Endocrinol Metab.  2021 Aug;36(4):865-874. 10.3803/EnM.2021.1108.

Aldosterone Inhibits In Vitro Myogenesis by Increasing Intracellular Oxidative Stress via Mineralocorticoid Receptor

• Lee JY
• Kim DA
• Choi E
• Lee YS
• Park SJ
• Kim BJ

• KMID: 2519674
• DOI: http://doi.org/10.3803/EnM.2021.1108

Abstract

Background
Despite clinical evidence indicating poor muscle health in subjects with primary aldosteronism (PA), it is still unclear whether the role of aldosterone in muscle metabolism is direct or mediated indirectly via factors, such as electrolyte imbalance or impaired glucose uptake. As one approach to clarify this issue, we investigated the effect of aldosterone on in vitro myogenesis and the potential mechanism explaining it.
Methods
Myogenesis was induced in mouse C2C12 myoblasts with 2% horse serum. Immunofluorescence, quantitative reversetranscription polymerase chain reaction, Western blot, viability, and migration analyses were performed for experimental research.
Results
Recombinant aldosterone treatment suppressed muscle differentiation from mouse C2C12 myoblasts in a dose-dependent manner, and consistently reduced the expression of myogenic differentiation markers. Furthermore, aldosterone significantly increased intracellular reactive oxygen species (ROS) levels in myotubes, and treatment with N-acetyl cysteine, a potent biological thiol antioxidant, reversed the decrease of myotube area, myotube area per myotube, nucleus number per myotube, and fusion index due to aldosterone through decreasing oxidative stress. A binding enzyme-linked immunosorbent assay confirmed that mineralocorticoid receptor (MR) interacted with aldosterone in C2C12 myoblasts, while eplerenone, an MR inhibitor, blocked aldosterone-stimulated intracellular ROS generation during myogenesis and markedly attenuated the suppression of in vitro myogenesis by aldosterone.
Conclusion
These findings support the hypothesis that hypersecretion of aldosterone, like PA, directly contributes to muscular deterioration and suggest that antioxidants and/or MR antagonists could be effective therapeutic options to reduce the risk of sarcopenia in these patients.

Keyword

Aldosterone; Sarcopenia; Muscle development; Receptors; mineralocorticoid; Oxidative stress

Figure

• 

  Fig. 1 Recombinant aldosterone (Aldo) inhibits in vitro myogenesis. (A) Mouse C2C12 myoblasts (MBs) were differentiated into myotubes (MTs) with 2% horse serum after exposure to the indicated concentrations of recombinant Aldo for 3 days. MTs were stained with anti-myosin heavy chain (MyHC) antibody while nuclei were counterstained with 4,6-diamidino-2-phenyindole (DAPI). Quantitative results per field are presented (n=4). (B) Quantitative reverse-transcription polymerase chain reaction and (C) Western blot analyses of myogenin, myocyte enhancer factor 2C (Mef2C), and/or MyHC in C2C12 cells without or with 2% horse serum in the presence of 100 nM Aldo for 3 days (n=3). (D) The directional migration and (E) viability of mouse C2C12 MBs were assessed by a Boyden chamber system and cell counting kit-8 (CCK-8) assay after exposure to the indicated concentrations of Aldo for 6 and 24 hours, respectively (n=5). Scale bars: 100 μm (A) and 50 μm (D). Data are expressed as mean±standard error of the mean. OD, optical density. aP<0.05 vs. control or MB; bP<0.05 vs. untreated MT.

• 

  Fig. 2 The inhibitory effects of aldosterone (Aldo) on myogenesis are mediated by increased intracellular reactive oxygen species (ROS) generation. (A) Mouse C2C12 myoblasts were differentiated into myotubes with 2% horse serum after exposure to the indicated concentrations of recombinant Aldo for 3 days. Intracellular ROS levels were measured using a fluorescent probe, chloromethyl derivative of 2′,7′-dichlorofluorescein diacetate (CM-H2DCFDA; n=5). (B, C) Mouse C2C12 myoblasts were differentiated into myotubes with 2% horse serum in the presence or absence of 100 nM Aldo and/or 1 mM N-acetyl-L-cysteine (NAC) for 3 days. (B) Intracellular ROS levels were measured using H2DCFDA (n=3). (C) Myotubes were stained with anti-myosin heavy chain (MyHC) antibody while nuclei were counterstained with 4,6-diamidino-2-phenyindole (DAPI). Quantitative results per field are presented (n=3). Scale bars: 100 μm (B) and 100 μm (C). aP<0.05 vs. control; bP< 0.05 vs. 100 nM Aldo.

• 

  Fig. 3 Mineralocorticoid receptor (MR) is the major receptor for aldosterone (Aldo) in muscle cells. (A) Interaction between Aldo and MR in mouse C2C12 myoblasts (MBs) using binding enzyme-linked immunosorbent assay (ELISA). Varying amounts of cell lysates were incubated in Aldo- or bovine serum albumin (BSA)-coated wells. Thereafter, the level of MR was determined by ELISA (n=4). (B) Western blot analysis to determine the MR expression in mouse C2C12 cells without or with 2% horse serum for 2 days (n=3). (C, D) Mouse C2C12 MBs were differentiated into myotubes (MTs) with 2% horse serum in the presence or absence of 100 nM Aldo and/or 1 μM eplerenone (Eple) for 3 days. (C) Intracellular reactive oxygen species levels were measured using a fluorescent probe, chloromethyl derivative of 2′,7′-dichlorofluorescein diacetate (CM-H2DCFDA; n=3). (D) MTs were stained with anti-MyHC antibody while nuclei were counterstained with 4,6-diamidino-2-phenyindole (DAPI). Quantitative results per field are presented (n=3). Scale bars: 100 μm (C) and 100 μm (D). OD, optical density. aP<0.05 vs. control; bP<0.05 vs. 100 nM Aldo.

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