Korean J Physiol Pharmacol.  2015 May;19(3):203-210. 10.4196/kjpp.2015.19.3.203.

EGCG Blocked Phenylephrin-Induced Hypertrophy in H9C2 Cardiomyocytes, by Activating AMPK-Dependent Pathway

• Cai Y
• Zhao L
• Qin Y
• Wu XQ

• KMID: 1791420
• DOI: http://doi.org/10.4196/kjpp.2015.19.3.203

Abstract

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism. Previous studies have shown that activation of AMPK results in suppression of cardiac myocyte hypertrophy via inhibition of the p70S6 kinase (p70S6K) and eukaryotic elongation factor-2 (eEF2) signaling pathways. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea, possesses multiple protective effects on the cardiovascular system including cardiac hypertrophy. However, the molecular mechanisms has not been well investigated. In this study, we found that EGCG could significantly reduce natriuretic peptides type A (Nppa), brain natriuretic polypeptide (BNP) mRNA expression and decrease cell surface area in H9C2 cardiomyocytes stimulated with phenylephrine (PE). Moreover, we showed that AMPK is activated in H9C2 cardiomyocytes by EGCG, and AMPK-dependent pathway participates in the inhibitory effects of EGCG on cardiac hypertrophy. Taken together, our findings provide the first evidence that the effect of EGCG against cardiac hypertrophy may be attributed to its activation on AMPK-dependent signaling pathway, suggesting the therapeutic potential of EGCG on the prevention of cardiac remodeling in patients with pressure overload hypertrophy.

Keyword

AMP-activated protein kinase; Epigallocatechin-3-gallate; H9C2 cardiomyocytes; Phenylephrine

MeSH Terms

AMP-Activated Protein Kinases
Brain
Cardiomegaly
Cardiovascular System
Energy Metabolism
Humans
Hypertrophy*
Myocytes, Cardiac*
Natriuretic Peptides
Phenylephrine
Phosphotransferases
RNA, Messenger
Tea
AMP-Activated Protein Kinases
Natriuretic Peptides
Phenylephrine
Phosphotransferases
RNA, Messenger
Tea

Figure

• 

  Fig. 1 EGCG blocks cardiac hypertrophy in H9C2 cardiomyocytes. H9C2 cardiomyocytes were pretreated with EGCG (25µM) for 1 h followed by stimulation with 100µM PE for 24 h. (A) Cellular hypertrophy was demonstrated by changes in mRNA levels of hypertrophic biomarkers Nppa and BNP. (B and C) Cardiomyocytes were stained with rhodamine-phalloidin followed by cell surface area quantization. *p<0.05 vs. the control group, #p<0.05 vs. the PE group, n=3.

• 

  Fig. 2 EGCG treatment increases AMPK phosphorylation. (A and B) H9C2 cardiomyocytes were treated with various concentrations of EGCG for 24 h. The levels of phosphor-/total AMPK were detected. (C and D) Cells were treated with 25µM EGCG for indicated time points and the protein expression of phosphor-/total AMPK were assessed by western blotting. (E and F) Cells were preincubated with 25µM EGCG for 1 h followed by stimulation with 100µM PE for 24 h, and then the Levels of phosphor-/total AMPK were measured by western blotting. *p<0.05 vs. the group without treatment, #p<0.05 vs. the group treated with PE, n=3.

• 

  Fig. 3 EGCG decreases the p70S6K phosphorylation and increases the eEF2 phosphorylation. Cells were preincubated with 25µM EGCG for 1 h followed by stimulation with 100µM PE for 24 h. (A and B) The Levels of phospho-/total p70S6K were measured by western blotting. (C and D) The phospho-/total eEF2 were detected by western blotting. *p<0.05 vs. the group without treatment, #p<0.05 vs. the group treated with PE, n=3.

• 

  Fig. 4 AMPK participates in the inhibitory effects of EGCG on cardiac hypertrophy in H9C2 cardiomyocytes. H9C2 cardiomyocytes treated with Compound C were pretreated with 25µM EGCG for 1 h followed by stimulation with PE for 24 h. (A) Levels of phospho-/total AMPK were measured by western blotting. (B) Nppa and BNP mRNA levels were measured by quantitative RT-PCR with GAPDH as endogenous control. (C and D) Cardiomyocytes were stained with rhodamine-phalloidin followed by cell surface area quantization. (E) The protein expression of phosphor-/total p70S6K and phosphor-/total eEF2 were detected by western blotting. (F) H9C2 cardiomyocytes were preincubated with a catalase (200 U/ml) or L-NAME (5 mM) for 1 h and subsequently incubated with the indicated concentrations of EGCG for 24 h. Levels of phosphor-/total AMPK were measured by western blotting. (G) Model for EGCG inhibiting cardiac hypertrophy. *p<0.05 vs. the group without treatment, #p<0.05 vs. the group treated with PE alone, and $p<0.05 vs. the group treated with PE plus EGCG, n=3.

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