Epigenetic revival of a dead cardiomyocyte through mitochondrial interventions

Mitochondrial epigenetics involved in cardiac diseases

Epigenetic analysis via mitochondrial DNA methylation is scarce in the literature and unseen in cardiac-specific studies. DNA methylation is observed as a potential factor and marker of disease. Here we unravel our novel hypothesis to the study of cardiovascular diseases (CVDs) due to alterations in methylation patterns and its effects on mitochondrial damage (Figure 1). Baccarelli et al. have reported that patients with CVD have significantly higher methylation of the mtDNA than healthy controls (30). The mtDNA methylation was measured in platelets, and the authors speculate that it could serve as a non-invasive marker for the etiology of CVD. Methylation of the mitochondrial gene PGC-1α contributes to cardiomyopathy in male rats (10). Potentially speaking, since the mitochondria are inherited maternally, changes in the mitochondrial DNA methylation markers during the mother’s lifetime (pre-conception) could make a direct influence on the child’s health and susceptibility to disease. Specifically in cardiac diseases, mitochondria play a vital role in cardiac bioenergetics, maintenance of calcium and ROS production. Obesity, insulin resistance and diabetes, which pose cardiovascular risk, are marked by abnormalities in epigenetic regulation. HDACs, for example, affect mitochondrial energy metabolism and, thus, function (31). Inhibition of class I and II HDACs improved energy metabolism in high-fat diet fed mice (31). Sirtuins, which behave as histone deacetylases, are induced in the heart (SIRT3) by exercise and protect against adverse cardiac remodeling following pressure overload (32). Hypoxia-inducible factors (HIFs), which are regulated by mitochondrial complex 3, are methylated at the CpG islands in diabetes and obesity (33, 34). For the mtDNA expression and maintenance, there is high crosstalk between nucleus and the mitochondria. The precise molecular mechanisms underlying these processes and the ones that affect cardiac function are so far to be deciphered.

Figure 1:

The effect of environmental factors like smoking.

Environmental factors like smoking, diet and pollutants alter the mtDNA methylation pattern leading to increased chromatin exposure, increase potential mutation and eventually mitochondrial damage.

Mitochondria and heart failure

As stated above, the mitochondria is a vital component of metabolic function. Impeding its action through damage from various sources leads to mitochondrial and cardiomyocyte decline. The basic regulatory function of the mitochondria relies on transporters such as Na⁺/K⁺-ATPase, Na⁺/Ca²⁺ exchanger, and MCU (mitochondrial calcium uniport). Mitochondrial consumption of calcium is a factor in the heart muscle relaxation, and when damaged, the mitochondria inefficiently take up calcium, causing cytosolic calcium increase and increased protease activity (40). The Na⁺/K⁺-ATPase is an active transport mechanism that pumps three sodium ions out of the mitochondria while pumping two potassium ions in. This action maintains the mitochondrial membrane potential and ratio of potassium/calcium in the mitochondria (41).

Li and colleagues analyzed the function of sodium/potassium pump (NKA) by inhibiting it with cardiac glycosides, leading to proarrhythmic behavior due to impaired mitochondrial bioenergetics. Cardiac glycosides block sodium extrusion causing calcium increase via reversing the Na⁺/Ca²⁺ exchanger (NCX). It was revealed that inhibiting the NKA caused decreased mitochondrial calcium uptake and increased oxidative phosphorylation activity resulting in increased ROS production (40). In the preservation of cardiomyocytes a channel known as Trpm2 mitigates ROS production, acting to stabilize the mitochondria. Hoffman et al. experimented with Trpm2 knockout (KO) mice and revealed that after ischemic-reperfusion the Trpm2 Ca²⁺ influx channel reduces mitochondrial ROS production. Also, the Trpm2 channel protects against doxorubicin cardiomyopathy, since there is a decrease in the survival rate of Trpm2 KO mice (42). Yancey et al. analyzed the effects of a mitochondrial-targeted antioxidant known as mitoubiqnone (MitoQ) on Sprague-Dawley rat cardiomyocytes. Eight weeks post aortocaval fistula (ACF), mitochondrial ROS production increased and mitochondrial membrane potential decreased. MitoQ substantially improved the effects of ACF in these rats but did not attenuate left ventricular dilation or fractional shortening (43).

Bondarenko et al. revealed the role of the mitochondrial calcium uniporter in mice lacking MCU expression. MCU formerly known as protein CCDC109A is an essential transporter focused on calcium uptake into the mitochondria (44). MCU^-/- mice had significant alterations, such as the functional impairment of the ability to perform strenuous exercise and minor decreases in strength. All apoptotic markers and metabolic changes were the same among the mutant and wild type (WT) mice. MCU^-/- mice had a 25% decrease in matrix calcium compared to the WT. Therefore, the MCU uniporter has an essential role in mitochondrial calcium uptake. Bondarenko et al. presented a link between the permeability transition pore (PTP) and the MCU transporter. MCU is the mediator of PTP opening, and when open, PTP depolarizes the membrane and inhibits ATP production. Michels et al. has noted the deficiency of two Ca²⁺ selective channels (mCa1 and mCa2) in the failing heart. These channels assist in calcium uptake into the mitochondria (45).

Bondarenko et al. revealed the coexistence of MCU-dependent and MCU-independent Ca²⁺ channels in the inner mitochondrial membrane. Mitochondrial calcium currents were assessed in MCU knockdown HeLa cells. Interestingly, the extralarge conductance of mitochondrial (Ca²⁺) current (xl-MCC) was upregulated 2.3-fold, and the bursting currents remained unchanged. Therefore, these currents are not MCU dependent. The literature is lacking a follow-up article on the actions of the xl-MCC in HF patients. We suspect that the channel sustaining this current has met its maximum calcium uptake in the HF patient, leading to a decrease in the patient’s status (44). Essentially, mitochondrial calcium transport channels sustain the stability of the mitochondria and cardiomyocytes. The cardiac degradative process within a HF patient is dependent upon increased calcium and ROS production by the unstable mitochondria. The distortion of channels and currents as mentioned above affects protease activity by ROS formation and decreased calcium handling. ROS products such as superoxide (O₂) and hydrogen peroxide (H₂O₂) are produced within the mitochondria under two conditions: when mitochondria are not producing enough ATP resulting in a high proton motive force and reduced coenzyme Q and when there is a high NADH/NAD⁺ ratio in the mitochondrial matrix (46). The literature has noted that the deficiencies producing ROS lie within the supercomplexes of the electron transport chain. The leakage of electrons in the electron transport chain causes the superoxide (ROS) production. Maranzana et al. focused their experimentation on complex I by disrupting the association of complexes I-III. The investigation resulted in a substantial increase in ROS from complex I (47). Interestingly, the mental health drug haloperidol, a psychopharmacology drug, inhibits mitochondrial complex I in the brain tissue. The effects of this drug on cardiomyocytes are limited, but further investigation would be compelling (48).

Huang and coworkers presented a novel haloperidol derivative called N-n butyl haloperidol iodide (F2) that was shown to inhibit H₂O₂-induced sodium/calcium exchanger activation in rat cardiomyocytes. F2 protects against cardiac I/R injury by directly inhibiting the MEK (extracellular signal regulated kinase-kinase)/ERK (extracellular signal regulated kinase) pathway and Na⁺/H⁺ exchanger (NHE) activation (49). The cardiomyocyte NCX exchanger was shown by Eigel and colleagues to induce cardiomyocyte apoptosis post hypoxia in guinea-pig ventricular cardiomyocytes (50). Hinata et al. examined the mechanism by which H₂O₂ activates NCX in guinea-pig ventricular cardiomyocytes using an NHE inhibitor. They found an increase in the NCX current via H₂O₂ activation by two pathways, one of which activates NHE by a PI3K-dependent mechanism and the other by the activation of a tyrosine kinase (Src family) (51). NCLX, the sodium/calcium exchanger respective to the mitochondria, maintains the same antiport function within the mitochondria. The mitochondrial H⁺/Ca²⁺ exchanger (Letm1) and NCLX are both factors in the mitochondrial membrane potential and act as apoptotic regulators. Although it is believed that Letm1 releases Ca²⁺ from the mitochondria when NCLX activity is low, the topic is controversial (52). De Marchi et al. clashes with the above statement. This group presents that NCLX is the mediator of calcium extrusion from the mitochondria, not Letm1, and support the idea that NCLX has a protective function by standardizing NAD(P)H production and regulating ROS. Increased mtCa²⁺ showed increased auto-fluorescence of NAD(P)H, which was drastically diminished by NCLX overexpression (53).

Cardiac arrhythmias are a major factor represented in cardiomyopathies such as HF. Dysregulation of calcium is a direct cause of myocardial arrhythmogenic activity. Takeuchi et al. present NCLX as a regulator of automaticity in HL-1 cardiomyocytes. They knocked down NCLX with siRNA, and this knockdown resulted in prolonged action potential generation due to decreased SR calcium leak (54). Therefore, overall observation of calcium extrusion via MCU, NCLX and potentially Letm1 demonstrates that it maintains the normal cardiac physiological state. When there is dysregulation of calcium such as in HF, the transport systems decline in function. As a result, apoptotic signals are triggered resulting in degradation and decline.

Mitophagy and heart failure

Hemodynamic stress induces mitochondria to act as ‘angels of death’ promoting apoptosis by producing excess ROS with pro-death proteins and inhibiting ATP synthesis. Autophagy is the catabolic cellular digestion eradicating damaged cellular components using the lysosome. Mitophagy is mitochondrial-driven autophagy, which is a cardioprotective response acting as a beneficial adaptation to early induced stress. In the healthy myocardium a balance between mitophagy and mitochondrial biogenesis is present (55). The myocardium is composed of a variety of accumulated ‘old’ post-mitotic cells including defective mitochondria and aggregated irregular proteins known as lipofuscin. The catabolic nature of autophagocytosis and cellular digestion by proteases seems to be lacking in the myocardium. Lipofuscin proteins act to restrict the release of ROS from deficient mitochondria due to a lysosomal compartment. With the progression of age, lipofuscin-filled cardiomyocytes become overloaded with mitochondria causing increased oxidative stress and HF development. Dysregulation of deficient mitochondria is present in various cardiomyopathies, and some of them are caused by genetic deficiencies in mitochondrial DNA. Hemochromatosis, a genetic iron overload disease causing degradation of iron-saturated ferritin in lysosomes, leads to lysosome sensitivity by oxidative stress causing increased redox-active iron. Lysosomal activity is pertinent in the HF model, and up-regulation of lysosomal activity may degrade damaged mitochondria and lead to prolonged cardiomyopathy and congestive heart failure (CHF) development. Potentially, this is a target for future therapeutics and may increase the life span of CHF patients (56).

Cardiac health is dependent on increased mitophagy during stress. There seems to be a deficiency in research focused on the beneficial aspects of exercise-induced mitophagy. The cardioprotective nature of mitophagy would lead to the assumption that increased mitophagy would occur within the myocardium during exercise-induced stress. Regular physical training could lead to ’mitophagy exercise’ within the myocardium and would increase mitophagy activity in daily life, preventing cardiomyopathies by degrading deficient mitochondria and limiting ROS production, especially in an older population. Selective mitophagy occurs within the diabetic heart, specifically in insulin-resistant and insulin-deficient diabetes. Both conditions are accompanied by hyperglycemia. As noted by Kobayashi and colleagues, high levels of glucose and high fat diets directly inhibit cardiomyocyte autophagy. Cardiomyocyte autophagy tends to express inverse roles in type 1 and 2 diabetes. Diminished autophagy is proposed to be cardioprotective in type-1 diabetic model, whereas decreased autophagy in type 2 diabetes contributes to cardiac injury (57). In type 1 diabetic model, attenuation of cardiac injury and decreased ROS production was observed in beclin-1 and Atg 16 deficient mice. Beclin-1 is a protein that is essential in the autophagy cascade. Xu et al. crossed beclin knockouts with OVE26 diabetic mice, and in this diabetic model beclin-1 deficiency attenuated cardiac injury (58). Therefore, mitochondrial clearance via mitophagy is beclin-1 dependent (59).

Increasing the rate of mitochondrial consumption by mitophagy causes an increase in efficient mitochondria to be present in the sarcoplasm, allowing for increased calcium uptake by mitochondria and decreasing the detrimental effects of high calcium. Therefore, we can assume that mitophagy is tied to the protease calpain and other calcium-dependent proteases. Efficient mitophaghy within the sarcoplasm would degrade malignant mitochondria allowing for increased ‘good’ mitochondria leading to decreased calcium levels and calpain activity. Calpain activity may be tied to mitophagy, which is not a readily studied area. Mitochondrial impairment results in a loss of ATP production; thus, the vast bioenergetic needs of the heart may not be met. Mitochondrial fission is a beneficial factor segregating (non-repairable) impaired mitochondria to be lysed. Do impaired mitochondria induce a secondary messenger, signaling impaired properties to healthy mitochondria, thereby increasing the number of impaired mitochondria? If so, this could be a factor in the degradative effects seen in HF. Another conjecture is the possibility that increased mitophagy could alleviate arryhthmogenic responses in the myocardium by clearing the mitochondria which are not impaired.

Studies by Xue and coworkers revealed that calpain activity is induced during atrial fibrillation in the dog heart model, resulting in atrial remodeling. Though the focus of their study was not mitochondrial dynamics, it is stated that the cardiomyocyte mitochondria were misshaped and differentially sized (60). We assume that this change in shape may lead to mitochondrial impairment. Also, use of calpain inhibitors led to decreased calpain activity and reversed structural remodeling. A hunch may be proposed that mitochondrial degradative activity is linked to calpain and therefore HF.

Hyperhomocysteinemia and mitophagy

In our laboratory we have found that within the HHcy mouse model the cardiac-specific knockout of NMDAR1 resulted in decreased levels of mitochondrial MMP-9. Mitochondrial MMP-9 within the HHcy mouse model has been found to induce mitophagy. Deletion of the NMDAR1 receptor prevents Hcy from activating MMP-9 and translocation of connexin-43. Translocation of the gap junction protein connexin-43 leads to a mitophagic response (61). In addition, we found that there is a decrease in the mitochondrial fusion/fission ratio, i.e. there is more fission than fusion, and it promotes mitophagy (see Figure 2) (62, 63). There is an increase in calcium uptake, increased ROS production and increased cytochrome c leakage which is detrimental to the cell. The use of mitochondrial division inhibitor was able to ameliorate the damage done by pressure overload-induced HF (63, 64). Mitophagy is related to increased calcium uptake and intracellular calcium concentration. We have reviewed that homocysteine induces intracellular calcium by (1) agonizing the NMDAR1 receptor which draws more calcium; (2) decreasing expression of peroxisome proliferator activator receptor (PPAR) which impairs NCX1 and hence calcium handling; and (3) impairing the SR Ca²⁺ ATPase (SERCA2a) (65).

Figure 2:

The role of mitochondrial fusion and fission in mitophagy.

Mitochondrial fusion is promoted by Mfn2 gene, while Drp1 promotes mitochondrial fission. If the ratio of Mfn2 and Drp1 is altered, there is abnormal increase in the fission which leads to mitophagy. We have reported that mitochondrial division inhibitor has beneficial effects in pressure overload induced heart failure (63) and abnormal Mfn2/Drp1 ratio can lead to endothelial dysfunction (62).

Intra-mitochondrial Lon protease

Lon protease is an ATP-dependent protease and a key enzyme involved in the stabilization of the eukaryotic mitochondrial matrix. The mitochondrial Lon protease acts to eliminate oxidatively modified proteins and is seen to be upregulated in various cancers but is relatively unexplored in CVDs. It is noted for its actions as a chaperone, due to its ability to repair misfolded proteins within the mitochondria. Pinti et al. confirmed the Lon protease as a stress regulatory protein. Upregulation of D-deoxyribose and stavudine (d4T) activates the release of Lon protease in the presence of ROS. Mechanistic analysis concluded that variation in Lon transcriptional promoter regions allows for increased or decreased expression, and d4T upregulates Lon promoter activity in all cell lines (66). Kao and colleagues found that the Lon protease is a regulator of apoptosis through its association with heat shock proteins (HSP); HSP60-mtHSP70 complex (67). This interaction was confirmed by co-immunoprecipitation and immunofluorescence co-localization assay. Their findings included the identification of 76 Lon-associated proteins participating in the mitochondrial chaperone system. Also, since HSP60 binds to p53 (known as a significant factor in apoptosis), it inhibits apoptosis. This action is Lon dependent because the Lon protease acts to degrade proteins, maintaining the stability of the HSP60-mtHSP70 complex (67). Decreased Lon protease activity leads to increased apoptotic signaling and mitochondrial instability within the cardiomyocyte. This is detrimental, and embryonic death occurs in animal models of Lon protease knockouts as observed by Quiros et al. (68).

Lon protease and TFAM

Interestingly, a link between the Lon protease and mtDNA has been established in the literature. Matsushima et al. have demonstrated a role of the Lon protease in mtDNA metabolism in Drosophila Schneider cells. This group showed that RNAi knockdown of Lon increases TFAM (human mitochondrial transcription factor A) expression and mtDNA copy number. Upregulation of Lon causes the reverse reaction. Lu et al. has also described a correlation of TFAM and the Lon protease via phosphorylation. More specifically, phosphorylation of TFAM within the high-mobility group box 1 inactivates transcription. Lu et al. also showed that unbound TFAM is degraded by the Lon protease. Interestingly, the anticancer drug bortezomib decreases Lon protease activity (69). Potentially, use of this drug could allow for increased intracellular ROS production due to Lon protease inhibition, potentially resulting in mitochondrial and cardiomyocyte decline. Additionally, Watanabe and coworkers found that TFAM and TFB2M regulate SERCA2a gene transcription. This group established that mtDNA-specific transcription factors have the potential to regulate nuclear DNA. This was shown through TFAM and TFB2M binding to the SERCA2a gene promoter of rat neonatal cardiomyocytes. Watanabe et al. also stated that TFAM and TFB2M levels correlated with SERCA2a levels in the rat MI model (7). Knowing this correlation between TFAM and SERCA2a transcription, we can speculate that the Lon protease which regulates TFAM will indirectly regulate SERCA2a. Knowing that activation of SERCA2a would cause increased calcium uptake from the sarcoplasm, we can propose that the mitochondrial Lon protease is a major player in maintaining cardiac health (see Figure 3). Potentially, if maternal cigarette smoking alters mitochondrial TFAM methylation, this may correlate to the transcriptional activation of TFAM causing alterations in Lon protease and SERCA2a activity.

Figure 3:

The role of altered mtDNA methylation.

mtDNA methylation can shut down certain beneficial genes like Lon protease which can lead to intramitochondrial ROS increase. On the other hand, hypomethylation of TFAM can lead to its increased expression and binding to SERCA2a gene, which subsequently leads to calcium mishandling. All these events together lead to mitochondrial dysfunction.

In a clinical study performed by Jerkins et al., it was revealed that bortezomib induces congestive HF (70). Could the underlying molecular mechanism be tied to decreased Lon protease activity and dysregulation of TFAM/mtDNA ratio and SERCA2a. Lu et al. showed that Lon depletion increased TFAM levels and mtDNA content. Therefore, this group presents a critical regulation activity performed by the Lon protease (69). Palacin et al. has performed a study analyzing the incidence of early onset of MI and its relation to SNPs within the TFAM gene. This study analyzed the H haplogroup and found four polymorphisms. Overall, this study concluded that H-haplogroup male tobacco smokers had a higher incidence of early-onset MI (8). This haplogroup type may be more susceptible to tobacco smoke intake and its effects on mitochondrial DNA. Smoking may play a vital role in TFAM gene regulation among all populations.

Lon protease and COX

Cytochrome c activation is associated with apoptotic signal pathways and is released from the mitochondria as leakage. Under hypoxic conditions the Lon protease is characterized as the protease that degrades COX 4 in the electron transport chain (71). Cytochrome c release may be indirectly linked to cytochrome oxidase 4 degradation. In relation to cardiac disease, cardiac hypoxia may be a factor in HF. A link is established between cardiac ischemia and cytochrome c release as performed by Borutaite et al. (9). The activation of the Lon protease is driven by increased intramitochondrial ROS production. Yang et al. showed that CSE (cigarette smoke extract) treatment decreased COX subunit II mRNA and protein levels in vascular endothelial cells (72). In an analysis by Fukuda and colleagues it was found that under hypoxic conditions HIF-1 regulates COX IV subunit expression. This is performed via transcriptional activation of the Lon gene, which degrades COX4-1 via protease release (73). Pawlak et al. also noted the decreased COX IV expression in a steroidogenesis study involving cigarette smoking (74). Kawashima et al. observed the effects of maternal cigarette smoking on placental gene expression. The study showed that the placental gene expression of placental growth factor (PGF) and HIF1A was significantly higher and the BAX/BCL2 mRNA ratio was significantly higher than in the non-smoker group (75). As we know BCL2 is a major factor in cytochrome c release.

Is it possible that decreased methylation markers seen on the COX subunit due to smoking allow for decreased Lon activity driving cardiomyocyte apoptosis via upregulated intracellular ROS? If a compromised mitophagic response and increased mitochondrial decline occurs, this may drive the significant increase in protease activity seen in HF patients, due to the vast ROS production and calcium mishandling. Although performed in WI-38 VA-13 human lung fibroblasts, Bota and coworkers showed that Lon downregulation results in significant caspase 3 activation and apoptotic death due to mitochondrial decline (76). In the earlier studies of Lon protease function Bota et al. described the degradative abilities of Lon protease protein. Focusing on the mitochondrial oxidized aconitase, decreased presence of Lon protease results in increased oxidative aconitase, consequently resulting in protein aggregation (77). Therefore, Lon protease activity to degrade ROS production is essential because increased ROS drives increased oxidation of proteins leading to aggregation. ROS mainly caused by electron leak from the mitochondrial complexes of the electron transport chain can further oxidize proteins resulting in aggregation. As we know that Lon protease reduces oxidative stress and clears oxidized proteins, Ngo et al. proposed that Lon protease activation is biphasic and therefore upregulated in transient stress and downregulated in chronic stress (78). Although this may be true, it is likely that mtDNA transcription factors and signaling are interrupted causing the Lon protease decreased expression. Hart et al. showed that in high-capacity runner rats, antioxidant resveratrol increased aerobic performance and upper limb strength. We speculate that molecularly this antioxidant assisted in the degradation of ROS production allowing for increased healthy mitochondria. The study also showed that resveratrol activated AMP-activated protein kinase, SIRT-1 and TFAM. The authors propose that this increase in aerobic performance is due to activation of the AMPK-SIRT-1-PGC-1alpha pathway (79). The Lon protease plays a significant mitochondrial role, and we speculate that cigarette smoking will compromise the Lon protease at the mtDNA level.

Calpains and matrix metalloproteinases

Calpains are a family of intracellular cytosolic cysteine proteases activated by calcium and localized within the cytosol and mitochondria. Calpain 1 in particular has been implemented as a major degradative protease and factor in cardiomyopathies. Secondarily, MMPs are zinc-dependent endopeptidases, capable of degrading the ECM causing cardiac remodeling. This family of proteases has two well-studied members within the cardiac system: MMP2 and MMP9, which play a role in HF. MMPs are regulated by TIMPs. This family of proteases is noted for their involvement in cardiac remodeling and atherosclerotic plaque formation. Both proteolytic families play major roles in the degradative process of HF.

Interestingly, Vindis et al. analyzed an oxidized low-density lipoprotein apoptotic pathway driven by calpain activation. Upregulation of calpain caused Bid cleavage allowing for cytochrome c release through the mitochondrial transition pore. The release of cytochrome c activates caspase 3 (80). Elaboration on the previous discussion of Lon protease about indirect release of cytochrome c, this route may be driven by calpain. It is known that Bax proapoptotic protein is translocated from the cytosol to the mitochondria, which stimulates cytochrome c release. Gao et al. found that mitochondrial calpain cleaves the N-terminus of Bax creating the Bax/p18 fragment, which is stated to be a potent proapoptotic fragment that stimulates cytochrome c release independent of Bcl-2 (81). Therefore, mitochondrial calpain is a mediator of cytochrome c release.

Arrington and colleagues found that Calpain 10, the intra-mitochondrial protease, resides in the mitochondrial compartments. This group observed the cleavage of electron transport chain complex I subunits ND6 and NDUFV2 by calpain 10. Arrington et al. proposed that increased cytosolic calcium causes increased mitochondrial transport via the ruthenium red sensitive calcium channel uniporter. The increased mitochondrial matrix calcium activates calpain 10 causing cleavage of complex I (82). Although Arrington et al. have proposed this as a beneficial factor, Maranzana et al. noted that disrupting the association of complexes I-III resulted in a substantial increase in ROS from complex I (47). There may be a causative relationship between calpain 10 cleavage of the electron transport chain and decline of the mitochondria. We are considering the possibility that in pathological situations, Calpain 10 may cleave additional sections of the electron transport chain. This speculation is based on ROS production, when breaks in the complexes I-III occur leading to mtDNA modifications, as previously discussed. Our lab has previously established a link between HHcy and mitochondrial damage via activation of MMP-9. We showed that Hcy activates calpain-1 and induces its translocation from the cytosol to within the mitochondria, leading to MMP-9 activation through intra-mitochondrial oxidative bursts (83). It is possible that the mitochondrial calpain discussed by Gao et al. may be calpain 1 and is found within the mitochondria due to Hcy translocation.

DNA/mtDNA methylation and cigarette smoking

In recent years, cardiovascular epigenetics is a rising topic for discussion and a new area of unventured discovery. The growing public health concern centered around cigarette smoking raises the epigenetic conversation of how this self-induced pathogen affects our cardiovascular health. The literature has a focus on how maternal smoking changes nuclear DNA methylation patterns in infants and newborns. Ivorra et al. has identified 185 CpG islands with altered methylation patterns in the infants of mothers who smoked during pregnancy. These changes correspond to 110 gene regions, of which 10 are newly identified regions including FRMD4A, ATP9A, GALNT2 and MEG3 and are significant in embryonic development. Ivorra et al. observed DNA methylation patterns in umbilical cord blood as compared to maternal peripheral venous blood. Findings of statistically significant differences were noted on 31 CpG islands that were associated with 25 genes. Analysis was performed using an Illumina Infinium Human Methylation 450Beadchip. Ivorra et al. observed that CpG islands of the exposed group had 90.3% higher methylation, with three hypomethylated sites (92).

Alterations in methylation pattern of nuclear DNA have also been noted in a massive study by Richmond and coworkers. This group observed 800 mother-offspring pairs involved in prenatal smoking. DNA methylation was observed at three time points, at birth, age 7 years and 17 years. Of the 7 gene regions and 15 CpG sites analyzed, 4 genes (AHRR, MYO1G, CYP1A1, CNTNAP2) maintained abnormal DNA methylation patterns throughout the time points. There was strong maternal association between maternal and offspring DNA methylation alterations as compared to paternal smoking, primarily due to the in utero cigarette smoke exposure (93). Intriguingly, this study links the in utero DNA methylation alterations to a potentially long-term effect of in utero cigarette exposure. Nielsen et al. analyzed maternal smoking during pregnancy and observed three DNA methylation time points: (1) during pregnancy in placenta, (2) at birth in cord blood and (3) buccal epithelium tissue. It is noted that an altered DNA methylation pattern occurred at specific genes: CYP1A1, AhRR, FOXP3, TSLP, IGF2, AXL, PTPRO, C11orf52, FRMD4A and BDNF (94). Zhang et al. observed a protective effect of demethylation treatment on CSE-induced mouse emphysema model. Analyzing mitochondrial transcription factor A (mtTFA) in various tested groups showed a 4-fold increased methylation in the CSE group compared to the control. The use of DNA methylation inhibitor (Aza) prevented this hypermethylation. Increased methylation correlated with a 3-fold decrease in mtTFA and COX subunit II mRNA and protein levels. Treatment with Aza decreased methylation and improved emphysema and mitochondrial COX activity (95).

mtDNA methylation is correlated to the exposure of chromatin and mutation in the genome. Mutation, although not always directly related to disease, may result in genetic modifications. Some mutations in the mitochondrial DNA may be associated with mitochondrial apoptosis creating increased ROS. This is an unfound area of study, because if the mother’s mitochondrial epigenetic methylation markers are changed before giving birth to her offspring, then those specific markers may be passed to their young ones. This could potentially increase the rate of mutation within the mitochondria leading to the damage of mtDNA and mitochondrial proteins resulting in increased proteolytic activity. The same can be said for the healthy female who exercises regularly thereby passing ’good mitochondria’ to her young. Therefore, does the brunt of cardiac disease stem from the stability of the mitochondria? Could methylation marker changes lead to increased mitochondrial fission and fusion? A recently published public health review article supports the potential detrimental effects that maternal pre-conception cigarette smoking may have on epigenetic changes (96). Pre-conception alterations in mitochondrial DNA methylation may play a greater role in today’s cardiomyopathies than is readily studied.

Reviving a dead myocyte: the mitochondrial puzzle pieces of heart failure

Whether a dead myocyte can be revived is a big question. We have highlighted two aspects that underline myocyte death: mitochondrial dysfunction and proteases. Mitochondrial dysfunction comprises epigenetic modifications of the mtDNA and abnormalities in the calcium transport system, the electron transport chain and the ATP synthesis machinery. The epigenetic modifications of the mtDNA which includes DNA methylation at CpG islands affects genes like TFAM, Lon protease and COX. These genes can induce the expression of calpains and MMPs, which degrade the intracellular machinery of the myocytes and lead to myocyte death. We speculate that a dead myocyte can be revived if protected from the adverse effect of mitochondrial and cytosolic proteases like calpain and MMPs (Figure 4). We have published that mir133a regulates the differential methylation of DNA and affects the expression of MMP9 gene (35). Mir133a is one of the potential candidates to revive impaired myocytes. Use of calpain inhibitors like calpastatin which are endogenous can reduce mitochondrial calpain levels and prolong myocyte death. Mitochondrial MMP9 can also be inhibited by the use of endogenous inhibitors like TIMPs. Mir27a and Mir27b can target TFAM and regulate its levels, hence regulating the levels of SERCA2a and calcium balance. Similarly, mir203 can be employed to regulate the levels of COX in the mitochondria. Finally, since dietary habits like methionine intake and smoking along with environmental factors like pollutants affect mtDNA methylation, these factors should be managed that can help resuscitate myocyte health.

Figure 4:

Proposed mechanisms for reviving a dead myocyte.

Myocytes can be revived by two proposed mechanisms: (1) mtDNA alterations and (2) proteolytic interventions. mtDNA methylation is altered by smoking, diet and environmental factors that lead to altered expression of some genes like TFAM, which transcriptionally regulates Lon protease and SERCA2a. Lon protease degrades COX to release cytochrome c and causes mitochondrial dysfunction. Lon protease can also lead to increase in the expression of SERCA2a and eventually calcium mishandling. Calcium increase induces calpain activation that can degrade contractile proteins. All these events can lead to myocyte death. Diet, smoking and environmental pollutants can cause altered DNA methylation and increase in ROS production. It can activate MMPs and cause cardiac remodeling. Strategies to revive a dead myocyte can involve managing TFAM (mir27), COX (mir203), ROS, Lon protease, MMP9 (mir133a), calpain and mtDNA methylations.