LPS-induced autophagy is mediated by oxidative signaling in cardiomyocytes and is associated with cytoprotection

Abstract

Bacterial endotoxin lipopolysaccharide (LPS) is responsible for the multiorgan dysfunction that characterizes septic shock and is causal in the myocardial depression that is a common feature of endotoxemia in patients. In this setting the myocardial dysfunction appears to be due, in part, to the production of proinflammatory cytokines. A line of evidence also indicates that LPS stimulates autophagy in cardiomyocytes. However, the signal transduction pathway leading to autophagy and its role in the heart are incompletely characterized. In this work, we wished to determine the effect of LPS on autophagy and the physiological significance of the autophagic response. Autophagy was monitored morphologically and biochemically in HL-1 cardiomyocytes, neonatal rat cardiomyocytes, and transgenic mouse hearts after the administration of bacterial LPS or TNF-alpha. We observed that autophagy was increased after exposure to LPS or TNF-alpha, which is induced by LPS. The inhibition of TNF-alpha production by AG126 significantly reduced the accumulation of autophagosomes both in cell culture and in vivo. The inhibition of p38 MAPK or nitric oxide synthase by pharmacological inhibitors also reduced autophagy. Nitric oxide or H(2)O(2) induced autophagy in cardiomyocytes, whereas N-acetyl-cysteine, a potent antioxidant, suppressed autophagy. LPS resulted in increased reactive oxygen species (ROS) production and decreased total glutathione. To test the hypothesis that autophagy might serve as a damage control mechanism to limit further ROS production, we induced autophagy with rapamycin before LPS exposure. The activation of autophagy by rapamycin suppressed LPS-mediated ROS production and protected cells against LPS toxicity. These findings support the notion that autophagy is a cytoprotective response to LPS-induced cardiomyocyte injury; additional studies are needed to determine the therapeutic implications.

Fig. 1.

LPS induced autophagy in cardiac cells. Green fluorescent protein (GFP)-microtubule-associated protein light chain 3 (LC3)-expressing HL-1 cells were exposed to LPS in medium, and autophagy was assessed. A: representative images of GFP-LC3-labeled puncta in HL-1 cells 4 h after exposure to 1 μg/ml LPS. B: histogram showing the number of autophagosomes per cell under basal conditions and after LPS exposure (x-axis, number of autophagosomes per cell; y-axis, number of cells observed with the indicated number of autophagosomes). C and D: autophagic flux in HL-1 cells was determined in the absence (black bars) or presence (hatched bars) of 100 nM Bafilomycin A1 (C) for 4 h or 3 μM chloroquine (hatched bars; D) for 2 h. The percentage of cells with numerous GFP-LC3 dots is represented as “%autophagy.” E: HL-1 cells were treated with 1 μg/ml LPS for 4 h or starved (stv) for 3.5 h in Krebs-Henseleit buffer in the absence or presence (+) of Bafilomycin A1. Cell lysates were separated on 10–20% SDS-PAGE and analyzed by Western blot using anti-LC3 antibody. F: GFP-LC3 expressing cells were treated with indicated concentrations of LPS for 4 h. Data represented as means ± SE from at least 3 independent experiments. *P < 0.01; **P < 0.001. Scale bar, 10 μm. Con, control.

Fig. 2.

LPS induced autophagy via TNF-α production. Cells were cultured under the same conditions as described in Fig. 1 and treated with 50 ng/ml TNF-α. A: representative images showing activation of autophagy in HL-1 cells 4 h after addition of 50 ng/ml TNF-α. Scale bar, 10 μm. B: population distribution of cells containing various numbers of autophagosomes after exposure to TNF-α. C: determination of autophagic flux in response to TNF-α in the absence (black bars) or presence (hatched bars) of Bafilomycin A1. *P < 0.01. D: Western blot of LC3-I/LC3-II processing under the same conditions. E: dose-dependent stimulation of autophagy by TNF-α. F: effect of tyrosine kinase inhibitor tyrphostin AG126 on LPS- or TNF-α-induced autophagy. HL-1 cells were incubated with LPS or TNF-α in the absence or presence of 30 μM AG126 for 4 h in the presence of Bafilomycin A1, and autophagy was scored by fluorescence microscopy. ns, Not significant. *P < 0.01.

Fig. 3.

MAPK pathway and nitric oxide synthase (NOS) activity is involved in LPS-mediated autophagy. A: p38 MAPK is activated by LPS. HL-1 cells were preincubated with 10 μM SB203580 (SB) for 2 h followed by 1 μg/ml LPS for the indicated time. Cell lysates were resolved on 10–20% SDS-PAGE and blotted with anti-phospho (p)-p38 or anti-p38 antibodies. B: effect of p38 MAPK inhibitor and NOS inhibitor on autophagy. HL-1 cells were pretreated with 10 μM SB or 10 μM N^G-monomethyl-l-arginine(l-NMMA) for 2 h followed by LPS for 4 h in the absence (black bars) or presence (hatched bars) of Bafilomycin A1. *P < 0.001 compared with control; **P < 0.001 compared with LPS treated. C: effect of p38 MAPK inhibitor and NOS inhibitor on TNF-α induced autophagy in HL-1 cells. *P < 0.01 compared with control; **P < 0.001 compared with TNF-α.

Fig. 4.

LPS-induced autophagy is mediated by oxidative stress. A: GFP-LC3-expressing HL-1 cells were preincubated with 2 mM N-acetyl-cysteine (NAC) for 2 h before 4 h incubation with 1 μg/ml LPS. Cells were fixed, and GFP-positive cells were scored for autophagy. Data are means ± SE of 3 independent experiments. *P < 0.01 compared with control; **P < 0.001 compared with LPS. B: GFP-LC3 HL-1 cells were preincubated without or with NAC before addition of 100 μM H₂O₂ or 200 μM sodium nitroprusside (SNP) for 4 h. Data are means ± SE of 3 independent experiments. *P < 0.01; **P < 0.001 compared with H₂O₂ or SNP.

Fig. 5.

Rapamycin (Rap or Rapa) inhibits reactive oxygen species (ROS) production and protects against LPS-mediated cell death. A: HL-1 cells were treated with LPS with or without 5 μM rapamycin for 4 h, and ROS production was determined using 5-(and 6-) chloromethyl-2′,7′-dichlorohydrofluoroscein diacetate acetyl ester. B: HL-1 cells were incubated without or with LPS for 4 h, and then total glutathione (GSH) was measured spectrophotometrically. C: HL-1 cells were exposed to LPS with or without 5 μM rapamycin pretreatment. After 48 h, cell death was scored with propidium iodide (PI) and Hoechst 33342. Representative images of nuclear staining are shown. D: quantification of cell death. Results are presented as fold increase compared with untreated cells. Data are means ± SE of 3 independent experiments. *P < 0.01 compared with control; **P < 0.001 compared with LPS. E: neonatal rat cardiac myocytes were cultured in serum-free media in the presence or the absence of wortmannin (Wm; 10⁻⁷ M) and/or LPS (10⁻⁴ M) as indicated for 48 h. Values are means of 4 independent experiments. **P < 0.001 compared with control; *P < 0.01 compared with LPS. TUNEL Pos, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling positive; RFU, relative fluorescent units.

Fig. 6.

LPS causes mitochondrial damage in heart tissue. Mice received LPS (1.5 mg/kg ip), and hearts were removed 4 h later and processed for electron microscopy. Representative images show swollen mitochondria with ruptured outer membranes (arrowheads) and autophagosomes (arrows). Control heart tissue is shown for comparison.

Fig. 7.

LPS stimulated autophagic flux and upregulated cathepsin D in vivo. Hearts of mCherry-LC3 transgenic mice were harvested 4 h after LPS injection (1.5 mg/kg ip injection) with or without concurrent administration of chloroquine (CQ) to prevent lysosomal degradation of autophagosomes. A: fluorescence micrographs of heart tissues under indicated conditions. Bright dots indicate autophagosomes. B: quantitative analysis of mCherry puncta from each condition. *P < 0.001 compared with control. C: immunoblot of LC3 in heart tissue lysates prepared from mice treated as described above. D: immunodetection of cathepsin D in sections of mCherry-LC3 transgenic mouse hearts treated as above (no CQ). Cathepsin D (green) colocalized with mCherry-LC3 puncta (red) as indicated by arrows. SS, steady state.

Fig. 8.

Effect of AG126 on induction of autophagy by LPS in vivo. mCherry-LC3 transgenic mice were pretreated with the tyrosine kinase inhibitor AG126 (0.5 mg/kg ip) or vehicle 1 h before LPS (1.5 mg/kg ip); hearts were harvested 4 h later. A: fluorescence micrographs of heart tissues under indicated conditions. Bright dots indicate autophagosomes. B: quantitative analysis of mCherry puncta from each condition, normalized to the number of nuclei in each field. *P < 0.001 compared with control; **P < 0.0001 compared with LPS.

Fig. 9.

Scheme of LPS-induced autophagy in cardiomyocytes. In cardiomyocytes, LPS induces activation of p38 MAPK and induces the expression of TNF-α and NOS. Multiple signaling pathways converge to ROS/reactive nitrogen species (RNS) production, which results in activation of autophagy through Atg4.