The role of the Hsp90/Akt pathway in myocardial calpain-induced caspase-3 activation and apoptosis during sepsis
© Li et al; licensee BioMed Central Ltd. 2013
Received: 19 October 2012
Accepted: 18 February 2013
Published: 20 February 2013
Recent studies have demonstrated that myocardial calpain triggers caspase-3 activation and myocardial apoptosis in models of sepsis, whereas the inhibition of calpain activity down-regulates myocardial caspase-3 activation and apoptosis. However, the mechanism underlying this pathological process is unclear. Therefore, in this study, our aim was to explore whether the Hsp90/Akt signaling pathway plays a role in the induction of myocardial calpain activity, caspase-3 activation and apoptosis in the septic mice.
Adult male C57 mice were injected with lipopolysaccharide (LPS, 4 mg/kg, i.p.) to induce sepsis. Next, myocardial caspase-3 activity and the levels of Hsp90/p-Akt (phospho-Akt) proteins were detected, and apoptotic cells were assessed by performing the TUNEL assay.
In the septic mice, there was an increase in myocardial calpain and caspase-3 activity in addition to an increase in the number of apoptotic cells; however, there was a time-dependent decrease in myocardial Hsp90/p-Akt protein levels. The administration of calpain inhibitors (calpain inhibitor-Ш or PD150606) prevented the LPS-induced degradation of myocardial Hsp90/p-Akt protein and its expression in cardiomyocytes in addition to inhibiting myocardial caspase-3 activation and apoptosis. The inhibition of Hsp90 by pretreatment with 17-AAG induced p-Akt degradation, and the inhibition of Akt activity by pretreatment with wortmannin resulted in caspase-3 activation in wildtype C57 murine heart tissues.
Myocardial calpain induces myocardial caspase-3 activation and apoptosis in septic mice via the activation of the Hsp90/Akt pathway.
KeywordsCalpain Hsp90/Akt Caspase-3 activation Apoptosis Sepsis
Endotoxins or bacterial lipopolysaccharides depress myocardial contractility in laboratory animals and humans [1, 2]. Although, the molecular and cellular mechanisms that mediate the pathogenesis of septic cardiomyopathy are still unclear, several lines of evidence suggest that myocardial caspase-3 activation plays a major role in myocardial dysfunction [3–6].
The blockade of myocardial caspase-3 activation significantly attenuates myocardial dysfunction and improves the survival rate during sepsis [4–6], and therefore, the mechanism involved in LPS-induced caspase-3 activation has been explored in cardiomyocytes [7–9]. In a recent study that we published, an increase in myocardial calpain activity in the septic mouse was noted [7, 8], and in addition, over-expression of calpastatin, a specific inhibitor of calpain, or treatment with pharmacological inhibitors of calpain prevented myocardial caspase-3 activation during endotoxemia. These results suggest that calpain is involved in the activation of caspase-3 during sepsis . However, the mechanisms involved in calpain-induced caspase-3 activation have not been completely defined in septic cardiomyocytes.
Akt, a serine/threonine and prosurvival kinase, is involved in the regulation of caspase-3 activation and apoptosis [10–13]. Heat shock protein 90 (Hsp90), a molecular chaperone, is essential for the proper functioning of Akt because it forms a chaperone-substrate protein complex, and a reduction in Hsp90-Akt binding results in Akt inactivation . Therefore, it is possible that activated calpain induces caspase-3 activation and apoptosis via cleavage of its substrate Hsp90, a key Akt regulator protein, and inhibition of Akt activation [15, 16]. Therefore, we hypothesized that calpain activation would adversely affect the Hsp90/Akt signaling pathway and induce caspase-3 activation and apoptosis during sepsis.
In this study, we have determined the role of the Hsp90/Akt pathway in lipopolysaccharide (LPS)-induced myocardial caspase-3 activation and apoptosis. We observed that the inhibition of calpain reduced Hsp90 degradation and increased Akt activity, thereby preventing caspase-3 activation and apoptosis in septic mice. These results indicate that the Hsp90/Akt pathway negatively regulates LPS-induced myocardial caspase-3 activation and apoptosis.
Pathogen-free and wild-type adult C57BL/6 mice (male, 6–8 weeks, 25–30 g) were used. Animals were housed under a 12 h light–dark cycle with food and water available ad libitum. All of the experimental procedures were approved by the Institutional Animal Ethics Committee of Peking Union Medical College.
In this study, a total of 90 mice were divided into six different groups with 15 mice in each group). The control mice (sham group) were injected intraperitoneally (i.p) with 100 μl PBS solution, and the LPS-treated mice were injected with LPS (4 mg/kg, i.p), which was isolated from Escherichia coli serotype 055:B5 (Sigma, St. Louis, MO) and dissolved in 100 μl PBS solution. Calpain inhibitor-Ш (10 mg/kg, i.p) or PD150606 (3 mg/kg, i.p) plus LPS treated mice were injected i.p, and the calpain inhibitors-III or PD150606 were dissolved in 80 μl DMSO. The mice were injected i.p with either calpain inhibitor-III or PD150606 alone 30 minutes before injecting LPS, and all of the mice were subjected to biological and physiological experiments at 4 h post-treatments. In addition, the time course experiments were performed at 0, 1, 2, 4, and 6 h after LPS injection, and 5 mice were used for each time point.
Calpain activity assay
Calpain activity was measured using the fluorescence substrate, N-succinyl- LLVY-AMC (Cedarlane Laboratories, Burlington, NC, USA), as previously described . This assay measures the fluorescence intensity of AMC when it is cleaved from a peptide substrate. The fluorescence intensity of the cleaved AMC was quantified by using a multilabel reader (excitation, 360 nm; emission, 460 nm, Wallac 1420, PerkinElmer, Turku, Finland), and calpain activity was determined by measuring the difference between calcium-dependent and calcium-independent fluorescence. All experiments were conducted in duplicate.
Caspase-3 activity assay
Myocardial caspase-3 activity was measured using a caspase-3 fluorescent assay kit according to the manufacturer’s protocol (BIOMOL Research Laboratories) . Briefly, the whole hearts were isolated from mice and homogenized. Duplicate sets of protein samples were incubated with either Ac-DEVD-AMC, a caspase-3 substrate, or Ac-DEVD-AMC plus the inhibitor, ACDEVD-CHO, at 37°C for 2 h before the measurements were obtained by using a fluorescent spectrophotometer (excitation at 380 nm, emission at 405 nm, Wallac 1420, PerkinElmer, Turku, Finland). The signals obtained from the inhibitor-treated samples served as the background.
Western blotting analysis
The proteins (40 μg each lane) from each sample were subjected to SDS-PAGE using a 10% gel and subsequently electrotransferred onto membranes. The expression levels of Hsp90, p-Akt/Akt and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins were determined by first probing the blots with specific antibodies (1:1000, Cell Signaling, Danvers, MA) and then by performing enhanced chemiluminescence detection.
In situdetection of apoptotic cells
To identify and quantitatively assess the number of cells that underwent apoptosis in the heart, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay was performed on the paraffin-embedded sections of murine heart tissues using an in situ apoptosis detection kit (Roche Molecular Biochemicals), according to the manufacturer’s instructions, based on our previous report . All of the sections were analyzed using a Leica microscope.
Histological preparation and immunohistochemistry
The murine heart tissues were fixed in a 4% paraformaldehyde-PBS solution over a period of 24 h and subjected to standard histological procedures for paraffin-embedded sections. Paraffin sections (5 μm thickness) were sliced for performing immunohistochemical experiments and incubated with rabbit anti-mouse Hsp90 and p-Akt antibodies (1:100, Cell Signaling) overnight at 4°C. The expression levels of Hsp90 and p-Akt in the heart tissues were visualized by employing routine immunoperoxidase techniques. The sections were first counterstained with hematoxylin and then dehydrated and mounted using routine methodologies. The secondary goat polyclonal antibody (IgG) was purchased from Jackson (USA).
All of the data are presented as the mean ± SE. The statistical differences between the two groups were compared using unpaired Student’s t-test. One-way ANOVA followed by the Student-Newman-Keuls test was used for performing multigroup comparisons. P < 0.05 was considered to be statistically significant.
Myocardial calpain activities increased in septic mice
Decrease in the myocardial p-Akt and Hsp90 proteins in LPS-challenged C57 mice
The Hsp90/Akt pathway is a well-known signaling pathway that is anti-apoptotic and promotes cell survival in a variety of conditions including sepsis [16, 19–22]. It has been recently demonstrated that calpain decreased phospho-Akt (p-Akt, Thy308) levels and inhibited the Akt pathway . In the LPS-challenged C57 mice, the total Akt protein content did not change; however, there was a significant time-dependent reduction in the amounts of p-Akt and Hsp90, the decrease in myocardial Hsp90/p-Akt expression was maximal at approximately 4 h. Blockade of calpain activation by either calpain inhibitor-β or PD150606 prevented the down-regulation of Hsp90 protein and promoted Akt activation; this was demonstrated by an increase in p-Akt protein levels (Figure 1A, B). Therefore, these results indicate that the observed decrease in Hsp90/p-Akt protein was mediated by calpain. Meanwhile, the caspase-3 activity was accordingly increased in the LPS-challenged mice (Figure 1C), and the calpain inhibitors, calpain inhibitor-Ш and PD150606, prevented the activation of caspase-3.
Localization of Hsp90/p-Akt protein in the myocardium
Myocardial caspase-3 activation and apoptosis in the septic heart
Blockade of Akt by the PI3K inhibitor induces caspase-3 activation
To our knowledge, this is the first study that suggests the existence of a direct link between the activation of calpain and the subsequent activation of caspase-3 with the Hsp90/Akt pathway. We observed that myocardial calpain induces caspase-3 activation and apoptosis. The underlying mechanism involves a myocardial calpain-induced decrease in Hsp90/p-Akt protein levels and inhibition of Akt signaling, which increases caspase-3 activity and apoptosis during sepsis.
It has been demonstrated that calpain activation during sepsis [7–9, 17, 23] plays an important role in caspase-3 activation and cellular apoptosis via the cleavage of pro- or anti-apoptotic proteins [24, 25]. It was observed that caspase-3 activation was involved in the process of myocardial dysfunction via the cleavage of α-actin, α-actinin, and TnT, which produced direct functional effects on the myofilament activation and contractile function . However, in our limited study we failed to notice the cleavage of Bcl-2 and Bad in response to myocardial calpain activation in the septic mouse .
Akt, a serine/threonine kinase, is downstream of phosphatidylinositol 3-kinase (PI3 kinase) and is involved in the regulation of caspase-3 activation and apoptosis. This enzyme becomes phosphorylated and activated by a number of growth factors, cytokines and hormones, inhibits caspases and exerts anti-apoptotic effects by inactivating GSK-3β , the latter activating p53 , inducing stress to the endoplasmic reticulum , phosphorylation and translocation of Bax to the mitochondria . In addition, Akt inhibits the activation of caspases and apoptosis by inhibiting Bid (the deactivator of caspase-9) and retaining cytochrome c in the mitochondria [10, 12, 13]. In our laboratory, we observed that there was a decrease in the level of p-Akt protein in LPS-treated cardiomyocytes . In this study, we observed that in the septic mice, calpain was activated and p-Akt was decreased. Further, the inhibition Akt signaling by wortmannin induced myocardial caspase-3 activation in wildtype C57 mice. These data indicate that Akt signaling plays an important role in the activation of myocardial caspase-3 during sepsis.
To investigate potential mechanisms of calpain-mediated Akt inhibition, we next determined whether calpain activation altered Hsp90 protein content and/or the interaction between Hsp90 and Akt proteins. Akt is one of Hsp90’s substrates, and therefore, Hsp90 contributes to the functional stabilization of Akt, activation of PI3K/Akt signaling pathway and cell survival. In addition, Hsp90 regulates Akt activity by inhibiting its dephosphorylation and proteosomal degradation [31, 32]. The Hsp90/Akt pathway is an important survival and antiapoptic pathway in a variety cells and settings because the cleavage of Hsp90 in Akt/Hsp90 complex appears to be very important in the destabilization of the Akt/Hsp90 complex and in the triggering of apoptotic signals [20–22]. As Hsp90 has been demonstrated to be a substrate of calpain in the diaphragm muscle of the rat, calpain activation by supplementation with Ca2+ in vitro led to the cleavage of Hsp90 and caused inhibition of the Akt signaling pathway . These results suggest that calpain activation may diminish Hsp90-Akt binding and consequently inactivate the Akt signaling pathway. In this study, the expression levels of the myocardial Hsp90 protein were decreased in response to calpain activation, suggesting that myocardial calpain cleaved Hsp90, which then induced p-Akt degradation and inhibition of Akt signaling in septic mice.
In this study, we found that the Hsp90/Akt signaling pathway plays a role in the induction of myocardial calpain activity, caspase-3 activation and apoptosis in the septic mice. The activated calpain induces caspase-3 activation and apoptosis via cleavage of Hsp90, an Akt molecular chaperone protein, and inhibition of Akt activation indicated by the decrease in myocardial p-Akt protein levels, which induces caspase-3 activity and apoptosis during sepsis.
Myocardial calpain and caspase-3 activity increased in the septic mice.
There was a time-dependent decrease in myocardial Hsp90/p-Akt protein levels in the septic mice.
Calpain inhibitors prevented the LPS-induced degradation of myocardial Hsp90/p-Akt protein and its expression in cardiomyocytes
The inhibition of Hsp90 by pretreatment with 17-AAG induced p-Akt degradation.
The inhibition of Akt activity by pretreatment with wortmannin resulted in caspase-3 activation in wildtype C57 murine heart tissues.
Heat shock protein 90
Tdt-Mediated Dutp Nick-End Labeling
This study was supported by grants from the National Natural Science Foundation of China (no. 81000104 and no. 81160141), the China Postdoctoral Science Foundation (2012 M510356) and the Postdoctoral Fellows Foundation of the Chinese Academy of Medical Sciences (2011-XH-9).
- Natanson C, Eichenholz PW, Danner RL, Eichacker PQ, Hoffman WD, Kuo GC, Banks SM, MacVittie TJ, Parrillo JE: Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock. J Exp Med. 1989, 169: 823-832. 10.1084/jem.169.3.823.View ArticlePubMedGoogle Scholar
- Suffredini AF, Fromm RE, Parker MM, Brenner M, Kovacs JA, Wesley RA, Parrillo JE: The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med. 1989, 321: 280-287. 10.1056/NEJM198908033210503.View ArticlePubMedGoogle Scholar
- Carlson DL, Willis MS, White DJ, Horton JW, Giroir BP: Tumor necrosis factor-alpha-induced caspase activation mediates endotoxin-related cardiac dysfunction. Crit Care Med. 2005, 33: 1021-1028. 10.1097/01.CCM.0000163398.79679.66.View ArticlePubMedGoogle Scholar
- Lancel S, Joulin O, Favory R, Goossens JF, Kluza J, Chopin C, Formstecher P, Marchetti P, Neviere R: Ventricular myocyte caspases are directly responsible for endotoxin-induced cardiac dysfunction. Circulation. 2005, 111: 2596-2604. 10.1161/CIRCULATIONAHA.104.490979.View ArticlePubMedGoogle Scholar
- Fauvel H, Marchetti P, Chopin C, Formstecher P, Neviere R: Differential effects of caspase inhibitors on endotoxin-induced myocardial dysfunction and heart apoptosis. Am J Physiol Heart Circ Physiol. 2001, 280: H1608-H1614.PubMedGoogle Scholar
- Larche J, Lancel S, Hassoun SM, Favory R, Decoster B, Marchetti P, Chopin C, Neviere R: Inhibition of mitochondrial permeability transition prevents sepsis-induced myocardial dysfunction and mortality. J Am Coll Cardiol. 2006, 48: 377-385. 10.1016/j.jacc.2006.02.069.View ArticlePubMedGoogle Scholar
- Li X, Li Y, Shan L, Shen E, Chen R, Peng T: Over-expression of calpastatin inhibits calpain activation and attenuates myocardial dysfunction during endotoxaemia. Cardiovasc Res. 2009, 83: 72-79. 10.1093/cvr/cvp100.View ArticlePubMedGoogle Scholar
- Li XP, Li L, Chen RZ, Liu TW, Wu WF, Shen E, Yang YZ, Chen HZ: Association between myocardial calpain activation and apoptosis in lipopolysaccharide-induced septic mouse model. Zhonghua Xin Xue Guan Bing Za Zhi. 2010, 38: 834-838.PubMedGoogle Scholar
- Li HL, Suzuki J, Bayna E, Zhang FM, Dalle Molle E, Clark A, Engler RL, Lew WY: Lipopolysa- ccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT(1) receptors. Am J Physiol Heart Circ Physiol. 2002, 283: H461-H467.View ArticlePubMedGoogle Scholar
- Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME: Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell. 1997, 91: 231-241. 10.1016/S0092-8674(00)80405-5.View ArticlePubMedGoogle Scholar
- Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, Donner DB: NF-κB activation by tumour necrosis factor requires the Akt serinethreonine kinase. Nature. 1999, 401: 82-85. 10.1038/43466.View ArticlePubMedGoogle Scholar
- Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC: Regulation of cell death protease caspase-9 by phosphorylation. Science. 1998, 282: 1318-1321.View ArticlePubMedGoogle Scholar
- Kennedy SG, Kandel ES, Cross TK, Hay N: Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol Cell Biol. 1999, 19: 5800-5810.View ArticlePubMedPubMed CentralGoogle Scholar
- Sato S, Fujita N, Tsuruo T: Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci USA. 2000, 97: 10832-10837. 10.1073/pnas.170276797.View ArticlePubMedPubMed CentralGoogle Scholar
- Stalker TJ, Skvarka CB, Scalia R: A novel role for calpains in the endothelial dysfunction of hyperglycemia. FASEB J. 2003, 17: 1511-1513.PubMedGoogle Scholar
- Smith IJ, Dodd SL: Calpain activation causes a proteasome- dependent increase in protein degradation and inhibits the Akt signalling pathway in rat diaphragm muscle. Exp Physiol. 2007, 92: 561-573. 10.1113/expphysiol.2006.035790.View ArticlePubMedGoogle Scholar
- Hu H, Li X, Li Y, Wang L, Mehta S, Feng Q, Chen R, Peng T: Calpain-1 induces apoptosis in pulmonary microvascular endothelial cells under septic conditions. Microvasc Res. 2009, 78: 33-39. 10.1016/j.mvr.2009.04.005.View ArticlePubMedGoogle Scholar
- Song W, Lu X, Feng Q: Tumor necrosis factor-α induces apoptosis via inducible nitric oxide synthase in neonatal mouse cardiomyocytes. Cardiovasc Res. 2000, 45: 595-602. 10.1016/S0008-6363(99)00395-8.View ArticlePubMedGoogle Scholar
- Shen E, Fan J, Peng T: Glycogen synthase kinase-3 beta suppresses tumor necrosis factor-alpha expression in cardiomyocytes during lipopolysaccharide stimulation. J Cell Biochem. 2008, 104: 329-338. 10.1002/jcb.21629.View ArticlePubMedGoogle Scholar
- Tuttle RL, Gill NS, Pugh W, Lee JP, Koeberlein B, Furth EE, Polonsky KS, Naji A, Birnbaum MJ: Regulation of pancreatic B-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha. Nat Med. 2001, 7: 1133-1137. 10.1038/nm1001-1133.View ArticlePubMedGoogle Scholar
- Stoica BA, Movsesyan VA, Lea PM, Faden AI: Ceramide-induced neuronal apoptosis is associated with dephosphorylation of Akt, BAD, FKHR, GSK-3beta, and induction of the mitochondrial- dependent intrinsic caspase pathway. Mol Cell Neurosci. 2003, 22: 365-382. 10.1016/S1044-7431(02)00028-3.View ArticlePubMedGoogle Scholar
- King TD, Bijur GN, Jope RS: Caspase-3 activation induced by inhibition of mitochondrial complex I is facilitated by glycogen synthase kinase-3beta and attenuated by lithium. Brain Res. 2001, 919: 106-114. 10.1016/S0006-8993(01)03005-0.View ArticlePubMedGoogle Scholar
- Zafrani L, Gerotziafas G, Byrnes C, Hu X, Perez J, Lévi C, Placier S, Letavernier E, Leelahavanichkul A, Haymann JP, Elalamy I, Miller JL, Star RA, Yuen PS, Baud L: Calpastatin controls polymicrobial sepsis by limiting procoagulant microparticle release. Am J Respir Crit Care Med. 2012, 185: 744-755. 10.1164/rccm.201109-1686OC.View ArticlePubMedPubMed CentralGoogle Scholar
- Tan Y, Wu C, De Veyra T, Greer PA: Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli. J Biol Chem. 2006, 281: 17689-17698. 10.1074/jbc.M601978200.View ArticlePubMedGoogle Scholar
- Goll DE, Thompson VF, Li H, Wei W, Cong J: The calpain system. Physiol Rev. 2003, 83: 731-801.View ArticlePubMedGoogle Scholar
- Communal C, Sumandea M, de Tombe P, Narula J, Solaro RJ, Hajjar RJ: Functional consequences of caspase activation in cardiac myocytes. PNAS. 2002, 99: 6252-6256. 10.1073/pnas.092022999.View ArticlePubMedPubMed CentralGoogle Scholar
- Pap M, Cooper GM: Role of translation initiation factor 2B in control of cell survival by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 β signaling pathway. Mol Cell Biol. 2002, 22: 578-586. 10.1128/MCB.22.2.578-586.2002.View ArticlePubMedPubMed CentralGoogle Scholar
- Watcharasit P, Bijur GN, Song L, Zhu J, Chen X, Jope RS: Glycogen synthase kinase-3 β(GSK3 β) binds to and promotes the actions of p53. J Biol Chem. 2003, 278: 48872-48879. 10.1074/jbc.M305870200.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim AJ, Shi Y, Austin RC, Werstuck GH: Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3. J Cell Sci. 2005, 118: 89-99. 10.1242/jcs.01562.View ArticlePubMedGoogle Scholar
- Linseman DA, Butts BD, Precht TA, Phelps RA, Le SS, Laessig TA, Bouchard RJ, Florez-McClure ML, Heidenreich KA: Glycogen synthase kinase-3 β phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis. J Neurosci. 2004, 24: 9993-10002. 10.1523/JNEUROSCI.2057-04.2004.View ArticlePubMedGoogle Scholar
- Neckers L: HSP90 Inhibitor as novel cancer chemotherapeutic agent. Trends Mol Med. 2002, 8: S55-S61. 10.1016/S1471-4914(02)02316-X.View ArticlePubMedGoogle Scholar
- Jeon YK, Park CH, Kim KY, Li YC, Kim J, Kim YA, Paik JH, Park BK, Kim CW, Kim YN: The heat-shock protein 90-inhibitor, geldanamy- cin induces apoptosis cell death in Epstein-Barr virus-positive NK/T-cell lymphoma by Akt down-regulation. J Pathol. 2007, 213: 170-179. 10.1002/path.2219.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/13/8/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.