The inhibitor of cyclin-dependent kinase 4a signaling pathway induces aging in human skeletal muscle myoblasts and decreases the mitochondrial membrane potential

Objective To study the effect of the inhibitor of cyclin-dependent kinase 4a (INK4a) signaling pathway on myoblastic aging. Methods We transfected human skeletal muscle myoblasts with a recombinant lentiviral vector, pLVX-p16INK4a, encoding the p16INK4a gene, and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to identify p16INK4a gene transcription and protein expression. Senescence-associated β-galactosidase staining was used to assess the degree of cell senescence. The mitochondrial membrane potential was analyzed by JC-1 staining and flow cytometry. Results We demonstrated that a senescence phenotype was evident in myoblasts transfected with p16INK4a, the analysis of mitochondrial membrane potential after JC-1 staining by flow cytometry showed a marked decrease in p16 group (-40.287±12.663) mV, while it was (-9.267±1.332) mV in CV group and (-6.967±2.031) mV in NC group. The relative amount of p16 protein and mRNA in p16 group was 45.25±8.21 and 33.89±7.87, which was more than that in CV (10.18±2.69 and 5.26±1.92)and NC (9.41±1.70 and 7.16±1.86) groups (P=0.000). Conclusion These findings indicated that upregulation of the INK4a signaling pathway directly induced aging in human skeletal muscle myoblasts. Moreover, INK4a signaling pathway activates the mitochondrial pro-aging pathway by reducing the mitochondrial membrane potential, which indirectly accelerates aging in myoblasts. Key words: Aging; Human skeletal muscle myoblast; Inhibitor of cyclin-dependent kinase 4a; Mitochondrial membrane potential