CRISPR/Cas9-mediated MSTN gene editing induced mitochondrial alterations in C2C12 myoblast cells

Background: Myostatin (MSTN) negatively regulates muscle mass and is a potent regulator of energy metabolism. However, MSTN knockout have affect mitochondrial function. This research assessed the mitochondrial energy metabolism of Mstn−/+ KO cells, and wondered whether the mitochondria biogenesis are affected. Results: In this study, we successfully achieved Mstn knockout in skeletal muscle C2C12 cells using a CRISPR/Cas9 system and measured proliferation and differentiation using the Cell-Counting Kit-8 assay and qPCR, respectively. We found that MSTN dysfunction could promote proliferation and differentiation compared with the behaviour of wild-type cells. Moreover, Mstn KO induced an increase in KIF5B expression. The mitochondrial content was significantly increased in Mstn KO C2C12 cells, apparently associated with the increases in PGC-1α, Cox1, Cox2, ND1 and ND2 expression. However, no differences were observed in glucose consumption and lactate production. Interestingly, Mstn KO C2C12 cells showed an increase in IL6 and a decrease in TNF-1α levels. Conclusion: These findings indicate that MSTN regulates mitochondrial biogenesis and metabolism. This gene-editing cells provided favourable evidence for animal breeding and metabolic diseases.

Apoptosis and autophagy of retinal ganglion cell-5 cells induced by hydrostatic pressure / 中华实验眼科杂志

Background Researches showed that the mitochondrial-mediated apoptosis and autophagy play an important role in the survival of retinal ganglion cells (RGCs) in glaucoma.However,whether high pressure will lead to mitochondrial-mediated apoptosis and autophagy is not elucidated.Objective This study was conducted to explore whether elevated pressure can directly induce the mitochondrial-mediated apoptosis and autophagy in cultured RGC-5 cells in vitro.Methods RGC-5 cells were exposed to self-made pressure device and treated by 0,20,40 and 60 mmHg pressure for four hours in pressurized bottles.RGC-5 cells were incubated simultaneously in sealed incubator bottles and served as normal control.The morphological changes of the cells were examined under the inverted phase-contrast microscope.The apoptosis percentages of the cells were detected by flow cytometry.Mitochondrial membrane potentials of the cells were assessed using the JC-1,a fluorescent dye.And the expressions of cytochrome C (Cyt-c),microtubule associated protein light chain 3 (LC3) and Beclin-1 protein in the cells was detected by Western blot.Results Cultured cells showed fusiform shape in the normal control group and 0 mmHg group with more dendritic process.The cell density was obviously reduced and the number of dead cells was increased in the 20,40 and 60 mmHg groups.The apoptotic percentage was (15.69 ± 0.77)％,(15.77 ± 1.14)％,(18.30± 1.07) ％,(23.28 ± 1.33)％ and (34.47± 1.17)％ in the normal control group and 0,20,40 and 60 mmHg groups,respectively,showing a significant intergroup difference (F =150.90,P＜0.001),and the apoptotic percentage in the 40 mmHg group and 60 mmHg group was significantly increased in comparison with the normal control group (both at P＜0.01).The mitochondrial membrane potential was high in the normal control group and 0 mmHg group,with the reddish fluorescence in the cells,and the reddish fluorescence was weakened in the 20 mmHg group and 40 mmHg group.The lowing of mitochondrial membrane potential was seen in the 60 mmHg group,with the green fluorescence.Compared with the normal control group,the expression of Cyt-c,LC3-Ⅱ and Beclin-1 proteins in pressured groups was correspondingly increased (all at P＜0.05).Conclusions Elevated pressure induces the morphologic change of RGC-5 cells,results in mitochondrial membrane potential reduction and mitochondrial-mediated apoptosis and autophagy.

Improvement of RPE cells growth and metabolism abilities by mitochondrial transfer of neural stem cells / 中华实验眼科杂志

Background Researches showed that stem cells can rescue damaged cells through mitochondrial transfer.This mode has been used to regenerative cell-based therapy.Retinal pigment degeneration is an eye disease of retinal pigment epithelial (RPE) cell apoptosis as pathogenetic mechanism.Whether stem cells can repair the target cells by above mechanism has not been clarified.Objective This study was to investigate the influence of mitochondrial transfer on the function of RPE cells.Methods The RPE cells of Long-Evans rats were isolated and cultured and the third generation of cells were used in sequential experiment.The cells were identified by detecting the expressions of RPE65 and Bestrophin proteins with immunofluorescence stain.Mouse neural stem cells (NSCs) (C17.2 strain) with green fluorescence protein (GFP) and without GFP were cultured.Mitotracker-green and Mitotracker-red staining were separately used to labeled the mitochondria of RPE cells and NSCs.RPE cells were cocultured with NSCs,and wheat germ agglutinin (WGA) was used to mark the tunneling nanotubes (TNT) between the two kinds of cells,and then the mitochondrial migration in TNT was exhibited by the laser scanning confocal microscope.The proportion of RPE cells in different cycles was assayed after marked with propidium iodide (PI) by flow cytometry.The contents of ATP,ADP and AMP in RPE cells were detected by high performance liquid chromatography (HPLC).Results The third-generation of RPE cells grew well with the RPE65-and Bestrophinpositive rate ＞85％.The Mitotracker-red-labeled rates of NSCs and RPE cells were no less than 95％.TNT structure was seen to appear the blue fluorescence between RPE cells and NSCs 24 hours after co-cultured and the red dye mitochondria from NSCs migrated toward red dye mitochondria from RPEs with the lapse of time.The RPE cell proportion reduced in G1 phase and increased by 5％ and 2％ in the S phase and G2/M phase respectively after mitochondrial transfer than before (P=0.016,0.114,0.189).The contents of ATP,ADP and AMP in the RPE cells were (8.77 ±3.68),(2.76±0.92) and (1.07 ±0.65) μg/mg after cell co-culture,and those before co-culture were (11.29±2.29),(3.12±0.95) and (1.59± 1.22) μg/mg,without significant differences between them (P =0.370,0.668,0.553).Conclusions NSCs can transfer normal mitochondria to co-cultured RPEs via TNT structure.Mitochondrial exchange might be one of therapeutic mechanisms of NSCs recuing damaged RPE cells.