Calsarcin-2 May Play a Compensatory Role in the Development of Obese Sarcopenia

Abstract

Although obese sarcopenia is a major public health problem with increasing prevalence worldwide, the factors that contribute to the development of obese sarcopenia are still obscure. In order to clarify this issue, a high-fat-diet-induced obese sarcopenia mouse model was utilized. After being fed with a high-fat diet for 24 weeks, decreased motor functions and muscle mass ratios were found in the C57BL/6 mice. In addition, the expression of calsarcin-2 was significantly increased in their skeletal muscle, which was determined by a microarray analysis. In order to clarify the role of calsarcin-2 in muscle, lentiviral vectors containing the calsarcin-2 gene or short hairpin RNA targeted to calsarcin-2 were used to manipulate calsarcin-2 expressions in L6 myoblasts. We found that an overexpression of calsarcin-2 facilitated L6 myoblast differentiation, whereas a calsarcin-2 knockdown delayed myoblast differentiation, as determined by the expression of myogenin. However, the calsarcin-2 knockdown showed no significant effects on myoblast proliferation. In addition, to clarify the relationship between serum calsarcin-2 and sarcopenia, the bilateral gastrocnemius muscle mass per body weight in mice and appendicular skeletal muscle mass index in humans were measured. Although calsarcin-2 facilitated myoblast differentiation, the serum calsarcin-2 concentration was negatively related to skeletal muscle mass index in mice and human subjects. Taken together, calsarcin-2 might facilitate myoblast differentiation and appear to play a compensatory role in sarcopenia.

Keywords: calsarcin-2; high-fat diet; myoblast differentiation; myocyte; sarcopenia.

Figure 1

High-fat-diet-induced sarcopenia mouse model. Eight-week-old C57BL/6J male mice were fed a high-fat diet (HFD) or chow diet for 24 weeks. After a 12 h starvation period, grip strength (A) and latency to fall in the rotarod test were assessed (B). At the end of the experiments, the total mass of bilateral gastrocnemius muscle divided by body weight was measured (C). The cross-sectional area of gastrocnemius muscle fibers was determined using ImageJ (D). The black dots in the figures represent the assay values for each individual mouse. Quantification was performed on samples from 6 to 8 animals and is expressed as mean ± SEM. *** p < 0.001, as compared with chow diet group.

Figure 2

Calsarcin-2 is upregulated in sarcopenia mouse model. Eight-week-old C57BL/6J male mice were fed a high-fat diet (HFD) or chow diet for 24 weeks. After a 12 h starvation period, the skeletal muscle tissues were removed, the changes in gene profile were determined through a microarray, and the genes clusters were analyzed (A). The calsarcin-2 (CS2) mRNA expression via real-time PCR (B) and protein levels (C) via Western blots were determined. In addition, plasma samples were collected for measurements of CS2 concentrations by ELISA (D). The black dots in the figures represent the assay values for each individual mouse. Data are expressed as mean ± SEM. *** p < 0.001, as compared with chow diet group.

Figure 3

Overexpression of calsarcin-2 promotes myoblast differentiation. Overexpression of calsarcin-2 (CS2) in L6 myoblasts was accomplished using lentiviral vectors containing the CS2 gene (A). KEGG classification of differentially expressed genes in CS2-overexpressing L6 cells was performed (B). The differentiation of L6 myoblasts was induced, and protein lysates were harvested at indicated times for determination of CS2 and myogenin (MYOG) levels by Western blotting (C). The MYOG expression levels were determined in CS2-overexpressing L6 myoblasts (D). The expression of myosin heavy chain 2 (MYH) was determined in CS2-overexpressing L6 cells using immunohistochemistry (magnification 200×) (E). The black dots in the figures represent the assay values for each individual cell. Data are expressed as mean ± SEM. ** p < 0.01 and *** p < 0.001, as compared with indicated groups or control group.

Figure 4

Knockdown of calsarcin-2 decelerated L6 myoblast differentiation. Knockdown of calsarcin-2 (CS2) in L6 myoblasts was accomplished using lentiviral vectors containing short hairpin RNA targeted to CS2 (A). Cell proliferation was assessed with the MTT assay (B). The MYOG expression levels were determined in CS2-knockdowned L6 myoblasts (C). Data are expressed as mean ± SEM. The black dots in the figures represent the assay values for each individual cell. ** p < 0.01 and *** p < 0.001, as compared with indicated groups or control group.

Figure 5

Serum calsarcin-2 concentration was negatively associated with skeletal muscle mass index in mice and humans. Eight-week-old C57BL/6J male mice were fed an HFD or chow diet for 24 weeks. The serum calsarcin-2 (CS2) concentrations were determined by enzyme-linked immunosorbent assay, and the total mass of bilateral gastrocnemius muscle mass was measured. The relationship between serum CS2 concentration and skeletal muscle mass per body weight (%) was analyzed using a linear regression analysis. The black dots in the figure represent the assay values for each individual mouse (A). A total of seventy-six human subjects were enrolled. The CS2 concentrations were determined by enzyme-linked immunosorbent assay, and the appendicular skeletal muscle mass index (ASMI) was determined by limb skeletal muscle mass (kg)/height² (m²). The relationship between serum CS2 concentration and ASMI was analyzed using a linear regression analysis. The black dots in the figure represent the assay values for each individual human (B).