Palmitate induces H9c2 cell apoptosis by increasing reactive oxygen species generation and activation of the ERK1/2 signaling pathway

Abstract

Cardiac myocytes undergo apoptosis under conditions of high free fatty acid concentrations, including palmitate, which is implicated in lipotoxic cardiomyopathy. However, the underlying mechanisms remain unknown. The aim of the present study was to understand the role of reactive oxygen species (ROS) production and the extracellular signal‑regulated kinase 1/2 (ERK1/2) signaling pathway in palmitate‑induced apoptosis in H9c2 cells. H9c2 cells were exposed to palmitate for 12 h. The effect on the cell viability of H9c2 cells was evaluated using the 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) assay and cell apoptosis was determined by Hoechst 33342 staining. Levels of intracellular ROS were determined using a peroxide‑sensitive fluorescent probe, 2',7'‑dichlorofluorescein diacetate. Protein expression was measured by western blot analysis. Following treatment with palmitate for 12 h, H9c2 cells apoptosis was demonstrated as increased brightly condensed chromatin or unclear fragments by staining with Hoechst 33342, which was associated with increasing levels of active caspase‑3 and cleaved poly (ADP-ribose) polymerase (PARP). In this model of treatment with palmitate, H9c2 cell apoptosis correlated with increased levels of p53 and Bax expression and reduced levels of Bcl-2 expression. Palmitate‑induced apoptosis was observed to increase levels of intracellular ROS production and p‑ERK1/2 and decrease p‑Akt significantly. Consistent with these results, palmitate‑induced apoptosis was attenuated by the ERK1/2 inhibitor, U0126, through partial reduction of intracellular ROS generation. Collectively, these results indicate that palmitate‑induced apoptosis in H9c2 cells is mediated by activation of the ERK1/2 signaling pathway and increased ROS generation.

Inhibition of SH2-domain-containing inositol 5-phosphatase (SHIP2) ameliorates palmitate induced-apoptosis through regulating Akt/FOXO1 pathway and ROS production in HepG2 cells

“…However, the role of ROS in stimulating lipoapoptosis appears to be cell-type dependent as palmitate-treated neonatal cardiomyocytes undergo apoptosis independently of the oxidative stress [33]. In the current study, ROS production was identified at increased levels following palmitate-induced apoptosis in HepG2 cells, consistent with previous studies [21,32,34]. We also found that ascorbic acid co-treatment simultaneously normalized palmitate-induced ROS …”

“…We also observed an increased FOXO-1 phosphorylation in parallel with Another mechanism linking SHIP2 to apoptosis is oxidative stress. A growing body of evidence suggests that the oxidative stress due to excessive production of ROS is the main contributor to the pathophysiology of diabetic complications [21,30]. Increasing ROS production has been demonstrated to account for apoptosis in response to palmitate in numerous cell lines [31,32].…”

“…It has been previously shown that palmitate induces oxidative stress in different cells [17,21]. To investigate whether the effects of SHIP2 on apoptosis is mediated through the ROS production, HepG2 cells were co-incubated with 0.5mM palmitate and 50µM ascorbic acid as a ROS scavenger.…”

Palmitate promotes autophagy and apoptosis through ROS-dependent JNK and p38 MAPK

“…In neonatal rat ventricular myocytes, NAC and MAPK inhibitors failed to rescue PA-induced apoptosis. However, another study using H9c2 cells as the research object supports our conclusion, which showed that ERK inhibitor reduced PA-induced 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 apoptosis through partial suppression of intracellular ROS generation, suggesting important roles of MAPKs and ROS in H9c2 cells [4]. Thus, we speculate that the effects of PA may be cell type specific and the exact roles of ROS and MAPKs need to be verified in vivo.…”

“…Several studies have proposed that PA promotes ROS production in different cardiac cell types [4e8]. Although Wei et al reported that U0126, an inhibitor of extracellular signal-regulated kinase (ERK), reduced PA-induced apoptosis through partial suppression of intracellular ROS generation, there was no direct evidence to prove the involvement of ROS [4]. A recent study demonstrated that autophagy was induced by PA as an adaptive response to ER stress since it was sensitive to ER stress inhibition in H9c2 cells [9], however, whether ROS plays a role in PA-induced autophagy and apoptosis remains unknown.…”