Mitochondrial DNA Hypomethylation Is a Biomarker Associated with Induced Senescence in Human Fetal Heart Mesenchymal Stem Cells

Figure 3

Altered mtDNA methylation in senescent MSCs. (a) Schematic diagram of the mitochondrial genes and the location of CpG islands. In order to detect mtDNA methylation in senescent cells, we designed 11 pairs of methylation-specific primers located on different genes on mitochondrion. (b) Comparison of mtDNA methylation between the control and senescent HMSCs. mtDNA methylation was measured by combined bisulfite restriction analysis (COBRA). PCR products from mtDNA of control and senescent HMSCs were digested by TaqI or HpyCH4IV (HPY) to separate the unmethylated and methylated DNAs. Taq I and HpyCH4IV recognize and digest the methylated ACGT and TCGA sites, respectively. After treatment with sodium bisulfate, unmethylated cytosines were converted to uracils, and the TTGA and ATGT sites are not digested by these two enzymes. After digestion, unmethylated and methylated DNA were separated on 3% agarose gels. Only the data for CpG islands 4, 2, and 1 are presented here. (c) Differential mtDNA methylation between the control and senescent SMSCs. (d) Altered mtDNA methylation in human neonatal and adult fibroblasts. (e) Quantitation of mtDNA CpG methylation. The methylated and unmethylated bands were scanned. The status of CpG methylation was calculated as the relative percentage of DNA methylation using the untreated MSCs as 100. as compared with that in untreated MSC control cells. Note the decrease in mtDNA methylation in senescent MSCs.

(a) mtDNA CpG islands

(b) mtDNA methylation in HMSCs

(c) mtDNA methylation in SMSCs

(d) mtDNA methylation in fibroblasts

(e) Quantitation of methylation