Leptin protects H9c2 rat cardiomyocytes from H 2 O 2 -induced apoptosis Megumi Eguchi*, Yuantao Liu*, Eyun-Jung Shin and Gary Sweeney Department of Biology, York University, Toronto, Canada The rapid increase in the prevalence of obesity to epi- demic proportions is a serious concern, as obesity is associated with the development of many complications including type 2 diabetes, hypertension and heart failure [1]. Heart failure is a leading cause of mortality in indus- trialized countries, and is accompanied by progressive left ventricular remodeling characterized by hypertro- phy of the myocytes, impaired vascularization in the heart, abnormal extracellular matrix composition (fibro- sis) and elevated cardiomyocyte cell death [2]. However, in addition to increasing the risk for initial myocardial infarction, obesity may confer protective effects that limit cardiac remodeling post-infarction, the so-called obesity paradox [3]. Necrosis was initially viewed as the major pathway by which cardiomyocytes are lost during remodeling; however, research in the past 10–15 years has indicated that apoptosis has important pathophysio- logical consequences in the development and progres- sion of heart failure [4,5]. Indeed, the apoptotic rate is significantly increased (from 0.001% to 0.08%) in the failing heart [2]. Adipokines, collectively referring to factors derived from adipose tissue, have attracted tremendous research interest in recent years as an important mech- anistic link between obesity and various associated complications [6]. The circulating adipokine profile is altered in obese individuals, and it is now clear that development of heart failure can be directly influenced by adipokines [1]. Leptin is the product of the obese (ob) gene, and its main function is to control appetite and energy expenditure by acting on the hypothalamus [7]. There is a positive correlation between circulating leptin concentration and the body mass of an indiv- idual. This suggests the existence of leptin resistance in Keywords apoptosis; caspase; heart failure; leptin; mitochondria Correspondence G. Sweeney, Department of Biology, York University, Toronto, M3J 1P3 Canada Fax: +1 416 736 5698 Tel: +1 416 736 2100 ext. 66635 E-mail: gsweeney@yorku.ca *These authors contributed equally to this work (Received 23 January 2008, revised 25 March 2008, accepted 14 April 2008) doi:10.1111/j.1742-4658.2008.06465.x Obesity is a known risk factor for induction of myocardial infarction, but, paradoxically, may also confer a protective effect against subsequent remodeling leading to heart failure. In this study, we investigated the effect of leptin, the product of the obese (ob) gene, on cardiomyocyte apoptosis, a well-characterized component of cardiac remodeling after myocardial infarction. Exposing H9c2 cells to H 2 O 2 decreased cell viability, and this was attenuated by pretreating cells with leptin for 1 h, but not 24 h. Leptin also attenuated the ability of H 2 O 2 to increase phosphatidylserine exposure and annexin V binding. Further investigation of underlying mechanisms of leptin’s protective effect demonstrated that the H 2 O 2 -induced decrease in mitochondrial membrane potential (Y) leading to cytochrome c release was attenuated by leptin pretreatment, and this was associated with reduced translocation of the pro-apoptotic Bax protein to the mitochondrial mem- brane. Finally, leptin prevented H 2 O 2 -induced increases in caspase-3 cleav- age and activity, although again 24 h leptin pretreatment did not confer significant protection. In summary, we have demonstrated that acute leptin pretreatment mediates anti-apoptotic effects in H9c2 rat cardiomyocytes, which may be of significance in clarifying the direct impact of leptin on the heart. Abbreviations JC-1, 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazoyl carbocyanide iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PS, phosphatidylserine. 3136 FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS the hypothalamus of such individuals; however, whether the heart is leptin-resistant is still controversial [8,9]. Leptin’s action is mediated by six isoforms of leptin receptors [10]. These receptors can be classified as secreted (Ob-Re), short (Ob-Ra, c, d and f) and long (Ob-Rb) forms. The adult heart has been shown to express both long and short ObR isoforms, but pre- dominantly short forms of the receptor [11], and it has also been shown that the heart is a site of leptin production [12]. The local expression of leptin and its receptors in the heart further suggests that leptin can potentially affect cardiac function by directly acting on the heart, and this has been confirmed by several recent studies [13–15]. Exposure of cardiomyocytes to H 2 O 2 and other reac- tive oxygen species is increased in the heart, especially after short ischemia ⁄ reperfusion, and excessive oxida- tive stress contributes to the pathogenesis of heart fail- ure [16]. A high circulating leptin concentration is seen in the majority of obese individuals. In this study, we have investigated the effects of leptin on H 2 O 2 -induced cell death in H9c2 cells, the most appropriate in vitro model of cardiomyocytes currently available. This was accomplished by analyses of apoptotis [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and annexin V binding], together with investigation of the mechanistic role played by the intrinsic pathway of apoptosis (change in mitochon- drial membrane potential (Y), cytochrome c release and caspase-3 activity). Results Leptin treatment for 1 h but not 24 h protects H9c2 cells from H 2 O 2 -induced decreases in cell viability The effect of H 2 O 2 treatment on the cell viability of H9c2 cells was measured by the uptake and reduction of MTT to an insoluble formazan dye. H 2 O 2 treatment for 5 h significantly reduced the cell viability as expected, and this effect was attenuated upon 1 h pre- incubation with 6 nm leptin but not 24 h pre-incuba- tion (Fig. 1). The dose of leptin was chosen based on preliminary experiments, previous work by ourselves and others in vitro [17,18], and because it is relevant to the circulating levels observed in obesity [19]. H 2 O 2 -induced phosphatidylserine exposure is decreased by leptin treatment Appearance of phosphatidylserine (PS) in the outer leaflet of the phospholipid bilayer without disrupted integrity of the membrane is one of the earliest char- acteristics of apoptotic cells. In order to study the effect of leptin on H 2 O 2 -induced PS exposure, cells were incubated with appropriate treatments as indi- cated, and analyzed for the degree of annexin V binding to the surface of intact cells (Fig. 2A,B). Cells were counterstained with propidium iodide to allow distinction between apoptosis and necrosis. Leptin treatment alone did not affect PS exposure, but an increase in annexin V binding was observed after as little as 2 h H 2 O 2 treatment. No increase in propidium iodide staining was apparent under these conditions, but was seen in positive control experi- ments (data not shown), indicating that the cell death was predominantly due to apoptosis. Quanti- tative assessment of fluorescence (Fig. 2C) showed that 1 h leptin pretreatment significantly attenuated the level of annexin V binding detected in response to H 2 O 2 . Although apparently decreasing the effects of H 2 O 2 , 24 h leptin pretreatment did not have a significant effect. Leptin pretreatment attenuates H 2 O 2 -induced loss of mitochondrial membrane potential The mitochondrial membrane potential (Y) is a critical factor in maintaining the integrity of mitochondria and subsequent regulation of apoptosis. Loss of mito- chondrial membrane potential will lead to release of the cytochrome c from mitochondria, which in turn 0 1 24 0 1 24 Leptin (h) H 2 O 2 –+++–– Cell viability (fold over control) 0 0.2 0.4 0.6 0.8 1 1.2 * Fig. 1. Leptin pretreatment for 1 h but not 24 h attenuates the abil- ity of H 2 O 2 to decrease cell viability. H9c2 cells were treated with or without 6 n M leptin for 1 h or 24 h prior to exposure to H 2 O 2 (400 lM) for 5 h, and cell viability was measured using the MTT assay. Data represent mean ± SEM (n = 4). The asterisk indicates a statistically significant difference from H 2 O 2 treatment alone (P < 0.05). M. Eguchi et al. Regulation of cardiomyocyte apoptosis by leptin FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS 3137 activates downstream caspases to cause apoptosis [20,21]. 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimi- dazoyl carbocyanide iodide (JC-1) accumulates as aggregates in the normal hyperpolarized mitochondria, resulting in red fluorescence, but JC-1 exists in the monomeric form in apoptotic cells and stains cells green. Here we observed that untreated control cells exhibit numerous brightly stained mitochondria that emit red fluorescence (Fig. 3). Cells treated with H 2 O 2 exhibited fewer red JC-1 aggregates, and more green fluorescence of monomers appeared in the cytoplasm, indicating dissipation of the mitochondrial membrane potential. Leptin pretreatment attenuated these H 2 O 2 - induced changes (Fig. 3). Leptin pretreatment reduces cytochrome c release from mitochondria Release of cytochrome c from mitochondria is a criti- cal step in progression of the intrinsic apoptotic path- way [20,21]. H 2 O 2 treatment for 2 h increased the release of cytochrome c from mitochondria, as can be seen by the loss of co-localization of cytochrome c and mitochondria (Fig. 4). The effect of H 2 O 2 was again attenuated by preincubation with leptin for 1 h (Fig. 4). Co-localization was unaffected in cells treated with leptin alone. The mechanism whereby leptin attenuates the intrinsic pathway of apoptosis involves reduced Bax integration in the mitochondrial membrane To assess translocation of the pro-apoptotic Bax pro- tein to the mitochondrial membrane, we utilized an approach exploiting the observation that the N-termi- nal domain is only exposed and recognized by a spe- cific antibody when this protein translocates and integrates into the membrane [22]. In viable control cells, or those treated with leptin, little or no Bax immunofluorescence was observed (Fig. 5). However, when cells were exposed to H 2 O 2 , we observed pro- nounced staining for Bax, with a maximal effect after 4 h, and this was clearly attenuated in cells pretreated with leptin for 1 h (Fig. 5). A B C Fig. 2. H 2 O 2 -induced annexin V binding to the cell surface decreases with leptin pretreatment. Phosphatidylserine externali- zation was assessed via annexin V binding in the absence (A) or presence (B) of 2 h H 2 O 2 (400 lM) treatment with or without leptin pretreatment (6 n M, 1 h or 24 h). Cells were treated to allow detection of both ann- exin V (green) and propidium iodide (red), and images representative of those obtained for at least eight independent experiments are shown for each condition. The results from all experiments (n > 3) were quanti- fied, and (C) shows the mean fluorescence (±SEM). The asterisk indicates a significant difference compared with H 2 O 2 alone (P < 0.05). Regulation of cardiomyocyte apoptosis by leptin M. Eguchi et al. 3138 FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS H 2 O 2 -induced increases in caspase-3 cleavage and activity are attenuated by leptin pretreatment Caspase-3 is an executioner of apoptosis, and is involved in many important events that lead to the completion of apoptosis [23]. Cleavage of caspase-3 is indicative of activation, and in cells treated with leptin alone there was no change in the cleavage of caspase-3 compared to control. H 2 O 2 treatment increased gener- ation of the cleaved form of caspase-3, and this was attenuated by 1 h leptin pretreatment (Fig. 6A). The levels of cleaved caspase-3 correlated well with enzy- matic activity, which was increased 1.8-fold compared to control upon H 2 O 2 treatment. This effect of H 2 O 2 was again significantly reduced by leptin 1 h pretreat- ment, but not significantly by 24 h pretreatment (Fig. 6B). In order to determine the functional conse- quences of the above findings, we examined whether the protective effect of leptin on cell viability was observed after a prolonged time period subsequent to H 2 O 2 exposure. When the number of living cells, as determined by trypan blue exclusion, was counted three days after exposure to H 2 O 2 , over 2.2-fold more cells were viable when pretreated with leptin for 1 h as opposed to exposure to H 2 O 2 alone (data not shown). Discussion There has been great interest in the relationship between circulating leptin levels and the development of cardiovascular diseases, but the precise role of leptin is still controversial [24]. Hyperleptinemia, which is commonly seen in obese individuals, has been pro- posed to play a role in the development of various car- diovascular diseases [25,26]. Heart failure is a common end-stage event resulting from various cardiovascular diseases, and it is now well established that cardiomyo- cyte apoptosis is an important component of cardiac remodeling, ultimately leading to heart failure. An excellent recent study suggested that leptin can prevent the increased levels of apoptosis observed upon ageing in ob ⁄ ob mice [13]. However, the direct effect of leptin on cardiomyocyte apoptosis and the intracellular mechanisms involved are still unclear. H9c2 cells, together with use of H 2 O 2 to induce apop- tosis, have been used on many occasions as a model system to study regulation of cardiomyocyte cell death [27–29]. Here we used this model system to show the effects of short-term (1 h) and long-term (24 h) exposure of H9c2 cells to leptin on H 2 O 2 -induced cell death. Our results indicate that 1 h pretreatment with leptin is able to significantly decrease the apoptotic effects of H 2 O 2 on H9c2 cells and thus protect them from death. How- ever, when 24 h preincubation was used, a protective effect was not observed. This is not entirely without precedent, as we have previously shown that acute and chronic leptin treatments have distinct effects on insulin signaling and subsequent regulation of glucose uptake in skeletal muscle cells [30,31]. These results suggest that transient intracellular effects stimulated by acute leptin treatment play an important role in the cardioprotective role of leptin, and that the enhanced lipid accumulation found after 24 h treatment with leptin [18] may convey deleterious effects [32,33]. The effects observed after a short period of leptin exposure may be of physiological relevance given the fact that circulating leptin levels fluctuate with diurnal rhythm and are not consistently high for 24 h [34]. We have shown here that leptin’s cardioprotective effect against H 2 O 2 -induced apoptosis occurs through the prevention of activation of apoptotic markers at an early stage, including PS exposure to the outer membrane – one of the first detectable signs of apopto- sis [35]. The lack of significant propidium iodide stain- ing in our annexin V binding studies suggests that 2 h treatment with H 2 O 2 does not induce significant necro- sis in these cells. Furthermore, upon investigation of the mechanisms underlying H 2 O 2 -induced apoptosis and their regulation by leptin, we observed changes in Fig. 3. Leptin attenuates H 2 O 2 -induced mitochondrial membrane potential loss in H9c2 cells. Quiescent H9c2 cells with or without 1 h leptin (6 n M) pretreatment were exposed to 0.4 lM H 2 O 2 for 30 min. JC-1 fluorescence was measured by confocal microscopy, assessing the emission shift from green (530 nm) to red (590 nm) using 488 nm excitation. Composite red and green fluorescence is shown. Results are representative of those from three separate experiments. M. Eguchi et al. Regulation of cardiomyocyte apoptosis by leptin FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS 3139 major components of the intrinsic pathway of apopto- sis. Notably, the mechanism whereby leptin prevents activation of the intrinsic pathway of apoptosis appears to involve prevention of the H 2 O 2 -induced change in the cellular localization and activity level of the pro-apoptotic Bax protein [36] detected by immu- nofluorescence microscopy using a conformation-sensi- tive antibody [22]. Accordingly, attenuation of a decrease in mitochondrial membrane potential, and of the subsequently increased cytochrome c release and caspase-3 activation was also observed in cells pre- treated with leptin. The theory of selective leptin resistance occurring in obese individuals has been suggested based upon observations that, while the effects of leptin on satiety and energy metabolism were blunted, the sympatho- excitatory effects were maintained in obese individuals [9,37]. Whether enhanced or suppressed myocardial leptin action, either direct or centrally mediated, exists pre- and post-myocardial infarction in obese MergedCytochrome c Mito Tracker A B C D Fig. 4. Leptin decreases H 2 O 2 -induced cyto- chrome c release from mitochondria. Confo- cal analysis of H9c2 cells treated with or without leptin (6 n M, 1 h) prior to exposure to H 2 O 2 (400 lM) for 2 h shows immuno- staining of cytochrome c (green), Mitotrac- ker staining of mitochondria (red), and merged images of the two showing co-local- ization in yellow. Upon release of cyto- chrome c from mitochondria, green fluorescence can be seen independently. (A) Control, (B) leptin treatment for 1 h, (C) H 2 O 2 treatment, (D) H 2 O 2 treatment with 1 h leptin pretreatment. Images shown are representative of four independent experiments. Fig. 5. Leptin attenuates H 2 O 2 -induced exposure of the Bax N-ter- minus. Immunofluorescence staining (green) of Bax using Bax N-terminal (N20) antibody, which only detects Bax localized in mito- chondrial membrane. The results are for cells after 4 h exposure to H 2 O 2 (400 lM) with or without leptin pretreatment (6 nM, 1 h). Images are representative of three independent experiments. Regulation of cardiomyocyte apoptosis by leptin M. Eguchi et al. 3140 FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS individuals is still a matter of some debate. Our study clearly indicates a direct, as opposed to systemic or centrally mediated, role for leptin in mediating cardio- myocyte apoptosis, and reinforces data from in vivo studies suggesting a cardioprotective role for leptin via mediation of anti-apoptotic effects. As mentioned above, leptin- or leptin receptor-deficient rodents dis- play an increased rate of cardiac apoptosis. The increase in apoptotic rate and mortality was abolished upon leptin injection in ob ⁄ ob mice but not db ⁄ db mice, indicating that leptin plays an important role in cardioprotection [13]. Furthermore, it has recently been shown that perfusion of the heart with leptin dur- ing a short reperfusion period (35 min) significantly decreased mitochondrial membrane pore opening and the infarct size induced by ischemia ⁄ reperfusion [38]. In summary, our current in vitro study suggests that leptin exerts a protective effect against H 2 O 2 -induced apoptosis in H9c2 rat cardiomyocytes by preventing activation of components of the mitochondrial-depen- dent intrinsic pathway of apoptosis. This is in keeping with other recent data [13], butt the effect mediated by leptin in vivo may depend on the development of leptin resistance, the stage in progression of heart failure or other variables. Overall, the direct influence of leptin on cardiac structure and function is still uncertain, but appears to be of growing importance. Experimental procedures Culture of H9c2 rat cardiomyocytes The rat embryonic ventricular myocardial cell line H9c2 was maintained as described previously [39] in DMEM with 4.5 gÆL )1 glucose supplemented with 10% (v ⁄ v) fetal bovine serum and 1% penicillin ⁄ streptomycin (v ⁄ v). Cells were routinely grown to 80% confluence in 75 cm 2 flasks at 37 °C with an atmosphere of 5% CO 2 prior to passage and seeding for experiments. All cell-culture materials were pur- chased from Wisent (Quebec, Canada). For the induction of cell death, cells were exposed to H 2 O 2 (400 lm, Sigma- Aldrich, St Louis, MO, USA) for various time periods as indicated following treatment with leptin (6 nm). We analyzed ObR expression in these cells by PCR, and found expression of both long (ObRb) and short (ObRa) receptor isoforms (data not shown). Determination of cell viability The MTT assay was performed as described previously [40] as a measure of cell viability. In addition, trypan blue exclusion was used in some experiments, and the number of trypan blue-negative cells was counted using a hemocytom- eter 3 days after the end of H 2 O 2 treatment. Annexin V binding assay Annexin V Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) was used to detect PS exposure to the outer sur- face of the cell membrane according to the manufacturer’s protocol. Briefly, cells were grown in a 12-well plate with cover slips in each well. They were treated with H 2 O 2 follow- ing incubation with leptin. Then the cells were washed with cold NaCl ⁄ P i and 1· binding buffer (10 mm Hepes pH 7.4, 140 mm NaCl, 2.5 mm CaCl 2 ). Cells were then incubated with annexin V Alexa Fluor 488 (1 : 20 dilution) and 1 lgÆmL )1 propidium iodide diluted in 1· binding buffer for 15 min. After incubation, cells were washed twice in 1· bind- ing buffer before mounting the cover slips on glass slides using DAKO fluorescent mounting medium (DakoCytoma- tion, Missisauga, Canada). Annexin V Alexa Fluor 488 was H 2 O 2 Leptin (24h) + + + + – – – – Cleaved caspase 3 To ta l caspase 3 Leptin (1h) H 2 O 2 + + + + – – – – Cleaved caspase 3 To ta l caspase 3 0 0.5 1 1.5 2 2.5 H 2 O 2 Leptin (h) + + + – 0 – – 0 1 24 24 1 Caspase 3 activity (fold above control) * A B Fig. 6. H 2 O 2 -induced cleavage and activa- tion of caspase-3 are reduced in leptin pre- treated cells. (A) Representative western blots of cell lysates prepared after H 2 O 2 treatment (400 lM, 4 h) with or without lep- tin pretreatment (6 n M, 1 h or 24 h). Levels of the cleaved form of caspase-3 (17 ⁄ 19 kDa) as well as changes in total cas- pase-3 levels (35 kDa) were analysed by western blotting. (B) Quantitative analysis of the activity of caspase-3 measured using a specific caspase-3 activity assay kit (mean- s ± SEM, n = 3). The asterisk indicates a statistically significant difference from H 2 O 2 treatment alone (P < 0.05). M. Eguchi et al. Regulation of cardiomyocyte apoptosis by leptin FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS 3141 excited at 495 nm, and the fluorescence of cells was deter- mined using a confocal microscope (Olympus Fluoview Center Valley, PA, USA). Quantification was performed by analyzing the fluorescence intensity per cell, and the data shown are means ± SEM of all experiments, in which two cover slips were used per condition and nine fields of view from each cover slip were quantified. Immunofluorescent detection of conformational changes in Bax (N-terminal exposure) by confocal microscopy For analysis of Bax immunofluorescence, cells grown on cover slips were washed twice with NaCl ⁄ P i , fixed with 4% paraformaldehyde in NaCl ⁄ P i for 15 min, permeabilized with 0.2% Triton X-100 for 5 min and blocked using 3% BSA in NaCl ⁄ P i for 1 h at room temperature. Cells were then incubated for 1 h at 37 °C with anti-Bax N-terminal IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 150) in blocking buffer. The unique feature of this assay is that the N-terminal epitope is not detected when Bax is retained in the cytosol, but is exposed and detected upon Bax insertion into the mitochondrial membrane [22]. The cells were washed three times in NaCl ⁄ P i , and incubated for 1 h at room temperature in anti-rabbit IgG Alexa Fluor 488 serum (Molecular Probes; 1 : 2000). After wash- ing, cells were mounted using DAKO mounting medium and confocal images were analysed as above. Measurement of mitochondrial membrane potential (Y) using JC-1 H9c2 cells were grown on cover-slips, treated as indicated in Fig. 3, and then washed twice with NaCl ⁄ P i . The cells were incubated with 5 lm JC-1 dye (Molecular Probes) in serum-free medium for 15 min at 37 °C. The medium was then removed, and the cells were washed three times with NaCl ⁄ P i . The cells were examined immediately under a con- focal microscope. JC-1 fluorescence was measured to assess the emission shift from green (530 nm) to red (590 nm) in polarized mitochondria at 488 nm excitation. Immunofluorescent detection of intracellular cytochrome c localization by confocal microscopy To detect cytochrome c release from the mitochondria, cells grown on cover slips were first treated to stain mitochondria by incubation for 10 min at room temperature with 10 nm MitoTracker CMTMRos dye (Molecular Probes) in NaCl ⁄ P i . Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked using 3% serum dissolved in NaCl ⁄ P i for 30 min at room temperature. Cells were then probed with monoclonal anti-cytochrome c IgG (BD Biosciences Pharmingen, Oakville, Canada; 1 : 250 dilution in blocking solution) for 1 h at room temperature, followed by staining with goat anti-mouse Alexa Fluor 488 (Molecular Probes; 1 : 1000) for 1 h at room temperature. After washing, cells were mounted using DAKO mounting medium, and ana- lyzed by confocal microscopy. Caspase-3 activity assay Caspase-3 activity was measured using an Apo-ONE homo- geneous caspase-3 assay kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Briefly, cells grown on 96-well plates were treated with H 2 O 2 with or without leptin pretreatment. After exposure to H 2 O 2 , Apo-ONE caspase-3 ⁄ 7 reagent was added, and the mixture incubated at room temperature for up to 18 h. The level of fluorescence was measured using a Wallac 1420 Victor 3 apparatus (Perkin Elmer, Waltham, MA, USA) with excita- tion ⁄ emission at 499 ⁄ 521 nm. Immunoblotting for total and cleaved forms of caspase-3 After appropriate treatment of cells, they were washed in NaCl ⁄ P i and lysed using lysis buffer (0.5 m Tris ⁄ HCl pH 6.8, 2% v ⁄ v SDS, 15% v ⁄ v glycerol 10% v ⁄ v 2-mercaptoethanol, 0.2 mm phenylmethanesulfonyl fluoride, 10 lgÆmL )1 leupep- tin, 1 mm pepstatin A, 0.5 mm Na 3 VO 4 , 0.2 mm E64, 2 mm okadoic acid, a few grains of bromophenol blue). Centri- fugation at 1500 g was used to precipitate floating cells, which were collected and lysed with the cells growing in culture dish. Each lysate was collected and transferred to Eppendorf tubes, which were heated to 65 °C for 15 min, and the cells were further lysed by passing five times through a 25-gauge needle ⁄ syringe. After centrifuging each sample at 12 000 g for 2 min at 4 °C, 35 lL aliquots were loaded onto a 10% SDS–PAGE gel. After protein transfer to poly(vinyli- dene difluoride) membrane, the membrane was incubated with the primary caspase-3 antibody solution (1 : 1000, Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. The antibody detects both total (35 kDa) and cleaved (17 ⁄ 19 kDa) forms of caspase-3. Then the membrane was incubated in horseradish peroxidase-linked secondary anti- body solution (1 : 10 000) for 1 h and analyzed by enhanced chemilunenescence. The b-actin content was routinely checked to confirm the accuracy of protein loading on gels (data not shown). Quantification of band intensity upon wes- tern blotting was conducted using nih image software (National Institutes of Health, Bethesda, MD, USA). Statistical analysis All data presented are expressed as means ± SEM. Sta- tistical analysis was undertaken using Student’s t-test. Regulation of cardiomyocyte apoptosis by leptin M. Eguchi et al. 3142 FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS Differences between groups were considered significant at P < 0.05. Acknowledgements Funding for this work was provided by the Canadian Institutes of Health Research (CIHR) via an operating grant and a New Investigator award to GS. References 1 Abel ED, Litwin SE & Sweeney G (2008) Cardiac remodeling in obesity. Physiol Rev 88, 389–419. 2 Hilfiker-Kleiner D, Landmesser U & Drexler H (2006) Molecular mechanisms in heart failure focus on cardiac hypertrophy, inflammation, angiogenesis, and apoptosis. J Am Coll Cardiol 48, A56–A66. 3 Hall JA, French TK, Rasmusson KD, Vesty JC, Roberts CA, Rimmasch HL, Kfoury AG & Renlund DG (2005) The paradox of obesity in patients with heart failure. J Am Acad Nurse Pract 17, 542–546. 4 Kunapuli S, Rosanio S & Schwarz ER (2006) ‘How do cardiomyocytes die?’ Apoptosis and autophagic cell death in cardiac myocytes. J Card Fail 12, 381–391. 5 Reeve JL, Duffy AM, O’Brien T & Samali A (2005) Don’t lose heart – therapeutic value of apoptosis prevention in the treatment of cardiovascular disease. J Cell Mol Med 9, 609–622. 6 Kobayashi K (2005) Adipokines: therapeutic targets for metabolic syndrome. Curr Drug Targets 6, 525–529. 7 Ahima RS & Flier JS (2000) Leptin. Annu Rev Physiol 62, 413–437. 8 Rahmouni K, Morgan DA, Morgan GM, Mark AL & Haynes WG (2005) Role of selective leptin resistance in diet-induced obesity hypertension. Diabetes 54, 2012– 2018. 9 Mark AL, Correia ML, Rahmouni K & Haynes WG (2002) Selective leptin resistance: a new concept in leptin physiology with cardiovascular implications. J Hypertens 20, 1245–1250. 10 Sweeney G (2002) Leptin signaling. Cell Signal 14, 655– 663. 11 Matsui H, Motooka M, Koike H, Inoue M, Iwasaki T, Suzuki T, Kurabayashi M & Yokoyama T (2007) Ischemia ⁄ reperfusion in rat heart induces leptin and leptin receptor gene expression. Life Sci 80, 672– 680. 12 Purdham DM, Zou MX, Rajapurohitam V & Karmazyn M (2004) Rat heart is a site of leptin production and action. Am J Physiol Heart Circ Physiol 287, H2877–H2884. 13 Barouch LA, Gao D, Chen L, Miller KL, Xu W, Phan AC, Kittleson MM, Minhas KM, Berkowitz DE, Wei C et al. (2006) Cardiac myocyte apoptosis is associated with increased DNA damage and decreased survival in murine models of obesity. Circ Res 98, 119–124. 14 Madani S, De Girolamo S, Munoz DM, Li RK & Sweeney G (2006) Direct effects of leptin on size and extracellular matrix components of human pediatric ventricular myocytes. Cardiovasc Res 69, 716–725. 15 Ren J & Relling DP (2006) Leptin-induced suppression of cardiomyocyte contraction is amplified by ceramide. Peptides 27, 1415–1419. 16 Sawyer DB, Siwik DA, Xiao L, Pimentel DR, Singh K & Colucci WS (2002) Role of oxidative stress in myo- cardial hypertrophy and failure. J Mol Cell Cardiol 34, 379–388. 17 Tajmir P, Ceddia RB, Li RK, Coe IR & Sweeney G (2004) Leptin increases cardiomyocyte hyperplasia via extracellular signal-regulated kinase- and phosphatidyl- inositol 3-kinase-dependent signaling pathways. Endocri- nology 145, 1550–1555. 18 Palanivel R, Eguchi M, Shuralyova I, Coe I & Sweeney G (2006) Distinct effects of short- and long-term leptin treatment on glucose and fatty acid uptake and metabo- lism in HL-1 cardiomyocytes. Metabolism 55, 1067– 1075. 19 Caro JF, Kolaczynski JW, Nyce MR, Ohannesian JP, Opentanova I, Goldman WH, Lynn RB, Zhang PL, Sinha MK & Considine RV (1996) Decreased cerebro- spinal-fluid ⁄ serum leptin ratio in obesity: a possible mechanism for leptin resistance. Lancet 348, 159–161. 20 Suleiman MS, Halestrap AP & Griffiths EJ (2001) Mitochondria: a target for myocardial protection. Phar- macol Ther 89, 29–46. 21 Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J 341, 233–249. 22 Desbiens KM, Deschesnes RG, Labrie MM, Desfosses Y, Lambert H, Landry J & Bellmann K (2003) c-Myc potentiates the mitochondrial pathway of apoptosis by acting upstream of apoptosis signal-regulating kinase 1 (Ask1) in the p38 signalling cascade. Biochem J 372, 631–641. 23 Clerk A, Cole SM, Cullingford TE, Harrison JG, Jor- makka M & Valks DM (2003) Regulation of cardiac myocyte cell death. Pharmacol Ther 97, 223–261. 24 Ren J (2004) Leptin and hyperleptinemia – from friend to foe for cardiovascular function. J Endocrinol 181, 1–10. 25 Schulze PC & Kratzsch J (2005) Leptin as a new diag- nostic tool in chronic heart failure. Clin Chim Acta 362, 1–11. 26 Schulze PC, Kratzsch J, Linke A, Schoene N, Adams V, Gielen S, Erbs S, Moebius-Winkler S & Schuler G (2003) Elevated serum levels of leptin and soluble leptin receptor in patients with advanced chronic heart failure. Eur J Heart Fail 5, 33–40. M. Eguchi et al. Regulation of cardiomyocyte apoptosis by leptin FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS 3143 27 Yasuoka C, Ihara Y, Ikeda S, Miyahara Y, Kondo T & Kohno S (2004) Antiapoptotic activity of Akt is down- regulated by Ca 2+ in myocardiac H9c2 cells. Evidence of Ca(2+)-dependent regulation of protein phosphatase 2Ac. J Biol Chem 279, 51182–51192. 28 Murata H, Ihara Y, Nakamura H, Yodoi J, Sumikawa K & Kondo T (2003) Glutaredoxin exerts an antiapop- totic effect by regulating the redox state of Akt. J Biol Chem 278, 50226–50233. 29 Han H, Long H, Wang H, Wang J, Zhang Y & Wang Z (2004) Progressive apoptotic cell death trig- gered by transient oxidative insult in H9c2 rat ven- tricular cells: a novel pattern of apoptosis and the mechanisms. Am J Physiol Heart Circ Physiol 286, H2169–H2182. 30 Tajmir P, Kwan JJ, Kessas M, Mozammel S & Sweeney G (2003) Acute and chronic leptin treatment mediate contrasting effects on signaling, glucose uptake, and GLUT4 translocation in L6–GLUT4myc myotubes. J Cell Physiol 197, 122–130. 31 Sweeney G, Keen J, Somwar R, Konrad D, Garg R & Klip A (2001) High leptin levels acutely inhibit insulin- stimulated glucose uptake without affecting glucose transporter 4 translocation in L6 rat skeletal muscle cells. Endocrinology 142, 4806–4812. 32 McGavock JM, Victor RG, Unger RH & Szczepaniak LS (2006) Adiposity of the heart, revisited. Ann Intern Med 144, 517–524. 33 Ghosh S & Rodrigues B (2006) Cardiac cell death in early diabetes and its modulation by dietary fatty acids. Biochim Biophys Acta 1761, 1148–1162. 34 Wagner R, Oberste-Berghaus C, Herpertz S, Blum WF, Pelz B, Hebebrand J, Senf W, Mann K & Albers N (2000) Time relationship between circadian variation of serum levels of leptin, insulin and cortisol in healthy subjects. Horm Res 54, 174–180. 35 Laimer M, Ebenbichler CF, Kaser S, Sandhofer A, Weiss H, Nehoda H, Aigner F & Patsch JR (2002) Weight loss increases soluble leptin receptor levels and the soluble receptor bound fraction of leptin. Obes Res 10, 597–601. 36 Cook SA, Sugden PH & Clerk A (1999) Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: associ- ation with changes in mitochondrial membrane poten- tial. Circ Res 85, 940–949. 37 Correia ML, Haynes WG, Rahmouni K, Morgan DA, Sivitz WI & Mark AL (2002) The concept of selective leptin resistance: evidence from agouti yellow obese mice. Diabetes 51, 439–442. 38 Smith CC, Mocanu MM, Davidson SM, Wynne AM, Simpkin JC & Yellon DM (2006) Leptin, the obesity- associated hormone, exhibits direct cardioprotective effects. Br J Pharmacol 149, 5–13. 39 Wang L, Ma W, Markovich R, Lee WL & Wang PH (1998) Insulin-like growth factor I modulates induction of apoptotic signaling in H9C2 cardiac muscle cells. Endocrinology 139, 1354–1360. 40 Eguchi M, Gillis LC, Liu Y, Lyakhovsky N, Du M, McDermott JC & Sweeney G (2007) Regulation of SOCS-3 expression by leptin and its co-localization with insulin receptor in rat skeletal muscle cells. Mol Cell Endocrinol 267, 38–45. Regulation of cardiomyocyte apoptosis by leptin M. Eguchi et al. 3144 FEBS Journal 275 (2008) 3136–3144 ª 2008 The Authors Journal compilation ª 2008 FEBS . Leptin protects H9c2 rat cardiomyocytes from H 2 O 2 -induced apoptosis Megumi Eguchi*, Yuantao Liu*, Eyun-Jung. mentioned above, leptin- or leptin receptor-deficient rodents dis- play an increased rate of cardiac apoptosis. The increase in apoptotic rate and mortality