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Modulation of palmitate-induced cardiomyocyte cell death by interventions that alter intracellular calcium
Prostaglandins, Leukotrienes and Essential Fatty Acids (1999) 61(3), 195–201 © 1999 Harcourt Publishers Ltd Article no. plef.1999.0090
Modulation of palmitate-induced cardiomyocyte cell death by interventions that alter intracellular calcium S. W. Rabkin,1 M. Huber,2 G. Krystal2 1
Cardiology, 2Terry Fox Laboratory, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
Summary The objective of this study was to investigate whether palmitate-induced cell death in cardiomyocytes was dependent on alterations of intracellular calcium ([Ca2+)I). Specifically, we sought to determine whether palmitate might produce a cellular calcium overload by increasing calcium influx into the cell or by altering sarcoplasmic reticulum (SR) calcium transport. We also determined whether palmitate’s effects might be modulated by agents that alter [Ca2+]I. Treatment of chick embryonic cardiomyocytes in culture with palmitate (100 uM) produced a significant (P < 0.05) and 42.9 ± 5.3% reduction in cell survival or increase in cell death. As determined by FURA-2 measurement of [Ca2+]I, the cytotoxicity of palmitate on cardiomyocytes did not appear to be mediated through acute increases in [Ca2+]I. In contrast, the unsaturated fatty acid, arachidonic acid increased [Ca2+]I. The calcium ionophore ionomycin significantly (P < 0.05) increased palmitate-induced cardiomyocyte cell death. The effects of ionomycin and palmitate, however, were additive, suggesting palmitate and ionomycin acted in an independent manner to induce cell death. Furthermore, in contrast to palmitate, an ionomycin-induced increase in [Ca2+]I was demonstrated in these cells. Inhibition of SR calcium reuptake by thapsigargin, which acutely increases [Ca2+]I, also significantly (P < 0.05) increased palmitateinduced cardiomyocyte death. Again, these two agents most likely acted in an independent manner because of the additive nature of the effect of palmitate and thapsigargin on cell viability. Pamitate-induced cardiotoxicity was not mediated through release of [Ca2+]I from SR or through voltage-operated channels on plasma membranes, as neither SR calcium depletion by low concentrations of ryanodine nor blockade of the voltage-operated calcium channel with nifedipine significantly altered palmitate-induced cardiomyocyte death. These data suggest that palmitate-induced cardiac cell death is enhanced by increases in [Ca2+]I and highlights the potential adverse effect of a combination of palmitate with conditions that increase [Ca2+]I in cardiomyocytes. © 1999 Harcourt Publishers Ltd INTRODUCTION Elevated plasma concentrations of free fatty acids accompany acute myocardial infarction.1,2 These fatty acids are recognized to increase the extent and severity of myocardial ischemic damage,2–4 but the cellular and molecular mechanisms by which fatty acids exert these deleterious effects still remain undefined. Several possible mechanisms for fatty-acid-induced cardiotoxicity have been proposed and these have mainly focused on alterations in Received 24 May 1999 Accepted 1 June 1999 Correspondence to: S. W. Rabkin, University of British Columbia, D410 2733 Heather St, Vancouver, British Columbia, Canada V5Z 3J5. Tel.: +1 604 875 5847; Fax: +1 604 875 5849; E-mail: [email protected]
cellular glucose and fatty acid metabolism.2,5–7 The potential of unsaturated fatty acids to cause cellular calcium overload as a mechanism to explain fatty-acid-induced cell death has also been proposed.8 Evidence for such a mechanism includes the finding that free polyunsaturated fatty acids of both the f-3 and the f-6 series induce a rapid increase in the cytosolic free Ca2+ concentration ([Ca2+]I) in the JURKAT T cell line.9 As well, arachidonic acid increases [Ca2+]I in single pancreatic islet beta cells10 and in GH3 pituitary cells.11 In addition, oleic acid or arachidonic acid have been shown to augment [Ca2+]I in Ehrlich ascites tumor cells from a thapsigargin-sensitive Ca2+ pool.12 In neonatal rat cardiomyocytes, arachidonic acid is known to produce a rapid increase in [Ca2+]I.8 While it appears from the above-noted data that unsaturated fatty acids may indeed cause cell damage, at least in part 195
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through increasing [Ca2+]I, there is no data as yet whether palmitate, an abundant saturated fatty acid in the body, exerts its deleterious effects through increasing [Ca2+]I, although this has been proposed.13 More investigation in this area is required since fatty-acid-induced activation of inward calcium currents by increasing [Ca2+]I is well recognized to contribute to ischemia-induced pathogenic events in the heart.14 Excessive increase in [Ca2+]I is one of the mechanisms underlying cell death in cardiac cells as well as other cell types.15–18 Increased levels of [Ca2+]I may trigger activation of Ca2+-dependent enzymes culminating in cell death.19 The fatty acid palmitate has been demonstrated to produce apoptotic cell death in the murine cell lines LyD9 and in WEHI-231,20 as well as in cardiomyocytes.13 Cell death has been linked to regulation of [Ca2+]I because of an observed increase in nuclear Ca2+ transport and activation of nuclear Ca2+-dependent kinases.16–18 Intracellular Ca2+ is regulated by calcium entry and efflux across the plasmalemma as well as by [Ca2+]I pools such as the sarcoplasmic reticulum (SR).21–22 In this regard, the role of SR Ca2+ in modulating cell death processes is becoming more appreciated.18 Recently, several molecular probes have been identified that modulate SR-based calcium fluxes. Thapsigargin, for instance, a sesquiterpene gamma-lactone which selectively inhibits the sarcoplasmic and endoplasmic reticulum Ca2+-dependent ATPase family of intracellular ‘Ca2+-pumping’ ATPases23 produces a three- to four-fold elevation in the levels of [Ca2+]I.24 Thapsigargin’s action is selective for the SR Ca2+ ATPase in intact cardiac myocytes and is a valuable probe to examine the importance of SR Ca2+ pools and the role of the SR Ca2+ ATPase.25 Polyunsaturated fatty acids are known to stimulate the release of Ca2+ from the SR,9,12 but whether saturated fatty acids such as palmitate also alter SR-based calcium fluxes remains to be explored. The purpose of this study was to elucidate the potential of palmitate to alter [Ca2+]I and determine whether modulation of [Ca2+]I does influence palmitate-induced cell death in cardiomyocytes. Considering the critical role that SR calcium plays in excitation–contraction coupling of cardiac muscle,21 we sought to examine the hypothesis that SR Ca2+ modulates palmitate-induced cell death in cardiomyocytes.
MATERIALS AND METHODS Cell cultures Chick embryonic ventricular cells were cultured from 7-day chick embryos from white Leghorn eggs using previously described methods.26 The protocol was approved by the University committee on use of animals for research. Myocytes were maintained in culture in
medium 818A [73% DBSK, 20% M199, 6% fetal calf serum and 1% antibiotic–antimycotic (10 000 mg/ml streptomycin sulphate, 10 000 U/ml penicillin G sodium and 25 mg/ml amphotericin B) for 72 h prior to experimentation. The proportion of cells showing spontaneous contraction or displaying muscle-specific markers on immunohistologic examination was in excess of 90% at this time. Cell viability
MTT assay The MTT assay, an index of cell viability and cell growth, is based on the ability of viable cells to reduce MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) from a yellow, water-soluble dye to a dark blue, insoluble formazan product.27 Cardiomyocytes were seeded in multiwell microtitre plates (Falcon #3072, Becton Dickinson, Lincoln Park, NJ, USA) at 20 000 cells per well for 72 h. Then, cardiomyocytes were treated with palmitate, diluent, or other agents. MTT dye was added to each well for the last 4 h of palmitate treatment at 37°C for 4 h. The reaction was stopped with the addition of solubilization reagent (Promega, Madison, WI, USA) and the absorbance (optical density) was determined at 570 nm on a multiwell plate reader (BioRad model #3550, BioRad, Mississauga, Canada). Background absorbance of medium in the absence of cells was subtracted. All samples were assayed in duplicate and the mean for each experiment was calculated. The relationship of the absorbance of MTT to cell number was verified in separate experiments in which 1200 to 37 000 cardiomyocytes were added to different wells of a microtitre plate. There was a significant (P < 0.01) linear relationship between cell number and absorbance with a correlation coefficient of 0.98. Trypan blue exclusion To assess cell viability, cardiomyocytes were grown on coverslips. After 72 h in culture, palmitate or its diluent was added to the media. At predetermined times, the coverslips were removed, stained with 0.4% trypan blue and examined microscopically in a hemocytometer to determine the total number of cells and the number of dead cells, i.e. those retaining trypan blue. Four representative high-power fields were selected and counted and the mean was calculated. Measurement of [Ca2+]I Calcium fluxes were measured according to Huber et al.28 In brief, chick cardiomyocytes were incubated with 2 uM fura-2/AM in Tyrode’s buffer (130 mM NaCl, 5 mM KCl, 1.4 mM MCaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA in 10 mM Hepes, pH 7.4) at 23°C for 45 min. Then,
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the cells were washed. resuspended in 1 ml of the same buffer at 5 × 105 cells/ml in a stirring cuvette. Following stimulation by ionomycin (10 uM) or palmitate (100 uM), cytosolic calcium was measured by monitoring fluorescence intensity at 510 nm, after excitation of the sample with two different wavelengths (340 and 380 nm) using an MC200 monochromator from SLM AMINCO with a 8100 V3.0 software program. Materials All cell culture media components were from Gibco/BRL Life Sciences (Burlington, Ontario, Canada). Palmitate, arachidonic acid, thapsigargin, ryanodine and nifedipine were from Sigma Chemicals Ltd (St Louis, MO, USA). Palmitate was usually dissolved in warm ethanol and was freshly prepared for each experiment. Arachidonic acid was kept under N2, diluted in methanol and used shortly thereafter. For comparative studies with arachidonic acid, palmitate was similarly dissolved in methanol. Trypan blue was from BDH Chemicals Ltd (Poole, England). The MTT assay kit was from Promega (Madison, WI, USA). Fura-2/AM was from Molecular Probes (Eugene, OR, USA). All other chemicals were from Fischer Scientific (Ottawa, Ontario, Canada). Data analysis The data are presented as the mean±SEM. Hypothesis testing used a one-way analysis of variance. Examination of a pair of groups used Kruskal–Wallis multiple–comparison test. Linear least-squares regression was also performed. The null hypothesis was rejected if the probability of a Type I error was less than 5% (P < 0.05). RESULTS Palmitate reduces cardiomyocyte survival Cardiomyocytes treated with palmitate (100 uM) for 24 h displayed a marked and significant (P < 0.001) reduction in MTT absorbance compared to vehicle treated (control) cells (Fig. 1). There was little overlap in absorbance between palmitate treated and diluent treated cells. The effect of palmitate cannot be attributed to a non-specific effect of a fatty acid, since the C12 saturated fatty acid, laurate (100 uM) did not affect cell viability. Based on the ability of viable cells to reduce MTT and the linear relationship between cardiomyocyte cell number and MTT absorbance, palmitate reduced cell viability or increased cell death. Compared to control, palmitate (100 uM) produced a significant (P < 0.001 ) and 42.9± 5.3% increase in cell death. Palmitate-induced cardiomyocyte cell death was confirmed by the trypan blue exclusion assay which demonstrated a significant © 1999 Harcourt Publishers Ltd
Fig. 1 The effect of palmitate on the MTT absorbance. Cardiomyocytes were seeded in multiwell microtitre at 20 000 cells per well. After 72 h, cells were treated with palmitate, laurate or the diluent (ethanol) for 24 h. MTT dye was added to each well for the last 4 h of treatment, the reaction was stopped and the absorbance (optical density) was determined at 570 nm. Background absorbance of medium in the absence of cells was subtracted. The graph is a box plot that plots the median (horizontal bar) and the 90th percentile of the distribution for all values. The results are from independent experiments, done in duplicate, for cells treated with palmitate (N = 14), laurate (N = 5) or the diluent (ethanol) (N = 14).
(P < 0.05), 3.6 ± 1.3-fold, increase in the ratio of dead to alive cells from 0.25 ± 0.01 to 0.88 ± 0.30 for, respectively, diluent and palmitate (100 uM) treatment for 24 h. To assess the role of increased [Ca2+]I in palmitateinduced cell death, cardiomyocytes were treated with the calcium ionophore ionomycin (10 uM) alone or in combination with palmitate (100 uM) for 24 h. Inonomycin significantly (P < 0.05) increased cardiomyocyte cell death. Cells treated with palmitate plus ionomycin showed significantly more cell death compared to cells treated with either ionomycin or palmitate alone (Fig. 2). Palmitate and [Ca2+]I Based on these observations, we asked whether palmitate was capable of modulating the calcium response induced by ionomycin. Measurement of [Ca2+]I using the fluorescent probe Fura-2/AM confirmed the ability of ionomycin to strongly increase [Ca2+]I (Fig. 3). The pattern of the [Ca2+]I response was consistent with a sustained increase in [Ca2+]I rather than a transient Ca2+ increase associated with receptor-operated Ca2+ influx. To assess the modulating capacity of palmitate, this fatty acid was added 200 s after ionomycin. The addition of palmitate did not further enhance the [Ca2+]I response initiated by ionomycin (Fig. 3). Furthermore, palmitate alone did not induce an increase in [Ca2+]I nor was the increase in [Ca2+]I produced by ionomycin enhanced by palmitate pretreat-
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Fig. 2 The effect of ionomycin on palmitate-induced cell viability or cell death was assessed by the MTT assay. Cardiomyocytes were seeded in multiwell microtitre at 20 000 cells per well. After 72 h, cells were treated with palmitate, 100 uM, (n = 14), ionomycin 10 uM (n = 5), the combination of both (n = 5) or the diluent (control) (n = 14) for 24 h. MTT dye was added to each well for the last 4 h of treatment, the reaction was stopped and the absorbance (optical density) was determined at 570 nm. Background absorbance of medium in the absence of cells was subtracted. Each sample was done in duplicate. The data are expressed as a percentage of their relevant control, in the same plate and represent the mean±S.E.M.
Fig. 3 Effect of ionomycin and palmitate on intracellular calcium mobilization. Intracellular calcium concentration was measured in chick cardiomyocytes using Fura-2/AM as described in Materials and Methods. Ionomycin (10 uM) was added at 100 s and palmitate (100 uM) at 300 s.
ment (Fig. 4). In contrast to palmitate, arachidonic acid produced a sustained increase in [Ca2+]I (Fig. 5). Modulation of [Ca2+]I and cardiomyocyte cell death Recognizing that calcium channel blockers produce only a small decrease in [Ca2+]I,29 we nevertheless sought to determine if the calcium channel blocker nifedipine would ameliorate the cardiotoxicity of palmitate. Nifedicine is a relatively specific calcium channel blocker in these cardiomyocytes30 and these agents can protect against calcium-overload-mediated cell death.31 There was no significant reduction in palmitate-induced cardiomyocyte
Fig. 4 Palmitate does not alter ionomycin-induced calcium flux. Fura-2/AM-preloaded chick cardiomyocytes were stimulated with palmitate (100 uM) at 100 s and with ionomycin (10 uM) at 300 s, and intracellular calcium concentrations were measured according to Materials and Methods. The slight increases after palmitate stimulation is due to the diluent of the fatty acid, ethanol.
Fig. 5 Arachadonic acid but not palmitate increases intracellular calcium. Fura-2/AM-preloaded chick cardiomyocytes were stimulated with palmitate (200 uM) or arachadonic acid (200 uM) or their diluent methanol and intracellular calcium concentrations were measured according to Materials and Methods.
death with nifedipine (10 uM) suggesting that palmitate’s action is calcium-independent or at least not mediated through or modulated by voltage-operated calcium channels (Fig. 6). Palmitate-induced cell death was examined further by employing the SR Ca2+ ATPase inhibitor thapsigargin. This tumor-promoting sesquiterpene lactone is known to induce release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Specifically we compared the cell death induced by thapsigargin (10–9 M), palmitate alone (100 uM) and their combination. The two agents together resulted in an additive effect on cell death (Fig. 7) verifying our data with the ionophore ionomycin (Fig. 2). The increase in cell death produced by thapsigargin plus palmitate (63.3 ± 7.6%) was similar to the arithmetic
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Fig. 6 Effect of nifedipine on palmitate-induced cell death in cardiomyocytes. The bar graph showing the effect of nifedipine 0.1 uM (n = 8), or 1.0 (n = 8) or 10.0 uM (n = 9) as well as the combination of palmitate with nifedipine 0.1 uM (n = 8), or 1.0 (n = 8) or 10.0 uM (n = 9). The palmitate data is repeated from Figure 2 for ease of comparison. Results are the change from control and are expressed as the mean±S.E.M.
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Fig. 8 Effect of ryanodine on palmitate-induced cell death on cardiomyocytes. The bar graph showing the effect of ryanodine 1 uM (n = 6), ryanodine 3 uM (n = 6), the combination of palmitate 100 uM plus ryanodine 10 uM (n = 6) or ryanodine 3 uM (n = 6). The palmitate data is repeated from Figure 2 for ease of comparison. Results are the change from control and are expressed as the mean±S.E.M.
uptake and there seems to be no interdependence of the signals generated by these agents. DICUSSION
Fig. 7 The effect of thapsigargin on palmitate-induced reduction cell viability. Cardiomyocytes were maintained in multiwell plates for 72 h and then treated with palmitate, 100 uM, thapsigargin 10–9 M (n = 6), the combination of both (n = 6) or the diluent (control) for 24 h. The difference in absorbance at 570 nm was calculated and subtracted from background absorbance of the medium in the absence of cells. The palmitate data is repeated from Figure 2 for ease of comparison. The data are expressed as a percentage of their relevant control, in the same plate and represent the mean±S.E.M. of separate experiments.
combination of palmitate (42.9 ± 5.3%) and thapsigargin (15.5 ± 6.1%). Thapsigargin was verified to increase [Ca2+]I in our chick cardiomyocytes (data not shown). To explore further the potential of calcium release from SR to modulate palmitate-induced cell death, cardiomyocytes were treated with ryanodine alone or in combination with palmitate. In contrast to thapsigargin, low-micromolar concentrations of ryanodine, a SR Ca2+-release channel activator, depletes SR Ca2+ storage and blunts the release of Ca2+ from the SR.22 There was no increase in cell death produced by the combination of ryanodine and palmitate compared to palmitate alone (Fig. 8). Our experiments in total suggest that the cell-death-initiating actions of palmitate are augmented by agents that increase transsarcolemmal calcium influx or inhibit SR calcium re© 1999 Harcourt Publishers Ltd
Fatty acids are the major substrate for myocardial energy production; however, high extracellular concentrations of the 16-carbon saturated fatty acid, palmitate, induce myocardial cell death. The amount of cell death produced by palmitate in embryonic cardiomyocytes in this study was consistent with the data in neonatal rat cardiomyocytes reported by DeVries et al.13 The potential mechanisms speculated to be responsible for palmitate-induced cardiomyocyte cell death include rapid increases in [Ca2+]I with attendant calcium overload.8 The results of our study question this possibility, as there was no change in [Ca2+]I in response to palmitate. The present study confirms the data of Vacher et al.11 in GH3 cells that palmitate does not increase [Ca2+]I and extends it to cardiomyocytes. Our study provides data to support the theoretical argument against calcium as a mediator of palmitate-induced cardiomyocyte death.13 In contrast to palmitate, we found that the unsaturated fatty acid arachidonic acid increases [Ca2+]I; a finding consistent with the data of other investigators in other cell types.8,11 In GH3 cells, arachidonic acid increases [Ca2+]I in part through stimulation of ICa.11 In contrast to arachidonic acid, the saturated fatty acid stearic acid does not alter ICa through voltage-gated L-channels in neonatal rat cardiomyocytes.32 A major finding of this study is that palmitate-induced cell death is augmented by ionomycin. This is consistent with different mechanisms of action for these agents, as ionomycin increased while palmitate did not increase [Ca2+]I. Considering the deleterious role of myocardial calcium overload in conditions such myocardial infarction,
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the present study highlights the adverse effects of elevated plasma concentrations of free fatty acids that accompany acute myocardial infarction.1–4 The combination of increased extracellular palmitate and calcium overload would be especially damaging to the myocardium. Thapsigargin in combination with palmitate increased cardiomyocyte cell death to levels greater than what was induced by palmitate or thapsigargin alone. Thapsigargin which produces an initial increase in [Ca2+]I through inhibition of SR calcium reuptake,22,24,25 produced a small amount of cardiomyocyte cell death. In other cell types, thapsigargin appears to induce more cell death.18,24,33 While the most likely explanation for the adverse effect of the combination of thapsigargin plus palmitate is similar to ionomycin as thapsigargin increases [Ca2+]I,22,24,25 an alternative explanation exists. Thapsigargin depletes the intracellular SR calcium pool and cell death has been attributed to SR calcium depletion in neuroblastoma cell lines, fetal rat cerebrocortical cells in cultures and the S49 cell line.34,35 This is a less attractive explanation because low (micromolar) concentrations of ryanodine, a SR Ca2+-release channel activator, depletes SR Ca2+ stores and blunts the release of Ca2+ from the SR.22 Yet we found no increase in cell death produced by the combination of ryanodine and palmitate compared to palmitate alone. Exogenous fatty acids contribute about 90% of the fatty acid carbon present in cellular triglyceride and phospholipid.36 The presence of fatty acids in excess of that required for energy or phospholipid synthesis results in excessive triacylglycerol synthesis, leading to structural alterations of the cell.36 One of the putative explanations for palmitate-induced cell death is precipitation of triacylglycerol, from the excess available palmitate, at the site of its synthesis in SR with resulting impairment in SR function.13 Data from the present study do not support this hypothesis, as there was no synergistic effect of palmitate with thapsigargin or ryanodine, both of which alter SR calcium handling. In contrast to palmitate, the unsaturated fatty acids oleic acid and arachidonic acid, increase [Ca2+]I in Ehrlich tumor cells from a thapsigarginsensitive Ca2+ pool.12 The present study used embryonic chick cardiomyocytes. While the origin of the cell type is important in the extrapolation of the data, this cell type has several advantages. First, components of cell death pathways in this cell type have similarities to humans.37 Second, intracellular calcium in the embryonic chick heart is distributed in three kinetically different compartments similar to adult mammalian hearts.38 Third, the response to arachidonic acid is similar to mammalian cardiomyocytes.8 In summary, palmitate induces cardiomyocyte cell death through a mechanism that does not involve direct increases in [Ca2+]I. Ionomycin, which increased [Ca2+]I, augmented palmitate-induced cell death despite the
absence of enhancement of ionomycin-induced elevation [Ca2+]I, by palmitate. The ability of palmitate to induce cardiomyocyte cell death in the presence of thapsigargin or ryanodine suggests that palmitate-induced cardiomyocyte death is independent of an action on SR calcium flux. The implication of these findings are that in situations of elevated circulating palmitate, cardiomyocytes are vulnerable to enhanced cell death in conditions associated with increased [Ca2+]I.38,39 ACKNOWLEDGMENTS This study was supported in part by a grant from the Heart and Stroke Foundation of British Columbia and the Yukon. The authors thank Carol Smythe for her technical assistance.
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R. K., Krystal G. Targeted disruption of SHIP leads to steel factor-induced degranulation of mast cells. EMBO J 1998; 17: 7311–7319. Zaugg C. E., Kojima S., Wu S. T., Wikman-Coffelt J., Parmley W. W., Buser P. T. Intracellular calcium transients underlying interval-force relationship in whole rat hearts: effects of calcium antagonists. Cardiovascular Res 1995; 30: 212–221. Sippel H., Stauffert I., Estler C. J. Protective effect of various calcium antagonists against an experimentally induced calcium overload in isolated hepatocytes. Biochem Pharm 1993; 46: 1937–1944. Rabkin S. W., Freestone N., Quamme G. Nifedipine does not alter the increased cystolic free magnesium during inhibition of mitochondrial function in isolated cardiac myocytes. Gen Pharmacol 1994; 25: 1483–1491. Xiao Y. F., Gomez A. M., Morgan J. P., Lederer W. J., Leaf A. Suppression of voltage-gated L-type Ca2+ currents by polyunsaturated fatty acids in adult and neonatal rat ventricular myocytes. Proc Natl Acad Sci USA 1997; 94: 4182–4187. Escargueil-Blanc I., Salvayre R., Negre-Salvayre A. Necrosis and apoptosis induced by oxidized low density lipoproteins occur through two calcium-dependent pathways in lymphoblastoid cells. FASEB J 1994; 8: 1075–1080. Nath R., Raser K. J., Hajimohammadreza I., Wanag K. K. Thapsigargin induces apoptosis in SH-SY5Y neuroblastoma cells and cerebrocortical cultures. Biochem Mol Biol Intl 1997; 43: 197–205. Bian X., Hughes F. M. Jr, Huang Y., Cidlowski J. A., Putney J. W. Jr. Roles of cytoplasmic Ca2+ and intracellular Ca2+ stores in induction and suppression of apoptosis in S49 cells. Am J Physiol 1997; 272: C1241–C1249. MacKenzie C. G., Mackenzie J. B., Reiss O. K. In: Rothblat G. H., Kritchevsky D. (eds). Lipid metabolism in tissue culture cells. Wistar Symposium. Philadephia: Wistar Institute; 1967: 63. Eguchi Y., Ewert D. L., Tsujimoto Y. Isolation and characterization of the chicken bcl-2 gene: expression in a variety of tissue including lymphoid and neuronal organs in adult and embryo. Nucleic Acids Res 1992; 220: 4187–4192. Prakash P., Meera P., Tripathi O. 45Ca-efflux in embryonic heart and its modification by caffeine and ryanodine. J Develop Physiol 1992; 18: 285–293. Ver Donck L., Borgers M., Verdonck F. Inhibition of sodium and calcium overload pathology in the myocardium: a new cytoprotective principle. Cardiovas Res 1993; 27: 349–357. Bolli R. Basic and clinical aspects of myocardial stunning. Prog Cardiovascular Dis 1998; 40: 477–516.
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Modulation of palmitate-induced cardiomyocyte cell death by interventions that alter intracellular calcium S. W. Rabkin,1 M. Huber,2 G. Krystal2 1
Cardiology, 2Terry Fox Laboratory, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
Summary The objective of this study was to investigate whether palmitate-induced cell death in cardiomyocytes was dependent on alterations of intracellular calcium ([Ca2+)I). Specifically, we sought to determine whether palmitate might produce a cellular calcium overload by increasing calcium influx into the cell or by altering sarcoplasmic reticulum (SR) calcium transport. We also determined whether palmitate’s effects might be modulated by agents that alter [Ca2+]I. Treatment of chick embryonic cardiomyocytes in culture with palmitate (100 uM) produced a significant (P < 0.05) and 42.9 ± 5.3% reduction in cell survival or increase in cell death. As determined by FURA-2 measurement of [Ca2+]I, the cytotoxicity of palmitate on cardiomyocytes did not appear to be mediated through acute increases in [Ca2+]I. In contrast, the unsaturated fatty acid, arachidonic acid increased [Ca2+]I. The calcium ionophore ionomycin significantly (P < 0.05) increased palmitate-induced cardiomyocyte cell death. The effects of ionomycin and palmitate, however, were additive, suggesting palmitate and ionomycin acted in an independent manner to induce cell death. Furthermore, in contrast to palmitate, an ionomycin-induced increase in [Ca2+]I was demonstrated in these cells. Inhibition of SR calcium reuptake by thapsigargin, which acutely increases [Ca2+]I, also significantly (P < 0.05) increased palmitateinduced cardiomyocyte death. Again, these two agents most likely acted in an independent manner because of the additive nature of the effect of palmitate and thapsigargin on cell viability. Pamitate-induced cardiotoxicity was not mediated through release of [Ca2+]I from SR or through voltage-operated channels on plasma membranes, as neither SR calcium depletion by low concentrations of ryanodine nor blockade of the voltage-operated calcium channel with nifedipine significantly altered palmitate-induced cardiomyocyte death. These data suggest that palmitate-induced cardiac cell death is enhanced by increases in [Ca2+]I and highlights the potential adverse effect of a combination of palmitate with conditions that increase [Ca2+]I in cardiomyocytes. © 1999 Harcourt Publishers Ltd INTRODUCTION Elevated plasma concentrations of free fatty acids accompany acute myocardial infarction.1,2 These fatty acids are recognized to increase the extent and severity of myocardial ischemic damage,2–4 but the cellular and molecular mechanisms by which fatty acids exert these deleterious effects still remain undefined. Several possible mechanisms for fatty-acid-induced cardiotoxicity have been proposed and these have mainly focused on alterations in Received 24 May 1999 Accepted 1 June 1999 Correspondence to: S. W. Rabkin, University of British Columbia, D410 2733 Heather St, Vancouver, British Columbia, Canada V5Z 3J5. Tel.: +1 604 875 5847; Fax: +1 604 875 5849; E-mail: [email protected]
cellular glucose and fatty acid metabolism.2,5–7 The potential of unsaturated fatty acids to cause cellular calcium overload as a mechanism to explain fatty-acid-induced cell death has also been proposed.8 Evidence for such a mechanism includes the finding that free polyunsaturated fatty acids of both the f-3 and the f-6 series induce a rapid increase in the cytosolic free Ca2+ concentration ([Ca2+]I) in the JURKAT T cell line.9 As well, arachidonic acid increases [Ca2+]I in single pancreatic islet beta cells10 and in GH3 pituitary cells.11 In addition, oleic acid or arachidonic acid have been shown to augment [Ca2+]I in Ehrlich ascites tumor cells from a thapsigargin-sensitive Ca2+ pool.12 In neonatal rat cardiomyocytes, arachidonic acid is known to produce a rapid increase in [Ca2+]I.8 While it appears from the above-noted data that unsaturated fatty acids may indeed cause cell damage, at least in part 195
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through increasing [Ca2+]I, there is no data as yet whether palmitate, an abundant saturated fatty acid in the body, exerts its deleterious effects through increasing [Ca2+]I, although this has been proposed.13 More investigation in this area is required since fatty-acid-induced activation of inward calcium currents by increasing [Ca2+]I is well recognized to contribute to ischemia-induced pathogenic events in the heart.14 Excessive increase in [Ca2+]I is one of the mechanisms underlying cell death in cardiac cells as well as other cell types.15–18 Increased levels of [Ca2+]I may trigger activation of Ca2+-dependent enzymes culminating in cell death.19 The fatty acid palmitate has been demonstrated to produce apoptotic cell death in the murine cell lines LyD9 and in WEHI-231,20 as well as in cardiomyocytes.13 Cell death has been linked to regulation of [Ca2+]I because of an observed increase in nuclear Ca2+ transport and activation of nuclear Ca2+-dependent kinases.16–18 Intracellular Ca2+ is regulated by calcium entry and efflux across the plasmalemma as well as by [Ca2+]I pools such as the sarcoplasmic reticulum (SR).21–22 In this regard, the role of SR Ca2+ in modulating cell death processes is becoming more appreciated.18 Recently, several molecular probes have been identified that modulate SR-based calcium fluxes. Thapsigargin, for instance, a sesquiterpene gamma-lactone which selectively inhibits the sarcoplasmic and endoplasmic reticulum Ca2+-dependent ATPase family of intracellular ‘Ca2+-pumping’ ATPases23 produces a three- to four-fold elevation in the levels of [Ca2+]I.24 Thapsigargin’s action is selective for the SR Ca2+ ATPase in intact cardiac myocytes and is a valuable probe to examine the importance of SR Ca2+ pools and the role of the SR Ca2+ ATPase.25 Polyunsaturated fatty acids are known to stimulate the release of Ca2+ from the SR,9,12 but whether saturated fatty acids such as palmitate also alter SR-based calcium fluxes remains to be explored. The purpose of this study was to elucidate the potential of palmitate to alter [Ca2+]I and determine whether modulation of [Ca2+]I does influence palmitate-induced cell death in cardiomyocytes. Considering the critical role that SR calcium plays in excitation–contraction coupling of cardiac muscle,21 we sought to examine the hypothesis that SR Ca2+ modulates palmitate-induced cell death in cardiomyocytes.
MATERIALS AND METHODS Cell cultures Chick embryonic ventricular cells were cultured from 7-day chick embryos from white Leghorn eggs using previously described methods.26 The protocol was approved by the University committee on use of animals for research. Myocytes were maintained in culture in
medium 818A [73% DBSK, 20% M199, 6% fetal calf serum and 1% antibiotic–antimycotic (10 000 mg/ml streptomycin sulphate, 10 000 U/ml penicillin G sodium and 25 mg/ml amphotericin B) for 72 h prior to experimentation. The proportion of cells showing spontaneous contraction or displaying muscle-specific markers on immunohistologic examination was in excess of 90% at this time. Cell viability
MTT assay The MTT assay, an index of cell viability and cell growth, is based on the ability of viable cells to reduce MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) from a yellow, water-soluble dye to a dark blue, insoluble formazan product.27 Cardiomyocytes were seeded in multiwell microtitre plates (Falcon #3072, Becton Dickinson, Lincoln Park, NJ, USA) at 20 000 cells per well for 72 h. Then, cardiomyocytes were treated with palmitate, diluent, or other agents. MTT dye was added to each well for the last 4 h of palmitate treatment at 37°C for 4 h. The reaction was stopped with the addition of solubilization reagent (Promega, Madison, WI, USA) and the absorbance (optical density) was determined at 570 nm on a multiwell plate reader (BioRad model #3550, BioRad, Mississauga, Canada). Background absorbance of medium in the absence of cells was subtracted. All samples were assayed in duplicate and the mean for each experiment was calculated. The relationship of the absorbance of MTT to cell number was verified in separate experiments in which 1200 to 37 000 cardiomyocytes were added to different wells of a microtitre plate. There was a significant (P < 0.01) linear relationship between cell number and absorbance with a correlation coefficient of 0.98. Trypan blue exclusion To assess cell viability, cardiomyocytes were grown on coverslips. After 72 h in culture, palmitate or its diluent was added to the media. At predetermined times, the coverslips were removed, stained with 0.4% trypan blue and examined microscopically in a hemocytometer to determine the total number of cells and the number of dead cells, i.e. those retaining trypan blue. Four representative high-power fields were selected and counted and the mean was calculated. Measurement of [Ca2+]I Calcium fluxes were measured according to Huber et al.28 In brief, chick cardiomyocytes were incubated with 2 uM fura-2/AM in Tyrode’s buffer (130 mM NaCl, 5 mM KCl, 1.4 mM MCaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.1% BSA in 10 mM Hepes, pH 7.4) at 23°C for 45 min. Then,
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the cells were washed. resuspended in 1 ml of the same buffer at 5 × 105 cells/ml in a stirring cuvette. Following stimulation by ionomycin (10 uM) or palmitate (100 uM), cytosolic calcium was measured by monitoring fluorescence intensity at 510 nm, after excitation of the sample with two different wavelengths (340 and 380 nm) using an MC200 monochromator from SLM AMINCO with a 8100 V3.0 software program. Materials All cell culture media components were from Gibco/BRL Life Sciences (Burlington, Ontario, Canada). Palmitate, arachidonic acid, thapsigargin, ryanodine and nifedipine were from Sigma Chemicals Ltd (St Louis, MO, USA). Palmitate was usually dissolved in warm ethanol and was freshly prepared for each experiment. Arachidonic acid was kept under N2, diluted in methanol and used shortly thereafter. For comparative studies with arachidonic acid, palmitate was similarly dissolved in methanol. Trypan blue was from BDH Chemicals Ltd (Poole, England). The MTT assay kit was from Promega (Madison, WI, USA). Fura-2/AM was from Molecular Probes (Eugene, OR, USA). All other chemicals were from Fischer Scientific (Ottawa, Ontario, Canada). Data analysis The data are presented as the mean±SEM. Hypothesis testing used a one-way analysis of variance. Examination of a pair of groups used Kruskal–Wallis multiple–comparison test. Linear least-squares regression was also performed. The null hypothesis was rejected if the probability of a Type I error was less than 5% (P < 0.05). RESULTS Palmitate reduces cardiomyocyte survival Cardiomyocytes treated with palmitate (100 uM) for 24 h displayed a marked and significant (P < 0.001) reduction in MTT absorbance compared to vehicle treated (control) cells (Fig. 1). There was little overlap in absorbance between palmitate treated and diluent treated cells. The effect of palmitate cannot be attributed to a non-specific effect of a fatty acid, since the C12 saturated fatty acid, laurate (100 uM) did not affect cell viability. Based on the ability of viable cells to reduce MTT and the linear relationship between cardiomyocyte cell number and MTT absorbance, palmitate reduced cell viability or increased cell death. Compared to control, palmitate (100 uM) produced a significant (P < 0.001 ) and 42.9± 5.3% increase in cell death. Palmitate-induced cardiomyocyte cell death was confirmed by the trypan blue exclusion assay which demonstrated a significant © 1999 Harcourt Publishers Ltd
Fig. 1 The effect of palmitate on the MTT absorbance. Cardiomyocytes were seeded in multiwell microtitre at 20 000 cells per well. After 72 h, cells were treated with palmitate, laurate or the diluent (ethanol) for 24 h. MTT dye was added to each well for the last 4 h of treatment, the reaction was stopped and the absorbance (optical density) was determined at 570 nm. Background absorbance of medium in the absence of cells was subtracted. The graph is a box plot that plots the median (horizontal bar) and the 90th percentile of the distribution for all values. The results are from independent experiments, done in duplicate, for cells treated with palmitate (N = 14), laurate (N = 5) or the diluent (ethanol) (N = 14).
(P < 0.05), 3.6 ± 1.3-fold, increase in the ratio of dead to alive cells from 0.25 ± 0.01 to 0.88 ± 0.30 for, respectively, diluent and palmitate (100 uM) treatment for 24 h. To assess the role of increased [Ca2+]I in palmitateinduced cell death, cardiomyocytes were treated with the calcium ionophore ionomycin (10 uM) alone or in combination with palmitate (100 uM) for 24 h. Inonomycin significantly (P < 0.05) increased cardiomyocyte cell death. Cells treated with palmitate plus ionomycin showed significantly more cell death compared to cells treated with either ionomycin or palmitate alone (Fig. 2). Palmitate and [Ca2+]I Based on these observations, we asked whether palmitate was capable of modulating the calcium response induced by ionomycin. Measurement of [Ca2+]I using the fluorescent probe Fura-2/AM confirmed the ability of ionomycin to strongly increase [Ca2+]I (Fig. 3). The pattern of the [Ca2+]I response was consistent with a sustained increase in [Ca2+]I rather than a transient Ca2+ increase associated with receptor-operated Ca2+ influx. To assess the modulating capacity of palmitate, this fatty acid was added 200 s after ionomycin. The addition of palmitate did not further enhance the [Ca2+]I response initiated by ionomycin (Fig. 3). Furthermore, palmitate alone did not induce an increase in [Ca2+]I nor was the increase in [Ca2+]I produced by ionomycin enhanced by palmitate pretreat-
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Fig. 2 The effect of ionomycin on palmitate-induced cell viability or cell death was assessed by the MTT assay. Cardiomyocytes were seeded in multiwell microtitre at 20 000 cells per well. After 72 h, cells were treated with palmitate, 100 uM, (n = 14), ionomycin 10 uM (n = 5), the combination of both (n = 5) or the diluent (control) (n = 14) for 24 h. MTT dye was added to each well for the last 4 h of treatment, the reaction was stopped and the absorbance (optical density) was determined at 570 nm. Background absorbance of medium in the absence of cells was subtracted. Each sample was done in duplicate. The data are expressed as a percentage of their relevant control, in the same plate and represent the mean±S.E.M.
Fig. 3 Effect of ionomycin and palmitate on intracellular calcium mobilization. Intracellular calcium concentration was measured in chick cardiomyocytes using Fura-2/AM as described in Materials and Methods. Ionomycin (10 uM) was added at 100 s and palmitate (100 uM) at 300 s.
ment (Fig. 4). In contrast to palmitate, arachidonic acid produced a sustained increase in [Ca2+]I (Fig. 5). Modulation of [Ca2+]I and cardiomyocyte cell death Recognizing that calcium channel blockers produce only a small decrease in [Ca2+]I,29 we nevertheless sought to determine if the calcium channel blocker nifedipine would ameliorate the cardiotoxicity of palmitate. Nifedicine is a relatively specific calcium channel blocker in these cardiomyocytes30 and these agents can protect against calcium-overload-mediated cell death.31 There was no significant reduction in palmitate-induced cardiomyocyte
Fig. 4 Palmitate does not alter ionomycin-induced calcium flux. Fura-2/AM-preloaded chick cardiomyocytes were stimulated with palmitate (100 uM) at 100 s and with ionomycin (10 uM) at 300 s, and intracellular calcium concentrations were measured according to Materials and Methods. The slight increases after palmitate stimulation is due to the diluent of the fatty acid, ethanol.
Fig. 5 Arachadonic acid but not palmitate increases intracellular calcium. Fura-2/AM-preloaded chick cardiomyocytes were stimulated with palmitate (200 uM) or arachadonic acid (200 uM) or their diluent methanol and intracellular calcium concentrations were measured according to Materials and Methods.
death with nifedipine (10 uM) suggesting that palmitate’s action is calcium-independent or at least not mediated through or modulated by voltage-operated calcium channels (Fig. 6). Palmitate-induced cell death was examined further by employing the SR Ca2+ ATPase inhibitor thapsigargin. This tumor-promoting sesquiterpene lactone is known to induce release of Ca2+ from the endoplasmic and sarcoplasmic reticulum. Specifically we compared the cell death induced by thapsigargin (10–9 M), palmitate alone (100 uM) and their combination. The two agents together resulted in an additive effect on cell death (Fig. 7) verifying our data with the ionophore ionomycin (Fig. 2). The increase in cell death produced by thapsigargin plus palmitate (63.3 ± 7.6%) was similar to the arithmetic
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Fig. 6 Effect of nifedipine on palmitate-induced cell death in cardiomyocytes. The bar graph showing the effect of nifedipine 0.1 uM (n = 8), or 1.0 (n = 8) or 10.0 uM (n = 9) as well as the combination of palmitate with nifedipine 0.1 uM (n = 8), or 1.0 (n = 8) or 10.0 uM (n = 9). The palmitate data is repeated from Figure 2 for ease of comparison. Results are the change from control and are expressed as the mean±S.E.M.
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Fig. 8 Effect of ryanodine on palmitate-induced cell death on cardiomyocytes. The bar graph showing the effect of ryanodine 1 uM (n = 6), ryanodine 3 uM (n = 6), the combination of palmitate 100 uM plus ryanodine 10 uM (n = 6) or ryanodine 3 uM (n = 6). The palmitate data is repeated from Figure 2 for ease of comparison. Results are the change from control and are expressed as the mean±S.E.M.
uptake and there seems to be no interdependence of the signals generated by these agents. DICUSSION
Fig. 7 The effect of thapsigargin on palmitate-induced reduction cell viability. Cardiomyocytes were maintained in multiwell plates for 72 h and then treated with palmitate, 100 uM, thapsigargin 10–9 M (n = 6), the combination of both (n = 6) or the diluent (control) for 24 h. The difference in absorbance at 570 nm was calculated and subtracted from background absorbance of the medium in the absence of cells. The palmitate data is repeated from Figure 2 for ease of comparison. The data are expressed as a percentage of their relevant control, in the same plate and represent the mean±S.E.M. of separate experiments.
combination of palmitate (42.9 ± 5.3%) and thapsigargin (15.5 ± 6.1%). Thapsigargin was verified to increase [Ca2+]I in our chick cardiomyocytes (data not shown). To explore further the potential of calcium release from SR to modulate palmitate-induced cell death, cardiomyocytes were treated with ryanodine alone or in combination with palmitate. In contrast to thapsigargin, low-micromolar concentrations of ryanodine, a SR Ca2+-release channel activator, depletes SR Ca2+ storage and blunts the release of Ca2+ from the SR.22 There was no increase in cell death produced by the combination of ryanodine and palmitate compared to palmitate alone (Fig. 8). Our experiments in total suggest that the cell-death-initiating actions of palmitate are augmented by agents that increase transsarcolemmal calcium influx or inhibit SR calcium re© 1999 Harcourt Publishers Ltd
Fatty acids are the major substrate for myocardial energy production; however, high extracellular concentrations of the 16-carbon saturated fatty acid, palmitate, induce myocardial cell death. The amount of cell death produced by palmitate in embryonic cardiomyocytes in this study was consistent with the data in neonatal rat cardiomyocytes reported by DeVries et al.13 The potential mechanisms speculated to be responsible for palmitate-induced cardiomyocyte cell death include rapid increases in [Ca2+]I with attendant calcium overload.8 The results of our study question this possibility, as there was no change in [Ca2+]I in response to palmitate. The present study confirms the data of Vacher et al.11 in GH3 cells that palmitate does not increase [Ca2+]I and extends it to cardiomyocytes. Our study provides data to support the theoretical argument against calcium as a mediator of palmitate-induced cardiomyocyte death.13 In contrast to palmitate, we found that the unsaturated fatty acid arachidonic acid increases [Ca2+]I; a finding consistent with the data of other investigators in other cell types.8,11 In GH3 cells, arachidonic acid increases [Ca2+]I in part through stimulation of ICa.11 In contrast to arachidonic acid, the saturated fatty acid stearic acid does not alter ICa through voltage-gated L-channels in neonatal rat cardiomyocytes.32 A major finding of this study is that palmitate-induced cell death is augmented by ionomycin. This is consistent with different mechanisms of action for these agents, as ionomycin increased while palmitate did not increase [Ca2+]I. Considering the deleterious role of myocardial calcium overload in conditions such myocardial infarction,
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the present study highlights the adverse effects of elevated plasma concentrations of free fatty acids that accompany acute myocardial infarction.1–4 The combination of increased extracellular palmitate and calcium overload would be especially damaging to the myocardium. Thapsigargin in combination with palmitate increased cardiomyocyte cell death to levels greater than what was induced by palmitate or thapsigargin alone. Thapsigargin which produces an initial increase in [Ca2+]I through inhibition of SR calcium reuptake,22,24,25 produced a small amount of cardiomyocyte cell death. In other cell types, thapsigargin appears to induce more cell death.18,24,33 While the most likely explanation for the adverse effect of the combination of thapsigargin plus palmitate is similar to ionomycin as thapsigargin increases [Ca2+]I,22,24,25 an alternative explanation exists. Thapsigargin depletes the intracellular SR calcium pool and cell death has been attributed to SR calcium depletion in neuroblastoma cell lines, fetal rat cerebrocortical cells in cultures and the S49 cell line.34,35 This is a less attractive explanation because low (micromolar) concentrations of ryanodine, a SR Ca2+-release channel activator, depletes SR Ca2+ stores and blunts the release of Ca2+ from the SR.22 Yet we found no increase in cell death produced by the combination of ryanodine and palmitate compared to palmitate alone. Exogenous fatty acids contribute about 90% of the fatty acid carbon present in cellular triglyceride and phospholipid.36 The presence of fatty acids in excess of that required for energy or phospholipid synthesis results in excessive triacylglycerol synthesis, leading to structural alterations of the cell.36 One of the putative explanations for palmitate-induced cell death is precipitation of triacylglycerol, from the excess available palmitate, at the site of its synthesis in SR with resulting impairment in SR function.13 Data from the present study do not support this hypothesis, as there was no synergistic effect of palmitate with thapsigargin or ryanodine, both of which alter SR calcium handling. In contrast to palmitate, the unsaturated fatty acids oleic acid and arachidonic acid, increase [Ca2+]I in Ehrlich tumor cells from a thapsigarginsensitive Ca2+ pool.12 The present study used embryonic chick cardiomyocytes. While the origin of the cell type is important in the extrapolation of the data, this cell type has several advantages. First, components of cell death pathways in this cell type have similarities to humans.37 Second, intracellular calcium in the embryonic chick heart is distributed in three kinetically different compartments similar to adult mammalian hearts.38 Third, the response to arachidonic acid is similar to mammalian cardiomyocytes.8 In summary, palmitate induces cardiomyocyte cell death through a mechanism that does not involve direct increases in [Ca2+]I. Ionomycin, which increased [Ca2+]I, augmented palmitate-induced cell death despite the
absence of enhancement of ionomycin-induced elevation [Ca2+]I, by palmitate. The ability of palmitate to induce cardiomyocyte cell death in the presence of thapsigargin or ryanodine suggests that palmitate-induced cardiomyocyte death is independent of an action on SR calcium flux. The implication of these findings are that in situations of elevated circulating palmitate, cardiomyocytes are vulnerable to enhanced cell death in conditions associated with increased [Ca2+]I.38,39 ACKNOWLEDGMENTS This study was supported in part by a grant from the Heart and Stroke Foundation of British Columbia and the Yukon. The authors thank Carol Smythe for her technical assistance.
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