**4. Epigenetics in HF**

In the last decade, and this is still an evolving field of research, evidence showed the involvement of epigenetics signature in HF. Although it is known that many factors affect epigenetics patterns (environmental, diet, lifestyle, pollutants) [190], and, in turn, epigenetics may be the cause of given pathological states (including HF) [191], we would summarize how mitochondrial function intersects with epigenetics in HF. In detail, the consequences of mitochondrial alterations or metabolites produced during the development of HF are emphasized accordingly to the topic of this review.

Epigenetics programs include DNA methylation, histone acetylation/methylation, and noncoding RNAs [192]. Chromatin acetylation involves acetyltransferases and deacetylases which make it more or less accessible to DNA binding elements, respectively. The role of this couple of enzymes was strongly matched to irreversible cardiac remodeling due to either the activation (when acetylated) or depletion (when deacetylated) of GATA Binding Protein 4 (GATA4) and Myocyte Enhancer binding Factor 2 (MEF2), transcription factors responsible for the gene expression rewiring during cardiac hypertrophy [193–195]. Chromatin methylations are reversible and can be related to activation and inhibition of the

transcriptional activity; Kaneda et al. demonstrated the significant relationship between a triple addition of the methyl group to lysine 4 or 9 of histone H3 and HF [196]. Interestingly, three genes located close to these modifications are RYR2, CACNB2 (encoding for the subunit beta 2 of the voltage-dependent calcium channel), and CACNA2D1 (encoding for the alpha-2 and delta subunits of the voltage-dependent calcium channel complex), responsible for intracellular Ca2+ cycling [196]. Instead, DNA methylation is usually synonymous with gene expression "shutdown". In animal models of pressure overload and cardiac hypertrophy, increased DNA methylation led to significant repression of the transcriptional activity in the left ventricle and allowing for cardiac contractility [197,198]. Otherwise, noncoding RNAs alter gene expression in a wide variety of modes which are not the focus of this review and are reviewed elsewhere [199].

It is clear that changes in metabolism play a crucial role in HF. Additionally, metabolites' availability influences epigenetics patterns, because most of them act as cofactors for enzymes inducing chromatin modifications and control the transcriptional program as a consequence [200]. Of interest, some of these metabolites are part of those intermediates becoming altered and concurring in HF development; these are ketones (such as βOHB, α-ketoglutarate), Acetyl-CoA, NAD+, FAD+, and mitochondrial ROS.

Acetyl-CoA is one of the main regulators of protein acetylation due to the possibility to supply acetyl groups to enzymes in charge of modifying histones [201]. Thus, the rate of FAO (and its changes during HF) would be associated to either higher or lower gene expression on the basis of the Acetyl-CoA produced [202]. This indeed constitutes a critical factor in cycles of acetylation/deacetylation. βOHB also increases acetylation of proteins being a potent inhibitor of histone deacetylases [203]; this action can lead to beneficial effects in HF due to the activation of neighboring genes such as Forkhead Box O3 (FOXO3), which confers resistance to oxidative stress and sirtuins, already known to have strong cardioprotective roles [204]. Moreover, NAD+, an essential cofactor for many enzymes involved in energy production in cardiomyocytes [205,206] and found to be decreased during HF stages, is also essential as a cofactor for sirtuins function [207]. Sirtuins are a family of proteins able to deacetylate chromatin with crucial roles. In this context, the absence of NAD+ has negative effects on the expression of sirtuins which determine no longer protective roles to counteract HF [208]. NAD+ levels are further finely regulated by Nicotinamide Nucleotide Adenylyltransferase 2 (NMNAT2), whose upregulation protect from hypertrophy [205]. During HF, a significant amount of mitochondrial ROS are produced; as well as the role already addressed, they alter both DNA and histone methylation [200], the first one by oxidizing guanosine in 8-oxo-20-deoxyguanosine and producing as a consequence a hypomethylation state of the DNA; this triggers the onset of inflammation and oxidative stress due to the activation of gene expression involved in those pathways [209]; the second, by reducing S-adenosil metionina (SAM), which is responsible for methylation of histones [210].

#### **5. Conclusions and Future Perspectives**

HF is caused by a multitude of risk factors and is involved in many cardiovascular pathologies such as coronary artery disease, hypertension, faulty heart valves, and congenital defects. For this reason, it is of great interest and involves a considerable percentage of patients. Cardiology has made great strides worldwide in prevention and patient care, but many steps of basic research are still poorly understood, and translational approaches should be improved. If on the one hand, the metabolic switch and related consequences during the adaptative stages and advanced HF are fairly known, further research about Ca2+ cycling, handling, signaling, and interconnections with mitochondrial fusion-fission machinery deserves attention. In particular, a focus on the Ca2+ spatio-temporal route and which proteins are involved in it, from the extracellular milieu to the cytosol, would be noteworthy. Then, the contribution of mitochondria as organelles closely related to SR and able to buffer Ca2+ to orchestrate a plethora of downstream events would also be noteworthy. Although animal models for the in vivo study of MAMs are lacking, and

investigations on patients continue to be limited, efforts in this way may improve the creation of targeted therapeutic strategies to recover mitochondrial and SR function in HF.

**Author Contributions:** Conceptualization, G.M. and P.P.; software, V.A.M.V.; validation, G.M., P.P. and C.G.; investigation, G.M., V.A.M.V. and E.B.; resources, G.M., V.A.M.V. and E.B.; writing original draft preparation, V.A.M.V. and E.B.; writing—review and editing, G.M.; visualization, G.M.; supervision, G.M.; funding acquisition, P.P., C.G. and G.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Italian Association for Cancer Research (IG-23670 to P.P. and IG-19803 to C.G.), Progetti di Rilevante Interesse Nazionale (PRIN2017E5L5P3 to P.P. and PRIN20177E9EPY to C.G.), Italian Ministry of Health (GR-2013-02356747 to C.G. and GR-2019- 12369862 to G.M.), local funds from the University of Ferrara, the European Research Council Grant (853057-InflaPML to C.G.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** P.P. is grateful to Camilla degli Scrovegni for the continuous support.

**Conflicts of Interest:** The authors have no conflicts of interests.

#### **References**

