Aldosterone Excess Induced Mitochondria Decrease and Dysfunction via Mineralocorticoid Receptor and Oxidative Stress In Vitro and In Vivo

Cheng-Hsuan Tsai, Chien-Ting Pan, Yi-Yao Chang, Shih-Yuan Peng, Po-Chin Lee, Che-Wei Liao, Chia-Tung Shun, Po-Ting Li, Vin-Cent Wu, Chia-Hung Chou, I-Jung Tsai, Chi-Sheng Hung, Yen-Hung Lin, Cheng-Hsuan Tsai, Chien-Ting Pan, Yi-Yao Chang, Shih-Yuan Peng, Po-Chin Lee, Che-Wei Liao, Chia-Tung Shun, Po-Ting Li, Vin-Cent Wu, Chia-Hung Chou, I-Jung Tsai, Chi-Sheng Hung, Yen-Hung Lin

Abstract

Aldosterone excess plays a major role in the progression of cardiac dysfunction and remodeling in clinical diseases such as primary aldosteronism and heart failure. However, the effect of aldosterone excess on cardiac mitochondria is unclear. In this study, we investigated the effect of aldosterone excess on cardiac mitochondrial dysfunction and its mechanisms in vitro and in vivo. We used H9c2 cardiomyocytes to investigate the effect and mechanism of aldosterone excess on cardiac mitochondria, and further investigated them in an aldosterone-infused ICR mice model. The results of the cell study showed that aldosterone excess decreased mitochondrial DNA, COX IV and SOD2 protein expressions, and mitochondria ATP production. These effects were abolished or attenuated by treatment with a mineralocorticoid receptor (MR) antagonist and antioxidant. With regard to the signal transduction pathway, aldosterone suppressed cardiac mitochondria through an MR/MAPK/p38/reactive oxygen species pathway. In the mouse model, aldosterone infusion decreased the amount of cardiac mitochondrial DNA and COX IV protein, and the effects were also attenuated by treatment with an MR antagonist and antioxidant. In conclusion, aldosterone excess induced a decrease in mitochondria and mitochondrial dysfunction via MRs and oxidative stress in vitro and in vivo.

Keywords: aldosterone; heart failure; mitochondrial dysfunction; oxidative stress; primary aldosteronism.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Aldosterone suppressed mitochondrial DNA and protein in a dose-dependent manner. (A) The effects of different dosages of aldosterone on H9c2 cells. H9c2 cells were treated with different concentrations of aldosterone (10−10, 10−9, 10−8 and 10−7 M) and vehicle (equal volume of DMSO) for 72 h. The mitochondrial DNA copy number was quantified by qPCR. (B) The effects of different durations of aldosterone treatment on H9c2 cells. H9c2 cells were treated with 10−7 M aldosterone and the mitochondrial copy number was quantified by qPCR at 0, 8, 24, 48 and 72 h. (C) The effects of different durations of aldosterone treatment on SOD2. H9c2 cells were treated with 10−7 M aldosterone, and the expression of SOD2 was determined by ELISA. (D) The dose effect of aldosterone on mitochondrial COX IV protein. H9c2 cells were treated with different concentrations of aldosterone (vehicle, 10−10, 10−9, 10−8 and 10−7 M) for 72 h. COX IV was stained with anti-COX IV antibodies. The fluorescence intensity of COX IV was measured using a fluorescence microscope. (E) H9c2 cells were treated with different concentrations of aldosterone (10−10, 10−9, 10−8 and 10−7 M) for 72 h. COX IV was stained with anti-COX IV antibodies (red), and nuclear DNA was stained with DAPI (blue). The representative images were captured using a fluorescence microscope; magnification ×400. (F) The effects of aldosterone on mitochondria (COX IV) and cytosolic (α-Tubulin and GAPDH) protein in H9c2 cells. H9c2 cells were treated with different concentrations of aldosterone (vehicle, 10−10, 10−9, 10−8 and 10−7 M) for 72 h. COX IV, α–Tubulin and GAPDH were determined using Western blot analysis. # p < 0.05 and * p < 0.01, compared between the two groups indicated by the line underneath.
Figure 2
Figure 2
Aldosterone-associated reduction in mitochondrial DNA and protein via MR and MAPK/P38 pathway. (A,B) Role of MRs and glucocorticoid receptors on mitochondrial DNA and SOD2 protein. H9c2 cells were treated with 10−7 M eplerenone (an MR antagonist), 10−7 M mifepristone (a glucocorticoid receptor antagonist) and vehicle (equal volume of DMSO) for 1 h prior to 10−7 M aldosterone treatment. After 72 h, mitochondrial DNA was determined by qPCR and the expression of SOD2 was determined by ELISA. (C,D) Role of signaling mediators on mitochondrial DNA and SOD2. H9c2 cells were treated with 5 µg/mL SB203580 (an MAPK/p38 inhibitor), 50 µg/mL PD98059 (an MEK/ERK inhibitor), 50 µg/mL LY294002 (a PI3K/ AKT inhibitor) or vehicle (equal volume of DMSO) for 1 h prior to 10−7 M aldosterone treatment. After 72 h, mitochondrial DNA was determined by qPCR and the expression of SOD2 was determined by ELISA. * p < 0.01, compared between the two groups indicated by the line underneath.
Figure 3
Figure 3
The MAPK/p38 signal pathway was associated with ROS production in aldosterone-treated H9c2 cells. (A) Aldosterone-induced ROS production in H9c2 cells. H9c2 cells were treated with 10−7 and 10−10 M aldosterone, and ROS regeneration was detected by reading the relative fluorescence from 0 to 5 h. The ROS fluorescence image of H9c2 cells treated with 10−7 and 10−10 M aldosterone at 45 min. The cell morphology under a light microscope was also examined; magnification ×400. (B) H9c2 cells were treated with 10−7 M eplerenone, 5 µg/mL SB203580 or vehicle (equal volume of DMSO) for 1 h prior to 10−7 M aldosterone treatment. After 1 h, ROS regeneration was detected by reading the relative fluorescence. (C) H9c2 cells were treated with 5 mM NAC (antioxidant, N–acetyl–L–cysteine) or vehicle (equal volume of ddH2O) for 1 h prior to 10−7 M aldosterone treatment. After 72 h, the ATP formation capability was determined using a Seahorse XF–24 extracellular flux analyzer. (D,E) The effects of aldosterone on fission (Drp1) and fusion (Mfn2) mRNA expression in H9c2 cells. H9c2 cells were treated with different concentrations of aldosterone (vehicle, 10−10, 10−9, 10−8 and 10−7 M) for 72 h. mRNA expression of Drp1 and Mfn2 were determined using quantitative RT-PCR. (F,G) The effects of aldosterone on fission (Drp1) and fusion (Mfn2) protein in H9c2 cells. H9c2 cells were treated with different concentrations of aldosterone (vehicle, 10−10, 10−9, 10−8 and 10−7 M) for 72 h. Drp1 and Mfn2 were determined using Western blot analysis. * p < 0.01 compared between the two groups indicated by the line underneath.
Figure 4
Figure 4
In vivo effect of aldosterone on mitochondrial DNA and COX IV protein in ICR mice. (A) Time course effect of aldosterone on heart tissue mitochondrial DNA in the aldosterone-infused ICR mice and controls. (B) Effect of an MR antagonist (eplerenone) and antioxidant (NAC, N–acetyl–L–cysteine) on cardiac mitochondrial DNA. (C) Immunohistochemical staining of cardiac mitochondrial COX IV protein after 0, 2, 4 and 6 weeks of aldosterone infusion; magnification ×400. (D) Western blot of cardiac COX IV protein and the corresponding quantitative results after 0, 2, 4 and 6 weeks of aldosterone infusion. (E) Immunohistochemical staining of cardiac mitochondrial COX IV protein after 6 weeks of aldosterone infusion in control and aldosterone-infused ICR mice treated with eplerenone, NAC and vehicle; magnification ×400. (F) Western blot of cardiac COX IV protein and the corresponding quantitative results after 6 weeks of aldosterone infusion in control and aldosterone-infused ICR mice treated with eplerenone, NAC and vehicle. # p < 0.05 and * p < 0.01, compared between the two groups indicated by the line underneath.
Figure 5
Figure 5
Schematic of the signaling of aldosterone-induced cardiac mitochondrial dysfunction in H9c2 cells. Aldosterone-induced cardiac mitochondrial dysfunction through MR/MAPK/p38 and ROS pathways. Mitochondrial DNA, SOD2, COX IV protein and ATP production were suppressed.

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