mTOR-STAT3-notch signalling contributes to ALDH2-induced protection against cardiac contractile dysfunction and autophagy under alcoholism - PubMed (original) (raw)

mTOR-STAT3-notch signalling contributes to ALDH2-induced protection against cardiac contractile dysfunction and autophagy under alcoholism

Wei Ge et al. J Cell Mol Med. 2012 Mar.

Abstract

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) has been shown to benefit myopathic changes following alcohol intake, although the precise mechanism is still unclear. This study was designed to evaluate the role of ALDH2 on chronic alcohol intake-induced myocardial geometric and functional damage with a focus on autophagic signalling. Wild-type friendly virus B (FVB) and transgenic mice overexpressing ALDH2 driven by chicken β-actin promoter were fed a 4% alcohol liquid diet for 12 weeks. Cardiac geometry and function were assessed using echocardiographic and IonOptix systems. Western blot analysis was used to evaluate the essential autophagy markers, Akt and AMP-dependent protein kinase (AMPK) as well as their downstream signalling mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3). Alcohol intake altered cardiac geometry and function as demonstrated by lessened LV wall and septal thickness, enlarged end systolic and diastolic diameters, decreased fractional shortening and cell shortening, the effects of which were mitigated by ALDH2 transgene. Chronic alcohol intake triggered myocardial autophagy as shown by LC3B II isoform switch, as well as decreased phosphorylation of mTOR, the effects of which were ablated by ALDH2. Chronic alcohol intake suppressed phosphorylation of Akt and AMPK, which was reconciled by ALDH2. Levels of Notch1 and STAT3 phosphorylation were dampened by chronic alcohol intake in FVB but not ALDH2 myocardium. Moreover, the γ-secretase Notch inhibitor N\xE2\x80\x90[N-(3,5-difluorophenacetyl)-1-alany1]-S-phenyglycine t-butyl ester exacerbated ethanol-induced cardiomyocyte contractile dysfunction, apoptosis and autophagy. In summary, these findings suggested that ALDH2 elicits cardioprotection against chronic alcohol intake-induced cardiac geometric and functional anomalies by inhibition of autophagy possibly via restoring the Akt-mTOR-STAT3-Notch signalling cascade.

© 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

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Figures

Fig 1

Fig 1

Effect of 12 week alcohol intake on echocardiographic indices in FVB and ALDH2 mice. (A) Heart rate; (B) LV wall thickness; (C) intraventricular (IV) septal thickness; (D) LV EDD; (E) LV ESD and (F) fractional shortening. Mean ± S.E.M., n = 6–11 mice per group, *P < 0.05 versus FVB group, #P < 0.05 versus FVB-ethanol group.

Fig 2

Fig 2

Effect of 12 week alcohol intake on cardiomyocyte shortening in myocytes from FVB and ALDH2 mice. (A) Resting cell length; (B) peak cell shortening (normalized to cell length); (C) maximal velocity of shortening (+d_L_/d_t_); (D) maximal velocity of relengthening (–d_L_/d_t_); (E) TPS and (F) TR90. Mean ± S.E.M., n = 66–67 cells from three mice per group, *P < 0.05 versus FVB group, #P < 0.05 versus FVB-ethanol group.

Fig 3

Fig 3

Effect of 12 week alcohol intake on autophagy markers in myocardium from FVB and ALDH2 mice. (A) Beclin-1; (B) LC3B-II; (C) LC3B-I and (D) LC3B-II-to-LC3B-I ratio. Insets: representative immunoblots of Belin-1, LC3B-I, LC3B-II and GAPDH (loading control) using specific antibodies. Mean ± S.E.M., n = 4–5, *P < 0.05 versus FVB group, #P < 0.05 versus FVB-ethanol group.

Fig 4

Fig 4

Effect of 12 week alcohol intake on Akt and AMPK signalling in myocardium from FVB and ALDH2 mice. (A) Akt; (B) AMPK; (C) phosphorylated Akt (pAkt); (D) Phosphorylated AMPK (pAMPK); (E) pAkt-to-Akt ratio and (F) pAMPK-to-AMPK ratio. Insets: representative gel blots of pan and pAkt and AMPK using specific antibodies (GAPDH as loading control). Mean ± S.E.M., n = 4–5, *P < 0.05 versus FVB group, #P < 0.05 versus FVB-ethanol group.

Fig 5

Fig 5

Effect of 12 week alcohol intake on mTOR signalling in myocardium from FVB and ALDH2 mice. (A) Representative gel blots depicting pan and phosphorylated mTOR as well as α-tubulin proteins using specific antibodies; (B) mTOR; (C) pmTOR and (D) pmTOR-to-mTOR ratio. Mean ± S.E.M., n = 4–5, *P < 0.05 versus FVB group, #P < 0.05 versus FVB-ethanol group.

Fig 6

Fig 6

Effect of 12 week alcohol intake on Notch1 and STAT3 signalling in myocardium from FVB and ALDH2 mice. (A) Notch1; (B) STAT3; (C) phosphorylated STAT3 (pSTAT) and (D) pSTAT-to-STAT ratio. Representative gel blots depicting Notch1, STAT3, pSTAT3, α-tubulin and GAPDH (loading controls) using specific antibodies; Mean ± S.E.M., n = 4–5, *P < 0.05 versus FVB group, #P < 0.05 versus FVB-ethanol group.

Fig 7

Fig 7

Effect of the Notch inhibitor DAPT on ethanol toxicity-induced cardiomyocyte contractile dysfunction. Cardiomyocytes from FVB mice were incubated with ethanol (240 mg/dl) for four hrs in the absence and presence of DAPT (20 μM) before mechanical function was assessed. (A) Resting cell length; (B) peak cell shortening (normalized to cell length); (C) maximal velocity of shortening (+d_L_/d_t_); (D) maximal velocity of relengthening (–d_L_/d_t_); (E) TPS and (F) TR90. Mean ± S.E.M., n = 46 cells per group, *P < 0.05 versus control group, #P < 0.05 versus ethanol group.

Fig 8

Fig 8

Effect of the Notch inhibitor DAPT on ethanol-induced apoptosis and autophagy. Cardiomyocytes from FVB mice were incubated with ethanol (240 mg/dl) for four hrs in the absence and presence of DAPT (20 μM) prior to assessment of apoptosis and autophagy. (A) Caspase-3 activity; (B) LC3B-1 expression; (C) LC3B-II expression and (D) LC3B-II-to-LC3B-I ratio. Insets: representative gel blots of LC3B-I, LC3B-II and GAPDH (loading control) using specific antibodies. Mean ± S.E.M., n = 3–4 mice per group, *P < 0.05 versus control, #P < 0.05 versus ethanol group.

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