in the heart (Figure 4C). Stimulation of β-adrenergic receptors and PKA activates ROS production by mitochondria26 and promotes accumulation of intracellular Ca2+ ([Ca2+]i).27 Meanwhile, it has been found that ROS may trigger PKC activation28 and increase in [Ca2+]i could also activate PKC via direct Ca2+-dependent activation or via a G-protein activated by Ca2+-dependent phospholipase C.29
The cardioprotective effect of PKC activation (primarily PKCε) is well established7 and we have also shown recently that urocortin-induced reduction of oxidative stress is mediated by PKC resulting in MPTP inhibition during reperfusion.30 How this is achieved remains unclear. It has been reported that PKCε may phosphorylate the voltage-dependent anion channel in the outer mitochondrial membrane or prevent binding of cyclophilin D to adenine nucleotide translocase which results in inhibition of MPTP,31 although our own data failed to detect these changes in IP.3 PKC may also phosphorylate the BH3-only protein Bad32 which increases the availability of Bcl-2 for antioxidant and anti-apoptotic functions.33 Whatever the mechanism, it appears that PKCε activation prevents ROS production and MPTP opening during ischaemia and reperfusion.
We have previously found that the PKC inhibitor chelerythrine abrogated TP-induced cardioprotection,2 and here, we show (Table 4) that chelerythrine completely abolished the protective effect of adenosine and considerably reduced cardioprotection afforded by the consecutive isoproterenol/adenosine treatment. However, chelerythrine had little effect on the protective effect of isoproterenol. These results suggest that PKA-induced cardioprotection in our experiments did not rely exclusively on PKC activation but was associated with other mechanisms including glycogen depletion prior to ischaemia. Our data also show that the strong protective effect of the consecutive isoproterenol/adenosine treatment was rather a result of the synergic action of both PKA and PKC than PKC being the only effector in the signalling mechanisms of this pharmacological treatment.
We demonstrated previously that protection by TP involves inhibition of MPTP opening.2 Here, we show that consecutive treatment of the heart with isoproterenol and adenosine also dramatically reduced calcium-induced mitochondria swelling, an indicator of MPTP opening (Figure 5A). Treatment with isoproterenol or adenosine alone also gave a significant, but smaller, reduction in calcium-induced mitochondria swelling. This is of interest because it was demonstrated more than 30 years ago that mitochondria isolated from livers treated with glucagon, dibutyryl-cAMP or α-adrenergic agonists (PKC activating) retained accumulated calcium for longer than those from control livers.34–36 This increase in ‘calcium retention time’ is now known to reflect an inhibition of MPTP opening and thus it appears likely that a similar cAMP-dependent protective mechanism to that seen in the heart also operates in liver.
For both IP and TP, inhibition of the MPTP in mitochondria isolated at the end of ischaemia or during reperfusion correlates with a decreased oxidative stress as reflected in protein carbonylation,2,3 and here, we show that the powerful protective effect of the sequential isoproterenol + adenosine treatment was also accompanied by a significant decrease in protein carbonylation (Fig 5B). Treatment with each agent on its own also showed a slight reduction in protein carbonylation but this was not statistically significant. No published data are available on the effects of glucagon or α-adrenergic agonists on liver mitochondrial protein carbonylation, but glucagon was found to decrease mitochondrial lysophospholipid accumulation37 consistent with reduced lipid peroxidation,38 another indicator of oxidative stress. Thus, it is possible that the inhibition of MPTP opening by cAMP-dependent mechanisms in liver, as well as in TP and particularly isoproterenol + adenosine-treated hearts, involves a decrease in oxidative stress.
The novel findings of our study are as follows. First, PKA activation, like PKC activation, is a very important link in the signalling mechanism of TP with PKA activation being upstream of PKC activation and mediated in part by β-adrenergic stimulation. Secondly, pharmacologically induced consecutive PKA and PKC activation provides potent cardioprotection. Thirdly, this cardioprotection is associated with depletion of myocardial glycogen prior to ischaemia which may lead to less calcium loading during ischaemia. This, together with the reduction of oxidative stress during ischaemia, will lead to less MPTP opening and thus better recovery during reperfusion. This pharmacological strategy may represent a promising protocol for heart protection during prolonged ischaemia and reperfusion such as in open heart surgery or transplantation.
This work was supported by a grant from the British Heart Foundation (PG/07/080/23613). Funding to pay the Open Access publication charges for this article was provided by British Heart Foundation.
The authors would like to thank Professor Saadeh Suleiman, Elinor Griffiths, and Philippe Pasdois for helpful discussions and Marieangela C. Wilson for technical advice.
Conflict of interest: none declared.