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Aliskiren ameliorates pressure overload-induced heart hypertrophy and fibrosis in mice.

Weng LQ, Zhang WB, Ye Y, Yin PP, Yuan J, Wang XX, Kang L, Jiang SS, You JY, Wu J, Gong H, Ge JB, Zou YZ - Acta Pharmacol. Sin. (2014)

Bottom Line: The levels of signaling proteins were measured using Western blotting, while the expression of the relevant genes was analyzed using real-time QRT-PCR.These pathological alterations in TAC-mice were significantly ameliorated or blocked by ALK administration.In mechanically stretched cardiomyocytes, CGP53353 (a PKCβI inhibitor) prevented ERK phosphorylation and autophagic responses, while U0126 (an ERK inhibitor) blocked autophagic responses.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital and Institute of Biomedical Science, Fudan University, Shanghai 200032, China.

ABSTRACT

Aim: Aliskiren (ALK) is a renin inhibitor that has been used in the treatment of hypertension. The aim of this study was to determine whether ALK could ameliorate pressure overload-induced heart hypertrophy and fibrosis, and to elucidate the mechanisms of action.

Methods: Transverse aortic constriction (TAC) was performed in mice to induce heart pressure overload. ALK (150 mg·kg(-1)·d(-1), po), the autophagy inhibitor 3-methyladenine (10 mg·kg(-1) per week, ip) or the PKCβI inhibitor LY333531 (1 mg·kg(-1)·d-(1), po) was administered to the mice for 4 weeks. Heart hypertrophy, fibrosis and function were evaluated based on echocardiography, histological and biochemical measurements. Mechanically stretched cardiomyocytes of rats were used for in vitro experiments. The levels of signaling proteins were measured using Western blotting, while the expression of the relevant genes was analyzed using real-time QRT-PCR.

Results: TAC induced marked heart hypertrophy and fibrosis, accompanied by high levels of Ang II in plasma and heart, and by PKCβI/α and ERK1/2 phosphorylation in heart. Meanwhile, TAC induced autophagic responses in heart, i.e. increases in autophagic structures, expression of Atg5 and Atg16 L1 mRNAs and LC3-II and Beclin-1 proteins. These pathological alterations in TAC-mice were significantly ameliorated or blocked by ALK administration. In TAC-mice, 3-methyladenine administration also ameliorated heart hypertrophy, fibrosis and dysfunction, while LY333531 administration inhibited ERK phosphorylation and autophagy in heart. In mechanically stretched cardiomyocytes, CGP53353 (a PKCβI inhibitor) prevented ERK phosphorylation and autophagic responses, while U0126 (an ERK inhibitor) blocked autophagic responses.

Conclusion: ALK ameliorates heart hypertrophy, fibrosis and dysfunction in the mouse model in setting of chronic pressure overload, via suppressing Ang II-PKCβI-ERK1/2-regulated autophagy.

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Relationship among PKCβI/α, ERK, and autophagy. (A) Relationship among PKCα, pERK1/2 and autophagy. Representative gel blots and quantitative analysis of pPKCα, pPKCβI, pERK, LC3-II, and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs Ro. St, Stretch by 20% for 24 h. Ro, Ro-31-0432 (a PKCα inhibitor, 200 pmol/mL). (B) Relationship among PKCβI, pERK1/2, and autophagy. Representative gel blots and quantitative analysis of pPKCα, pPKCβI, pERK, LC3-II, and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs CGP. St, Stretch by 20% for 24 h. CGP, CGP53353 (a PKCβI inhibitor, 6.0 nmol/mL). (C) Relationship between pERK1/2 and autophagy. Representative gel blots and quantitative analysis of pERK, LC3-II and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs U0126. St, Stretch by 20% for 24 h. U0126, an ERK inhibitor (10 μmol/L). (D) TEM images and (E) quantitative analysis of autophagic structures on cardiac tissue sections, scale bar: 500 nm, arrows indicating autophagic structures. (F) Quantitative analysis of autophagic genes ATG5 and ATG16 L1.
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fig5A: Relationship among PKCβI/α, ERK, and autophagy. (A) Relationship among PKCα, pERK1/2 and autophagy. Representative gel blots and quantitative analysis of pPKCα, pPKCβI, pERK, LC3-II, and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs Ro. St, Stretch by 20% for 24 h. Ro, Ro-31-0432 (a PKCα inhibitor, 200 pmol/mL). (B) Relationship among PKCβI, pERK1/2, and autophagy. Representative gel blots and quantitative analysis of pPKCα, pPKCβI, pERK, LC3-II, and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs CGP. St, Stretch by 20% for 24 h. CGP, CGP53353 (a PKCβI inhibitor, 6.0 nmol/mL). (C) Relationship between pERK1/2 and autophagy. Representative gel blots and quantitative analysis of pERK, LC3-II and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs U0126. St, Stretch by 20% for 24 h. U0126, an ERK inhibitor (10 μmol/L). (D) TEM images and (E) quantitative analysis of autophagic structures on cardiac tissue sections, scale bar: 500 nm, arrows indicating autophagic structures. (F) Quantitative analysis of autophagic genes ATG5 and ATG16 L1.

Mentions: To test whether PKC isoforms participate in TAC-induced autophagy, mechanically stretched-cardiomyocytes were pretreated with a PKCβI/α inhibitor. As indicated by the results of the Western blot, mechanical stress significantly enhanced the levels of pERK1/2 and autophagic flux, an outcome that was prevented by pretreatment with the PKCβI inhibitor, CGP53353, but not the PKCα inhibitor, Ro-31-0432 (Figure 5A, 5B). To examine the effect of ERK inhibition on cardiomyocyte autophagy, cardiomyocytes were pretreated with the ERK inhibitor, U0126. Our data revealed that U0126 notably attenuated mechanical stress-induced elevations in LC3-II and Beclin-1 (Figure 5C). Taken together, our in vitro results revealed that mechanical stress-induced autophagy was partly mediated by the PKCβI-ERK1/2 dependent pathway.


Aliskiren ameliorates pressure overload-induced heart hypertrophy and fibrosis in mice.

Weng LQ, Zhang WB, Ye Y, Yin PP, Yuan J, Wang XX, Kang L, Jiang SS, You JY, Wu J, Gong H, Ge JB, Zou YZ - Acta Pharmacol. Sin. (2014)

Relationship among PKCβI/α, ERK, and autophagy. (A) Relationship among PKCα, pERK1/2 and autophagy. Representative gel blots and quantitative analysis of pPKCα, pPKCβI, pERK, LC3-II, and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs Ro. St, Stretch by 20% for 24 h. Ro, Ro-31-0432 (a PKCα inhibitor, 200 pmol/mL). (B) Relationship among PKCβI, pERK1/2, and autophagy. Representative gel blots and quantitative analysis of pPKCα, pPKCβI, pERK, LC3-II, and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs CGP. St, Stretch by 20% for 24 h. CGP, CGP53353 (a PKCβI inhibitor, 6.0 nmol/mL). (C) Relationship between pERK1/2 and autophagy. Representative gel blots and quantitative analysis of pERK, LC3-II and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs U0126. St, Stretch by 20% for 24 h. U0126, an ERK inhibitor (10 μmol/L). (D) TEM images and (E) quantitative analysis of autophagic structures on cardiac tissue sections, scale bar: 500 nm, arrows indicating autophagic structures. (F) Quantitative analysis of autophagic genes ATG5 and ATG16 L1.
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fig5A: Relationship among PKCβI/α, ERK, and autophagy. (A) Relationship among PKCα, pERK1/2 and autophagy. Representative gel blots and quantitative analysis of pPKCα, pPKCβI, pERK, LC3-II, and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs Ro. St, Stretch by 20% for 24 h. Ro, Ro-31-0432 (a PKCα inhibitor, 200 pmol/mL). (B) Relationship among PKCβI, pERK1/2, and autophagy. Representative gel blots and quantitative analysis of pPKCα, pPKCβI, pERK, LC3-II, and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs CGP. St, Stretch by 20% for 24 h. CGP, CGP53353 (a PKCβI inhibitor, 6.0 nmol/mL). (C) Relationship between pERK1/2 and autophagy. Representative gel blots and quantitative analysis of pERK, LC3-II and Beclin-1 are shown. Four parallel experiments were indicated. Mean±SEM. bP<0.05 vs Ctrl. eP<0.05 vs St. hP<0.05 vs U0126. St, Stretch by 20% for 24 h. U0126, an ERK inhibitor (10 μmol/L). (D) TEM images and (E) quantitative analysis of autophagic structures on cardiac tissue sections, scale bar: 500 nm, arrows indicating autophagic structures. (F) Quantitative analysis of autophagic genes ATG5 and ATG16 L1.
Mentions: To test whether PKC isoforms participate in TAC-induced autophagy, mechanically stretched-cardiomyocytes were pretreated with a PKCβI/α inhibitor. As indicated by the results of the Western blot, mechanical stress significantly enhanced the levels of pERK1/2 and autophagic flux, an outcome that was prevented by pretreatment with the PKCβI inhibitor, CGP53353, but not the PKCα inhibitor, Ro-31-0432 (Figure 5A, 5B). To examine the effect of ERK inhibition on cardiomyocyte autophagy, cardiomyocytes were pretreated with the ERK inhibitor, U0126. Our data revealed that U0126 notably attenuated mechanical stress-induced elevations in LC3-II and Beclin-1 (Figure 5C). Taken together, our in vitro results revealed that mechanical stress-induced autophagy was partly mediated by the PKCβI-ERK1/2 dependent pathway.

Bottom Line: The levels of signaling proteins were measured using Western blotting, while the expression of the relevant genes was analyzed using real-time QRT-PCR.These pathological alterations in TAC-mice were significantly ameliorated or blocked by ALK administration.In mechanically stretched cardiomyocytes, CGP53353 (a PKCβI inhibitor) prevented ERK phosphorylation and autophagic responses, while U0126 (an ERK inhibitor) blocked autophagic responses.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital and Institute of Biomedical Science, Fudan University, Shanghai 200032, China.

ABSTRACT

Aim: Aliskiren (ALK) is a renin inhibitor that has been used in the treatment of hypertension. The aim of this study was to determine whether ALK could ameliorate pressure overload-induced heart hypertrophy and fibrosis, and to elucidate the mechanisms of action.

Methods: Transverse aortic constriction (TAC) was performed in mice to induce heart pressure overload. ALK (150 mg·kg(-1)·d(-1), po), the autophagy inhibitor 3-methyladenine (10 mg·kg(-1) per week, ip) or the PKCβI inhibitor LY333531 (1 mg·kg(-1)·d-(1), po) was administered to the mice for 4 weeks. Heart hypertrophy, fibrosis and function were evaluated based on echocardiography, histological and biochemical measurements. Mechanically stretched cardiomyocytes of rats were used for in vitro experiments. The levels of signaling proteins were measured using Western blotting, while the expression of the relevant genes was analyzed using real-time QRT-PCR.

Results: TAC induced marked heart hypertrophy and fibrosis, accompanied by high levels of Ang II in plasma and heart, and by PKCβI/α and ERK1/2 phosphorylation in heart. Meanwhile, TAC induced autophagic responses in heart, i.e. increases in autophagic structures, expression of Atg5 and Atg16 L1 mRNAs and LC3-II and Beclin-1 proteins. These pathological alterations in TAC-mice were significantly ameliorated or blocked by ALK administration. In TAC-mice, 3-methyladenine administration also ameliorated heart hypertrophy, fibrosis and dysfunction, while LY333531 administration inhibited ERK phosphorylation and autophagy in heart. In mechanically stretched cardiomyocytes, CGP53353 (a PKCβI inhibitor) prevented ERK phosphorylation and autophagic responses, while U0126 (an ERK inhibitor) blocked autophagic responses.

Conclusion: ALK ameliorates heart hypertrophy, fibrosis and dysfunction in the mouse model in setting of chronic pressure overload, via suppressing Ang II-PKCβI-ERK1/2-regulated autophagy.

Show MeSH
Related in: MedlinePlus