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TNF-α-mediated cardiomyocyte apoptosis involves caspase-12 and calpain
BBRC Biochemical and Biophysical Research Communications 345 (2006) 1558–1564 www.elsevier.com/locate/ybbrc
TNF-a-mediated cardiomyocyte apoptosis involves caspase-12 and calpain Gagan Bajaj, Rajendra K. Sharma
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Department of Pathology and Laboratory Medicine, College of Medicine, Cancer Research Unit, Saskatchewan Cancer Agency, 20 Campus Drive, University of Saskatchewan, Saskatoon, Sask., Canada S7N 4H4 Received 13 April 2006 Available online 17 May 2006
Abstract Following ischemia-reperfusion, there is a sustained increase of TNF-a both locally in the heart as well as in circulating levels in blood. While TNF-a has been implicated in cardiomyocyte apoptosis which occurs in several cardiomyopathies, the molecular pathways by which TNF-a induces apoptosis in these cells are not fully elucidated. We investigated the role of the two families of cysteine proteases, caspases and calpains, which are known to participate in apoptotic cell death. The effect of the highly specific calpain inhibitor, Z-LLY-fmk, and the caspase pathways involved in TNF-a-mediated apoptosis of the HL-1 cardiomyocyte cell line were examined. Activation of the downstream caspase-3, and the cleavage of poly ADP-ribose polymerase (PARP) were observed in a time-dependent manner upon treatment with TNFa. Caspase-12, but not caspase-9, was activated in response to TNF-stimulation, indicating that an endoplasmic reticulum (ER)/calciumdependent pathway may be involved. In HL-1 cardiomyocytes, TNF-a-induced apoptosis appears to be mediated by calpain as apoptotic changes were abrogated in the presence of the highly specific calpain inhibitor, Z-LLY-fmk. In conclusion, our results suggest that TNFa-mediated apoptosis in HL-1 cardiomyocytes follows the caspase-12 apoptotic pathway that involves calpain. Ó 2006 Elsevier Inc. All rights reserved. Keywords: HL-1 cardiomyocytes; Apoptosis; TNF-a; Caspases; Calpains
Cardiomyocyte apoptosis contributes to the development of heart failure in ischemic and non-ischemic cardiomyopathies. Programmed cell death of myocardial cells has been correlated with the severity of cardiac arrest, chronic heart failure, ischemia, arrhythmogenic right ventricular dysplasia (characterized by the replacement of myocardial cells with fat and fibrous tissue), and viral myocarditis. The degree of myocardial apoptosis is a reflection of myocardial infarct size [1]. The pluripotent cytokine TNF-a has been implicated in complications arising subsequent to ischemic injury of cardiac tissue [1]. TNF-a can induce a wide range of biological effects including cell differentiation,proliferation, apoptosis, and multiple pro-inflammatory effects depending on the cell type and state of cellular activation [2].
*
Corresponding author. Fax: +1 3066552635. E-mail address: [email protected] (R.K. Sharma).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.059
In a short-term context, physiological concentrations of TNF-a are capable of inducing hypertrophy of the adult mammalian cardiomyocyte [3]. However, the sustained long-term overexpression of TNF-a may be maladaptive, provoking the induction of cardiac myocyte apoptosis [4]. Following ischemic stress, elevated levels of circulating TNF-a have been reported [5] raising the possibility of its direct involvement in subsequent heart failure [6]. Heart failure is also associated with an inflammatory state wherein myocardial and macrophage production of TNF-a can cause apoptosis of surrounding tissue. This localized inflammation is recognized as one of the contributing factors towards progression of heart failure associated with a poor prognosis. In fact, transgenic mice with cardiomyocyte-restricted overexpression of TNF-a develop myocardial inflammation, severe myocyte hypertrophy, and multiple signs of heart failure [7]. Proposed mechanisms by which TNF disrupts myocardial performance include cytotoxicity, oxidant stress,
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aberrant excitation–contraction coupling, myocyte apoptosis and the induction of other cardiac depressants such as IL-1, IL-2,and IL-6 [1]. TNF-a has been shown to affect cardiomyocyte contractility by disturbing calcium homeostasis [8] and eventually leading to both systolic and diastolic dysfunction. The direct effects of TNF-a on cardiomyocyte apoptosis have been reported to involve a sphingosine-dependent mechanism [9]. However, information on the caspases involved, and a more detailed picture of the actual TNF-a-mediated cardiomyocyte apoptosis needs to be clarified. Initiation and execution of apoptosis relies on a complex network of cysteine proteases named caspases which regulate proteolysis during apoptotic cell death. Functionally, caspases can be placed in two main groups: initiator and effector caspases. Initiator caspases are first to be activated upon the commitment of the cell to die. They are responsible for cleaving and activating the effector caspases-3, -6, and -7 which in turn act on specific cellular substrates. Three pathways known to activate the caspase cascade include (1) the ligand-activated caspase-8 pathway, (2) the mitochondrial pathway involving cytochrome c as well as the apoptosome-associated caspase-9 and -3, the pathway initiated by ER (endoplasmic reticulum) stress and activation of caspase-12 [10]. Different proapoptotic stimuli, including TNF, LPS, and FasL treatments can cause caspase-12 processing, which then actively contributes to the apoptotic progression [11]. In particular, caspase-12 activation has been associated with cerebral ischemia [12]. Even though activation of the caspase family of proteases represents a hallmark of the apoptotic process, apoptosis may also occur in the absence of measurable caspase activity, suggesting that other non-caspase proteases, including granzymes, calpains, and cathepsins, may orchestrate the induction of apoptosis [13]. Apoptosis induced by the disruption of intracellular calcium homeostasis has been shown to involve m-calpain mediated caspase-12 cleavage and activation [14]. Interestingly, m-calpain cleaves the apoptotic inhibitor Bcl-xL, and treatment with calpain inhibitors I and II, E64d can block cleavage and arrest apoptosis [14]. The significance of maintaining intracellular calcium homeostasis is especially relevant in the case of cardiomyocytes. Calcium homeostasis in these cells is a complex process regulated by a well-developed sarcoplasmic reticulum (SR) and associated calcium channels, exchangers, and pumps, whose activity may be altered during apoptosis. Calpains are calcium-dependent cysteine proteases that have been implicated in post-ischemic cell death. Calpain inhibitors have been reported to protect the heart from ischemia-reperfusion injury [15,16]. Caspase-12 is responsible for ER stress-induced but not mitochondria-mediated cell death. Since there have been some reports on cross-talk between the two families,we decided to examine the effect of the highly specific calpain inhibitor Z-LLY-fmk [17] on the induction of apoptosis by TNF-a in HL1 cardiomyocytes. The murine cardiomyocyte cell line HL-1, which
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maintains phenotypic characteristics of adult cardiomyocytes, was taken as our experimental system for studying the effects of short term as opposed to long-term stimulation by TNF-a. Another pathophysiological effect of TNF-a is to induce hypertrophy of cardiac myocytes. Previous reports indicate the involvement of the PI3-kinase-Akt/PKB pathway in the TNF-a-induced hypertrophic response in cardiacmyocytes [18,19]. Despite the number of reports on the cardiomyocyte response to TNF-a, the picture is still not clear regarding how one cytokine can induce two seemingly contradictory outcomes. In the present study, we also examined the induction of the PI3-kinase-Akt/PKB pathway in cultured HL-1 cardiacmyocytes to gain insight into the mechanism of TNF-a-induced apoptosis. Materials and methods Materials Claycomb medium was obtained from JRH Biosciences, Lenexa, KS (USA), fetal bovine serum, L-glutamine, and penicillin–streptomycin were purchased from Invitrogen (Canada). PVDF membrane and appropriate secondary antibodies conjugated to horseradish peroxidase were purchased from Bio-Rad Laboratories (Canada). Anti-caspase-3, -9, -12 and anti-cleaved caspase-3 (Asp 175) as well as anti-cleaved PARP (Asp 214) antibodies were obtained from cell signaling technology (Canada). Chemiluminescence reagent was purchased from NEN Life Science (USA). Protease inhibitor cocktail for mammalian cell culture and other analytical grade reagents were purchased from Sigma (Canada). Electrophoresis and Bradford protein assay reagents were obtained from Bio-Rad Laboratories (Canada). Calpain inhibitor Z-LLY-fmk was purchased from Bio Vision (USA). Methods Culture of HL-1 cells. HL-1 cells [20] were maintained as previously described [21] using Claycomb medium supplemented with 10% (vol/vol) fetal bovine serum 4 mM L-glutamine 10 lM noradrenaline (norepinephrine)and 1% (vol/vol) penicillin–streptomycin. Cells were plated onto fibronectin-gelatin-coated plates and grown at 37 °C in an atmosphere of 5% CO2. Treatments. TNF-a and Z-LLY-fmk treatments were carried out in serum-free media. Cells were incubated with 10 ng/ml TNF-a for varying durations to study the short and long-term effects. In order to test the efficiency of calpain inhibitor Z-LLY-fmk in limiting TNF-a-induced apoptosis, a pretreatment of 10 lM Z-LLY-fmk was carried out for 30 min in serum-free media, followed by a PBS wash, and further incubation with 10 ng/ml TNF-a (in serum-free media) for the desired durations. Western blot analysis. After the treatments, cells were washed with ice cold PBS and cells were lysed on ice using 1 mL lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM sucrose, 2 mM sodium orthovanadate, 80 mM b-glycerophosphate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM EGTA, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 1 mM phenylmethylsulphonyl fluoride, and 100 ll of protease inhibitor cocktail containing 8.0 lM aprotinin, 0.2 mM leupeptin, 0.4 mM bestatin, and 0.15 mM pepstatin. Cells were scraped off the plates and kept on ice for 30 min. Samples were then centrifuged at 13,000g for 2 min at 4 °C and supernatants were stored at 80 °C until use. Aliquots (20 lg protein) were resolved by SDS–PAGE and proteins were transferred on to a blotting grade PVDF membrane. The blots were blocked with 5% non-fat dried milk/0.02% PBS-Tween 20 for 1 h at room temperature and then probed with the appropriate antibody overnight in a cold room. The
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membranes were washed and incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase (1:10,000) for 1 h at room temperature. After further washing steps, visualization was carried out using a chemiluminescence reagent.
Results Short-term activation of Akt by TNF-a While TNF-a is essential for maintaining cellular homeostasis in the heart, elevated TNF-a levels can induce hypertrophy of myocytes [3]. In rats, TNF-a infusion causes a significant increase of left ventricular myocyte cross-sectional areas [22]. Treatment of cells with TNF-a can lead to enlargement of cell surface area and stimulation of protein synthesis by stimulating the PI3-kinase-Akt/PKB pathway [18]. However, the role of TNF-a in the myocardium remains controversial. In order to better understand the role of TNF-a in activating the PI3-kinase-Akt/PKB axis, TNF-a stimulation (10 ng/ml) of HL-1 cardiomyocytes was carried out for various periods of time. We assessed the effect of TNF-a on the phosphorylation state of Akt/ PKB at Ser-473 as a marker for PI3-kinase-Akt/PKB pathway activation. Stimulation of cardiac myocytes with TNFa caused a marked increase in the phosphorylation at Ser473. The effect of TNF-a on Akt/PKB phosphorylation was time-dependent, detected within 5 min after addition of TNF-a and reaching baseline levels by 30 min (Fig. 1A). These results were in accordance with previous studies performed in neonatal cardiac myocytes as well as other cell types such as HeLa cells or human cervical carcinoma cells [18,23]. A long-term stimulation of HL-1 cardiomyocytes with TNF-a (10 ng/ml) was also carried out spanning a range of 0–24 h (Fig. 1B), but no appreciable changes in Akt/PKB stimulation were observed. TNF-a-induced time-dependent activation of caspase-3 in cardiomyocytes We examined whether TNF-a could induce apoptosis of the HL-1 cell line. The caspase family of aspartate-specific cysteine proteases is the central executor of apoptosis. Activation of caspase-3 by proteolytic processing of pro-caspase-3 into activated 17 and 12 kDa subunits serves as an
early marker of apoptosis in a variety of cell types. We first determined whether caspase-3 was activated by TNF-a (10 ng/ml) in HL-1 cells. In this experiment, 10 ng/ml TNF-a was added to 60% confluent HL-1 cardiomyocytes, in serum-free media for 0, 2, 4, 8, 12, and 24 h. Immunoblots of the protein lysates from each of the time points were carried out with anti-caspase-3 (Fig. 2A) and anti-cleaved caspase-3 (Fig. 2B) antibodies, respectively. The 17 kDa cleaved caspase-3 fragment was detected faintly within 2 h of TNF-a treatment, with the intensity increasing steadily up to 24 h. To confirm the activity of caspase-3, we next monitored the cleavage of its substrate, the nuclear enzyme poly ADP-ribose polymerase (PARP). Caspase-3-directed proteolysis of the 113 kDa native PARP molecule enzyme is detected in immunoblots as an 89 kDa fragment, which acts as a marker of caspase-3 activity. Time-course experiments showed PARP cleavage within 2 h of addition of TNF-a treatment with a maximum intensity at 24 h (Fig. 3B). Caspase-9-mitochondrial pathway is not involved in TNF-a-induced HL-1 cardiomyocyte apoptosis Since the caspase-9 dependent mitochondrial pathway of apoptosis may activate caspase-3, we examined the expression and activation of caspase-9 proteins in immunoblots (Fig. 4). Though pro-caspase-9 was found in all samples, we did not detect the presence of p36, p18, or p10 caspase-9 activation products indicating that the mitochondrial apoptosis pathway was not involved in TNFa-induced HL-1 cardiomyocyte apoptosis. Caspase-12 is activated in TNF-a-induced HL-1 cardiomyocyte apoptosis Caspase-12, an endoplasmic reticulum (ER)-specific caspase, participates in apoptosis under ER stress. The activity of caspase-12 was measured by immunoblotting, and a notable increase in activated fragments of caspase12 was observed with 10 ng/ml TNF-a treatment. In Fig. 3A, cleaved caspase-12 can be seen on Western blots 12 and 24 h after treatment with TNF-a as compared to controls. There also appears to be upregulation in expression levels of the 60 kDa pro-caspase-12 by 12 h
Fig. 1. Effect of TNF-a on HL-1 cells. (A) HL-1 cells treated with 10 ng/ml TNF-a for the indicated time periods. (B) HL-1 cells treated with 10 ng/ml TNF-a for the duration of 0–24 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS–PAGE, transferred onto nitrocellulose membrane, and probed with phospho-specific monoclonal antibody against residue 473 of Akt as described under Materials and methods.
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Fig. 2. HL-1 cells treated with 10 ng/ml TNF-a for the duration of 0–24 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS– PAGE, transferred onto nitrocellulose membrane, and probed with caspase-3 and cleaved caspase-3 antibodies as described under Materials and methods. (A) caspase-3; (B) cleaved caspase-3; (C) shows equal loading of proteins in all lanes as assessed by probing the blots with b-actin antibody.
Fig. 3. HL-1 cells treated with 10 ng/ml TNF-a for the duration of 0–24 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS– PAGE, transferred onto nitrocellulose membrane, and probed with caspase-12 antibody and PARP antibody to show the pathway followed during apoptosis. (A) Caspase-12 antibody; (B) PARP antibody.
Fig. 4. HL-1 cells treated with 10 ng/ml TNF-a for the duration of 0–24 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS– PAGE, transferred onto nitrocellulose membrane, and probed with caspase-9 antibody. There were no appreciable changes in the caspase-9 levels showing that the pathway followed in apoptosis was specific to caspase-12 activation.
of TNF-a treatment (Fig. 3A). It has been reported earlier that ER stress specifically induces caspase-12 protein expression. [24] These data indicate that caspase-12 plays an important role in TNF-a-induced apoptosis in HL-1 cardiomyocytes.
Calpain inhibitor can partially inhibit TNF-a-induced apoptosis Since calpain, a Ca2+-dependent protease, is required for caspase-12 activation [14,25,26], we examined whether
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Fig. 5. HL-1 cells treated with either 10 ng/ml TNF-a, or pretreated with a calpain inhibitor 10 lM Z-LLY-fmk for 30 min and followed with the TNF treatment, for the duration of 0–12 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS–PAGE, transferred onto nitrocellulose membrane, and probed with cleaved caspase-3 and PARP antibody. (A) Cleaved caspase-3; (B) cleaved PARP; (C) shows equal loading of proteins in all lanes as assessed by probing the blots with b-actin antibody. Treatment with the calpain inhibitor brought down the degree of apoptotic damage as assessed by the cleaved caspase-3 and cleaved PARP products visible.
calpain/caspase-12 activation is involved in TNF-a-induced apoptosis in HL-1 cardiomyocytes. HL-1 cells were treated with either 10 ng/ml TNF-a or given a pretreatment with 10 lM Z-LLY-fmk, a specific calpain inhibitor for 30 min followed by a PBS wash and 10 ng/ml TNF-a treatment for the duration of 1, 2, 4, 8, and 12 h. Caspase-3 activation served as the end point marker for apoptosis and immunoblots was carried out on the protein lysates using anti-cleaved caspase-3 and anti-cleaved PARP antibodies. Pretreatment for 30 min with the calpain inhibitor Z-LLY-fmk resulted in reduction of TNF-a-mediated apoptosis in a time-dependent manner. Fig. 5A and B show that the calpain inhibitor, Z-LLY-fmk, reduced TNF-ainduced apoptosis. This was evident through diminished intensity of cleaved products of both caspase-3 (Fig. 5A) and PARP (Fig. 5B) in the lanes corresponding to treatment for 2, 4, and 8 h. By 12 h, however, the effect of the calpain inhibitor appeared to diminish because the intensity of both cleaved caspase-3 and PARP bands appeared similar to the corresponding TNF-a-treated control. Discussion This study examined the effect and signal transduction pathway of TNF-a-mediated apoptosis in the HL-1 cardiomyocyte cell line. The role of TNF-a in the myocardium is controversial, as TNF-a is considered responsible for causing the conflicting consequences of both hypertrophy and apoptosis. With these reports in mind, we tested the range of TNF-a exposures responsible for activating
either the Akt/PKB pathway responsible for growth and hypertrophy, or the programmed cell death pathway for apoptosis. First, we showed that the growth enhancing Akt/PKB pathway was rapidly stimulated within 5 min (Fig. 1A), peaked at 20 min, and then declined. These results were in accordance with earlier studies conducted in cardiac myocytes (18). However, the previous studies did not explore the long-term implications of TNF-a stimulation in their hypothesis. Under our experimental setup, longer exposure of up to 24 h to TNF-a did not show appreciable stimulation of the Akt/PKB pathway (Fig. 1B), indicating that sustained long-term exposure to TNF-a was not solely responsible for myocardial hypertrophy. Although previous studies have implicated TNF-a in the apoptosis of cardiomyocytes, the signal transduction pathways responsible were not defined [9,19,27,28]. This study aimed to resolve the pathways involved in TNFa-induced apoptosis of the HL-1 cardiomyocyte cell line. Sustained long-term exposure of up to 24 h to TNF-a was observed to induce apoptosis in the HL-1 cells as evidenced by the activation of caspase-3 and the PARP cleavage (Figs. 2A, B, and 3B). Caspase-3 has been identified as a key protease in the execution of apoptosis. There are several pathways by which caspase-3 could be activated, one possible route being the well described release of mitochondrial cytochrome c, formation of the apoptosome, and activation of caspase-9, which in turn activates the effector caspase-3. Interestingly, the initiator caspase-9 did not show any activation under similar circumstances (Fig. 4), indicating that
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the mitochondrial apoptotic pathway was not involved in this instance. MEFs from caspase-9 / knockout mice have been reported to be sensitive to TNF-a-directed apoptosis indicating that the TNF-induced pathway of apoptosis does not depend on caspase-9 [29]. Next, we identified that caspase-12 was activated upon TNF-a stimulation of HL-1 cardiomyocytes (Fig. 3A). TNF-a-directed apoptosis in HEK293T cells has been demonstrated to involve activated caspase-12 [30]. Several reports have demonstrated an essential role of the Ca2+ activated protease calpain in caspase-12 mediated apoptosis [14,31,32]. Apoptotic cell death induced by IFN-c/TNF-a treatment of MIN6N8 insulinoma cells has been shown to involve Ca2+ mediated calpain activation [33]. In our study pretreatment with the calpain specific inhibitor Z-LLY-fmk reduced the degree of apoptosis in HL-1 cells. This indicated the involvement of calpain in TNF-a-directed apoptosis (Fig. 5). An intriguing explanation for caspase-3 activation has been investigated in earlier reports exploring direct activation of caspase-3 by either caspase-12 or calpain [31,34]. Calcium-mediated activation of m-calpain in a cerebral hypoxia-ischemia rat model was found to be directly responsible for subsequent activation of caspase-3, thus representing a tentative pathway of ‘‘pathologicalapoptosis’’ [34]. In conclusion, this study provides the first demonstration of the essential role of the calpain/caspase-12 apoptotic pathway in TNF-a-induced cardiac apoptosis. Our observations on calpain participation in TNF-a-mediated cardiomyocyte apoptosis add another dimension to understanding the role of calpain in heart dysfunction. Several studies have suggested the use of calpain inhibitors in treating heart dysfunction [16,35,36]. The results have implications for understanding the mechanisms and efficiency of using calpain inhibitor drugs in treating heart dysfunction. Acknowledgments We are grateful to Dr. W.C. Claycomb of the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, LA, USA for the generous gift of HL-1 cell line. The excellent technical assistance of Ms. Theresa George is gratefully acknowledged. This work is supported by the Heart and Stroke Foundation of Saskatchewan. References [1] D.R. Meldrum, Tumor necrosis factor in the heart, Am. J. Physiol. 274 (1998) R577–R595. [2] B.B. Aggarwal, Signalling pathways of the TNF superfamily: a double-edged sword, Nat. Rev. Immunol. 3 (2003) 745–756. [3] T. Yokoyama, M. Nakano, J.L. Bednarczyk, B.W. McIntyre, M. Entman, D.L. Mann, Tumor necrosis factor-alpha provokes a hypertrophic growth response in adult cardiac myocytes, Circulation 95 (1997) 1247–1252.
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TNF-a-mediated cardiomyocyte apoptosis involves caspase-12 and calpain Gagan Bajaj, Rajendra K. Sharma
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Department of Pathology and Laboratory Medicine, College of Medicine, Cancer Research Unit, Saskatchewan Cancer Agency, 20 Campus Drive, University of Saskatchewan, Saskatoon, Sask., Canada S7N 4H4 Received 13 April 2006 Available online 17 May 2006
Abstract Following ischemia-reperfusion, there is a sustained increase of TNF-a both locally in the heart as well as in circulating levels in blood. While TNF-a has been implicated in cardiomyocyte apoptosis which occurs in several cardiomyopathies, the molecular pathways by which TNF-a induces apoptosis in these cells are not fully elucidated. We investigated the role of the two families of cysteine proteases, caspases and calpains, which are known to participate in apoptotic cell death. The effect of the highly specific calpain inhibitor, Z-LLY-fmk, and the caspase pathways involved in TNF-a-mediated apoptosis of the HL-1 cardiomyocyte cell line were examined. Activation of the downstream caspase-3, and the cleavage of poly ADP-ribose polymerase (PARP) were observed in a time-dependent manner upon treatment with TNFa. Caspase-12, but not caspase-9, was activated in response to TNF-stimulation, indicating that an endoplasmic reticulum (ER)/calciumdependent pathway may be involved. In HL-1 cardiomyocytes, TNF-a-induced apoptosis appears to be mediated by calpain as apoptotic changes were abrogated in the presence of the highly specific calpain inhibitor, Z-LLY-fmk. In conclusion, our results suggest that TNFa-mediated apoptosis in HL-1 cardiomyocytes follows the caspase-12 apoptotic pathway that involves calpain. Ó 2006 Elsevier Inc. All rights reserved. Keywords: HL-1 cardiomyocytes; Apoptosis; TNF-a; Caspases; Calpains
Cardiomyocyte apoptosis contributes to the development of heart failure in ischemic and non-ischemic cardiomyopathies. Programmed cell death of myocardial cells has been correlated with the severity of cardiac arrest, chronic heart failure, ischemia, arrhythmogenic right ventricular dysplasia (characterized by the replacement of myocardial cells with fat and fibrous tissue), and viral myocarditis. The degree of myocardial apoptosis is a reflection of myocardial infarct size [1]. The pluripotent cytokine TNF-a has been implicated in complications arising subsequent to ischemic injury of cardiac tissue [1]. TNF-a can induce a wide range of biological effects including cell differentiation,proliferation, apoptosis, and multiple pro-inflammatory effects depending on the cell type and state of cellular activation [2].
*
Corresponding author. Fax: +1 3066552635. E-mail address: [email protected] (R.K. Sharma).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.059
In a short-term context, physiological concentrations of TNF-a are capable of inducing hypertrophy of the adult mammalian cardiomyocyte [3]. However, the sustained long-term overexpression of TNF-a may be maladaptive, provoking the induction of cardiac myocyte apoptosis [4]. Following ischemic stress, elevated levels of circulating TNF-a have been reported [5] raising the possibility of its direct involvement in subsequent heart failure [6]. Heart failure is also associated with an inflammatory state wherein myocardial and macrophage production of TNF-a can cause apoptosis of surrounding tissue. This localized inflammation is recognized as one of the contributing factors towards progression of heart failure associated with a poor prognosis. In fact, transgenic mice with cardiomyocyte-restricted overexpression of TNF-a develop myocardial inflammation, severe myocyte hypertrophy, and multiple signs of heart failure [7]. Proposed mechanisms by which TNF disrupts myocardial performance include cytotoxicity, oxidant stress,
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aberrant excitation–contraction coupling, myocyte apoptosis and the induction of other cardiac depressants such as IL-1, IL-2,and IL-6 [1]. TNF-a has been shown to affect cardiomyocyte contractility by disturbing calcium homeostasis [8] and eventually leading to both systolic and diastolic dysfunction. The direct effects of TNF-a on cardiomyocyte apoptosis have been reported to involve a sphingosine-dependent mechanism [9]. However, information on the caspases involved, and a more detailed picture of the actual TNF-a-mediated cardiomyocyte apoptosis needs to be clarified. Initiation and execution of apoptosis relies on a complex network of cysteine proteases named caspases which regulate proteolysis during apoptotic cell death. Functionally, caspases can be placed in two main groups: initiator and effector caspases. Initiator caspases are first to be activated upon the commitment of the cell to die. They are responsible for cleaving and activating the effector caspases-3, -6, and -7 which in turn act on specific cellular substrates. Three pathways known to activate the caspase cascade include (1) the ligand-activated caspase-8 pathway, (2) the mitochondrial pathway involving cytochrome c as well as the apoptosome-associated caspase-9 and -3, the pathway initiated by ER (endoplasmic reticulum) stress and activation of caspase-12 [10]. Different proapoptotic stimuli, including TNF, LPS, and FasL treatments can cause caspase-12 processing, which then actively contributes to the apoptotic progression [11]. In particular, caspase-12 activation has been associated with cerebral ischemia [12]. Even though activation of the caspase family of proteases represents a hallmark of the apoptotic process, apoptosis may also occur in the absence of measurable caspase activity, suggesting that other non-caspase proteases, including granzymes, calpains, and cathepsins, may orchestrate the induction of apoptosis [13]. Apoptosis induced by the disruption of intracellular calcium homeostasis has been shown to involve m-calpain mediated caspase-12 cleavage and activation [14]. Interestingly, m-calpain cleaves the apoptotic inhibitor Bcl-xL, and treatment with calpain inhibitors I and II, E64d can block cleavage and arrest apoptosis [14]. The significance of maintaining intracellular calcium homeostasis is especially relevant in the case of cardiomyocytes. Calcium homeostasis in these cells is a complex process regulated by a well-developed sarcoplasmic reticulum (SR) and associated calcium channels, exchangers, and pumps, whose activity may be altered during apoptosis. Calpains are calcium-dependent cysteine proteases that have been implicated in post-ischemic cell death. Calpain inhibitors have been reported to protect the heart from ischemia-reperfusion injury [15,16]. Caspase-12 is responsible for ER stress-induced but not mitochondria-mediated cell death. Since there have been some reports on cross-talk between the two families,we decided to examine the effect of the highly specific calpain inhibitor Z-LLY-fmk [17] on the induction of apoptosis by TNF-a in HL1 cardiomyocytes. The murine cardiomyocyte cell line HL-1, which
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maintains phenotypic characteristics of adult cardiomyocytes, was taken as our experimental system for studying the effects of short term as opposed to long-term stimulation by TNF-a. Another pathophysiological effect of TNF-a is to induce hypertrophy of cardiac myocytes. Previous reports indicate the involvement of the PI3-kinase-Akt/PKB pathway in the TNF-a-induced hypertrophic response in cardiacmyocytes [18,19]. Despite the number of reports on the cardiomyocyte response to TNF-a, the picture is still not clear regarding how one cytokine can induce two seemingly contradictory outcomes. In the present study, we also examined the induction of the PI3-kinase-Akt/PKB pathway in cultured HL-1 cardiacmyocytes to gain insight into the mechanism of TNF-a-induced apoptosis. Materials and methods Materials Claycomb medium was obtained from JRH Biosciences, Lenexa, KS (USA), fetal bovine serum, L-glutamine, and penicillin–streptomycin were purchased from Invitrogen (Canada). PVDF membrane and appropriate secondary antibodies conjugated to horseradish peroxidase were purchased from Bio-Rad Laboratories (Canada). Anti-caspase-3, -9, -12 and anti-cleaved caspase-3 (Asp 175) as well as anti-cleaved PARP (Asp 214) antibodies were obtained from cell signaling technology (Canada). Chemiluminescence reagent was purchased from NEN Life Science (USA). Protease inhibitor cocktail for mammalian cell culture and other analytical grade reagents were purchased from Sigma (Canada). Electrophoresis and Bradford protein assay reagents were obtained from Bio-Rad Laboratories (Canada). Calpain inhibitor Z-LLY-fmk was purchased from Bio Vision (USA). Methods Culture of HL-1 cells. HL-1 cells [20] were maintained as previously described [21] using Claycomb medium supplemented with 10% (vol/vol) fetal bovine serum 4 mM L-glutamine 10 lM noradrenaline (norepinephrine)and 1% (vol/vol) penicillin–streptomycin. Cells were plated onto fibronectin-gelatin-coated plates and grown at 37 °C in an atmosphere of 5% CO2. Treatments. TNF-a and Z-LLY-fmk treatments were carried out in serum-free media. Cells were incubated with 10 ng/ml TNF-a for varying durations to study the short and long-term effects. In order to test the efficiency of calpain inhibitor Z-LLY-fmk in limiting TNF-a-induced apoptosis, a pretreatment of 10 lM Z-LLY-fmk was carried out for 30 min in serum-free media, followed by a PBS wash, and further incubation with 10 ng/ml TNF-a (in serum-free media) for the desired durations. Western blot analysis. After the treatments, cells were washed with ice cold PBS and cells were lysed on ice using 1 mL lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM sucrose, 2 mM sodium orthovanadate, 80 mM b-glycerophosphate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM EGTA, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 1 mM phenylmethylsulphonyl fluoride, and 100 ll of protease inhibitor cocktail containing 8.0 lM aprotinin, 0.2 mM leupeptin, 0.4 mM bestatin, and 0.15 mM pepstatin. Cells were scraped off the plates and kept on ice for 30 min. Samples were then centrifuged at 13,000g for 2 min at 4 °C and supernatants were stored at 80 °C until use. Aliquots (20 lg protein) were resolved by SDS–PAGE and proteins were transferred on to a blotting grade PVDF membrane. The blots were blocked with 5% non-fat dried milk/0.02% PBS-Tween 20 for 1 h at room temperature and then probed with the appropriate antibody overnight in a cold room. The
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membranes were washed and incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase (1:10,000) for 1 h at room temperature. After further washing steps, visualization was carried out using a chemiluminescence reagent.
Results Short-term activation of Akt by TNF-a While TNF-a is essential for maintaining cellular homeostasis in the heart, elevated TNF-a levels can induce hypertrophy of myocytes [3]. In rats, TNF-a infusion causes a significant increase of left ventricular myocyte cross-sectional areas [22]. Treatment of cells with TNF-a can lead to enlargement of cell surface area and stimulation of protein synthesis by stimulating the PI3-kinase-Akt/PKB pathway [18]. However, the role of TNF-a in the myocardium remains controversial. In order to better understand the role of TNF-a in activating the PI3-kinase-Akt/PKB axis, TNF-a stimulation (10 ng/ml) of HL-1 cardiomyocytes was carried out for various periods of time. We assessed the effect of TNF-a on the phosphorylation state of Akt/ PKB at Ser-473 as a marker for PI3-kinase-Akt/PKB pathway activation. Stimulation of cardiac myocytes with TNFa caused a marked increase in the phosphorylation at Ser473. The effect of TNF-a on Akt/PKB phosphorylation was time-dependent, detected within 5 min after addition of TNF-a and reaching baseline levels by 30 min (Fig. 1A). These results were in accordance with previous studies performed in neonatal cardiac myocytes as well as other cell types such as HeLa cells or human cervical carcinoma cells [18,23]. A long-term stimulation of HL-1 cardiomyocytes with TNF-a (10 ng/ml) was also carried out spanning a range of 0–24 h (Fig. 1B), but no appreciable changes in Akt/PKB stimulation were observed. TNF-a-induced time-dependent activation of caspase-3 in cardiomyocytes We examined whether TNF-a could induce apoptosis of the HL-1 cell line. The caspase family of aspartate-specific cysteine proteases is the central executor of apoptosis. Activation of caspase-3 by proteolytic processing of pro-caspase-3 into activated 17 and 12 kDa subunits serves as an
early marker of apoptosis in a variety of cell types. We first determined whether caspase-3 was activated by TNF-a (10 ng/ml) in HL-1 cells. In this experiment, 10 ng/ml TNF-a was added to 60% confluent HL-1 cardiomyocytes, in serum-free media for 0, 2, 4, 8, 12, and 24 h. Immunoblots of the protein lysates from each of the time points were carried out with anti-caspase-3 (Fig. 2A) and anti-cleaved caspase-3 (Fig. 2B) antibodies, respectively. The 17 kDa cleaved caspase-3 fragment was detected faintly within 2 h of TNF-a treatment, with the intensity increasing steadily up to 24 h. To confirm the activity of caspase-3, we next monitored the cleavage of its substrate, the nuclear enzyme poly ADP-ribose polymerase (PARP). Caspase-3-directed proteolysis of the 113 kDa native PARP molecule enzyme is detected in immunoblots as an 89 kDa fragment, which acts as a marker of caspase-3 activity. Time-course experiments showed PARP cleavage within 2 h of addition of TNF-a treatment with a maximum intensity at 24 h (Fig. 3B). Caspase-9-mitochondrial pathway is not involved in TNF-a-induced HL-1 cardiomyocyte apoptosis Since the caspase-9 dependent mitochondrial pathway of apoptosis may activate caspase-3, we examined the expression and activation of caspase-9 proteins in immunoblots (Fig. 4). Though pro-caspase-9 was found in all samples, we did not detect the presence of p36, p18, or p10 caspase-9 activation products indicating that the mitochondrial apoptosis pathway was not involved in TNFa-induced HL-1 cardiomyocyte apoptosis. Caspase-12 is activated in TNF-a-induced HL-1 cardiomyocyte apoptosis Caspase-12, an endoplasmic reticulum (ER)-specific caspase, participates in apoptosis under ER stress. The activity of caspase-12 was measured by immunoblotting, and a notable increase in activated fragments of caspase12 was observed with 10 ng/ml TNF-a treatment. In Fig. 3A, cleaved caspase-12 can be seen on Western blots 12 and 24 h after treatment with TNF-a as compared to controls. There also appears to be upregulation in expression levels of the 60 kDa pro-caspase-12 by 12 h
Fig. 1. Effect of TNF-a on HL-1 cells. (A) HL-1 cells treated with 10 ng/ml TNF-a for the indicated time periods. (B) HL-1 cells treated with 10 ng/ml TNF-a for the duration of 0–24 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS–PAGE, transferred onto nitrocellulose membrane, and probed with phospho-specific monoclonal antibody against residue 473 of Akt as described under Materials and methods.
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Fig. 2. HL-1 cells treated with 10 ng/ml TNF-a for the duration of 0–24 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS– PAGE, transferred onto nitrocellulose membrane, and probed with caspase-3 and cleaved caspase-3 antibodies as described under Materials and methods. (A) caspase-3; (B) cleaved caspase-3; (C) shows equal loading of proteins in all lanes as assessed by probing the blots with b-actin antibody.
Fig. 3. HL-1 cells treated with 10 ng/ml TNF-a for the duration of 0–24 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS– PAGE, transferred onto nitrocellulose membrane, and probed with caspase-12 antibody and PARP antibody to show the pathway followed during apoptosis. (A) Caspase-12 antibody; (B) PARP antibody.
Fig. 4. HL-1 cells treated with 10 ng/ml TNF-a for the duration of 0–24 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS– PAGE, transferred onto nitrocellulose membrane, and probed with caspase-9 antibody. There were no appreciable changes in the caspase-9 levels showing that the pathway followed in apoptosis was specific to caspase-12 activation.
of TNF-a treatment (Fig. 3A). It has been reported earlier that ER stress specifically induces caspase-12 protein expression. [24] These data indicate that caspase-12 plays an important role in TNF-a-induced apoptosis in HL-1 cardiomyocytes.
Calpain inhibitor can partially inhibit TNF-a-induced apoptosis Since calpain, a Ca2+-dependent protease, is required for caspase-12 activation [14,25,26], we examined whether
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Fig. 5. HL-1 cells treated with either 10 ng/ml TNF-a, or pretreated with a calpain inhibitor 10 lM Z-LLY-fmk for 30 min and followed with the TNF treatment, for the duration of 0–12 h. Twenty micrograms protein was loaded onto each lane, subjected to SDS–PAGE, transferred onto nitrocellulose membrane, and probed with cleaved caspase-3 and PARP antibody. (A) Cleaved caspase-3; (B) cleaved PARP; (C) shows equal loading of proteins in all lanes as assessed by probing the blots with b-actin antibody. Treatment with the calpain inhibitor brought down the degree of apoptotic damage as assessed by the cleaved caspase-3 and cleaved PARP products visible.
calpain/caspase-12 activation is involved in TNF-a-induced apoptosis in HL-1 cardiomyocytes. HL-1 cells were treated with either 10 ng/ml TNF-a or given a pretreatment with 10 lM Z-LLY-fmk, a specific calpain inhibitor for 30 min followed by a PBS wash and 10 ng/ml TNF-a treatment for the duration of 1, 2, 4, 8, and 12 h. Caspase-3 activation served as the end point marker for apoptosis and immunoblots was carried out on the protein lysates using anti-cleaved caspase-3 and anti-cleaved PARP antibodies. Pretreatment for 30 min with the calpain inhibitor Z-LLY-fmk resulted in reduction of TNF-a-mediated apoptosis in a time-dependent manner. Fig. 5A and B show that the calpain inhibitor, Z-LLY-fmk, reduced TNF-ainduced apoptosis. This was evident through diminished intensity of cleaved products of both caspase-3 (Fig. 5A) and PARP (Fig. 5B) in the lanes corresponding to treatment for 2, 4, and 8 h. By 12 h, however, the effect of the calpain inhibitor appeared to diminish because the intensity of both cleaved caspase-3 and PARP bands appeared similar to the corresponding TNF-a-treated control. Discussion This study examined the effect and signal transduction pathway of TNF-a-mediated apoptosis in the HL-1 cardiomyocyte cell line. The role of TNF-a in the myocardium is controversial, as TNF-a is considered responsible for causing the conflicting consequences of both hypertrophy and apoptosis. With these reports in mind, we tested the range of TNF-a exposures responsible for activating
either the Akt/PKB pathway responsible for growth and hypertrophy, or the programmed cell death pathway for apoptosis. First, we showed that the growth enhancing Akt/PKB pathway was rapidly stimulated within 5 min (Fig. 1A), peaked at 20 min, and then declined. These results were in accordance with earlier studies conducted in cardiac myocytes (18). However, the previous studies did not explore the long-term implications of TNF-a stimulation in their hypothesis. Under our experimental setup, longer exposure of up to 24 h to TNF-a did not show appreciable stimulation of the Akt/PKB pathway (Fig. 1B), indicating that sustained long-term exposure to TNF-a was not solely responsible for myocardial hypertrophy. Although previous studies have implicated TNF-a in the apoptosis of cardiomyocytes, the signal transduction pathways responsible were not defined [9,19,27,28]. This study aimed to resolve the pathways involved in TNFa-induced apoptosis of the HL-1 cardiomyocyte cell line. Sustained long-term exposure of up to 24 h to TNF-a was observed to induce apoptosis in the HL-1 cells as evidenced by the activation of caspase-3 and the PARP cleavage (Figs. 2A, B, and 3B). Caspase-3 has been identified as a key protease in the execution of apoptosis. There are several pathways by which caspase-3 could be activated, one possible route being the well described release of mitochondrial cytochrome c, formation of the apoptosome, and activation of caspase-9, which in turn activates the effector caspase-3. Interestingly, the initiator caspase-9 did not show any activation under similar circumstances (Fig. 4), indicating that
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the mitochondrial apoptotic pathway was not involved in this instance. MEFs from caspase-9 / knockout mice have been reported to be sensitive to TNF-a-directed apoptosis indicating that the TNF-induced pathway of apoptosis does not depend on caspase-9 [29]. Next, we identified that caspase-12 was activated upon TNF-a stimulation of HL-1 cardiomyocytes (Fig. 3A). TNF-a-directed apoptosis in HEK293T cells has been demonstrated to involve activated caspase-12 [30]. Several reports have demonstrated an essential role of the Ca2+ activated protease calpain in caspase-12 mediated apoptosis [14,31,32]. Apoptotic cell death induced by IFN-c/TNF-a treatment of MIN6N8 insulinoma cells has been shown to involve Ca2+ mediated calpain activation [33]. In our study pretreatment with the calpain specific inhibitor Z-LLY-fmk reduced the degree of apoptosis in HL-1 cells. This indicated the involvement of calpain in TNF-a-directed apoptosis (Fig. 5). An intriguing explanation for caspase-3 activation has been investigated in earlier reports exploring direct activation of caspase-3 by either caspase-12 or calpain [31,34]. Calcium-mediated activation of m-calpain in a cerebral hypoxia-ischemia rat model was found to be directly responsible for subsequent activation of caspase-3, thus representing a tentative pathway of ‘‘pathologicalapoptosis’’ [34]. In conclusion, this study provides the first demonstration of the essential role of the calpain/caspase-12 apoptotic pathway in TNF-a-induced cardiac apoptosis. Our observations on calpain participation in TNF-a-mediated cardiomyocyte apoptosis add another dimension to understanding the role of calpain in heart dysfunction. Several studies have suggested the use of calpain inhibitors in treating heart dysfunction [16,35,36]. The results have implications for understanding the mechanisms and efficiency of using calpain inhibitor drugs in treating heart dysfunction. Acknowledgments We are grateful to Dr. W.C. Claycomb of the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, LA, USA for the generous gift of HL-1 cell line. The excellent technical assistance of Ms. Theresa George is gratefully acknowledged. This work is supported by the Heart and Stroke Foundation of Saskatchewan. References [1] D.R. Meldrum, Tumor necrosis factor in the heart, Am. J. Physiol. 274 (1998) R577–R595. [2] B.B. Aggarwal, Signalling pathways of the TNF superfamily: a double-edged sword, Nat. Rev. Immunol. 3 (2003) 745–756. [3] T. Yokoyama, M. Nakano, J.L. Bednarczyk, B.W. McIntyre, M. Entman, D.L. Mann, Tumor necrosis factor-alpha provokes a hypertrophic growth response in adult cardiac myocytes, Circulation 95 (1997) 1247–1252.
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