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

Abstract

Background. Fetal heart can regenerate to restore its normal anatomy and function in response to injury, but this regenerative capacity is lost within the first week of postnatal life. Although the specific molecular mechanisms remain to be defined, it is presumed that aging of cardiac stem or progenitor cells may contribute to the loss of regenerative potential. Methods. To study this aging-related dysfunction, we cultured mesenchymal stem cells (MSCs) from human fetal heart tissues. Senescence was induced by exposing cells to chronic oxidative stress/low serum. Mitochondrial DNA methylation was examined during the period of senescence. Results. Senescent MSCs exhibited flattened and enlarged morphology and were positive for the senescence-associated beta-galactosidase (SA-β-Gal). By scanning the entire mitochondrial genome, we found that four CpG islands were hypomethylated in close association with senescence in MSCs. The mitochondrial COX1 gene, which encodes the main subunit of the cytochrome c oxidase complex and contains the differentially methylated CpG island 4, was upregulated in MSCs in parallel with the onset of senescence. Knockdown of DNA methyltransferases (DNMT1, DNMT3a, and DNMT3B) also upregulated COX1 expression and induced cellular senescence in MSCs. Conclusions. This study demonstrates that mitochondrial CpG hypomethylation may serve as a critical biomarker associated with cellular senescence induced by chronic oxidative stress.

Figure 1

Characterization of human fetal heart-derived mesenchymal stem cells (HMSCs). (a) The profile of stem cell markers in cultured HMSCs. Immunophenotypes of MSCs were determined by flow cytometry using labeled antibodies specific for the indicated human surface antigens. (b) Differentiation potential of HMSCs. Cells were stained by Alizarin Red for calcium deposits during osteogenic differentiation. Adipogenic differentiation was detected by Oil Red O staining (200x). (c) The “molecular memory of cardiac origin” of HMSCs. Left panel: schematic diagram of the published cardiac stem cell pathways [–8]. Right panel: expression of pathway genes in HMSCs. Total RNAs were isolated from HMSCs for RNA-Seq using a HiSeq4000 (Illumina). Colors represent from high (red) to low (blue) expression based on normalized FPKM values for each gene.

Figure 2

Induction of premature senescence in MSCs. (a) Induced senescence in MSCs. Senescence was induced by continuous exposure of HMSCs (heart-derived MSCs) and SMSCs (skin-derived MSCs) to a low dose of H₂O₂ (50 µM) and low serum (5% FBS) in the culturing medium. CT: control HMSCs treated with PBS; SN: senescent HMSCs treated with 50 µM H₂O₂ for 14 d (×10). (b) Quantitation of β-Gal cells. Cells were counted under microscopy. The results are expressed as the mean ± standard deviation of β-Gal positive cells per field. ^∗P < 0.05 as compared with the PBS control. (c) Senescent-related genes. Senescent HMSCs were harvested and RNA were extracted. RT-PCR was carried out to amplify senescence-related genes, including caveolin-1, apolipoprotein J, and OX 1. (d) Apoptosis-related genes. Expression of p53 and p21 genes was measured by RT-PCR. (e) Telomere length in senescent HMSCs. The relative length of telomere was estimated by qPCR as the ratio between the copies of telomere and the copies of β-globin (T/S). ^∗P < 0.05 as compared with the replicative senescence and high H₂O₂ groups.

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. ^∗P < 0.05 as compared with that in untreated MSC control cells. Note the decrease in mtDNA methylation in senescent MSCs.

Figure 4

Alteration of mitochondrial genes during cellular senescence. (a) Location of CpG islands in the mitochondrial genome. (b) Altered gene expression of mitochondrial COX1 and ND2 genes in senescent MSCs. (c) The enzyme activity of COX1 in control and senescent MSCs.

Figure 5

Knockdown of DNMTs induces senescence in MSCs. (a) Downregulation of DNMTs in senescent MSCs. After induction of cellular senescence, cells were harvested and the expression of DNMTs was determined by semiquantitative PCR. (b) Knockdown of DNMTs by shRNAs. Lentiviruses containing DNMT shRNAs (sh-DNMT1, sh-SNMT3a, and sh-DNMT3b) or scramble control (sh-CT) were transduced into HMSCs. After 72 h, transduction efficiency was assessed by the observation of GFP positive cells. Cells were harvested and RNA were extracted, and the expression of DNMTs was determined by RT-PCR. (c) Induction of cellular senescence by DNMT-shRNA knockdown. Left panel: HMSCs morphology taken 7 days after DNMT-shRNA transduction. Sh-CT: shRNA scramble control; sh-DNMTs: HMSCs were transducted by lentiviruses containing DNMT1, DNMT3a, and DNMT3b shRNAs. Middle panel: lentiviral transduction efficiency as shown by copGFP fluorescence of the shRNA vector. Right panel: senescent-associated β-Gal staining. After DNMT-shRNA knockdown, MSCs were stained for β-Gal activity. Note the occurrence of senescence in DNMT-knockdown MSCs in the absence of peroxide treatment. (d) Expression of COX and ND2 in DNMT-shRNA-treated MSCs. β-Actin was used as the internal control for PCR reaction. Cox1 was upregulated in DNMT-knockdown MSCs in parallel with cellular senescence.