Canadine from Corydalis turtschaninovii Stimulates Myoblast Differentiation and Protects against Myotube Atrophy

Abstract

Cachexia and sarcopenia are the main causes of muscle atrophy. These result in a reduction in the muscle fiber area, myo-protein content, and muscle strength, with various molecular modulators being involved. Although several reports have proposed potential therapeutic agents, no effective treatments have been found for muscle atrophy. We searched for myogenic modulators from medicinal plants to treat muscle diseases. We isolated six alkaloids from Corydalis turtschaninovii and evaluated their myogenic potential by using the MyoD reporter gene assay in C2C12 cells. Among the tested compounds, canadine showed the strongest transactivation of MyoD and increased MHC expression during myogenesis. The activation of p38 MAP kinase and Akt are major mechanisms that contribute to the myogenesis by canadine. Canadine increased the number of multinucleated and cylinder-shaped myotubes during myogenesis of C2C12 myoblasts. To determine the preventive effect of canadine in cancer-induced muscle wasting, differentiated C2C12 myotubes were treated with conditioned media from CT26 colon carcinoma culture (CT26 CM) in the presence of canadine. Canadine ameliorated the muscle protein degradation caused by CT26-CM by down-regulating the muscle specific-E3 ligases, MAFbx/atrogin-1 and MuRF1. In this study, we found that canadine from C. turtschaninovii stimulates myogenesis and also inhibits muscle protein degradation. Therefore, we suggest canadine as a protective agent against muscle atrophy.

Keywords: Corydalis turtschaninovii; canadine; isoquinoline alkaloid; muscle atrophy; myoblast differentiation.

Figure 1

Structures of compounds 1–6 isolated from C. turtschaninovii.

Figure 2

The myogenic effect of CT compounds. (A) Effect of the compounds on transactivation of MyoD. C2C12 myoblasts were transiently transfected with MyoD-responsive reporter 4RTK-luciferase (RTK-Luc) vector and pBP-MyoD expression vector. Transfected myoblasts were treated with each compound (1 nM) for 24 h and the cell lysate was subjected to luciferase assay system. Data are presented as relative luciferase activity (RLU) divided by the β-galactosidase activity; (B) Effect of compounds on myogenesis. Myoblasts were differentiated in the presence of each compound for 3 days. Protein extracts were analyzed for myosin heavy chain (MHC) expression via Western blotting. Pan-cadherin was used as loading control. Data are expressed as mean ± standard deviation (SD). Images are the representative of three independent experiments that show similar results. NC, negative control; 1–6, compounds 1–6. * p < 0.001 vs. NC.

Figure 3

The myogenic activity of canadine. (A) Effect of canadine on myogenesis. Myoblasts were differentiated in the presence of canadine (0.01~10 nM) for 3 days. Western blotting was performed to determine the MHC and MyoD expression; (B) Effect of canadine on the formation of MHC-expressing multinucleated myotubes. Myoblasts were differentiated as described in Materials and Methods section. Images show the expression of immunofluorescence for MHC (red-colored) and 4′,6-diamidino-2-phenylindole (DAPI) (nuclei, blue-colored) in MHC-expressing multinucleated myotubes. Scale bar = 200 μm. The data present the relative number of MHC-expressing multinucleated cells compared with untreated control group (0 nM). Images are the representative of three independent experiments that show similar results. * p < 0.001 vs. 0 nM.

Figure 4

Effect of canadine on p38 MAPK and Akt activation during myogenesis. Myoblasts were treated with canadine (0.01~10 nM) for 3 days and cell lysates were prepared. Protein blots were incubated with an anti-phospho-p38 MAPK or -phospho-Akt antibody. Total p38 MAPK and Akt were used as loading controls. Data represent the mean ± SD of triplicate experiments. * p < 0.001 vs. 0 nM.

Figure 5

Canadine alleviated CT26 conditioned medium (CM)-induced muscle atrophy in vitro (A) Effect of canadine on the expression of MHC and MyoD in CT26 CM-induced muscle wasting. Differentiated myotubes were pre-treated with canadine (1~100 nM) for 3 h, followed by CT26 CM treatment for 24 h. The expression of myogenic markers was determined by Western blot analysis; (B) Immunofluorescence for MHC (red) and DAPI (blue) in MHC-expressing multinucleated myotube. Scale bar = 200 μm. Data expressed the relative number of MHC-expressing multinucleated cells compared with the control group (NC). # p < 0.001 vs. NC; * p < 0.001 vs. CM alone (0 nM); (C) Effect of canadine on CM-induced E3 ligase expression. Western blotting was performed to determine the MAFbx and Murf1 expression. Gene expressions of MAFbx and Murf1 were determined using RT-qPCR as described in Materials and Methods. Data are means ± SD of triplicate experiments. # p < 0.01 vs. NC; * p < 0.01 vs. CM alone (0 nM).