Korean Circ J.  2024 Apr;54(4):172-186. 10.4070/kcj.2023.0197.

Dronedarone Attenuates Ang II-Induced Myocardial Hypertrophy Through Regulating SIRT1/FOXO3/PKIA Axis

• Chen C
• Hu S
• Hu HJ
• Liu ZX
• Wu XT
• Zou T
• Su H

• KMID: 2555529
• DOI: http://doi.org/10.4070/kcj.2023.0197

Abstract

Background and Objectives
Long-term pathological myocardial hypertrophy (MH) seriously affects the normal function of the heart. Dronedarone was reported to attenuate left ventricular hypertrophy of mice. However, the molecular regulatory mechanism of dronedarone in MH is unclear.
Methods
Angiotensin II (Ang II) was used to induce cell hypertrophy of H9C2 cells. Transverse aortic constriction (TAC) surgery was performed to establish a rat model of MH. Cell size was evaluated using crystal violet staining and rhodamine phalloidin staining. Reverse transcription quantitative polymerase chain reaction and western blot were performed to detect the mRNA and protein expressions of genes. JASPAR and luciferase activity were conducted to predict and validate interaction between forkhead box O3 (FOXO3) and protein kinase inhibitor alpha (PKIA) promoter.
Results
Ang II treatment induced cell hypertrophy and inhibited sirtuin 1 (SIRT1) expression, which were reversed by dronedarone. SIRT1 overexpression or PKIA overexpression enhanced dronedarone-mediated suppression of cell hypertrophy in Ang II-induced H9C2 cells. Mechanistically, SIRT1 elevated FOXO3 expression through SIRT1-mediated deacetylation of FOXO3 and FOXO3 upregulated PKIA expression through interacting with PKIA promoter. Moreover, SIRT1 silencing compromised dronedaronemediated suppression of cell hypertrophy, while PKIA upregulation abolished the influences of SIRT1 silencing. More importantly, dronedarone improved TAC surgery-induced MH and impairment of cardiac function of rats via affecting SIRT1/FOXO3/PKIA axis.
Conclusions
Dronedarone alleviated MH through mediating SIRT1/FOXO3/PKIA axis, which provide more evidences for dronedarone against MH.

Keyword

Dronedarone; SIRT1; Hypertrophy

Figure

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  Figure 1 Ang II stimulation successfully established a cell model of MH and inhibited SIRT1 expression in H9C2 cells.H9C2 cells were treated with diffident concentrations of Ang II (50, 100, 150 nmol/L) for 48 hours. (A) Cell viability was detected using MTT assay. (B, C) Cell size was evaluated using crystal violet staining and rhodamine phalloidin staining. (D) The mRNA expression of ANP, BNP, and β-MHC were examined by RT-qPCR. (E) The protein levels of SIRT1, ANP, BNP, and β-MHC were examined by western blot.Ang II = angiotensin II; ANP = atrial natriuretic peptide; BNP = B type natriuretic peptide; MH = myocardial hypertrophy; RT-qPCR = reverse transcription quantitative polymerase chain reaction; SIRT1 = sirtuin 1; β-MHC = β-myosin heavy chain.*p<0.05, **p<0.01, ***p<0.001. All data were obtained from 3 independent replications.

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  Figure 2 Dronedarone attenuated Ang II-induced MH.Diffident concentrations of dronedarone (1, 5, 10 μM) were used to treat H9C2 cells upon 150 nmol/L Ang II induction for 6 hours. (A, B) Cell size was evaluated using crystal violet staining and rhodamine phalloidin staining. (C) The protein levels of SIRT1, ANP, BNP, and β-MHC were examined by western blot.Ang II = angiotensin II; ANP = atrial natriuretic peptide; BNP = B type natriuretic peptide; Dro = dronedarone; MH = myocardial hypertrophy; SIRT1 = sirtuin 1; β-MHC = β-myosin heavy chain.*p<0.05, **p<0.01, ***p<0.001. All data were obtained from 3 independent replications.

• 

  Figure 3 Dronedarone regulated FOXO3 acetylation to promote PKIA expression through promoting SIRT1 expression, attenuating Ang II-induced MH.(A) The overexpression efficiency of SIRT1 was measured using RT-qPCR. Oe-SIRT1 H9C2 cells were subjected to Ang II induction and dronedarone treatment. (B) The protein levels of SIRT1, FOXO3 and Ac-FOXO3 were examined by western blot. (C, D) Cell size was evaluated using crystal violet staining and rhodamine phalloidin staining. (E) JASPAR predicted the binding site between FOXO3 and PKIA promoter. RT-qPCR was used to detect the overexpression efficiency of FOXO3. Luciferase activity assay validated the interaction between FOXO3 and PKIA promoter. (F) The protein levels of PKIA, ANP, BNP and β-MHC were examined by western blot.Ang II = angiotensin II; ANP = atrial natriuretic peptide; BNP = B type natriuretic peptide; Dro = dronedarone; FOXO3 = forkhead box O3; MH = myocardial hypertrophy; NC = negative control; oe- = overexpression; PKIA = protein kinase inhibitor α; RT-qPCR = reverse transcription quantitative polymerase chain reaction; SIRT1 = sirtuin 1; β-MHC = β-myosin heavy chain.*p<0.05, **p<0.01, ***p<0.001. All data were obtained from 3 independent replications.

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  Figure 4 Dronedarone suppressed PKA pathway to restrain Ang II-induced MH through upregulating PKIA expression.(A) The overexpression efficiency of PKIA in H9C2 cells was measured using RT-qPCR. PKIA-overexpressed H9C2 cells were subjected to Ang II induction or together with dronedarone treatment. (B, C) Cell size was evaluated using crystal violet staining and rhodamine phalloidin staining. (D) The protein levels of PKIA, PKA, ANP, BNP, and β-MHC were examined by western blot.Ang II = angiotensin II; ANP = atrial natriuretic peptide; BNP = B type natriuretic peptide; Dro = dronedarone; MH = myocardial hypertrophy; NC = negative control; oe- = overexpression; PKA = protein kinase A; PKIA = protein kinase inhibitor alpha; RT-qPCR = reverse transcription quantitative polymerase chain reaction; β-MHC = β-myosin heavy chain.*p<0.05, **p<0.01, ***p<0.001. All data were obtained from 3 independent replications.

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  Figure 5 Dronedarone attenuated Ang II-induced MH through regulating SIRT1/FOXO3/PKIA pathway.H9C2 cells were transfected with sh-SIRT1 or together with oe-PKIA. (A, B) The mRNA and protein expression of SIRT1 and PKIA were evaluated using RT-qPCR and western blot. H9C2 cells were transfected with sh-SIRT1 or together with oe-PKIA and then followed Ang II induction and dronedarone treatment. (C, D) Cell size was evaluated using crystal violet staining and rhodamine phalloidin staining. (E) The protein levels of FOXO3, PKA, ANP, BNP, and β-MHC were examined by western blot.Ang II = angiotensin II; ANP = atrial natriuretic peptide; BNP = B type natriuretic peptide; Dro = dronedarone; FOXO3 = forkhead box O3; NC = negative control; oe- = overexpression; PKIA = protein kinase inhibitor alpha; RT-qPCR = reverse transcription quantitative polymerase chain reaction; sh- = short hairpin RNA targeting; SIRT1 = sirtuin 1; β-MHC = β-myosin heavy chain.*p<0.05, **p<0.01, ***p<0.001. All data were obtained from 3 independent replications.

• 

  Figure 6 Dronedarone alleviated TAC surgery-induced MH and impairment of cardiac function of rats through regulating SIRT1/FOXO3/PKIA axis.Rats received TAC surgery to establish an animal model of MH and 90 mg/kg of dronedarone was given daily by gavage for 8 weeks. (A) The images of hearts of rats. (B) Myocardial histomorphology was detected using H&E staining. (C) Representative images of M-mode echocardiography in different groups, EF, FS, and HW/BW were detected. (D) The protein levels of FOXO3, PKIA, ANP, BNP, and SIRT1 were examined by western blot.ANP = atrial natriuretic peptide; BNP = B type natriuretic peptide; Dro = dronedarone; EF = ejection fraction; FOXO3 = forkhead box O3; FS = fraction shortening; H&E = hematoxylin and eosin; HW/BW = heart weight to body weight ratio; MH = myocardial hypertrophy; PKIA = protein kinase inhibitor alpha; SIRT1 = sirtuin 1; TAC = transverse aortic constriction.*p<0.05, **p<0.01, ***p<0.001. All data were obtained from 3 independent replications.

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