Mol Cell Biochem (2014) 388:269–276 DOI 10.1007/s11010-013-1918-x
Mitigation of postischemic cardiac contractile dysfunction by CaMKII inhibition: effects on programmed necrotic and apoptotic cell death Adrian Szobi • Tomas Rajtik • Slavka Carnicka Tana Ravingerova • Adriana Adameova
•
Received: 16 July 2013 / Accepted: 6 December 2013 / Published online: 18 December 2013 Ó Springer Science+Business Media New York 2013
Abstract While Ca2?/calmodulin-dependent protein kinase II (CaMKII) has been suggested to be an important protein regulating heart function upon ischemia/reperfusion (I/R), the mechanisms responsible are not fully known. Furthermore, it is not known whether CaMKII activation can modulate necroptosis, a recently described form of programmed cell death. In order to investigate these issues, Langendroff-perfused rat hearts were subjected to global ischemia and reperfusion, and CaMKII inhibition was achieved by adding the CaMKII inhibitor KN-93 (0.5 lmol/dm3) to the perfusion solution before the induction of ischemia. Immunoblotting was used to detect changes in expression of proteins modulating both necroptotic and apoptotic cell death. CaMKII inhibition normalized I/R induced increases in expression of necroptotic RIP1 and caspase-8 along with proteins of the intrinsic apoptotic pathway, namely cytochrome c and caspase-9. In addition, it increased the Bcl-2/Bax ratio and reduced caspase-3 and cleaved PARP1 content suggesting reduction of cell death. These changes coexisted with improvement of postischemic contractile function. On the other hand, there was no correlation between levels of pT287-CaMKIId and LVDP recovery after I/R. These results demonstrate for the first time that CaMKII inhibition may mitigate cardiac contractile dysfunction, at least partially, by limiting the contents of not only apoptotic, but also necroptotic A. Szobi T. Rajtik A. Adameova (&) Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Odbojarov 10, 83232 Bratislava, Slovak Republic e-mail: [email protected] S. Carnicka T. Ravingerova Institute for Heart Research, Slovak Academy of Sciences, Dubravska cesta 9, 84005 Bratislava, Slovak Republic
proteins. Phosphorylation of CaMKII seems unlikely to determine the degree of postischemic recovery of contractile function. Keywords CaMKII Ischemia Reperfusion Myocardium Necroptosis Apoptosis
Introduction Restoration of blood supply to previously ischemic heart is known to result in loss of cardiomyocytes that mainly die by necrosis and programmed cell death processes, such as apoptosis and autophagy. The first two mentioned types of cell death, unregulated necrosis and caspase-dependent apoptosis, are detrimental and can promote cardiac dysfunction, while autophagy can be considered to be a survival mechanism by which cells recycle their proteins, lipids, and organelles [1]. Recently, the understanding of cell death in ischemic/reperfused heart (I/R) has advanced and programmed cell death with morphological features of necrosis, termed as necroptosis or programmed necrosis, has been identified [2–4]. Unlike apoptosis, this form of cell death is caspase independent; its execution does not require the presence of active caspases. It has been shown that this type of programmed cell death significantly determines the extent of injury in hearts subjected to I/R and contributes to cardiac remodeling [4–6]. Likewise, necroptosis has been also reported to participate in I/Rinduced injury of kidneys [7] and brain [8, 9]. Necroptosis, like apoptosis, is induced by different death receptor ligands, including TNF-a, TRAIL ,and FasL which upon stimulation of the respective death receptors induce the formation of the cytosolic cell death complex II consisting of, among other proteins, receptor-interacting
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protein kinase 1 (RIP1) and caspase-8. In fact, active caspase-8 cleaves RIP1 within this complex and can induce apoptosis, while under caspase deficient conditions, uncleaved RIP1 kinase is active and can form a multiprotein complex referred to as the necrosome, which propagates downstream effects of necroptotic signaling [10]. This underscores the fact that caspase-8 activity determines whether a cardiomyocyte will die by necroptosis or apoptosis. Although several mechanisms capable of triggering necroptosis can be hypothesized, the exact signaling pathways activated during myocardial I/R are unknown. Of all the pathological mechanisms of myocardial I/R, Ca2? homeostasis disruption has the greatest relevance as it underlies the development of arrhythmias and hypercontracture [11, 12]. In addition, altered Ca2? levels due to I/R results in Ca2?-activated proteolysis altered cellular energetics and mitochondrial permeability transition which participate in cell death induction [13]. Although a lot of attention has been paid to pathogenesis of I/R with respect to Ca2?-mediated signaling, it is evident that there are some not fully characterized proteins which directly or indirectly regulate Ca2? levels, and thereby can promote phenotypes of I/R injury. Namely, overactivation of Ca2?/ calmodulin-dependent protein kinase II (CaMKII) has been documented to play a crucial role in I/R-induced disturbances in excitation–contraction coupling and excitation– transcription coupling [14, 15]. On the other hand, pharmacological inhibition of CaMKII has been shown to possess cardioprotective effects evidenced by abolishment of electrical instability and decrease in infarct size [16–19]. As there is a link between I/R-induced cell death and cellular Ca2? overload, which is regulated by CaMKII, the main aim of this study was to examine whether CaMKII inhibition by KN-93 is capable of modulating the content of markers of necroptotic and apoptotic cell death [13, 18– 20] and whether these changes are associated with the degree of recovery of mechanical function of the heart. In addition, we investigated whether postischemic LVDP recovery could be explained by changes in measured levels of activated CaMKIId.
Materials and methods Animals The protocol of this research study has been approved by the Ethics Committee of the Faculty of Pharmacy, Comenius University. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH publication No 85-23, revised 1996) with a prior approval by the Animal Care and Use Committee of the
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Slovak Republic. Male Wistar rats (250–300 g) were housed under standard conditions with a constant 12:12 h light/dark cycle (lights on 06:00) and a temperature of 22 ± 2 °C. Animals were fed a standard pelleted diet with ad libitum water intake. In vitro myocardial ischemia/reperfusion injury model After animal anesthetization (sodium pentobarbitone, 60 mg/kg, i.p.), rat hearts were quickly excised and subsequently perfused in the Langendorff mode. The perfusion was performed for 30 min at a constant perfusion pressure and temperature in a modified Krebs-Henseleit buffer pH 7.4 gassed with 95 % O2 and 5 % CO2 with the following composition: 118 mM NaCl, 3.2 mM KCl, 1.2 mM MgSO4, 25 mM NaHCO3, 1.18 mM KH2PO4, 2.5 mM CaCl2, and 5.5 mM glucose. After stabilization, 30 min global ischemia was induced followed by 40 min of reperfusion. Sham procedure utilizing the same protocol, but with no ischemia induction was used instead for hearts of the non-ischemic group. Non-elastic water-filled balloon inserted into the LV cavity was used to monitor hemodynamic parameters. In experiments with CaMKII inhibition, a selective CaMKII inhibitor KN-93 (Sigma-Aldrich, USA) was present in the perfusion solution at a concentration of 0.5 lmol/dm3 during the last 10 min of stabilization, the whole period of ischemia and the first 10 min of reperfusion. DMSO was used as a vehicle and its final concentration in the perfusate was 0.1 % (w/w). In the case of hearts with uninhibited CaMKII, they were perfused with perfusion solution containing only DMSO. The used concentration of KN-93 was chosen based on the results of studies which observed that higher concentrations of KN93 [21, 22] unlike a lower one [19] can also inhibit kinases other than CaMKII as well as ion channels and thereby measurably depress heart function. Immunoblotting Immunoblotting was performed as described previously [19]. Primary antibodies against RIP1 (3493, Cell Signaling Technology, USA), RIP3 (PRS2283, Sigma-Aldrich, USA), pThr286-CaMKIId (NBP1-64741, Novus Biologicals, USA), caspase-8 (SAB3500404, Sigma-Aldrich, USA), caspase-9 (SAB3500405, Sigma-Aldrich, USA), cytochrome c (SAB4502234, Sigma-Aldrich, USA), caspase-3 (sc-98785, Santa Cruz Biotechnology, USA), Bcl-2 (SAB4500003, Sigma-Aldrich, USA), Bax (B3428, SigmaAldrich, USA), PARP1 (9532, Cell Signaling Technology, USA) or b-actin (A2066, Sigma-Aldrich, USA), and secondary HRP-conjugated donkey anti-rabbit antibody (NA934V, GE Healthcare Life Sciences, UK) were used to
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Data are presented as mean ± SEM. Group differences in variables with normal distribution were tested for by using ANOVA and two-tailed unpaired Student’s t test. Statistical significance of correlations between hemodynamic parameters and protein levels was tested by Pearson’s test. All statistical analysis was performed with GraphPad Prism version 6.00 for Windows (GraphPad Software, USA). Differences between groups were considered to be significant at p B 0.05.
Results CaMKII inhibition improves postischemic contractile recovery Postischemic recovery of all of the measured parameters of contractile function was greatly improved as a result of CaMKII inhibition (Fig. 1). After 40 min of reperfusion, significant increase in LVDP was observed in hearts
LVDP (mm Hg)
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In order to determine whether inhibition of CaMKII is capable of influencing processes of I/R-induced necroptotic cell death in the heart, we performed western blotting analysis of RIP1, RIP3, and caspase-8. Representative blots are shown in Fig. 2a and protein expression data of these proteins are summarized in Fig. 2b, c. I/R injury significantly increased the protein expression of RIP1 (p = 0.0000044), while CaMKII inhibition normalized these levels (p = 0.034) suggesting that CaMKII may modulate necroptosis. RIP3 protein content was not influenced by neither I/R nor by KN-93 treatment compared to non-ischemic controls and it did not correlate with RIP1 expression (data not shown). The protein content of caspase-8 showed the same pattern of changes as RIP1 expression (p = 0.0001). CaMKII inhibition downregulated the increased levels of caspase-8 in I/R hearts
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Fig. 1 Hemodynamic parameters of Langendorffperfused control (IR) and KN93 treated hearts (KN-93) after 40 min of reperfusion preceded by 30 min of global ischemia. a LVDP, left ventricular developed pressure; b LVEDP, left ventricular end-diastolic pressure; c ?(dP/dt)max, maximal rate of contraction; d -(dP/dt)max, maximal rate of relaxation. Values are mean ± SEM. from 4 to 6 hearts per group. *p B 0.05 versus untreated I/R hearts
Effects of CaMKII inhibition on RIP1, RIP3 and caspase-8 protein levels in left ventricles of hearts
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subjected to KN-93 treatment (p = 0.035). CaMKII inhibition also significantly reduced values of LVEDP in KN93-treated hearts indicating improved postischemic diastolic function and abolishment of cardiac contracture (p = 0.047). In like manner, KN-93-treated I/R hearts displayed significant increases in maximal recovery of indices of contraction and relaxation (p = 0.011, p = 0.037, respectively).
baseline post 40m IR
baseline post 40m IR
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-(dP/dt)max (mm Hg/s)
detect proteins of interest. Signals were obtained through enhanced chemiluminescence (Pierce ECL Western Blotting Substrate, Pierce, USA) and blots were quantified by scanning densitometry. b-actin was used as the loading control.
+(dP/dt) max
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(p = 0.024). Intriguingly, caspase-8 upregulation during I/R did not result in increased formation of the RIP1 cleavage fragment which was recognized as a band with a molecular weight of *30 kDa (Fig. 2a, b). Effects of CaMKII inhibition on Bcl-2, Bax, caspase-3, caspase-9, PARP1 and cytochrome c protein expression in left ventricles of hearts In addition to necroptotic proteins, protein content of Bcl-2, Bax, cytochrome c, PARP1 (full-length and p89), caspase-9, and caspase-3 was analyzed to assess potential effects of CaMKII inhibition on the apoptotic component of cell death (Fig. 3). There was no significant difference in the expression of antiapoptotic Bcl-2 among the groups; however, the protein content of proapoptotic Bax was reduced in KN-93-treated hearts (Fig. 3a, b) (p = 0.005). I/R did not affect the Bcl-2/Bax ratio, a marker of susceptibility to apoptotic cell death, compared to controls (Fig. 3c). On the other hand, Bcl-2/Bax ratio was significantly increased after CaMKII inhibition compared to ischemic hearts (p = 0.039) indicating antiapoptotic capability of CaMKII inhibition. Cytochrome c and caspase-9 protein expression was elevated in the I/R group (p = 0.008, p = 0.037, respectively), while CaMKII inhibition completely abolished this observed increase (p = 0.029, p = 0.008, respectively). Changes in the protein content of caspase-3 mirrored those of Bax; I/R had no effect, while KN-93 significantly reduced caspase-3 protein expression (p = 0.030) (Fig. 3d). Full-length PARP1
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Fig. 2 Effects of CaMKII inhibition on the content of necroptotic proteins. a Representative immunoblots of left ventricular tissue of RIP1, N-terminal cleavage fragment of RIP1 (RIP1N), RIP3 and caspase-8 in control, I/R and KN-93-treated I/R hearts. b Protein expression of RIP1, RIP1N, RIP3. c caspase-8. Values are mean ± SEM. from 6 to 8 hearts per group. *p B 0.05 versus control group, # p B 0.05 versus untreated I/R hearts
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IR KN-93
RIP3
expression did not differ among the groups, while its 89 kDa fragment, a marker of early stage apoptosis, was reduced in KN-93-treated I/R hearts compared to the untreated I/R group (p = 0.050) (Fig. 3e). There was also a trend for I/R to elevate p89 PARP1 compared to the control group (p = 0.083). Correlation between posttranslational modification of CaMKIId and LVDP recovery To investigate whether changes in posttranslational modification of CaMKIId correlate with postischemic recovery of myocardial function, we additionally measured phosphorylation of CaMKIId on threonine 287 which is known to result in CaMKIId activation [22]. pT287-CaMKIId was significantly decreased after 40 min of reperfusion in the I/R group (p = 0.004), and CaMKII inhibition non-significantly reduced these levels even further (Fig. 4a, b). However, pT287-CaMKIId did not correlate with postischemic recovery of LVDP (Fig. 4c).
Discussion Although it has been well established that oxygen and energy substrate depletion as a result of restricted blood flow during ischemia are responsible for the observed pathophysiology in affected hearts and promote cell death, the signaling pathways underlying irreversible cell damage are still a subject of intensive investigation [23, 24]. Until
Mol Cell Biochem (2014) 388:269–276
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Fig. 3 Effects of CaMKII inhibition on the content of apoptotic proteins. a Representative immunoblots of left ventricular tissue of Bax, Bcl-2, caspase-9, cytochrome c, PARP1, PARP1 (p89) and caspase-3 in control, I/R, and KN-93-treated I/R hearts. b Protein expression of Bcl-2 and Bax. c Bcl-2/Bax ratio. d Protein expression of cytochrome c, caspase-9 and caspase-3. e Protein expression of PARP1 and PARP1 (p89). Values are mean ± SEM. from 6 to 8 hearts per group. *p B 0.05 versus control group, # p B 0.05 versus untreated I/R hearts
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recently, apoptosis and autophagy have been considered to be the only types of programmed cell death, while necrosis has long been believed to be an uncontrolled, passive process. In spite of large number of papers dealing with the subject of cell death during I/R, the concept of cardiomyocyte injury during I/R has been fundamentally changed by the discovery of tightly regulated signaling platforms and their protein constituents that can trigger a cascade leading to cell death with morphological features of necrosis. In this study, we investigated the potential influence of CaMKII on the expression of necroptotic along with apoptotic proteins in hearts injured by I/R. At the same time, we tested the hypothesis that the extent of changes in CaMKII phosphorylation may underlie the degree of postischemic contractile function. We showed for the first time that CaMKII inhibition was capable of normalizing protein content of necroptosis-modulating RIP1 and caspase-8 in hearts subjected to irreversible I/R. In addition, CaMKII inhibition downregulated proteins involved in the intrinsic apoptotic cell death pathway, namely caspase-9,
caspase-3, and cytochrome c, and increased the Bcl-2/Bax ratio. Importantly, pT287-CaMKIId was found to be significantly depressed in all hearts subjected to I/R and there was no correlation between pT287-CaMKIId and LVDP recovery. As indicated earlier, myocardial I/R likely results in activation of pronecroptotic pathways inducing cardiomyocyte injury. Indeed, we and others [5] showed significantly increased RIP1 protein content after I/R (Fig. 2b). However, in contrast to the mentioned study, we did not detect changes in the protein content of RIP3 which did not correlate with RIP1 protein levels (data not shown). This discrepancy is possibly a result of a different I/R settings utilized. We used an in vitro protocol with global ischemia induction followed by 40 min of reperfusion, while the study referred to investigated the role of necroptosis upon a long (24 h) reperfusion in hearts of anesthetized mice. Thus, it is apparent that the duration of reperfusion might affect fate of necroptotic proteins and subsequently the degree of activation of a particular type of cell death.
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pT287-CaMKII Fig. 4 Relationship between pT287-CaMKII and contractile dysfunction. a Representative immunoblots of left ventricular tissue of pT287-CaMKIId in control, I/R, and KN-93-treated I/R hearts. b Effects of CaMKII inhibition on phosphorylation at T287 of CaMKIId. c Linear regression between recovery of postischemic LVDP and protein expression of pT287-CaMKIId. Values are mean ± SEM from 6 to 8 hearts per group. *p B 0.05 versus control group, #p B 0.05 versus untreated I/R hearts, n.s not significant
Interestingly, caspase-8 protein content was increased alongside RIP1 (Fig. 2c). It should be pointed out that caspase-8 activity is known to be an important negative regulator of necroptotic cell death by inactivating RIP1 through cleaving off the kinase domain of RIP1 required for downstream necroptotic signaling [25]. Surprisingly, this increase in caspase-8 protein content did not result in changes in the amount of cleaved RIP1 (Fig. 2b) as could be expected. Furthermore, protein content of caspase-3, which is formed by caspase-8-mediated cleavage of procaspase-3 [26, 27], was not increased in hearts of the I/R group (Fig. 3d). Thus, it seems that despite increased formation of active caspase-8 from its procaspase form this
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change was not sufficient to increase RIP1 or procaspase-3 cleavage. Recently, it has been shown that CaMKII inhibition limits cardiac cell death due to necrosis and apoptosis [18]. In our study, CaMKII inhibition during myocardial I/R downregulated the executioner caspase-3 as well as PARP1 89 kDa cleavage fragment with concomitant increase of the Bcl-2/Bax ratio indicating antiapoptotic effects of this pharmacological modulation. Interestingly, caspase-3 expression was not increased by I/R compared to the control group, however, PARP1 p89 expression showed a trend toward it being increased which does suggest that apoptosis was actually induced by the used I/R protocol. Thus, it is very likely that caspase-7, also known to cleave PARP1 [28], may be rather than caspase-3 responsible for these results. Data from our study also showed that CaMKII inhibition is capable of abrogating the observed increase in expression of cytochrome c. Although we did not determine the cytosolic and mitochondrial content of cytochrome c in I/R hearts separately, it is very likely that the cytosolic content of cytochrome c, which mediates apoptosis, was increased, while its expression within mitochondria was unchanged as this was already reported previously by Lundberg and Szweda [29]. Therefore, it can be hypothesized that downregulation of cytochrome c as result of CaMKII inhibition attenuated apoptotic processes. The main mechanism thought to be responsible for the observed antiapoptotic effects of CaMKII inhibition is amelioration of mitochondrial Ca2? overload. In fact, these disturbances in Ca2? homeostasis secondary to hyperphosphorylation of Ca2? cycling proteins, such as phospholamban, ryanodine receptor, Na?/Ca2? exchanger, and L-type voltage gated Ca2? channel [18, 30, 31] are known to stimulate the release of cytochrome c from the mitochondrial intermembrane space into the cytosol and subsequently activate the intrinsic apoptotic cascade [26, 27]. In this study, we extended the knowledge about the role of CaMKII in cell death and showed that a low concentration of KN-93 (0.5 lmol/dm3) present in the perfusion solution during ischemia and at the beginning of reperfusion is capable of normalizing the protein content of RIP1 and caspase-8 known to modulate execution of necroptotic cell death. Herewith, we indirectly suggested a link between CaMKII-modulated [Ca2?]i levels and necroptosis activation. While mechanistic explanation of CaMKII involvement in necroptosis cannot be determined from this study, speculatively it may involve members of the cellular FLICE-inhibitory protein (c-FLIP) family, which are capable of inhibiting caspase-8 activity [32]. Actually, some studies have shown that CaMKII activity has the ability to increase both c-FLIP protein expression as well as phosphorylation leading to caspase-8 inhibition [33, 34]. As CaMKII activity was reported to be notably elevated
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during the early stages of reperfusion [18], under the same conditions caspase-8 activity might be reduced as well. Oppositely, in late stages of reperfusion, when CaMKII phosphorylation is depressed, the inhibitory effect on caspase-8 might be abolished. However, based on the results of this study, it seems to be likely that this reduced inhibitory effect is not sufficient to promote increased RIP1 and procaspase-3 cleavage in spite of increased caspase-8 formation in the I/R hearts. In line with the positive modulation of markers of cell death during I/R, CaMKII inhibition significantly alleviated postischemic myocardial contractile dysfunction as evidenced by greatly improved recovery of both left ventricular systolic and diastolic pressure (Fig. 1). Furthermore, we observed no correlation between T287 phopshorylation of CaMKIId and LVDP. Actually, CaMKIId phosphorylation at T287 was significantly depressed in hearts subjected to I/R with or without CaMKII inhibition compared to control hearts (Fig. 4b). In the already referenced study by Vila-Petroff et al. [18], a significant increase in phospholamban T17 phosphorylation, a marker for CaMKII activation, was documented after I/R at the 3 min mark, but a significant depression after 60 min of reperfusion. These time course changes in CaMKII activity agree with the depressed CaMKIId T287 phosphorylation observed in our study after 40 min of reperfusion. Thus, it appears that protein kinases other than CaMKII are dominant in maintaining myocardial function in the late reperfusion phase. Moreover, it could be hypothesized that changes in the protein expression of apoptotic and necroptotic proteins detected in the CaMKII-inhibited I/R group could be a result of reduction of this CaMKII activation during early reperfusion. In conclusion, our results show that CaMKII inhibition during myocardial I/R can modulate content of crucial necroptotic as well as apoptotic proteins in addition to improving postischemic restoration of myocardial mechanical function. While this would suggest that by modulating these markers of cell death, CaMKII inhibition could be, at least partially, responsible for these observations, disturbances in calcium homeostasis, asynchronous excitation–contraction coupling due to electrical instability, and other molecular and cellular aspects of I/R injury cannot be ruled out. Although we have proposed a few mechanisms underlying such cardioprotective effects of CaMKII inhibition, studies specifically designed to test these hypotheses can give us more insight into the role of CaMKII in necroptotic cell death. As a whole, this study supports the notion that modulation of myocardial CaMKII activity is a potentially interesting therapeutic target, which might limit cardiomyocyte death by both necroptosis and apoptosis and thus increase recovery of contractile function in the settings of I/R.
275 Acknowledgments Authors would like to thank Mrs. V. Hassova for her indispensable help. This study was supported by Grants UK/ 430/2013, VEGA 1/0638/12, VEGA 2/0054/11, APVV 0102-11, and APVV 0523-10. Conflict of interest of interest.
The authors declare that there are no conflicts
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Mitigation of postischemic cardiac contractile dysfunction by CaMKII inhibition: effects on programmed necrotic and apoptotic cell death Adrian Szobi • Tomas Rajtik • Slavka Carnicka Tana Ravingerova • Adriana Adameova
•
Received: 16 July 2013 / Accepted: 6 December 2013 / Published online: 18 December 2013 Ó Springer Science+Business Media New York 2013
Abstract While Ca2?/calmodulin-dependent protein kinase II (CaMKII) has been suggested to be an important protein regulating heart function upon ischemia/reperfusion (I/R), the mechanisms responsible are not fully known. Furthermore, it is not known whether CaMKII activation can modulate necroptosis, a recently described form of programmed cell death. In order to investigate these issues, Langendroff-perfused rat hearts were subjected to global ischemia and reperfusion, and CaMKII inhibition was achieved by adding the CaMKII inhibitor KN-93 (0.5 lmol/dm3) to the perfusion solution before the induction of ischemia. Immunoblotting was used to detect changes in expression of proteins modulating both necroptotic and apoptotic cell death. CaMKII inhibition normalized I/R induced increases in expression of necroptotic RIP1 and caspase-8 along with proteins of the intrinsic apoptotic pathway, namely cytochrome c and caspase-9. In addition, it increased the Bcl-2/Bax ratio and reduced caspase-3 and cleaved PARP1 content suggesting reduction of cell death. These changes coexisted with improvement of postischemic contractile function. On the other hand, there was no correlation between levels of pT287-CaMKIId and LVDP recovery after I/R. These results demonstrate for the first time that CaMKII inhibition may mitigate cardiac contractile dysfunction, at least partially, by limiting the contents of not only apoptotic, but also necroptotic A. Szobi T. Rajtik A. Adameova (&) Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Odbojarov 10, 83232 Bratislava, Slovak Republic e-mail: [email protected] S. Carnicka T. Ravingerova Institute for Heart Research, Slovak Academy of Sciences, Dubravska cesta 9, 84005 Bratislava, Slovak Republic
proteins. Phosphorylation of CaMKII seems unlikely to determine the degree of postischemic recovery of contractile function. Keywords CaMKII Ischemia Reperfusion Myocardium Necroptosis Apoptosis
Introduction Restoration of blood supply to previously ischemic heart is known to result in loss of cardiomyocytes that mainly die by necrosis and programmed cell death processes, such as apoptosis and autophagy. The first two mentioned types of cell death, unregulated necrosis and caspase-dependent apoptosis, are detrimental and can promote cardiac dysfunction, while autophagy can be considered to be a survival mechanism by which cells recycle their proteins, lipids, and organelles [1]. Recently, the understanding of cell death in ischemic/reperfused heart (I/R) has advanced and programmed cell death with morphological features of necrosis, termed as necroptosis or programmed necrosis, has been identified [2–4]. Unlike apoptosis, this form of cell death is caspase independent; its execution does not require the presence of active caspases. It has been shown that this type of programmed cell death significantly determines the extent of injury in hearts subjected to I/R and contributes to cardiac remodeling [4–6]. Likewise, necroptosis has been also reported to participate in I/Rinduced injury of kidneys [7] and brain [8, 9]. Necroptosis, like apoptosis, is induced by different death receptor ligands, including TNF-a, TRAIL ,and FasL which upon stimulation of the respective death receptors induce the formation of the cytosolic cell death complex II consisting of, among other proteins, receptor-interacting
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protein kinase 1 (RIP1) and caspase-8. In fact, active caspase-8 cleaves RIP1 within this complex and can induce apoptosis, while under caspase deficient conditions, uncleaved RIP1 kinase is active and can form a multiprotein complex referred to as the necrosome, which propagates downstream effects of necroptotic signaling [10]. This underscores the fact that caspase-8 activity determines whether a cardiomyocyte will die by necroptosis or apoptosis. Although several mechanisms capable of triggering necroptosis can be hypothesized, the exact signaling pathways activated during myocardial I/R are unknown. Of all the pathological mechanisms of myocardial I/R, Ca2? homeostasis disruption has the greatest relevance as it underlies the development of arrhythmias and hypercontracture [11, 12]. In addition, altered Ca2? levels due to I/R results in Ca2?-activated proteolysis altered cellular energetics and mitochondrial permeability transition which participate in cell death induction [13]. Although a lot of attention has been paid to pathogenesis of I/R with respect to Ca2?-mediated signaling, it is evident that there are some not fully characterized proteins which directly or indirectly regulate Ca2? levels, and thereby can promote phenotypes of I/R injury. Namely, overactivation of Ca2?/ calmodulin-dependent protein kinase II (CaMKII) has been documented to play a crucial role in I/R-induced disturbances in excitation–contraction coupling and excitation– transcription coupling [14, 15]. On the other hand, pharmacological inhibition of CaMKII has been shown to possess cardioprotective effects evidenced by abolishment of electrical instability and decrease in infarct size [16–19]. As there is a link between I/R-induced cell death and cellular Ca2? overload, which is regulated by CaMKII, the main aim of this study was to examine whether CaMKII inhibition by KN-93 is capable of modulating the content of markers of necroptotic and apoptotic cell death [13, 18– 20] and whether these changes are associated with the degree of recovery of mechanical function of the heart. In addition, we investigated whether postischemic LVDP recovery could be explained by changes in measured levels of activated CaMKIId.
Materials and methods Animals The protocol of this research study has been approved by the Ethics Committee of the Faculty of Pharmacy, Comenius University. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health (NIH publication No 85-23, revised 1996) with a prior approval by the Animal Care and Use Committee of the
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Slovak Republic. Male Wistar rats (250–300 g) were housed under standard conditions with a constant 12:12 h light/dark cycle (lights on 06:00) and a temperature of 22 ± 2 °C. Animals were fed a standard pelleted diet with ad libitum water intake. In vitro myocardial ischemia/reperfusion injury model After animal anesthetization (sodium pentobarbitone, 60 mg/kg, i.p.), rat hearts were quickly excised and subsequently perfused in the Langendorff mode. The perfusion was performed for 30 min at a constant perfusion pressure and temperature in a modified Krebs-Henseleit buffer pH 7.4 gassed with 95 % O2 and 5 % CO2 with the following composition: 118 mM NaCl, 3.2 mM KCl, 1.2 mM MgSO4, 25 mM NaHCO3, 1.18 mM KH2PO4, 2.5 mM CaCl2, and 5.5 mM glucose. After stabilization, 30 min global ischemia was induced followed by 40 min of reperfusion. Sham procedure utilizing the same protocol, but with no ischemia induction was used instead for hearts of the non-ischemic group. Non-elastic water-filled balloon inserted into the LV cavity was used to monitor hemodynamic parameters. In experiments with CaMKII inhibition, a selective CaMKII inhibitor KN-93 (Sigma-Aldrich, USA) was present in the perfusion solution at a concentration of 0.5 lmol/dm3 during the last 10 min of stabilization, the whole period of ischemia and the first 10 min of reperfusion. DMSO was used as a vehicle and its final concentration in the perfusate was 0.1 % (w/w). In the case of hearts with uninhibited CaMKII, they were perfused with perfusion solution containing only DMSO. The used concentration of KN-93 was chosen based on the results of studies which observed that higher concentrations of KN93 [21, 22] unlike a lower one [19] can also inhibit kinases other than CaMKII as well as ion channels and thereby measurably depress heart function. Immunoblotting Immunoblotting was performed as described previously [19]. Primary antibodies against RIP1 (3493, Cell Signaling Technology, USA), RIP3 (PRS2283, Sigma-Aldrich, USA), pThr286-CaMKIId (NBP1-64741, Novus Biologicals, USA), caspase-8 (SAB3500404, Sigma-Aldrich, USA), caspase-9 (SAB3500405, Sigma-Aldrich, USA), cytochrome c (SAB4502234, Sigma-Aldrich, USA), caspase-3 (sc-98785, Santa Cruz Biotechnology, USA), Bcl-2 (SAB4500003, Sigma-Aldrich, USA), Bax (B3428, SigmaAldrich, USA), PARP1 (9532, Cell Signaling Technology, USA) or b-actin (A2066, Sigma-Aldrich, USA), and secondary HRP-conjugated donkey anti-rabbit antibody (NA934V, GE Healthcare Life Sciences, UK) were used to
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Data are presented as mean ± SEM. Group differences in variables with normal distribution were tested for by using ANOVA and two-tailed unpaired Student’s t test. Statistical significance of correlations between hemodynamic parameters and protein levels was tested by Pearson’s test. All statistical analysis was performed with GraphPad Prism version 6.00 for Windows (GraphPad Software, USA). Differences between groups were considered to be significant at p B 0.05.
Results CaMKII inhibition improves postischemic contractile recovery Postischemic recovery of all of the measured parameters of contractile function was greatly improved as a result of CaMKII inhibition (Fig. 1). After 40 min of reperfusion, significant increase in LVDP was observed in hearts
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In order to determine whether inhibition of CaMKII is capable of influencing processes of I/R-induced necroptotic cell death in the heart, we performed western blotting analysis of RIP1, RIP3, and caspase-8. Representative blots are shown in Fig. 2a and protein expression data of these proteins are summarized in Fig. 2b, c. I/R injury significantly increased the protein expression of RIP1 (p = 0.0000044), while CaMKII inhibition normalized these levels (p = 0.034) suggesting that CaMKII may modulate necroptosis. RIP3 protein content was not influenced by neither I/R nor by KN-93 treatment compared to non-ischemic controls and it did not correlate with RIP1 expression (data not shown). The protein content of caspase-8 showed the same pattern of changes as RIP1 expression (p = 0.0001). CaMKII inhibition downregulated the increased levels of caspase-8 in I/R hearts
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Fig. 1 Hemodynamic parameters of Langendorffperfused control (IR) and KN93 treated hearts (KN-93) after 40 min of reperfusion preceded by 30 min of global ischemia. a LVDP, left ventricular developed pressure; b LVEDP, left ventricular end-diastolic pressure; c ?(dP/dt)max, maximal rate of contraction; d -(dP/dt)max, maximal rate of relaxation. Values are mean ± SEM. from 4 to 6 hearts per group. *p B 0.05 versus untreated I/R hearts
Effects of CaMKII inhibition on RIP1, RIP3 and caspase-8 protein levels in left ventricles of hearts
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subjected to KN-93 treatment (p = 0.035). CaMKII inhibition also significantly reduced values of LVEDP in KN93-treated hearts indicating improved postischemic diastolic function and abolishment of cardiac contracture (p = 0.047). In like manner, KN-93-treated I/R hearts displayed significant increases in maximal recovery of indices of contraction and relaxation (p = 0.011, p = 0.037, respectively).
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detect proteins of interest. Signals were obtained through enhanced chemiluminescence (Pierce ECL Western Blotting Substrate, Pierce, USA) and blots were quantified by scanning densitometry. b-actin was used as the loading control.
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(p = 0.024). Intriguingly, caspase-8 upregulation during I/R did not result in increased formation of the RIP1 cleavage fragment which was recognized as a band with a molecular weight of *30 kDa (Fig. 2a, b). Effects of CaMKII inhibition on Bcl-2, Bax, caspase-3, caspase-9, PARP1 and cytochrome c protein expression in left ventricles of hearts In addition to necroptotic proteins, protein content of Bcl-2, Bax, cytochrome c, PARP1 (full-length and p89), caspase-9, and caspase-3 was analyzed to assess potential effects of CaMKII inhibition on the apoptotic component of cell death (Fig. 3). There was no significant difference in the expression of antiapoptotic Bcl-2 among the groups; however, the protein content of proapoptotic Bax was reduced in KN-93-treated hearts (Fig. 3a, b) (p = 0.005). I/R did not affect the Bcl-2/Bax ratio, a marker of susceptibility to apoptotic cell death, compared to controls (Fig. 3c). On the other hand, Bcl-2/Bax ratio was significantly increased after CaMKII inhibition compared to ischemic hearts (p = 0.039) indicating antiapoptotic capability of CaMKII inhibition. Cytochrome c and caspase-9 protein expression was elevated in the I/R group (p = 0.008, p = 0.037, respectively), while CaMKII inhibition completely abolished this observed increase (p = 0.029, p = 0.008, respectively). Changes in the protein content of caspase-3 mirrored those of Bax; I/R had no effect, while KN-93 significantly reduced caspase-3 protein expression (p = 0.030) (Fig. 3d). Full-length PARP1
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Fig. 2 Effects of CaMKII inhibition on the content of necroptotic proteins. a Representative immunoblots of left ventricular tissue of RIP1, N-terminal cleavage fragment of RIP1 (RIP1N), RIP3 and caspase-8 in control, I/R and KN-93-treated I/R hearts. b Protein expression of RIP1, RIP1N, RIP3. c caspase-8. Values are mean ± SEM. from 6 to 8 hearts per group. *p B 0.05 versus control group, # p B 0.05 versus untreated I/R hearts
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expression did not differ among the groups, while its 89 kDa fragment, a marker of early stage apoptosis, was reduced in KN-93-treated I/R hearts compared to the untreated I/R group (p = 0.050) (Fig. 3e). There was also a trend for I/R to elevate p89 PARP1 compared to the control group (p = 0.083). Correlation between posttranslational modification of CaMKIId and LVDP recovery To investigate whether changes in posttranslational modification of CaMKIId correlate with postischemic recovery of myocardial function, we additionally measured phosphorylation of CaMKIId on threonine 287 which is known to result in CaMKIId activation [22]. pT287-CaMKIId was significantly decreased after 40 min of reperfusion in the I/R group (p = 0.004), and CaMKII inhibition non-significantly reduced these levels even further (Fig. 4a, b). However, pT287-CaMKIId did not correlate with postischemic recovery of LVDP (Fig. 4c).
Discussion Although it has been well established that oxygen and energy substrate depletion as a result of restricted blood flow during ischemia are responsible for the observed pathophysiology in affected hearts and promote cell death, the signaling pathways underlying irreversible cell damage are still a subject of intensive investigation [23, 24]. Until
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Fig. 3 Effects of CaMKII inhibition on the content of apoptotic proteins. a Representative immunoblots of left ventricular tissue of Bax, Bcl-2, caspase-9, cytochrome c, PARP1, PARP1 (p89) and caspase-3 in control, I/R, and KN-93-treated I/R hearts. b Protein expression of Bcl-2 and Bax. c Bcl-2/Bax ratio. d Protein expression of cytochrome c, caspase-9 and caspase-3. e Protein expression of PARP1 and PARP1 (p89). Values are mean ± SEM. from 6 to 8 hearts per group. *p B 0.05 versus control group, # p B 0.05 versus untreated I/R hearts
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recently, apoptosis and autophagy have been considered to be the only types of programmed cell death, while necrosis has long been believed to be an uncontrolled, passive process. In spite of large number of papers dealing with the subject of cell death during I/R, the concept of cardiomyocyte injury during I/R has been fundamentally changed by the discovery of tightly regulated signaling platforms and their protein constituents that can trigger a cascade leading to cell death with morphological features of necrosis. In this study, we investigated the potential influence of CaMKII on the expression of necroptotic along with apoptotic proteins in hearts injured by I/R. At the same time, we tested the hypothesis that the extent of changes in CaMKII phosphorylation may underlie the degree of postischemic contractile function. We showed for the first time that CaMKII inhibition was capable of normalizing protein content of necroptosis-modulating RIP1 and caspase-8 in hearts subjected to irreversible I/R. In addition, CaMKII inhibition downregulated proteins involved in the intrinsic apoptotic cell death pathway, namely caspase-9,
caspase-3, and cytochrome c, and increased the Bcl-2/Bax ratio. Importantly, pT287-CaMKIId was found to be significantly depressed in all hearts subjected to I/R and there was no correlation between pT287-CaMKIId and LVDP recovery. As indicated earlier, myocardial I/R likely results in activation of pronecroptotic pathways inducing cardiomyocyte injury. Indeed, we and others [5] showed significantly increased RIP1 protein content after I/R (Fig. 2b). However, in contrast to the mentioned study, we did not detect changes in the protein content of RIP3 which did not correlate with RIP1 protein levels (data not shown). This discrepancy is possibly a result of a different I/R settings utilized. We used an in vitro protocol with global ischemia induction followed by 40 min of reperfusion, while the study referred to investigated the role of necroptosis upon a long (24 h) reperfusion in hearts of anesthetized mice. Thus, it is apparent that the duration of reperfusion might affect fate of necroptotic proteins and subsequently the degree of activation of a particular type of cell death.
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pT287-CaMKII Fig. 4 Relationship between pT287-CaMKII and contractile dysfunction. a Representative immunoblots of left ventricular tissue of pT287-CaMKIId in control, I/R, and KN-93-treated I/R hearts. b Effects of CaMKII inhibition on phosphorylation at T287 of CaMKIId. c Linear regression between recovery of postischemic LVDP and protein expression of pT287-CaMKIId. Values are mean ± SEM from 6 to 8 hearts per group. *p B 0.05 versus control group, #p B 0.05 versus untreated I/R hearts, n.s not significant
Interestingly, caspase-8 protein content was increased alongside RIP1 (Fig. 2c). It should be pointed out that caspase-8 activity is known to be an important negative regulator of necroptotic cell death by inactivating RIP1 through cleaving off the kinase domain of RIP1 required for downstream necroptotic signaling [25]. Surprisingly, this increase in caspase-8 protein content did not result in changes in the amount of cleaved RIP1 (Fig. 2b) as could be expected. Furthermore, protein content of caspase-3, which is formed by caspase-8-mediated cleavage of procaspase-3 [26, 27], was not increased in hearts of the I/R group (Fig. 3d). Thus, it seems that despite increased formation of active caspase-8 from its procaspase form this
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change was not sufficient to increase RIP1 or procaspase-3 cleavage. Recently, it has been shown that CaMKII inhibition limits cardiac cell death due to necrosis and apoptosis [18]. In our study, CaMKII inhibition during myocardial I/R downregulated the executioner caspase-3 as well as PARP1 89 kDa cleavage fragment with concomitant increase of the Bcl-2/Bax ratio indicating antiapoptotic effects of this pharmacological modulation. Interestingly, caspase-3 expression was not increased by I/R compared to the control group, however, PARP1 p89 expression showed a trend toward it being increased which does suggest that apoptosis was actually induced by the used I/R protocol. Thus, it is very likely that caspase-7, also known to cleave PARP1 [28], may be rather than caspase-3 responsible for these results. Data from our study also showed that CaMKII inhibition is capable of abrogating the observed increase in expression of cytochrome c. Although we did not determine the cytosolic and mitochondrial content of cytochrome c in I/R hearts separately, it is very likely that the cytosolic content of cytochrome c, which mediates apoptosis, was increased, while its expression within mitochondria was unchanged as this was already reported previously by Lundberg and Szweda [29]. Therefore, it can be hypothesized that downregulation of cytochrome c as result of CaMKII inhibition attenuated apoptotic processes. The main mechanism thought to be responsible for the observed antiapoptotic effects of CaMKII inhibition is amelioration of mitochondrial Ca2? overload. In fact, these disturbances in Ca2? homeostasis secondary to hyperphosphorylation of Ca2? cycling proteins, such as phospholamban, ryanodine receptor, Na?/Ca2? exchanger, and L-type voltage gated Ca2? channel [18, 30, 31] are known to stimulate the release of cytochrome c from the mitochondrial intermembrane space into the cytosol and subsequently activate the intrinsic apoptotic cascade [26, 27]. In this study, we extended the knowledge about the role of CaMKII in cell death and showed that a low concentration of KN-93 (0.5 lmol/dm3) present in the perfusion solution during ischemia and at the beginning of reperfusion is capable of normalizing the protein content of RIP1 and caspase-8 known to modulate execution of necroptotic cell death. Herewith, we indirectly suggested a link between CaMKII-modulated [Ca2?]i levels and necroptosis activation. While mechanistic explanation of CaMKII involvement in necroptosis cannot be determined from this study, speculatively it may involve members of the cellular FLICE-inhibitory protein (c-FLIP) family, which are capable of inhibiting caspase-8 activity [32]. Actually, some studies have shown that CaMKII activity has the ability to increase both c-FLIP protein expression as well as phosphorylation leading to caspase-8 inhibition [33, 34]. As CaMKII activity was reported to be notably elevated
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during the early stages of reperfusion [18], under the same conditions caspase-8 activity might be reduced as well. Oppositely, in late stages of reperfusion, when CaMKII phosphorylation is depressed, the inhibitory effect on caspase-8 might be abolished. However, based on the results of this study, it seems to be likely that this reduced inhibitory effect is not sufficient to promote increased RIP1 and procaspase-3 cleavage in spite of increased caspase-8 formation in the I/R hearts. In line with the positive modulation of markers of cell death during I/R, CaMKII inhibition significantly alleviated postischemic myocardial contractile dysfunction as evidenced by greatly improved recovery of both left ventricular systolic and diastolic pressure (Fig. 1). Furthermore, we observed no correlation between T287 phopshorylation of CaMKIId and LVDP. Actually, CaMKIId phosphorylation at T287 was significantly depressed in hearts subjected to I/R with or without CaMKII inhibition compared to control hearts (Fig. 4b). In the already referenced study by Vila-Petroff et al. [18], a significant increase in phospholamban T17 phosphorylation, a marker for CaMKII activation, was documented after I/R at the 3 min mark, but a significant depression after 60 min of reperfusion. These time course changes in CaMKII activity agree with the depressed CaMKIId T287 phosphorylation observed in our study after 40 min of reperfusion. Thus, it appears that protein kinases other than CaMKII are dominant in maintaining myocardial function in the late reperfusion phase. Moreover, it could be hypothesized that changes in the protein expression of apoptotic and necroptotic proteins detected in the CaMKII-inhibited I/R group could be a result of reduction of this CaMKII activation during early reperfusion. In conclusion, our results show that CaMKII inhibition during myocardial I/R can modulate content of crucial necroptotic as well as apoptotic proteins in addition to improving postischemic restoration of myocardial mechanical function. While this would suggest that by modulating these markers of cell death, CaMKII inhibition could be, at least partially, responsible for these observations, disturbances in calcium homeostasis, asynchronous excitation–contraction coupling due to electrical instability, and other molecular and cellular aspects of I/R injury cannot be ruled out. Although we have proposed a few mechanisms underlying such cardioprotective effects of CaMKII inhibition, studies specifically designed to test these hypotheses can give us more insight into the role of CaMKII in necroptotic cell death. As a whole, this study supports the notion that modulation of myocardial CaMKII activity is a potentially interesting therapeutic target, which might limit cardiomyocyte death by both necroptosis and apoptosis and thus increase recovery of contractile function in the settings of I/R.
275 Acknowledgments Authors would like to thank Mrs. V. Hassova for her indispensable help. This study was supported by Grants UK/ 430/2013, VEGA 1/0638/12, VEGA 2/0054/11, APVV 0102-11, and APVV 0523-10. Conflict of interest of interest.
The authors declare that there are no conflicts
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