TFAM plays an essential role in maintenance, expression, and organization of mitochondrial DNA. Indeed, it is required for efficient promoter recognition by mitochondrial RNA polymerase and binds and induces significant conformational changes in mtDNA. In muscles of aged flies, TFAM and mtDNA have been found to form a more condensed structure than in juveniles (52) impeding access to the mitochondrial transcriptomic machinery with a subsequent reduction of gene expression. In detail, from 1 day of age to 12 weeks of age, the size of nucleoids decreases while their number increases (52). Under normal conditions, overexpression of TFAM leads to the reduction of Drosophila lifespan, whereas in the presence of 1% H2O2, flies that up-regulate TFAM resist the environment more, revealing that TFAM is a central factor under severe oxidative stress and mitochondria maintenance (53). Despite the lack of research on TFAM and aging in mammals, the repression of mitochondrial gene transcription in the presence of a high level of TFAM protein is well known (54) suggesting a role of TFAM in aging.

Recently, it has been observed that DNA methyltransferase 3 alpha (DNMT3A) and ten-even-transposon 2 (TET2) regulates the TFAM expression in macrophages (55). Specifically, decreased DNMT3A and/or TET2 results in downregulation of TFAM with subsequent cytosolic mtDNA release (55). The presence of mtDNA in the cytosol is responsible for the activation of cyclic GMP–AMP synthase (cGAS) signalling that triggers interferon alpha (IFNα) production with subsequent increase in inflammation (55).

Notably, in aging is well known the increasing of inflammation state (named inflammaging) which involved several cytokines included IFN (56). This finding suggests that TFAM migh contribute to the burst of inflammation observed in aging. in aging is well known the increasing of inflammation state (named inflammaging) which involved several cytokines included IFN (56). In young mice, lymphocyte-specific Tfam knockout recapitulated the characteristics of mitochondrial dysfunction that occurs in aged (22-month-old) wild-type mice, and this mitochondrial decline is characterized by increased secretion of inflammatory cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) (57).

It is well known that nuclear translocation of p53 protein is associated with increased aging due to its inhibition of PPARγ co-activator 1α (PGC1α) and PGC1β (58). It is interesting to see that in skeletal cells after endurance exercise, the presence of nuclear p53 is reduced while its presence is increased in mitochondria where it serves to positively modulate the activity of the mitochondrial transcription factor TFAM (59). Based on this evidence, physical activity in the elderly could also improve mitochondrial function through the p53-TFAM interaction.

Another epigenetic modification that occurs in aging is the global decrease in the level of methylation in genomic DNA, which cooperates with the modification of histones (60). Despite the global decrease in 5mC, it has been observed that some specific CpGs are particularly hypo- or hyper-methylated. This signature has been used to develop so-called “epigenetic clocks” that are able to specifically detect the biological age of individuals (61–65).

Although the most representative methylated base in mtDNA is adenine, recent work shows a correlation between aging and 5 mC methylation of mtDNA in the brain, where some 5 mCs (in this case both CpG and non-CpG) were found to be modulated (66). Unlike genomic DNA, aged mtDNA reveals a pattern of global hypermethylation (66). This feature is likely due to the loss of the ability of ten-eleven transposons (TETs) to eliminate methylation on cytosines, caused by altered metabolism in which the reduction of α-ketoglutarate (the cofactor of TETs) was observed (67). In recent work, a significant increase in DNA methylation levels was found according to age and the administration of the low-calorie diet. Particularly, the increase in methylation level represses genes involved in mitochondrial biogenesis, suggesting that mitochondrial dynamics is driven by age and diet (68). During aging, mitochondrial increase in reactive-oxygenated species (ROS) has been reported to modulate mtDNA (69, 70). Specifically, ROS-induced damage to mtDNA reveals impaired production or mutation of enzymes involved in oxidative phosphorylation, exacerbating mitochondrial dysfunction and enhanced cellular senescence (69).

The correlation between miRNA and histone deacetylase has been demonstrated during aging. Specifically, miR-9 and miR-34a have been found to be up-regulated in aging and both target Sirt1 mRNA, which is found to be down-regulated contributing to enhanced gene expression (71). In addition, Sirt1 is also regulated by miR-181a, which is down-regulated during aging instead (72). Repression of miR-181a in the elderly has been associated with defects in T-cell activation (72). The lncRNAs are also modulated in aging. For example, H19 is down-regulated in senescent endothelial cells (73). Specifically, repression of H19 reflects increased phosphorylation of STAT3, which becomes active and transcribes p16 and p21 reflecting cell cycle inhibition (73). In addition, in cardiomyocytes (CM), lncRNA ENSMUST00000134285 has been observed to control apoptosis. In this case, in aged tissues, the expression of ENSMUST00000134285 is increased and inhibits MAPK11 activity, initiating the apoptotic process (74).

Modulation of ncRNAs has also been observed in aging, and the ncRNAome is tissue-specific. An example is miR-183-5p, miR-199b-5p, miR-205-5p, and miR-200b-3p, which are up-regulated in 3-month-old mice and are involved in the thymus regression process (75). Similarly, modulation of the lncRNAome during aging was found in the liver, where Meg3, Rian and Mirg were found to be up-regulated, revealing increased inflammation, repression of cell proliferation and metabolic changes (76). The lncRNA nuclear-enriched abundant transcript 1 (NEAT1) was found to be modulated in bone marrow mesenchymal stem cell (BMSC) senescence (77). Up-regulation of NEAT1 drives the transition from bone cells to fat cells by hindering mitochondrial function, while repression of NEAT1 reflects an increase in Sirt3 (77), which, as has been discussed previously, is responsible for deacetylating TFAM (28).

In conclusion, the ncRNAs that are transcribed by nuclear DNA and shifted to mitochondria have been found to modulate oxidative stress response, Sirt1-dependent deacetylation, inflammation, cell cycle and apoptosis confirming their involvement in mitochondrial homeostasis leading to aging process (Table 1).

Modulation of the mtDNA methylation of the mt-COX2 gene has been observed in human cardiac mesenchymal stem cells (HMSCs) (87). In particular, senescent HMSCs reveal hypermethylation of the COX2 gene, which appears to be downregulated (87). On the other hand, overexpression of mt-COX2 and inhibition of DNMT1 by 5-aza-2′-deoxycytidine delay the senescence process (87). It has also been shown that, in the rat model, depletion of mt-COX2 reduces ATP and acetyl-CoA production. Consistently, the expression of genes related to mitochondrial oxidation, were downregulated, while glycolytic hexokinase 1 (HK1) was upregulated. These observations indicate that COX2-deficient rats develop hypertension, heart failure, and increased thrombotic events, probably as a result of dysregulation of cardiac energy metabolism (88).

Analysis of mtDNA methylation was also performed in platelets from CVD patients, where an increase in 5mC methylation was found in comparison with healthy controls (89). In particular, an increase in the methylation level of the mt-COX1/2/3 gene and mitochondrial leucine 1 (mt-TL1) tRNA was found (89). In line with these findings, in vascular smooth muscle cells (VSMCs) platelet-derived growth factor-BB (PDGF-BB) triggers the translocation of DNMT1 from the nucleus to the mitochondria, where mtDNA methylation increases resulting in suppression of gene expression (90). These increased levels of methylation are also associated with mitochondrial dysfunction, altered contractility of VSMCs, and aberrant cell growth (90).

Mitoepigenetics also seems to play an important role in CAD. Indeed, comparing mtDNA methylation levels of peripheral blood leukocytes from patients with stable coronary artery disease (SCAD) and acute coronary syndrome (ACS), a lower level of global 5 mC mtDNA was found in patients with ACS. In line with these findings, hypermethylation of the D-loop region was also detected in these patients, which is associated with reduced mtDNA synthesis (91)⁠.

In myocardial infarction, alteration of physiological functions of mitochondria leads to modification of proteins and lipids and inhibition of energy production, contractile capacity, cell necrosis, or apoptosis. In the myocardium, oxidative stress caused by ischemia/reperfusion (I/R) injury triggers nuclear translocation of nuclear respiratory factor 1 (NRF1) and upregulation of PPARG coactivator 1 alpha (PGC-1α), which increase TFAM transcription and contribute to mitochondrial biogenesis and repair, acting as a compensatory mechanism(92). Interestingly, TFAM depletion reflects the loss of cellular ability to respond to I/R damage. In CMs after the decrease in oxygen level, the amount of TFAM increases as a compensatory mechanism. Thereafter, its level progressively decreases revealing the increase in ROS production and calcium mismanagement. This phenomenon has been particularly observed in the later stages of HF. Therefore, restoring TFAM levels by increasing mitochondrial biogenesis and reducing ROS production could protect cardiomyocytes from the oxidative damage of mtDNA induced by I/R-injury (93).

Mitochondrial dysfunction is also a feature of hypertension that leads to increase vascular oxidative stress. In particular, Sirt3 has been found to play a key role in maintaining endothelium function (94). In the aortas of hypertensive mice, Sirt3 down-regulation was found to result in worsened blood pressure, vascular relaxation, superoxide and nitric oxide production, as well as increased hypoxia-induced factor 1 alpha (HIF1α), pro-inflammatory gene expression as well as vascular permeability (94). In addition, reduction of Sirt3 leads to increased acetylation of superoxide dismutase 2 (SOD2), resulting in loss of its antioxidant activity (94).

Consistently, beneficial effects of Sirt3 have also been observed (94). In a rat model, activation of the AMP-activated protein kinase (AMPK)-PGC1α-Sirt3 signaling pathway was observed (94). Specifically, PGC1α increases the expression of Sirt3, which in turn deacetylates NRF1, the transcription factor responsible for TFAM transcription (94). On the other hand, Sirt3 also acts directly on TFAM allowing its translocation into mitochondria and subsequently transcription of mitochondrial genes.

Activation of this pathway, thus on the one hand enhances mitochondrial biogenesis and function while on the other hand reduces ROS-dependent cellular stress (94). Interestingly, this pathway can be stimulated by melatonin administration through AMPK activation (94).

Consistently, beneficial effects of Sirt3 have also been observed in myocardial I/R injury (95). In a rat model of I/R, activation of the AMP-activated protein kinase (AMPK)-PGC1α-Sirt3 signalling pathway has been observed (95).. Specifically, PGC1α increases the expression of Sirt3 which in turn deacetylates NRF1 the transcription factor responsible for TFAM transcription (95). Moreover, Sirt3 also acts directly to TFAM allowing its translocation into mitochondria and subsequently transcription of mitochondrial genes. As a result,the biogenesis and function are gained and ROS-dependent cellular stress is reduced (95). Interestingly, this pathway can be stimulated by melatonin administration, which acts by activating AMPK (95). Similarly, the PGC1α-Sirt3-TFAM/NRF1 pathway was also found to be repressed in a rat model of isoprotenerol-induced HF (96). Of note, administration of perindopril improves cardiac function through SIRT3 and PGC1α signalling pathway activation (96).

In CVDs, several mitomiRs have been found to be involved in the pathogenesis. An example is miR-181c, a nuclear-encoded miRNA, which is translocated from nucleus to mitochondria and targets mt-COX1 mRNA (97)⁠. To mimic the HF condition, miR-181c has been up-regulated targeting nanoparticles to heart (97)⁠. The miR-181c- treated rats revealed a significant decrease in left ventricular fractional shortening and markedly lower ejection fraction (97). MiR-181c has also been found to regulate calcium uptake in cardiomyocytes (98)⁠. The miR-181c loss can protect the heart from I/R injury by modulating calcium transport through the upregulation of mitochondrial calcium uptake 1 (MICU1) (98)⁠. In fact, in miR-181c^−/− mouse model, the molecular mechanism found is the involvement of mt-COX1 which up-regulates the transcription factor specificity protein 1 (Sp1) that in turn triggers the expression of MICU1 (98).

Several miRNAs are involved in the pathogenesis of CVD. One example is miR-181c, a nuclear-encoded miRNA that is translocated from the nucleus to the mitochondria and targets mt-COX1 mRNA (97). To recapitulate an HF phenotype, miR-181c was up-regulated by targeting nanoparticles to the heart (97). Rats treated with miR-181c revealed a significant decrease in fractional shortening and ejection fraction (97). It was also found that miR-181c regulates calcium uptake in cardiomyocytes (98). Thus, suppression of miR-181c may protect the heart from I/R injury by modulating calcium transport through upregulation of mitochondrial calcium uptake 1 (MICU1) (98). Indeed, in the miR-181c^−/− mouse model, the molecular mechanism found is the involvement of mt-COX1 regulating the transcription factor specificity protein 1 which in turn triggers the expression of MICU1 preserving Ca²⁺ uptake in CMs after I/R injury (98). Interestingly, in the human heart, up-regulation of miR-181c leads to ventricular septal defects (99). In this condition, the molecular mechanism of miR181c involves inhibition of bone morphogenetic protein type 2 receptor (BMPR2) (99), which is critical for septal formation and valvulogenesis (100).

Small RNA-seq performed in mitochondria isolated from mouse failing hearts, showed that mitochondria-enriched microRNAs in HF were associated with energy metabolism and oxidative stress pathway (101). Little is known about these mitomiRs in CVDs. However, miR-696 has been observed to down-regulate PGC1α with subsequent impairment of mitochondrial function in skeletal muscle (102)⁠. An analogous mechanism might occur also during I/R injury in CMs, however no information is currently available.

On the other hand, lncRNA growth arrest specific 5 (GAS5) is known to sponge miR-532-5p and is up-regulated during I/R (103). Specifically, this lncRNA is increased in I/R injury and is responsible for phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) downregulation, thus resulting in apoptosis (103). Overexpression of GAS5 lncRNA during ischemic injury prevents the development of an adverse cardiac phenotype (103). Similarly, during stroke, miR-532-5p expression is strongly reduced and is associated with increased infarct area, neuronal apoptosis, and worsening neurological score (104). The up-regulation of miR-532-5p enhances the PI3K/AKT pathway that increases cell viability by attenuating neurological damage (104).

In hypoxia, the potentiation of miR-210 and its involvement in CVD is well known (105). Iron-sulfur cluster assembly proteins 1 and 2 (ISCU1/2) have been found to be a target of miR-210 in mitochondria (106). Increased miR-210, under oxygen-deficient conditions, reduces the level of ISCU1/2, which is involved in iron-sulphur clusters for electron transport and mitochondrial redox reactions (106). Consequently, downregulation of ISCU1/2 impairs metabolism and cell survival.

MitomiRs are also involved in cardiac hypertrophy. MiR-485-5p has been found repressed in mouse hypertrophic heart where it interacts with the mitochondrial anchored protein ligase (MAPL). In turn, the downregulation of MAPL reveals the over-expression of the mitochondrial fusion protein 2 (MFN2) which induces the mitochondrial fission (107)⁠. In hypertrophic hearts, miR-485-5p up-regulation was shown to reduce LV wall thickness (107)⁠. miR-485-5p is also regulated by metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) (108)⁠ that is over-expressed in hypoxic condition (109)⁠. In CVD patients, high level of circulating MALAT1 has been observed in association with inflammation and hypoxia (110)⁠. Notably, in hepatocellular carcinoma it has been found that MALAT1 binds the mtDNA modifying the mitochondrial structure, reducing oxidative phosphorylation and ATP production, decreasing mtDNA copy number, and activating the apoptosis (43)⁠. Despite the increase of MALAT1 in CVDs, no work reports the modulation of mitochondrial gene expression upon MALAT1-mtDNA interaction.

Figure 2 reports a brief overview of mitochondrial epigenetics in cardiovascular diseases.