STAT3 balances myocyte hypertrophy vis-à-vis autophagy in response to Angiotensin II by modulating the AMPKα/mTOR axis

<div><p>Signal transducers and activators of transcription 3 (STAT3) is known to participate in various cardiovascular signal transduction pathways, including those responsible for cardiac hypertrophy and cytoprotection. However, the role of STAT3 signaling in cardiomyocyte autophagy remains unclear. We tested the hypothesis that Angiotensin II (Ang II)-induced cardiomyocyte hypertrophy is effected, at least in part, through STAT3-mediated inhibition of cellular autophagy. In H9c2 cells, Ang II treatment resulted in STAT3 activation and cellular hypertrophy in a dose-dependent manner. Ang II enhanced autophagy, albeit without impacting AMPKα/mTOR signaling or cellular ADP/ATP ratio. Pharmacologic inhibition of STAT3 with WP1066 suppressed Ang II-induced myocyte hypertrophy and mRNA expression of hypertrophy-related genes ANP and β-MHC. These molecular events were recapitulated in cells with STAT3 knockdown. Genetic or pharmacologic inhibition of STAT3 significantly increased myocyte ADP/ATP ratio and enhanced autophagy through AMPKα/mTOR signaling. Pharmacologic activation and inhibition of AMPKα attenuated and exaggerated, respectively, the effects of Ang II on ANP and β-MHC gene expression, while concomitant inhibition of STAT3 accentuated the inhibition of hypertrophy. Together, these data indicate that novel nongenomic effects of STAT3 influence myocyte energy status and modulate AMPKα/mTOR signaling and autophagy to balance the transcriptional hypertrophic response to Ang II stimulation. These findings may have significant relevance for various cardiovascular pathological processes mediated by Ang II signaling.</p></div

Induction of myocyte autophagy by Ang II and STAT3 inhibition.

<p>(A) GFP-LC3 puncta (green) in H9c2 cells transfected with GFP-LC3 and treated with vehicle (Control) or Ang II and WP1066 alone or in combination. Scale bar = 10 μm. (B) Quantitation of GFP-LC3 autophagosomes (green puncta) in H9c2 cells treated with vehicle (Control) or Ang II and WP1066 alone or in combination. (C) Protein expression of autophagy markers p62 and LC3 in H9c2 cells treated with Ang II or/and WP1066. (D) Densitometric analysis of p62 and LC3 protein expression levels. Data represent mean ± SEM (n = 3). *<i>P</i><0.05 vs. control; ^#<i>P</i><0.05 vs. Ang II only.</p

AMPKα signaling is critical for the mediation of STAT3 influence on hypertrophy.

<p>(A) WP1066 and AICAR treatment for 48 h suppressed Ang II-induced ANP and β-MHC mRNA expression. Data represent mean ± SEM (n = 4); *<i>P</i><0.01 vs. Control; ^†<i>P</i><0.01 vs. Ang II alone; ^#<i>P</i><0.01 vs. Ang II + WP1066; ^§<i>P</i><0.05 vs. Ang II + AICAR. (B) Inhibition of AMPK with Compound C reversed the suppression of hypertrophy marker expression by WP1066 following Ang II treatment of H9c2 cells. Data represent mean ± SEM (n = 4); *<i>P</i><0.01 vs. Control; ^†<i>P</i><0.01 vs. Ang II alone; ^#<i>P</i><0.01 vs. Ang II + WP1066; and ^§<i>P</i><0.05 vs. Ang II + Compound C.</p

Pharmacological inhibition of STAT3 attenuated Ang II-induced myocyte hypertrophy.

<p>(A) Representative photomicrographs of H9c2 cells stained with crystal violet. Magnification 20x; scale bar = 10 μm. (B) Cross-sectional cell surface area (n = 100 cells/group). (C and D) qRT-PCR analysis of ANP and β-MHC mRNA expression in Ang II-treated cells with or without STAT3 inhibition. Data represent mean ± SEM (n = 4), *<i>P</i><0.05 vs. control, ^#<i>P</i><0.05 vs. Ang II only.</p

Exposure to Ang II induced myocyte hypertrophy and activated STAT3.

<p>(A) Representative photomicrographs of H9c2 cells stained with crystal violet, magnification: 20x; scale bar = 10 μm. (B) Cross-sectional cell surface area (n = 100 cells/group). (C) Time-course of Ang II-induced Tyr705 and Ser727 phosphorylation of STAT3 in H9c2 cells. (D) Densitometric data from Western immunoblotting. Levels of p-STAT3 (Y705), p-STAT3 (S727), STAT3 and p-JAK2 (Y1007/1008) were quantified and normalized relative to β-Actin. Data represent mean ± SEM (n = 4), *<i>P</i><0.05 vs. baseline (0h).</p

STAT3 modulates autophagy in H9c2 cells via AMPKα/mTOR pathway.

<p>(A) Representative Western immunoblots showing p-AMPKα, AMPKα, p-mTOR, mTOR, and β-actin protein expression in H9c2 cells treated with vehicle control, Ang II (5 μM), WP1066 (4μM) alone, and Ang II + WP1066 for 48 h. (B) The densitometric quantitation of p-AMPKα, AMPKα, p-mTOR and mTOR protein levels in H9c2 cells. (C) ADP/ATP ratio in H9c2 cells following indicated treatments. Data represent mean ± SEM (n = 3).*<i>P</i><0.05 vs. control; ^#<i>P</i><0.05 vs. Ang II only. (D) Representative Western immunoblots showing STAT3, p-AMPKα, AMPKα, p-mTOR, mTOR, and β-actin protein expression in H9c2 cells treated with scramble peptide control, STAT3 shRNA, scramble RNA + Ang II, and STAT3 shRNA + Ang II for 48 h.(E) The densitometric quantitation of p-AMPKα, AMPKα, p-mTOR and mTOR protein levels in H9c2 cells. (F) ADP/ATP ratio in STAT3-KD cells following Ang II treatment. Data represent mean ± SEM (n = 3).*<i>P</i><0.05 vs. control (scramble RNA); ^#<i>P</i><0.05 vs. STAT3-KD only.</p