Table 3.

mtDNA methylation studies in senescence and aging.

Experimental Model	Method	mtDNA Region Investigated	Observation	Reference
Mouse brain samples	Enzyme-linked immunosorbent assay and glucosyltransferase assay	D-loop region and MT-ND2 and MT-ND5 genes’ 5-hmC content. Global mtDNA content of 5-hmC and 5-mC.	Decreased content of 5-hmC both at a global and a sequence-specific level in frontal cortex, but not in the cerebellum, of mice during aging. Transcript levels of mtDNA genes, including MT-ND2, MT-ND4, MT-ND4L and MT-ND5, increased during aging in the frontal cortex.	[52]
Peripheral blood of 381 individuals aged 38–107 years	Bisulfite sequencing	MT-RNR1 and MT-RNR2 genes	MT-RNR1 methylation levels positively associated with increasing age, particularly among women older than 85 years of age. Subjects with higher methylation levels exhibited a mortality risk higher than those with lower levels.	[89]
Replicative and senescent human and mouse endothelial cells	Bisulfite sequencing	D-loop region and MT-COI gene	D-loop was demethylated and mtDNA copy number increased in senescent cells with respect to proliferative endothelial cells	[53]
Peripheral blood of 82 individuals aged 18–91 years	Bisulfite sequencing	Methylation levels of 133 CpG sites of mtDNA	Methylation of two CpG sites located within the MT-RNR1 gene showed an inverse correlation with age	[90]
Senescent mesenchymal stem cells (MSCs) from human fetal heart tissues	Combined bisulfite restriction analysis	Eleven CpG sites located in different mtDNA genes	Three CpG sites were found hypomethylated in senescent cells. One of these CpG sites was located in the MT-CO1 gene which in turn was up-regulated in MSCs in parallel with the onset of senescence.	[57]
Mouse oocyte maturation, postovulatory oocyte aging and early embryo development	Bisulfite sequencing	D-loop region and Mt-Rnr1, Mt-rnr2 and ATP genes	Absence of mitochondrial DNA methylation in mouse oocyte maturation, aging and early embryo development	[91]
Two senescence models were constructed, replicative senescence and stress-induced premature senescence, using human heart mesenchymal stem cells (HMSCs)	Bisulfite sequencing	MT-CO2 gene	Along with the senescence of HMSCs, MT-CO2 gene methylation increased and its protein expression level significantly decreased. Treatment with 5-aza-2’-deoxycytidine inhibited COX2 methylation.	[92]