Toxic Mechanism Of Maduramicin Ammonium In H9c2 Cardiac Muscle Cells And C2C12 Skeletal Muscle Cells

This study was set to test the toxicity of maduramicin ammonium in H9c2 and C2C12 cells,determine the effect of maduramicin ammonium on cell cycle distribution,apoptosis and autophagy in both cell lines and investigate the effect of related cellular signaling pathways on the toxicity of maduramicin ammonium in H9c2 and C2C12 cells.The details are divided into eight parts as follows:1.To determine the toxicity of maduramicin ammonium in cardiac and skeletal muscle cells,H9c2 and C2C12 cells were chosen as experimental models.Cells were treated with maduramicin ammonium for 5 days or 24,48,72 h at different concentrations（H9c2:0～5μg/mL,C2C12:0～1 pg/mL）,followed by cell counting and morphological analysis or one solution assay and trypan blue exclusion assay.The results showed that maduramicin ammonium inhibited cell growth in a concentration-dependent manner,reduced cell viability and induced cell death in concentration-and time-dependent manner.It suggests that maduramicin ammonium is cytotoxic in both cell lines.2.To determine the effect of maduramicin ammonium on cell cycle distribution in cardiac and skeletal muscle cells,H9c2 and C2C12 cells were chosen as experimental models.Cells were treated with maduramicin ammonium for 24 h at different concentrations（H9c2:0～5μg/mL,C2C12:0～1μg/mL）,or for different time（H9c2:36,48 and 72 h,C2C12:24,48 and 72 h）at 0.5μg/mL,followed by PI staining and flow cytometry;cells were treated with maduramicin ammonium for 24 h at different concentrations（H9c2:0～5μg/mL,C2C12:0～1μg/mL）,followed by Western blotting for cell cycle related proteins.The results showed that S phase cell number increased in H9c2 cells treated with high concentration maduramicin ammonium for 24 h;0.5μg/mL maduramicin ammonium arrested H9c2 cells at G₀/G₁ phase in a time-dependent manner;manduramicin arrested C2C12 cells at G₀/G₁ phase in concentration-and time-dependent manner;maduramicin ammonium upregulated protein expression of cyclins,CDK6 and CDC25B in H9c2 cells;maduramicin ammonium downregulated protein expression of cyclin D1,CDK4,CDK6,and CDC25A,and upregulated expression of p21Cip1 and p27Kip1,resulting in hypophosphorylation of Rb in C2C12 cells.It suggests that maduramicin ammonium inhibits cell proliferation by inducing cell cycle arrest in both cell lines.3.To determine the effect of maduramicin ammonium on apoptosis in cardiac and skeletal muscle cells,H9c2 and C2C12 cells were chosen as experimental models.Cells were treated with maduramicin ammonium for 72 h at different concentrations（H9c2:0～5μg/mL,C2C12:0～1μg/mL）,followed by Annexin V-PI staining and flow cytometry;cells were treated with maduramicin ammonium for 24 h at different concentrations（H9c2:0～5 pg/mL,C2C12:0～1μg/mL）,followed by Western blotting for apoptosis related proteins;cells,pretreated with or without z-VAD-fmk for 1 h,were treated with maduramicin ammonium for 24 h at 0.5μg/mL and 1μg/mL,followed by Western blotting for cleaved caspase and cleaved PARP,or for 48 h at 0.5μg/mL and 1μg/mL,followed by trypan blue exclusion assay;cells were treated with maduramicin ammonium for 24 h at different concentrations（H9c2:0～5μg/mL,C2C12;0～1μg/mL）,followed by Western blotting and immunofluorescence staining with anti-AIF antibody.The results showed that maduramicin ammonium induced apoptosis in a concentration-dependent manner;maduramicin ammonium upregulated expression of DR4 and TRAIL,leading to activation of caspases 8 and 3 as well as cleavage of PARP in H9c2 cells;maduramicin ammonium upregulated expression of DR4,TRADD,TRAIL,BAK and BAD,leading to activation of caspases 8,9 and 3 as well as cleavage of PARP in C2C12 cells;pretreatment with z-VAD-fmk for 1 h almost completely blocked maduramicin ammonium-induced cleavage of caspase 3 and PARP,but only partially prevented maduramicin ammonium-induced cell death;maduramicin ammonium upregulated expression of AIF and increased AIF nucleus translocation in H9c2 cells;maduramicin ammonium did not change expression level of AIF but did increase AIF nucleus translocation in C2C12 cells.It suggests that maduramicin ammonium induces caspase-dependent and-independent apoptosis in both cell lines.4.To determine the effect of maduramicin ammonium on autophagy in cardiac and were treated with maduramicin ammonium for 24 h at different concentrations（H9c2:0-5 pg/mL,C2C12:0～1μg/mL）,followed by Western blotting for autophagy related proteins;cells,expression of GFP-tagged LC3 with adenovirus infection,were treated with maduramicin ammonium for 24 h at indicated concentrations,followed by imaging with a fluorescence microscope.The results showed that maduramicin ammonium upregulated expression of LC3Ⅱand p62;maduramicin ammonium induced fluorescence puncta formation in cells expressed GFP-tagged LC3.It suggests that maduramicin ammonium induces autophagy and inhibits autophagy flux in both cell lines.5.To determine the effect of maduramicin ammonium on MAPK pathway proteins and protein phosphatases expression in cardiac and skeletal muscle cells,H9c2 and C2C12 cells were chosen as experimental models.Cells were treated with 0～1μg/mL maduramicin ammonium for 24 h,followed by Western blotting for MAPK pathway proteins and protein phosphatases;H9c2 cells,expression of MKK1-R4F or dominant negative PP2A with adenovirus infection,were treated with 0.5μg/mL and 1μg/mL maduramicin ammonium for 24 h or 48 h,followed by Western blotting,morphological analysis and one solution assay;H9c2 cells,pretreated with Okadaic acid for 1 h,were treated with 0.5μg/mL and 1μg/mL maduramicin ammonium for 48 h,followed by morphological analysis and one solution assay;C2C12 cells,expression of dominant negative c-Jun or PP5 with adenovirus infection,were treated with 0.5μg/mL and 1μg/mL maduramicin ammonium for 24 h or 48 h,followed by Western blotting,morphological analysis and one solution assay;C2C12 cells,pretreated with SP600125 for 1 h,were treated with 0.5μg/mL and 1μg/mL maduramicin ammonium for 48 h,followed by morphological analysis and one solution assay.The results showed that maduramicin ammonium downregulated expression of p-ERK,p-PP2A,demethylated PP2A（De-PP2A）,upregulated expression of methylated PP2A（Me-PP2A）in H9c2 cells;maduramicin ammonium upregulated expression of p-JNK,p-c-Jun,downregulated expression of PP5 in C2C12 cells;expression of MKK1-R4F or dominant negative PP2A with adenovirus infection or pretreatment with Okadaic acid attenuated the toxicity of maduramicin ammonium in H9c2 cells;expression of dominant negative c-Jun or PP5 with adenovirus infection or pretreatment with SP600125 attenuated the toxicity of maduramicin ammonium in C2C12 cells.It suggests that maduramicin ammonium inhibits ERK by PP2A activation,resulting the toxic effect in H9c2 cells;maduramicin ammonium activates JNK and Jun by PP5 inhibition,resulting the toxic effect in C2C12 cells.6.To determine the effect of maduramicin ammonium on Aktl-Fox03a pathway in cardiac and skeletal muscle cells,H9c2 and C2C12 cells were chosen as experimental models.Cells were treated with 0～1μg/mL maduramicin ammonium for 24 h,followed by Western blotting with anti-Aktl antibody;cells were treated with maduramicin ammonium for 24 h,followed by Western blotting or immunofluorescence staining with anti-Fox03a antibody;cells,expression of constitutively active Akt with adenovirus infection,were treated with 0.5μg/mL and 1μg/mL maduramicin ammonium for 24 or 48 h,followed by Western blotting and immunofluorescence staining or morphological analysis and one solution assay.The results showed that maduramicin ammonium downregulated expression of p-Akt1（S473）,p-Akt1（T308）and p-Fox03a（Thr132）;increased the content of Fox03a in nucleus;expression of constitutively active Akt with adenovirus infection attenuated maduramicin ammonium-induced Fox03a cytoplasm translocation inhibition and toxicity.It suggests that maduramicin ammonium inhibits Akt1-Fox03a pathway,resulting the toxic effect in H9c2 and C2C12 cells.7.To determine the effect of maduramicin ammonium on AMPK activity in cardiac and skeletal muscle cells,H9c2 and C2C12 cells were chosen as experimental models.Cells were treated with maduramicin ammonium for 24 h,followed by Western blotting with anti-AMPK antibody;cells,expression of dominant negative AMPK with adenovirus infection,were treated with 0.5μg/mL and 1μg/mL maduramicin ammonium for 24 h or 48 h,followed by Western blotting,morphological analysis and one solution assay;cells,pretreated with Compound C for 1 h,were treated with 0.5μg/mL and 1μg/mL maduramicin ammonium for 48 h,followed by morphological analysis and one solution assay.The results showed that maduramicin ammonium upregulated expression of p-AMPK（Thr132）;expression of dominant negative AMPK with adenovirus infection or pretreatment with Compound C attenuated the toxicity of maduramicin ammonium.It suggests that maduramicin ammonium induces AMPK activity,resulting the toxic effect in H9c2 and C2C12 cells.8.To determine the effect of maduramicin ammonium on oxidative stress and ER stress in models.Cells were treated with maduramicin ammonium for 24 h or 48 h,followed by ROS level detection;cells were treated with maduramicin ammonium for 24 h,followed by Western blotting for ER stress related proteins.The results showed that maduramicin ammonium induced ROS generation and upregulated expression of p-PERK（Thr980）and p-eIF2α（Ser51）.It suggests that maduramicin ammonium induces oxidative stress and ER stress in H9c2 and C2C12 cells.