Apoptosis In Neonatal Cardiomyocytes Research Articles

Protective effect of eicosapentaenoic acid on palmitate-induced apoptosis in neonatal cardiomyocytes

Long chain polyunsaturated fatty acids (PUFAs) play an important role in cardioprotection. These effects have been largely attributed to membrane docosahexaenoic acid. Conversely, saturated fatty acids trigger apoptosis in cardiomyocytes, with modifications of mitochondrial properties including cardiolipin loss, cytochrome c release and caspase-3 activation. The purpose of this study was to investigate the chronic effect of eicosapentaenoic acid (EPA) on mitochondrial apoptosis induced by palmitate treatment and the associated signalling pathways. Confluent cultures of rat neonatal cardiomyocytes were treated for 2 days in media enriched with either EPA or arachidonic acid (AA) and then exposed to palmitate (0.5 mM) to induce apoptosis, in the absence of PUFA supplements. The EPA treatment resulted in significant membrane enrichment in n − 3 PUFAs, especially in docosapentaenoic acid (DPA), and a large decrease in AA. Both AA and EPA treatments prevented caspase-3 activation, translocation of Bax to the mitochondria and release of cytochrome c induced by palmitate treatment. Furthermore, EPA, but not AA prevented the loss of mitochondrial cardiolipin due to apoptosis. These results suggest that EPA supplementation is able to protect cardiomyocytes against palmitate-induced apoptosis via an implication of different mitochondrial elements, possibly through its elongation to DPA, which is very efficient in cardiomyocytes.

The Pro-angiogenic Cytokine Pleiotrophin Potentiates Cardiomyocyte Apoptosis through Inhibition of Endogenous AKT/PKB Activity

Pleiotrophin is a development-regulated cytokine and growth factor that can promote angiogenesis, cell proliferation, or differentiation, and it has been reported to have neovasculogenic effects in damaged heart. Developmentally, it is prominently expressed in fetal and neonatal hearts, but it is minimally expressed in normal adult heart. Conversely, we show in a rat model of myocardial infarction and in human dilated cardiomyopathy that pleiotrophin is markedly up-regulated. To elucidate the effects of pleiotrophin on cardiac contractile cells, we employed primary cultures of rat neonatal and adult cardiomyocytes. We show that pleiotrophin is released from cardiomyocytes in vitro in response to hypoxia and that the addition of recombinant pleiotrophin promotes caspase-mediated genomic DNA fragmentation in a dose- and time-dependent manner. Functionally, it potentiates the apoptotic response of neonatal cardiomyocytes to hypoxic stress and to ultraviolet irradiation and of adult cardiomyocytes to hypoxia-reoxygenation. Moreover, UV-induced apoptosis in neonatal cardiomyocytes can be partially inhibited by small interfering RNA-mediated knockdown of endogenous pleiotrophin. Mechanistically, pleiotrophin antagonizes IGF-1 associated Ser-473 phosphorylation of AKT/PKB, and it concomitantly decreases both BAD and GSK3beta phosphorylation. Adenoviral expression of constitutively active AKT and lithium chloride-mediated inhibition of GSK3beta reduce the potentiated programmed cell death elicited by pleiotrophin. These latter data indicate that pleiotrophin potentiates cardiomyocyte cell death, at least partially, through inhibition of AKT signaling. In conclusion, we have uncovered a novel function for pleiotrophin on heart cells following injury. It fosters cardiomyocyte programmed cell death in response to pro-apoptotic stress, which may be critical to myocardial injury repair.

Application of flow-cytometry in the study of apoptosis in neonatal rat cardiomyocytes

Depending on the concentration, catecholamines activate various intracellular signaling pathways and can induce apoptosis in cardiac myocytes. Although 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) has been previously used to study mitochondria in intact cardiomyocytes, there have been no reports on the detection of apoptosis in neonatal cardiomyocytes in combination with flow cytometry and confocal microscopy. In our study, neonatal rat cardiomyocytes were exposed to norepinephrine (NE) and isoproterenol (ISO) in concentrations of 1 and 10 microM for 48 h. NE concentrations of 1 and 10 microM decreased the number of viable cardiomyocytes by 18% (*p < 0.05) and 24% (**p = 0.01), respectively. ISO in a concentration of 1 microM increased the number of viable cardiomyocytes by 13% while 10 microM decreased the number of viable cardiomyocytes by 43% (***p < 0.001). Apoptotic cells were detected by flow cytometry and confocal microscopy. NE in concentrations of 1 and 10 microM increased the percentage of apoptotic cells by 12.2% and 34.3%, respectively, while ISO alone in a concentration of 10 microM increased the percentage of apoptotic cells by 11.3%. The results demonstrated that these two methods are reliable and suitable for the detection and study of apoptosis in cultures of neonatal cardiomyocytes.

PUMA is critical for neonatal cardiomyocyte apoptosis induced by endoplasmic reticulum stress

Puma (p53-upregulated modulator of apoptosis), a proapoptotic BH3-only member of the Bcl-2 protein family, has been implicated in the pathomechanism of several diseases, including cancer, AIDS, and ischemic brain disease. We have recently shown that Puma is required for cardiac cell death upon ischemia/reperfusion of mouse hearts. Since ischemia/reperfusion is also associated with endoplasmic reticulum (ER) stress, in the present study we investigated whether Puma contributes to the ER stress-dependent component of cardiomyocyte apoptosis. Primary cultures of rat and mouse neonatal cardiomyocytes were treated with 3 muM thapsigargin or 100 ng mL(-1) tunicamycin. Puma levels were suppressed by adenoviral delivery of shRNA or targeted deletion of the puma gene. Puma expression was detected by RT-PCR and Western blotting. Apoptosis was assessed by TUNEL assay, caspase-3 cleavage, and cytochrome c release. We have shown that in rat neonatal cardiac myocytes, thapsigargin or tunicamycin treatment led to ER-stress, transcriptional upregulation of Puma, and apoptosis. Most importantly, cardiac myocytes acquired resistance to ER stress-induced apoptosis if Puma expression was downregulated by adenoviral delivery of shRNA or eliminated by targeted deletion in knockout mice. Taken together, our data indicate that Puma is a critical component of ER stress-induced apoptosis in cardiac myocytes, and inhibition of Puma activity may be used to treat cardiac infarcts or prevent heart failure by blocking ER stress-induced apoptosis.

α-linolenic acid prevents TNF-α-induced apoptosis in neonatal cardiomyocytes

INTRODUCTION

THE RESPONSE OF NEONATAL RAT VENTRICULAR MYOCYTES TO LIPOPOLYSACCHARIDE-INDUCED STRESS

Sepsis induced by exposure to lipopolysaccharide (LPS) can be life-threatening and lead to multiple-organ dysfunction. Sepsis-associated cardiac dysfunction is a primary cause of mortality. The response of isolated cardiac myocytes to LPS exposure is poorly understood. Cultured neonatal rat ventricular cardiomyocytes were used to evaluate the response to LPS exposure. Other authors have reported that LPS exposure at doses sufficient to induce tumor necrosis factor alpha (TNF-alpha) production and apoptosis in adult cardiomyocytes do not induce apoptosis in neonatal cardiomyocytes. We therefore hypothesized that neonatal cardiomyocytes have innate protective mechanisms that protect from septic damage. Cultured neonatal rat ventricular cardiomyocytes were stimulated by exposure to LPS for varying lengths of time. NFkappaB signaling pathways, TNF-alpha production, and Akt activation were monitored. We also assessed the induction of apoptosis in these cells by monitoring caspase-3 activity. LPS rapidly stimulates nuclear translocation of NFkappaB and Akt activation. TNF-alpha production is also stimulated. However, high doses of LPS are unable to induce apoptosis in these cells, and protection is not a function of Akt activation. LPS treatment also stimulated the levels of cyclooxygenase-2 and the production of downstream metabolites, specifically PGE2 and 15deoxyDelta12-14PGJ2 (15dPGJ2). Specific inhibition of cyclooxygenase-2 activity induced apoptosis in the presence of LPS, whereas direct exposure to 15dPGJ2 at pharmacological levels induced apoptosis. Neonatal rat ventricular cardiomyocytes have innate protective mechanisms that prevent apoptotic cell death after LPS exposure. Metabolic products of arachidonic acid metabolized by the cyclooxygenase pathway can be potentially apoptotic or antiapoptotic. The balance of these products within these cells may define the cellular response to LPS exposure.

Interrelations between oxidative stress and calcineurin in the attenuation of cardiac apoptosis by eugenol

In view of the known involvement of oxidative stress and calcineurin (Ca(2+)-calmodulin dependent protein phosphatase) in beta-Adrenergic stimulated events, we examined the influence of eugenol (an antioxidant generally regarded as safe by the Food and Agricultural Organization of the United Nations) on isoproterenol-induced apoptosis in neonatal cardiomyocytes. In comparison to unstimulated controls, cardiomyocytes stimulated with 50 microM isoproterenol for 48 h demonstrated (a) increased intracellular Ca(2+) levels (b) oxidative stress involving enhanced reactive oxygen species, decreased GSH/GSSG ratio, enhanced lipid peroxidation, increased activities of superoxide dismutase and glutathione peroxidase (c) apoptosis, evidenced by increased number of annexin V/TUNEL positive cells, enhanced membrane fluidity, decreased mitochondrial membrane potential, increased activities of caspase 3 and 9 along with (d) increased calcineurin activity. Pre-incubation of cardiomyocytes with 100 microM eugenol for 1 h, followed by isoproterenol treatment for 48 h, led to reversal of enhanced intracellular Ca(2+) levels, oxidative stress, calcineurin activation and apoptosis caused by isoproterenol. In addition, similar treatment of cardiomyocytes with 10 nM FK506, a calcineurin inhibitor, could also attenuate isoproterenol-induced apoptosis. These results indicate the beneficial effects of eugenol in preventing cardiomyocyte apoptosis.

Palmitate-induced apoptosis in neonatal cardiomyocytes is not dependent on the generation of ROS.

The saturated fatty acid palmitate induces apoptosis in neonatal rat cardiomyocytes. This apoptosis is associated with early mitochondrial release of cytochrome c and a subsequent loss of mitochondrial membrane potential. Recent reports implicate a role for reactive oxygen species (ROS) in palmitate-induced apoptosis. We studied the role of ROS in palmitate-induced apoptosis in the neonatal rat cardiomyocyte and report no evidence of ROS involvement. ROS production, nitric oxide production, and nuclear factor-kappaB activation were not increased above those observed using the nonapoptotic fatty acid oleate. Indeed, the production of ROS was significantly higher in cells treated with oleate. Furthermore, the presence of antioxidants and ROS scavengers did not attenuate the induction of apoptosis by palmitate. Variations in the fatty acid-to-albumin ratio from 2:1 to 7:1 had no effect on the absence of ROS production or on the extent of apoptosis. No evidence was found for an increase in oxidative protein modification in palmitate-treated cells. Our results lead us to conclude that oxidative stress does not play a role in palmitate-induced apoptosis.

Effect of NF- κ B Inhibition on TNF- α -induced Apoptosis and Downstream Pathways in Cardiomyocytes

Heart-specific inhibition of survival pathway gp130 was recently shown to sensitize transgenic mice towards stress stimuli, resulting in rapid onset of cardiac dilatation and heart failure. In order to identify further survival pathways we evaluated the role of transcription factor nuclear factor- κ B (NF- κ B) in tumour necrosis factor- α (TNF- α)-induced apoptosis of cardiomyocytes. TNF- α stimulation (10 ng/ml) of both H9c2 cells and primary cardiomyocytes isolated from neonatal Wistar rats resulted in rapid nuclear translocation of NF-κ B complexes. The NF- κ B complexes consisted of rel-proteins p50 and p65, as revealed by supershift analysis. Addition of proteasome inhibitor MG132 or adenoviral expression of a truncated I κ B α (Iκ BΔN) inhibited TNF- α -induced NF- κ B nuclear translocation in a dose-dependent manner. Both neonatal cardiomyocytes and H9c2 cells were resistant to TNF-induced apoptosis. However, specific inhibition of NF-κ B activation by Ad5-I κ B αΔN (MOI=50) or MG132 (5 μ m) increased apoptosis as measured by subG1-assay (H9c2 cells) and annexin V binding/propidium iodide (neonatal cardiomyocytes, FACS-analysis: 7±2% to 26±5% annexin V positive/PI negative), respectively. TUNEL-assay double-stained with anti- α -sarcomeric actin confirmed apoptosis of neonatal cardiomyocytes. Furthermore, caspase-3 activation was increased by 52±7% in neonatal cardiomyocytes after TNF α+Ad5-I κ Bα ΔN compared to TNF α+Ad5-control treatment. Protein levels of hiAP1, hiAP2, x-iAP, bcl-2 and bcl-x(L) were neither downregulated by NF- κ B inhibition nor upregulated by TNF- α stimulation. In summary, cardiomyocytes utilize transcription factor NF- κ B to activate survival factors in the context of TNF-α stimulation. As locally increased levels of TNF- α have been detected in heart failure, NF-κ B activity is essential for cellular homeostasis in the heart.

Fatty acid-induced apoptosis in neonatal cardiomyocytes: redox signaling.

Exposure of neonatal rat cardiac myocytes to palmitate and glucose produces apoptosis as seen by cytochrome c release, caspase 3-like activation, DNA laddering, and poly(ADP-ribose) polymerase cleavage. The purpose of this study was to understand the role of reactive oxygen species in the initiation of programmed cell death by palmitate. We found that palmitate (but not oleate) produces inhibition of carnitine palmitoyltransferase I, accumulation of ceramide, and inhibition of electron transport complex III. These events are subsequent to cytochrome c release and loss of the mitochondrial membrane potential. No differences in H2O2 production or N-terminal c-Jun kinase phosphorylation were detected between myocytes incubated in palmitate and control myocytes (nonapoptotic) incubated in oleate. These results suggest that the palmitate-induced loss of the mitochondrial membrane potential is not associated with H2O2 synthesis and that a membrane potential is required to generate reactive oxygen species following ceramide inhibition of complex III.

Effect of vacuolar proton ATPase on pHi, Ca2+, and apoptosis in neonatal cardiomyocytes during metabolic inhibition/recovery.

Recently, we found that vacuolar proton ATPase (VPATPase) operates in cardiomyocytes as a complementary proton-extruding mechanism. Its activity was increased by preconditioning with resultant attenuation of intracellular acidification during ischemia. In this study, we examined whether VPATPase-mediated proton efflux during metabolic inhibition/recovery may spare Na+ overload via Na+-H+ exchange, attenuate Na+-Ca2+ exchange, and decrease apoptosis. Neonatal rat cardiomyocytes were subjected to 2- to 3-hour metabolic inhibition with cyanide and 2-deoxyglucose and 24-hour recovery. The effect of VPATPase inhibition by 50 nmol/L bafilomycin A1 on apoptosis, pHi, and [Ca2+]i was studied by flow cytometry with propidium iodide, seminaphthorhodafluor (SNARF)-1-AM, and indo-1-AM staining, respectively. VPATPase inhibition increased the amount of apoptosis measured after 24 hours of recovery and abrogated the protective effect of inhibition of Na+-H+ exchange by (5-N-ethyl-N-isopropyl)amiloride (EIPA). Dual blockade of VPATPase and Na+-H+ exchange was additive in effect with EIPA on pHi during metabolic inhibition/recovery and recovery from the acid challenge with sodium propionate. VPATPase blockade increased the rate of accumulation of intracellular Ca2+ at the beginning of metabolic inhibition and abrogated the delaying effect of EIPA on intracellular Ca2+ accumulation. These results indicate that VPATPase plays an important accessory role in cardiomyocyte protection by reducing acidosis and Na+-H+ exchange-induced Ca2+ overload.

Enhanced expression of p53 and apoptosis induced by blockade of the vacuolar proton ATPase in cardiomyocytes.

Activation of the vacuolar proton ATPase (VPATPase) has been implicated in the prevention of apoptosis in neutrophils and adult cardiac myocytes. To determine the role of the VPATPase in apoptosis of cardiac myocytes, we used a potent and specific inhibitor of the VPATPase, bafilomycin A1. Bafilomycin A1 alone caused increased DNA laddering of genomic DNA and increased nuclear staining for fragmented DNA in neonatal cardiomyocyte apoptosis in a dose- and time-dependent manner. Intracellular acidification in cardiac myocytes was also observed after 18 h of bafilomycin A1 treatment. Accordingly, bafilomycin A1-treated myocytes also showed increased accumulation of p53 protein and p53-dependent transactivation of gene expression, including a persistent upregulation of p21/wild-type p53 activated fragment 1/cyclin kinase inhibitor protein-1 mRNA. The bafilomycin A1-induced increase in p53 protein levels was accompanied by a marked increase in p53 mRNA accumulation. In contrast, cardiac fibroblasts treated with bafilomycin A1 showed no change in p53 protein expression or pHi and did not undergo apoptosis even after 24 h of treatment. Our data suggest that blockade of the VPATPase induces apoptotic cell death of cardiac myocytes and that this may occur through a p53-mediated apoptotic pathway.

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion.

Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its anti-apoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.