Experimental design
C57BL/6J mice of different ages were used for in vivo experiments. An ACE2-KO mouse model (C57BL/6J background) was also used for functional studies. All mice were bought from the Laboratory Animal Research Institute of the Chinese Academy of Medical Sciences. In vitro assays were performed using C2C12 cells. Experiments were performed to determine skeletal muscle function and the expression of Ang (1-7) and Ang II in old (20 months), middle-aged (12 months), and young (3 months) C57BL/6J mice. Further, the skeletal muscle function and the expression of myocyte enhancer factor 2A (MEF2A) were determined in young (4 months) and old (16 months) ACE2-KO mice and their wild-type aged-matched controls.
Old (16 months) ACE2-KO mice were subcutaneously injected with 400 ng/kg/min of Ang-(1-7) (APEXBio, Houston, TX, USA, product No. A1041) for 4 weeks. Skeletal muscle function and the expression of MEF2A were compared before and after treatment. In the control group, ACE2-KO mice were subcutaneously injected with 0.9% normal saline. C2C12 cells (prepared from the bank of the cell of the Chinese Academy of Sciences) were grown for 48 hours in DMEM milieu including 2% serum of horse (Gibco, Grand Island, NY, USA), supplemented with 10-8 mol/L of Ang-(1–7) or the same volume of PBS. Following the period of incubation, the cellular differentiation levels and the expression of glucose transporter type 4 (Glut4), MEF2A, myosin heavy chain (MHC), and creatine kinase, muscle (CKM) proteins were evaluated. All in vivo protocols were confirmed through the Animal Committee of West China Hospital, Sichuan University.
Testing of skeletal muscle function
The function of skeletal muscle was evaluated by measuring the grip forelimb strength and the number of falls in a treadmill test. The grip strength of the mouse forelimb was measured using a tester of grip strength (YLS-13A, Jinan Yiyan Technology Development Co., Ltd, Jinan, China.). For each mouse, 5 measurements were performed at 5-second intervals and the average value recorded. The treadmill test used an experimental animal treadmill (ZH-PT, Shanghai Kehuai Instrument Co., Ltd, Shanghai, China., Shanghai, China) with the track speed set at 9 m/min. The number of falls from the track within 5 minutes was recorded. For each mouse, measurements were recorded in triplicate and performed at 5-minute intervals. The average number of falls was recorded.
Preparation of mouse skeletal muscle
Mice were anesthetized by using pentobarbital (45 mg/kg) and the gastrocnemius and soleus muscles were readily anatomized and frozen in liquid nitrogen for subsequent analyses.
Determination of the levels of Ang-(1-7) and Ang II in mouse skeletal muscles
Frozen skeletal muscle samples were thawed and precooled. A homogenate buffer was added to the samples before being homogenization. Specimens were then incubated at 4℃ for 1 hour and centrifuged. The supernatant was utilized to measure the concentrations of Ang II and Ang-(1-7) using the kits of ELISA (Cloud-Clone Corp., Houston, TX, USA) Cat. No. CEA005Mu (Ang II) and CES085Mi (Ang-(1-7)). The levels of Ang II and Ang-(1-7) were measures in muscle samples obtained from five mice in each experimental group.
Determination of MEF2A protein levels in mouse skeletal muscle
The expression of MEF2A was determined by Western blotting. Frozen skeletal muscle samples were thawed, lysed by using a lysis buffer, and centrifuged. The protein concentration of the supernatant was quantified utilizing the BCA assessment. Proteins were dissociated via SDS-PAGE and transferred to the membranes of nitrocellulose. The incubation of the membranes was carried out with a blocking solution at 37℃ for 1 hour. Primary antibody was then added directed against MEF2A (Proteintech, Rosemont, IL, USA, Cat. No. 12382-1-AP) and the incubation of the membranes was carried out during the night at 4℃. Secondary antibody (HRP Goat Anti-Rabbit IgG) was then increased to membranes and the protein levels detected using a chemiluminescence detection kit. The absorbance of each protein band was determined using the Image-Pro Plus 6.0 computer program for a quantitative study.
C2C12 cell culture, proliferation, and differentiation
C2C12 cells were cultivated in the DMEM milieu including 2% serum of horse. To analyze cell proliferation and differentiation, Ang-(1-7) was enhanced and the incubation of the cells was performed at 37℃ for an additional 48 h. PBS was added to the control cultures.
Protein expression in differentiating C2C12 cells
The expression of MHC, creatine kinase, muscle CKM, Glut4, MEF2A proteins was determined by Western blotting in C2C12 cells. Following the differentiation assessment, the cells were collected, rinsed with PBS, incubated on ice by using the RIPA as the lysis buffer and centrifuged. The concentration of protein in the supernatant was quantified employing the BCA assessment. Proteins were dissociated via SDS-PAGE and transferred to the membranes of nitrocellulose. The membranes were blocked for 1 hour at 37℃ and subsequently incubated during the night at 4℃ using antibodies against MHC (Proteintech, Rosemont, IL, USA, Cat. No. 10799-1-AP), CKM (Proteintech, Cat. No. 18712-1-AP), Glut4 (Proteintech, Rosemont, IL, USA, Cat. No. 21048-1-AP), and MEF2A (Proteintech, Rosemont, IL, USA, Cat. No. 12382-1-AP). In addition, the incubation of the membranes was carried out by taking advantage of a secondary antibody and exposed using a chemiluminescence detection kit. The absorbance of each protein band was determined using the Image-Pro Plus 6.0 computer program for a quantitative study.
Statistical methods
All achieved outcomes were expressed as mean ± SD and analyzed utilizing the SPSS 20.0 computer program package. A Student’s t-test or a t-test was utilized for comparison among two groups. One-way ANOVA or the Kruskal-Wallis test were implemented for comparison between several groups. A P-value threshold of 0.05 was utilized to ascertain statistical significance.