HDACs and Their Inhibitors on Post-Translational Modifications: The Regulation of Cardiovascular Disease

Abstract

Cardiovascular diseases (CVD), such as myocardial hypertrophy, heart failure, atherosclerosis, and myocardial ischemia/reperfusion (I/R) injury, are among the major threats to human health worldwide. Post-translational modifications alter the function of proteins through dynamic chemical modification after synthesis. This mechanism not only plays an important role in maintaining homeostasis and plays a crucial role in maintaining normal cardiovascular function, but is also closely related to the pathological state of various diseases. Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression, and play important roles in post-translational modification by catalyzing the deacetylation of key lysine residues in nucleosomal histones, which are closely associated with the occurrence and development of cardiovascular diseases. Recent studies indicate that HDAC inhibitors (HDACis) may represent a new class of drugs for the treatment of cardiovascular diseases by influencing post-translational modifications. In this review, we systematically summarize the mechanism of action of HDACs and HDACis in post-translational modifications related to common cardiovascular diseases, providing new ideas for the treatment of CVD, and explore possible future research directions on the relationship between HDAC and HDACi in post-translational modifications and cardiovascular diseases.

1. Introduction

1.1. Cardiovascular Disease

According to the World Health Organization, cardiovascular diseases remain the leading cause of death and impose a tremendous economic burden on society. Studies predict that there will be a 90.0% increase in cardiovascular prevalence and 73.4% increase in the crude death rate between 2025 and 2050, with the number of cardiovascular deaths projected to reach 20.5 million in 2025. Ischemic heart disease will remain the leading cause of cardiovascular death, while high systolic blood pressure will be the major vascular risk factor for death [1]. In addition, as the global population ages [2], the United Nations predicts that by 2050, nearly one in six people will be over 65, while cardiovascular disease places a massive burden on elderly patients [3] and significantly affects their quality of life [4]. Cardiovascular disease involves the heart and blood vessels, including diseases such as atherosclerosis, myocardial remodeling, hypertension, and myocardial ischemia/reperfusion (I/R) injury. Several factors can influence the development and progression of cardiovascular disease, including age, gender, obesity, smoking, alcohol consumption, hypertension, and various psychosocial factors, among others. In recent years, epigenetic modifications have been shown to be involved in the pathophysiological processes of cardiovascular disease [5], including various post-translational modifications. Therefore, studying the relationship between cardiovascular diseases and post-translational modifications of proteins may yield valuable novel insights and therapeutic targets.

1.2. Post-Translational Modifications of Proteins

Post-translational modifications (PTMs) are covalent modifications of amino acid side chains in translated proteins, which are catalyzed by various enzymes after the completion of the translation process or during hydrolytic processing and folding [6]. PTM is also essential for epigenetic expression mechanisms, which are related to replication, transcription, organism development, and cell differentiation [7]. PTM enriches the proteome by covalently adding functional groups to one or more amino acid residues of a protein, thereby altering its function and orientation [8,9]. PTM plays a key role in several physiological and cellular processes, including cell differentiation [10], protein degradation [11], signaling [12], the regulation of gene expression [13], and protein–protein interactions [14,15]. Under physiological and pathological conditions, it can expand the functional diversity of proteins by regulating protein folding, activity, stability, localization, signal transduction, and binding [16]. The primary forms of PTMs include ubiquitination, phosphorylation, acetylation, methylation, and glycosylation [8], which have been shown to affect various metabolic pathways and are implicated in the development and progression of various diseases [17]. Recently, novel PTMs, such as succinylation and lactylation, have been discovered. As histones were the first discovered substrates to be modified by acetylation [18], the enzymes responsible for adding and removing acetyl groups are commonly referred to as histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. These broad classes of modifying enzymes regulate many different protein modification types, making them attractive targets for drug development.

1.3. Histone Deacetylases (HDACs)

As the structural units of chromatin, nucleosomes are composed of DNA and histones, which are important for DNA packaging in eukaryotic cells [19]. The nucleosome is composed of an octamer consisting of one H3-H4 tetramer and two H2A-H2B dimers, around which DNA is wrapped [19]. HAT-catalyzed lysine acetylation of histone tails protruding from nucleosomes leads to the relaxation of chromatin structure, creating a conducive environment for transcriptional activation. HDACs exert their catalytic role by removing acetyl groups from lysine residues in histone tails. Thus, when HDACs are more active, histones bind to DNA more tightly, making it difficult for the transcriptional machinery to access DNA, leading to an inhibition of gene transcription [20]. To date, 18 mammalian HDACs have been identified and categorized into four groups, as shown in Figure 1: class I HDACs (HDAC1, 2, 3, 8); class II HDACs (HDAC4, 5, 6, 7, 9, 10); class III (sirtuin family: sirt1-7); and class IV with a single member (HDAC11) [21]. Class II HDACs are further divided into two subgroups: Class IIa, which has a large C-terminal domain, and Class IIb, with two deacetylase domains. Class I, II, and IV HDACs are zinc-dependent enzymes. Class I HDACs are present in all cells and are homologous to Rpd3 in yeast, traditionally considered the nuclear histone deacetylases. Class I HDACs are closely associated with several other protein subunits, including Sin3 and N-CoR, with which they mediate histone deacetylation and transcriptional co-inhibition [22]. Class II HDACs are homologous to yeast HDA1, having an extended N-terminal and catalytic structural domain, and they exhibit increased expression in striated muscle and the brain [21]. Class IIa HDACs (4, 5, 7, 9) have the ability to shuttle between the nucleus and cytoplasm and are expressed in the brain, heart, and muscles [23]. Class IIb HDACs (6, 10) are mainly located in the cytoplasm [23], with one study indicating that HDAC6 is expressed in myotome [24]. Class III HDACs are niacinamide adenine dinucleotide (NAD⁺)-dependent enzymes [21] that are involved in many biological processes, including cellular metabolism, stress response, and aging [25]. SIRT stands for “silent mating type information regulator” and was originally identified and named as a gene silencer that controls mating type in yeast [26]. They are further subclassified based on sequence homology and subcellular localization: SIRT1 is mainly located in the nucleus and shuttles into the cytosol under certain conditions [27]. SIRT2 is primarily located in the cytoplasm but is also present in the nucleus during the G2-to-M phase transition of the cell cycle [28]. SIRT3-5 are mainly localized in the mitochondria due to the presence of mitochondrial targeting sequences [29]. SIRT6-7 are nuclear proteins. SIRT6 is localized in chromatin, while SIRT7 is mainly present in nucleoli. Finally, the only class IV HDAC11 has sequence homology with class I and II HDACs. Its functions involve maintaining the protein stability of DNA replicators CDT1 and IL10 [30], as well as regulating transcription when associated with bromodomain protein 2 [31]. HDACs are usually combined with other proteins to form macromolecular structures that act as corepressor complexes and play a role in the silencing of several genes related to processes such as survival, proliferation, cell growth, angiogenesis, and differentiation. HDACs play a crucial role in post-translational modifications associated with cardiovascular diseases, and the discovery of novel HDACi drugs may facilitate their treatment by modulating HDAC functions.

1.4. Histone Deacetylase Inhibitors (HDACis)

HDACis have been highlighted as anticancer drugs with unique effects of inducing growth arrest, differentiation, and apoptosis [32,33]. HDACis are a class of natural and synthetic compounds that promote histone acetylation and chromatin remodeling, facilitating proper nucleosome positioning and reverting gene expression [34]. In addition to histones, small-molecule HDACis have also been found to regulate the acetylation levels of non-histone proteins, which is potentially valuable for disease treatment [35]. Most zinc-dependent HDACs consist of three components: a zinc-binding group (ZBG) located in the active site of the HDAC, a recognition structure (the Cap structure) that interacts with the entry residues in the active site, and a linker structure that binds these two components [36]. Class III HDACs require NAD⁺ for intrinsic activity, whereas class I, II, and IV HDACs are zinc-dependent. Therefore, most class I and II HDACis target zinc ions. HDACis can be classified into four categories based on their chemical structure: short-chain fatty acids (such as sodium butyrate and valproic acid), cyclic peptides (such as Apicidin and Romidepsin), hydroxamic acids (such as Trichostatin A, Vorinostat, Belinostat, and Panobinostat), ad benzamides (such as Entinostat and Mocetinostat), as shown in

Table 1. Four classes of HDAC inhibitors, including their names, chemical structures, and action targets.

Classification	HDAC Inhibitor	Chemical Structure	The Target
Short-chain fatty acid	Sodium Butyrate (NaB)		Pan inhibitor
	Valproic acid (VPA)		Pan inhibitor
Cyclic peptide	Apicidin		Pan inhibitor
	Romidepsin		HDAC1/2/4/6
Hydroxamate	Trichostatin A (TSA)		Class I/II
	Vorinostat (SAHA)		Pan inhibitor
	Belinostat		Pan inhibitor
	Panobinostat		Pan inhibitor
Benzamide	Entinostat		ClassI
	Mocetinostat		Pan inhibitor

. In recent years, many novel inhibitors targeting various metal-binding regions with monosulfonamide and thioacetyl groups have also been discovered. Hydroxamic acids are the most studied class of drugs with the highest molecular weight and short half-life [37], which can inhibit Class I and Class II HDACs with nM potency. Cyclic peptides are the most complex class of HDACis, including Apicidin, Romidepsin, and a group of peptides containing cyclic hydroxamic acids, which are generally considered to be Class I HDACis. Benzamide is an orally bioavailable drug that effectively and selectively inhibits class I and IV HDAC enzymes [38]. Short-chain fatty acids are relatively modest HDACis with some limited class I HDAC selectivity. Recent studies have proved that the active ingredients of Chinese herbs, such as ginsenosides, cinnamic acid [39], etc., also have HDAC inhibitory effects multiple studies have shown that HDACis are beneficial for cardiovascular disease, so it is important to develop a greater diversity of HDACis.

In this review, we systematically analyze the effects between the four classes of HDACs in post-translational modifications and common cardiovascular diseases. Additionally, we explore the regulatory mechanisms of HDACis in pathophysiologically relevant post-translational modifications, offering novel insights for future therapeutic strategies targeting cardiovascular diseases.

2. HDACs and Post-Translational Modifications Are Closely Associated with Cardiovascular Diseases

2.1. Cardiac Hypertrophy

Cardiac hypertrophy occurs when the heart adapts to continuously elevated blood pressure or continuously increased blood volume by increasing the heart muscle mass [40]. Based on its functional significance, cardiac hypertrophy can be categorized into physiological and pathological types, which are closely related to the nature, duration, and magnitude of the increased cardiac workload [41]. While physiological cardiac hypertrophy is associated with normal cardiac physiological processes, its pathological equivalent ultimately leads to heart failure and is characterized by an absolute increase in ventricular mass in response to different stressors [41].

Class I HDACs are often thought to play a role in promoting cardiac hypertrophy. There is evidence that HDAC1 and HDAC2 contribute to inhibit cardioprotection and anti-hypertrophy inhibition [36]. Calmodulin-dependent protein kinase II (CaMKII) is a direct downstream target of β-adrenergic [42] as well as Gαq signaling [43] (endothelin and angiotensin II), and mediates cardiomyocyte death [44], cardiac hypertrophy [45], as well as fibrosis [46] driven by neurohormone overactivity. Zhang et al. demonstrated that CaMKII can directly phosphorylate HDAC1, 2, and 3, to enhance their deacetylase activity in vitro, which may be related to their role in severe cardiac hypertrophy. HDAC2 is regulated by a variety of post-translational modifications, including serine phosphorylation, lysine ubiquitination, as well as tyrosine and cysteine nitration. Hypertrophy stimulators induce physical interaction between heat shock protein 70 (HSP70) and HDAC2, thereby selectively targeting HDAC2 in the heart [47]. In addition, when myocardial cells are infected with acetylated mimic mutants of HDAC2, the antihypertrophic effects of HDAC5 and leptomycin B-induced nuclear retention or overexpression of HDAC5 are significantly weakened. Hypertrophy stimulation promotes the translocation of casein kinase 2 (CK2) into the nucleus, resulting in the phosphorylation of HDAC2 on serines 394, 411, 422, 424, and S394 is the key site, which ultimately promotes the growth of cardiomyocytes [48]. In addition, in cardiac-specific HDAC3 knockout mice, HDAC3 cooperates with SMRT/n-CoR to a reduce histone acetylation in the vicinity of myocyte enhancer factor 2 (MEF2) [49], resulting in abnormal energy metabolism and cardiac hypertrophy [50]. Myofibril growth during cardiac hypertrophy is regulated by phosphorylation and acetylation of the actin-capping protein CapZβ1, and phenylephrine (PE) treatment of cardiomyocytes reduces HDAC3 binding to myofibrils, which leads to the phosphorylation of CapZβ1 at the serine 204 site and acetylation of CapZβ1 at the lysine 199 site [51]. The overexpression of HDAC8 can stimulate cardiac hypertrophy, promote the phosphorylation of p38 MAPK as well as the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) proteins, and inhibit myocardial hypertrophy by blocking HDAC8 activity [52].

Class IIa HDACs are expressed in the heart. They can directly bind to other hypertrophy-promoting transcription factors to inhibit their target genes, including GATA binding protein 4 (GATA4) [53], MADS-box family member serum response factor (SRF) [54], MEF2 [55], and nuclear factor of activated T-cells (NFAT) [56]. HDAC IIa is generally regarded as a cardiac anti-hypertrophic molecule whose function depends on binding to MEF2 and subsequent inhibition [57]. MEF proteins are a family of myocyte-specific enhancers that are responsible for transcriptional regulation during cardiomyocyte development [58]. Under pathological conditions such as continuous β-adrenergic receptor stimulation, angiotensin II (Ang II) infusion, or stress overload, MEF can promote the transcriptional regulation of cardiac hypertrophy [59]. HDAC IIa is usually combined with MEF2C. When cardiomyocytes are stimulated by pathological stress, HDAC is transported out of the nucleus, where MEF2C recruits p300 to chromatin in the absence of HDAC II, thereby enhancing the transcription of hypertrophy-related genes. The HDAC IIa-MEF2 pathway plays an important role by inhibiting fetal gene transcription and preventing cardiac hypertrophy. The activity of the HDAC II-MEF2C complex is regulated by HDAC II phosphorylation. In addition, HDAC4 forms an inhibitory complex with SUV39H1 to maintain MEF2 in the H3 methylation state, thereby reducing the expression of ANP and BNP, resulting in cardiac protection [60]. Since SUV39H1 is a nuclear histone methyltransferase, this interaction reveals the crosstalk mechanism between histone deacetylation and histone methylation [61]. As a response to adverse stimuli, CaMKIIδB increases the phosphorylation of HDAC4 and its translocation from the nucleus to the cytoplasm, leading to a decrease in HDAC4 levels in the nucleus. This change causes the HDAC4/SUV39H1 complex to dissociate, triggering H3K9 demethylation, Mef2 transcriptional activation, and ultimately cardiac hypertrophy [61]. The N-terminal region of Class II HDACs contains two conserved CaMK phosphorylation sites [62]. Phosphorylation of Class II HDACs by CaMK and other kinases can break the close interaction with MEF2, decreasing transcriptional activity. HDAC5 and HDAC9 have similar functions as endogenous inhibitors of cardiac hypertrophy in vivo [55,63]. The mechanism by which their dysfunction promotes hypertrophy involves the phosphorylation of two conserved serine residues on either side of the nuclear localization signal (S259 and S498 in HDAC5 and S218 and S448 in HDAC9). Phosphorylation mutants of HDAC5 at serines 259 and 498 exhibit resistance to PKC-induced signaling and attenuated cardiac hypertrophy [64]. Phosphorylation promotes binding to the 14-3-3 protein, which shields the nuclear localization sequence and induces a conformational change that exposes the C-terminal nuclear output sequence for nuclear export via the chromosomal region maintenance 1 (CRM1) protein [65,66].

The mechanism of action of HDAC IIb has been discovered in recent years. Studies have shown that knockout of the HDAC5 and HDAC6 genes can block the COX2/PGE2 pathway in response to Ang II-induced cardiac hypertrophy. In addition, the HDAC inhibitor sodium butyrate (NaB) can effectively inhibit the expression of COX2/PGE2. Ang II increases the levels of ANP and phosphorylated ERK (pERK), whereas NaB reverses this effect both in vivo and in vitro [67]. In summary, Ang II can induce cardiac hypertrophy by triggering an HDAC5/HDAC6-dependent mechanism that can be reversed by NaB. However, until now, no specific studies have investigated the role of HDAC10 in cardiac hypertrophy.

The SIRT protein family plays an important role in maintaining cardiac homeostasis. Studies have shown that oxidative stress is a key metabolic characteristic of aging, whereby excessive ROS can trigger DNA damage and cell-cycle arrest [68], while SIRTs have cardioprotective functions that can effectively prevent oxidative damage and the development of age-related lesions [69]. During cardiac hypertrophy, the expression levels of SIRT1, SIRT3, and PGC-1α (peroxisome proliferator-activated receptor γ-coactivator 1α) are significantly reduced [70,71]. SIRT1 and SIRT3 protect cardiomyocytes by enhancing the deacetylation of PGC-1α, reducing oxidative stress, and inhibiting the occurrence of cardiac hypertrophy [72,73]. It has been shown that knockdown of SIRT1 increases the level of crotonylation of SERCA2a to decrease its activity, which in turn affects the expression of proteins in the PPAR pathway, leading to changes in energy metabolism and alterations in physiological states such as cardiac hypertrophy [74]. The knockdown of SIRT5 resulted in increased lysine succinylation of proteins in the heart, which exhibited defective fatty acid metabolism, reduced ATP production, and hypertrophic cardiomyopathy [75]. The knockout of SIRT3 induces the acetylation of long-chain acyl-CoA dehydrogenase at lysine 42 and blocks fatty acid oxidation, thereby downregulating ATP synthesis and other processes [76]. SIRT3 can also reduce the accumulation of hypertrophy-associated lipids in the heart through deacetylation of LCAD, thereby slowing the development of cardiac hypertrophy [77]. Reactive oxygen species (ROS) and oligomycin sensitivity-conferring proteins (OSCP) are associated with the acetylation of SIRT3 substrates, and both are key regulators of cardiac hypertrophy [78]. SIRT3 increases NADPH levels through the deacetylation of IDH2, thereby increasing GSH and decreasing ROS levels [79]. These results suggest that SIRT3 is an endogenous negative regulator of cardiac hypertrophy that protects the heart by reducing cardiomyocyte ROS levels. SIRT3 can deacetylate and thereby activate Mn-superoxide dismutase (MnSOD), which catalyzes the disproportionation of superoxide radicals. However, the overexpression of SIRT4 can inhibit the interaction of MnSOD with SIRT3, resulting in an increase in the acetylation level of MnSOD and a decrease in its activity, and ultimately promote angiotensin II-induced hypertrophic growth in mice [80]. In addition, SIRT2 [81] and SIRT6 [82] have also been shown to prevent cardiac hypertrophy, during which the IGF-AKT signaling pathway is continuously activated. It was found that SIRT6 acts as an endogenous negative regulator of this process in cardiomyocytes. SIRT6 deficiency leads to an elevated H3K9 acetylation level and enhanced interaction with the stress-responsive transcription factor c-Jun, further promoting IGF signaling and ultimately leading to cardiac hypertrophy [83]. It also protects the myocardium from hypertrophy by reducing the protein level of p300 and subsequently decreasing the acetylation and transcriptional activity of the NF-κB p65 subunit [84]. SIRT7 has been shown to play a role in cardiac hypertrophy and the associated reduced lifespan. According to Vakruhsheva et al., SIRT7 deacetylates p53, inactivating it and thus preventing apoptosis [85]. Nicotinamide mononucleotide adenylyltransferase is not only a key enzyme in the biosynthesis of NAD⁺, but is also closely related to the activation of SIRTs and has been shown to inhibit angiotensin II-induced cardiac hypertrophy [86]. Furthermore, studies have shown that in muscle cells, the activation of AMP-activated protein kinase (AMPK) increases intracellular NAD⁺ levels, enhances SIRT1-mediated protein deacetylation, and activates downstream targets such as PGC-1α and forkhead box protein O1 (FOXO1). At the same time, the AMPK/SNF1 pathway triggers histone acetylation by phosphorylating them and promoting the assembly of the HAT complex, which in turn enhances the transcriptional activity of specific genes [87]. In this context, the activation of SIRTs is closely related to the AMPK/SNF1 pathway, whereas AMPK inhibits the activation of HDAC IIa. Specifically, stronger AMPK activation leads to higher activation levels of SIRTs and more HDAC IIa being transferred to the cytoplasm, resulting in an inhibitory effect on cardiac hypertrophy.

Overall, HDACs and various associated post-translational modifications play important roles in cardiac hypertrophy, with phosphorylation being the most extensively studied. Post-translational modifications may regulate nuclear-cytoplasmic shuttling, such as the phosphorylation of HDAC-IIa by CaMKII under pathological stimulation, which releases the inhibition of MEF2 and allows the transcription of hypertrophic genes. In summary, it was found that various types of HDACs play different roles. Class IIa HDACs and Class III HDACs play a protective role, whereas some of Class I can promote cardiac hypertrophy, as shown in Figure 2. However, there are still many mechanisms that have not been elucidated and will require further research in the future.

2.2. Heart Failure

Heart failure (HF) is a disease caused by a blood-pumping disorder in the heart, resulting in systolic or diastolic dysfunction, followed by reduced circulation and insufficient arterial perfusion [88]. HF is the most common cause of death due to CVD, affecting an estimated 64.3 million people worldwide [89]. This is a very complex disease in which various molecules and cells undergo various changes during its onset and following chronic disease process [90]. These changes not only affect the heart’s structure but also impair its contractile function. Heart failure is characterized by various changes in the body, including alterations of cardiomyocyte apoptosis, the development of cardiac fibrosis, and altered gene expression. However, the underlying mechanisms are still not fully understood.

HDACs downregulate gene expression in heart failure by acting as transcriptional repressors. Methyltransferase-like protein 7B (METTL7B), a member of the methyltransferase-like family containing the methyltransferase structural domain, inhibits the expression of USP38 through m6A-dependent mRNA degradation, leading to increased ubiquitination of HDAC3, which maintains histone lactylation at a later stage to improve cardiac remodeling and thus prevent heart failure [91]. HDAC3 deacetylates DNA methyltransferase 1 (DNMT1) to inhibit ubiquitination-mediated degradation, which promotes the expression of DNMT1, which inhibits the expression of SHP-1 by methylation of the promoter region, thereby leading to heart failure induced by cardiomyocyte hypertrophy [92].

Class IIa histone deacetylases inhibit cardiomyocyte hypertrophy by binding to the hypertrophic transcription factor (TF) myocyte enhancer factor-2 (MEF-2). Class IIa HDAC4, 5, 7, and 9 are affected by signal-dependent phosphorylation of Ca²⁺/calmodulin-dependent protein kinase (CaMK) family members. The phosphorylation of HDAC7 by salt-inducible kinase 1 (SIK1), a member of the CaMK family, stabilizes the deacetylase, leading to an increase in c-Myc expression, which in turn promotes the progression of heart failure [93]. However, there is little research on class IIb HDACs in relationship to post-translational modifications. Previous studies have shown that HDAC6 is involved in cardiac hypertrophy and may also be involved in heart failure. HDAC10 interacts with thioredoxin to regulate the alteration of ROS signaling in cancer cells, so there may be many hidden mechanisms that we need to explore.

SIRTs play a beneficial role during the development of heart failure. SIRT1 plays an important role in the process of improving heart failure. For example, SIRT1 reduces ferroptosis and ameliorates heart failure by decreasing the K382 acetylation level of p53 and degradation of SLC7A11, while increasing GSH abundance and glutathione peroxidase 4 (GPX4) expression [94]. SIRT1 also increases the deacetylation of FOXO3a, which in turn prevents apoptosis and ameliorates heart failure [95]. SIRT1 was found to improve cardiac function, reduce ventricular mass, and decrease apoptosis in cardiomyocytes by inhibiting the acetylation of NF-κB p65, leading to a decrease in the expression of NF-κB p65, which ameliorates heart failure [96]. SIRT1 significantly protected cardiomyocytes by decreasing miR-138-5p by increasing the deacetylation level of p53 [97]. In clinical practice, low expression of SIRT1 may upregulate antioxidants and pro-apoptotic molecules by increasing p53 acetylation and decreasing FOXO1 translocation into the nucleus [98]. Gorski et al. reported that SIRT1 controls cardiac SERCA2a function by acetylating cardiac SERCA2a at K492, which is critical for the pharmacological activation of SIRT1-mediated deacetylation and normalization of SERCA2a activity [99]. SIRT3 activation and mitochondrial metabolic processes are mediated by acetylation and SUMO crosstalk, but more crosstalk still needs further exploration [100]. Transforming growth factor β-1 (TGF-β1) mediates the transformation of fibroblasts into myofibroblasts, regulates tissue fibrosis, and increases the synthesis of extracellular matrix components. The loss of SIRT3 leads to the expression of TGF-β1 and hyperacetylation of glycogen synthase kinase 3β (GSK3β) at Lys15, while the deacetylation of SIRT3 simultaneously activates GSK3β, thereby reducing the fibrosis of cardiac tissue [101]. SIRT3 also regulates ferroptosis and cardiac fibrosis by regulating the acetylation of p53 [102]. SIRT3 deficiency leads to mitochondrial protein hyperacetylation and mitochondrial dysfunction. For example, downregulated SIRT3 expression in the myocardium of HF patients with obesity and metabolic syndrome leads to cyclophilin D (CypD) hyperacetylation, mitochondrial permeability transition pore (mPTP) opening, and cardiac insufficiency [103]. Therefore, SIRT3 is a key regulator of cardiac energy metabolism and a potential mitochondrial target for targeted drugs. In addition, SIRT5 can reduce isocitrate dehydrogenase 2 (IDH2) succinylation levels, protect cell viability, maintain mitochondrial homeostasis, and ameliorate myocardial fibrosis, thereby reducing the incidence of heart failure [104].

HDAC11 has been identified as a negative regulator of the well-known anti-inflammatory cytokine IL-10 [105]. This cytokine is associated with active inflammation in the early stages of heart failure through association with DNA methylation, which downregulates IL-10 [106]. Further research is needed to explore the relationship between HDAC11 and HF.

The study of HDACs in heart failure is currently limited, especially in the context of various post-translational modifications, as shown in Figure 3. Various histone deacetylases (HDACs) regulated by post-translational modifications (PTMs) perform distinct functions by targeting different substrates. For instance, SIRT1 confers cardioprotection through the deacetylation of p53 within the nucleus, whereas HDAC3 facilitates heart failure by deacetylating DNMT1 in the nucleus. This area needs to be explored in depth in the future, and related studies are expected to reveal potential new directions for the treatment of heart failure.

2.3. Atherosclerosis

Atherosclerosis (AS) is characterized by the yellowish color of the lipids accumulated in the arterial lining and is one of the most common types of cardiometabolic disease [107]. Atherosclerosis plays a key role in the pathogenesis and progression of multiple cardiovascular diseases, including coronary and peripheral artery disease [108]. Its main feature is the thickening of arterial walls due to the formation of plaques under the endoderm, composed of fatty acids, cholesterol, calcium, fibrin, cell debris, and metabolic waste. These lesions can lead to varying degrees of narrowing of the arteries or even complete blockage of blood flow, leading to a lack of oxygen to the heart, brain, kidneys, pelvis, arms, or lower limbs [109]. As the plaque grows, it may become less stable and rupture, leading to the formation of a localized blood clot that can further block downstream veins or arteries. This condition is commonly referred to as thrombosis [110]. In severe cases, this can further lead to life-threatening complications such as myocardial infarction (MI) and stroke.

HDAC1-3 play different roles in various regulatory processes related to atherosclerosis. Increased expression of the vascular cell adhesion molecule VCAM-1 on the surface of activated arterial endothelial cells (EC) is closely related to the occurrence of atherosclerosis. Hu et al. demonstrated that HDAC1 and HDAC2 can further reduce the methylation of the GATA6 promoter by inhibiting the acetylation of STAT3 at lys685, thereby regulating the expression of VCAM-1 in endothelial cells and promoting the formation of atherosclerotic lesions [111]. Endogenously expressed microRNAs (miRNAs), small non-coding RNAs that regulate gene expression at the post-transcriptional level [112], have been demonstrated to play a pivotal role in diverse biological and cellular processes [113]. For example, miR-21, miR-23a, and miR-24 have been implicated in the regulation of the development of cardiac hypertrophy [114]. Recent studies have shown that miRNAs are significantly dysregulated in the pathological processes of atherosclerosis and cardiovascular diseases, involving multiple aspects, such as endothelial cell activity, macrophage function, and vascular smooth muscle cell behavior [115,116,117]. Wang et al. found that the overexpression of HDAC1 enhanced miR-224-3p-mediated FOSL2 inhibition and inhibited the progression of atherosclerosis by deacetylating HIF-1α [118]. Zhao et al. further explored how hyperhomocysteinemia (HHcy) promotes atherogenesis by altering histone acetylation patterns and regulating miRNA expression. Studies have shown that Hcy-induced atherosclerosis is mediated by the increased expression of HDAC1, and the upregulation of HDAC1 reduces the level of acetylation of histone H3 at the lys9 (H3K9ac) site, thereby inhibiting the expression of miR-34a. This in turn contributes to the accumulation of total cholesterol (TC), free cholesterol, and triglycerides, accelerating the process of atherosclerosis [119]. Matrix metalloproteinase-9 (MMP-9) is a zinc-dependent endopeptidase capable of degrading extracellular matrix components, and its increased expression is closely related to the occurrence of atherosclerosis. Interferon-β (IFN-β) can somewhat inhibit the progression of atherosclerosis by downregulating the mRNA expression of MMP-9. In the specific mechanism, IFN-β reduces the acetylation level of histone H3 by increasing the recruitment of HDAC-1 to the MMP-9 promoter region, thus inhibiting its transcriptional activity. The results also indicated that the proximal AP-1 site plays a key role in this inhibitory process. When the AP-1 site is inactivated by a point mutation, the IFN-β-mediated transcriptional inhibition effect disappears, thereby promoting the further development of atherosclerosis [120]. Macrophage-triggered chronic inflammation and smooth muscle cell-induced vascular remodeling are two main pathophysiological processes in the formation of atherosclerosis. The main histocompatibility class II (MHC II) transactivator (CIITA) is a transcriptional regulator of MHC II activation and type I collagen inhibition induced by interferon-γ (IFN-γ). CIITA and HDAC2 interact inside smooth muscle cells and macrophages. HDAC2 can reduce the interaction between CIITA and RFX5 by protein degradation and deacetylation of CIITA, thereby counteracting the activity of CIITA and promoting the development of atherosclerosis [121]. In another study, HDAC2 was shown to protect against atherosclerosis. In male mice, when HDAC2 is absent, there is an increase in acetylation on histone 3, an increase in GRX1 expression, as well as an association with increased MKP-1 activity and a decrease in monocyte derivation and macrophage recruitment [122]. P21 plays a pivotal role in vascular remodeling. The phosphorylation of HDAC2 facilitates its dissociation from RARα and enhances its interaction with KLF transcription factor 5 (Klf5), resulting in the deacetylation of Klf5. This process subsequently promotes the dissociation of Klf5 from the p21 promoter, thereby diminishing the inhibitory effect on the p21 promoter and ultimately contributing to the progression of atherosclerosis [123]. Vascular smooth muscle cell (VSMC) aging plays a critical role in the development of atherosclerosis. VSMC-specific TRAP1 deficiency alleviates VSMC aging and atherosclerosis via metabolic reprogramming. Conversely, TRAP1 significantly increased aerobic glycolysis, leading to elevated lactate production. Accumulated lactate promoted histone H4 lysine 12 lactylation (H4K12la) by down-regulating the unique histone lysine delactylase HDAC3, promoting senescence-associated secretory phenotype (SASP) expression. These findings may offer a novel therapeutic strategy for atherosclerosis [124].

Among enzymes of the HDAC IIa subtype, HDAC4 is capable of inhibiting the expression of miR-148b-3p and promoting the transcription of NCOR1 by reducing the levels of H3 and H4 acetylation in the promoter region of the miR-148b-3p/NCOR1 gene cluster. This process further promotes the overexpression of KLF7, which has a protective effect on the cells and effectively reduces the incidence of atherosclerotic lesions [125]. Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid compound with anti-inflammatory, metabolic modulatory, and cardiovascular protective properties. BA can upregulate the expression of the transcription factor KLF2, increase the intracellular Ca²⁺ level, activate the CaMKKβ, CaMKIIα, and AMPK signaling pathways, enhance the phosphorylation of ERK5, HDAC5, and MEF2C, and ultimately induce the expression of eNOS, thereby exerting a preventive effect against atherosclerosis [126]. By contrast, among the HDAC IIb isoforms, lncRNA NORAD enhances the deacetylation of H3K9 by recruiting HDAC6 to the promoter region of the VEGF gene, thereby inhibiting its transcription, leading to increased vascular endothelial cell injury and promoting the development of atherosclerosis [127].

SIRT1 plays a protective role in the development and progression of atherosclerosis [128]. Senescence leads to atherosclerosis, and LA ribonucleoprotein 7 (LARP7) is a senescence antagonist. When DNA damage-mediated activation of the ataxia telangiectasia mutated (ATM) gene triggers extracellular shuttling and the downregulation of LARP7, it inhibits the activity of SIRT1 deacetylase. This enhances the acetylation of p53 and NF-κB (p65) to increase their transcriptional activity, accelerating cellular aging and atherosclerotic progression [129]. S-adenosyl homocysteine (SAH) is a risk factor for cardiovascular disease. The inhibition of SAH hydrolase (SAHH) leads to the accumulation of SAH and the inhibition of DNMT3b (DNA methyltransferase 3b), which results in hypomethylation of the H19 promoter and reduced intracellular adenosine levels, combined with reduced activation of AMPK (AMP-activated protein kinase), which in turn inhibits SIRT1 mediated-hyperacetylation of histone H3 to promote atherosclerosis [130]. Long non-coding RNA (lincRNA) p21 is involved in the development of atherosclerosis, and competitively binds to miR-221 to promote the deacetylation of Pcsk9 by SIRT1, thereby reducing the progression of atherosclerosis [131]. It has been shown that senescent endothelial cells have low levels of nitric oxide synthase (eNO), while nitric oxide (NO) has vasodilator, antioxidant, and atheroprotective effects [132]. Mattagajasingh et al. showed that SIRT1 activates eNO during caloric restriction by increasing endothelial nitric oxide synthase levels through deacetylation. By contrast, decreased levels of SIRT1 lead to increased acetylation of eNO on lysine residues 496 and 506, inhibiting its activity [133]. Amp-activated protein kinase (AMPK)-mediated phosphorylation, SIRT-1-mediated coronin deacetylation, and SIRT-1-mediated eNOS deacetylation have atheroprotective effects in human umbilical vein endothelial cells (HUVECs) in vitro. At the same time, eNOS deacetylation of HDAC3 at K610 promotes atherosclerosis [133,134,135]. Jiang et al. showed that SIRT2 stabilizes the gluconeogenesis process during fasting through the deacetylation of phosphoenolpyruvate carboxykinase (PEPCK1), which inhibits its ubiquitination and degradation, thereby activating and maintaining its function in gluconeogenesis [136]. N ε-carboxymethyl lysine (CML), an active component of advanced glycation end-products (AGEs), significantly enhances the activity of nuclear factor of activated T-cells 1 (NFATc1) by mediating crosstalk between acetylation and phosphorylation. This process leads to the downregulation of SIRT3, increased acetylation of NFATc1 at lysine 549 (K549), and antagonizes the phosphorylation of NFATc1 at tyrosine 270 (Y270) by focal adhesion kinase (FAK). Consequently, these mechanisms promote the progression of atherosclerosis [137]. ROS play a key role in atherosclerosis, and SIRT3 can reduce oxidative stress by reducing mitochondrial ROS production through epigenetic regulation. SIRT3 deacetylates forkhead transcription factor O subfamily member 3a (FOXO3a) and protects mitochondria from oxidative stress, further exercising protective antioxidant properties [138]. SIRT6 is an atheroprotective factor that mediates the deubiquitination of the K37 and K532 sites of HIF-1α [139]. SIRT6 is the target of miR-92a-3p, which activates the MAPK signaling pathway in vitro by negatively regulating SIRT6 as well as increasing the apoptosis and phosphorylation levels of JNK and p38 MAPK, which in turn exerts an anti-atherosclerotic effect [140]. Oxidized low-density lipoprotein (oxLDL) is a known risk factor for atherosclerosis, and cardiac myosin-related transcription factor A (MRTF-A) induces ICAM-1 transcription in response to oxLDL. Furthermore, SIRT6 interacts with MRTF-A to regulate the acetylation of MRTF-A, and promotes the deacetylation of MRTF-A to phosphorylate serine 154, mediating the inhibition of SIRT6 [141]. In addition, SIRT6 can also interact with apoptosis-associated speck-like protein (ASC) to inhibit its acetylation, thereby reducing the interaction between ASC and NLRP3 and inhibiting the apoptosis of endothelial cells, thereby slowing the development of atherosclerosis [142]. Kawahara et al. showed that SIRT6 alters NF-κB expression through the deacetylation of histone H3K9, thereby preventing the binding of the NF-κB RELA (REL-associated protein) subunit to its target gene promoter, reducing inflammation [143]. The combination of inflammation, cellular aging, and high LDL cholesterol constitutes a vicious cycle of atherosclerosis, and SIRT6 may stop this cycle. SIRT7-mediated succinylation of protein arginine methyltransferase 5 (PRMT5) enhances its activity and induces arginine methylation of SREBP1a, which promotes the biogenesis of fatty acids, TAGs, and cholesterol, which is strongly associated with atherosclerosis [144]. This is because HDACs are involved in regulating a wide range of biological processes, such as endothelial cells, smooth muscle cells, cholesterol metabolism, and inflammation. Arteriosclerosis can be better combated by developing HDACis that target the catalytic domain of HDAC [145].

Consistent with the roles of various classes of HDACs in cardiac hypertrophy, each class of HDACs has its own role, with the most studied being acetylation and phosphorylation. Under acetylation, the same HDAC has different functions in different pathways. HDAC1 can enhance the expression of miR-224-3p and inhibit atherosclerosis, but it can also inhibit the expression of miR-34a and promote the development of atherosclerosis. The roles of various HDACs in atherosclerosis have received much attention, with HDAC1 and SIRT1 being the most studied, one promoting and the other inhibiting atherosclerosis, as shown in Figure 4. Various other HDACs may also have promoting or inhibiting effects on atherosclerosis, and the relevant mechanisms need to be further explored in the future.

2.4. Myocardial Ischemia/Reperfusion (I/R) Injury

I/R is a pathological process associated with a variety of diseases that can lead to cell death and organ damage. Ischemia arises when blood supply is restricted, leading to severe tissue hypoxia. Although restoring blood flow to ischemic tissues aims to alleviate ischemia, it may paradoxically induce further damage, a condition referred to as I/R injury. Myocardial I/R damage is widespread in ischemic heart disease, the leading cause of which is atherosclerosis [146]. When an atherosclerotic plaque becomes unstable and ruptures or erodes, it can trigger thrombosis, which may either cause the occlusion of the coronary artery lumen at the site of the plaque, or lead to distal coronary artery embolism [147]. In cases of acute myocardial infarction, early and successful revascularization can effectively prevent the loss of contractile myocardial mass, minimize infarct size, and enhance the prognosis [148]. Nevertheless, reperfusion may paradoxically result in increased and accelerated myocardial damage, a phenomenon referred to as myocardial I/R injury.

Class I HDACs play a protective role in I/R. Ischemic postprocessing (IPostC) has been proposed to reduce the risk of I/R injury. Autophagy is involved in I/R-induced myocardial injury in the elderly. By decreasing the binding of DNMT3b and HDAC2 to its promoter, DNA hypomethylation and H3K14 hyperacetylation of the miR-181a-2-3p promoter can be induced. This subsequently inhibits the binding of c-Myc to the miR-181a-2-3p promoter, ultimately alleviating I/R-induced myocardial injury [149]. GSK-3 is a serine/threonine kinase initially characterized as an enzyme capable of phosphorylating and downregulating glycogen synthase, which serves as the rate-limiting enzyme of glycogen metabolism. In addition, GSK-3β is also involved in I/R injury. HDAC3 regulates Ser9 phosphorylation of GSK-3β and thereby protects cells. The disruption of HDAC4 sumoylation resulted in HDAC4 accumulation in cardiomyocytes and impairment of HDAC4 ubiquitination, leading to a significant reduction in ROS levels. Therefore, SUMOylation of HDAC4 may be a prospective target for I/R therapy. HDAC6 levels are positively correlated with ROS production, while it protects against I/R injury by modulating the acetylation level of the antioxidant protein peroxiredoxin-1 (Prdx1) at the K197 site [150].

SIRT family members also play a protective role in myocardial tissue. SIRT1 enhances the transcriptional activity of forkhead protein O1 (FOXO1) by decreasing its acetylation levels, thereby upregulating the expression of ferritin heavy chain 1 (Fth1). As an inhibitor of ferroptosis, Fth1 suppresses the iron-dependent cell death of cardiomyocytes, safeguarding cardiac function against I/R injury [151]. The upregulation of SIRT1 and SIRT3 protein levels was found to result in decreased acetylation of p53 and PGC-1α, reduced phosphorylation of FOXO-1, elevated Bcl-2 levels and activity, as well as increased MnSOD levels. These changes collectively inhibit cardiomyocyte apoptosis and protect against I/R injury [152]. SIRT1 can regulate Nrf2 deacetylation, thereby upregulating the downstream signaling pathways of Nrf2 and enhancing its activity to protect cells. SIRT1 can regulate Nrf2 deacetylation, thereby upregulating the downstream signaling pathways of Nrf2 and enhancing its activity to protect cells [153]. In addition, SIRT1 prevents the phosphorylation of Akt and mitigates the reduction of Drp1 activation caused by anoxia/reoxygenation (A/R) injury, thereby inhibiting ischemia/reperfusion (I/R) damage [154]. By inhibiting hypoxia/reoxygenation (H/R) injury and modulating the phosphorylation levels of PI3K, AKT, and mTOR, it exerts protective effects against H/R-induced cardiomyocyte apoptosis and excessive autophagy [155]. SIRT1 deacetylates the p65 subunit of NF-κB at the lysine 310 site and blocks its transcriptional activity, thereby inhibiting oxidative stress and inflammatory responses. Isocitrate dehydrogenase 2 (IDH2) inhibits caspase-3 activation and alleviates mitochondrial damage via a SIRT3-dependent mechanism. In the IDH2 K413R mutant, this leads to enhanced enzymatic activity, decreased production of mitochondrial ROS, and mitigation of I/R injury [156]. Deficiencies in SIRT1 and SIRT3 result in the inactivation of AMPK and significant alterations of mitochondrial oxidative phosphorylation (OXPHOS), thereby impairing mitochondrial respiration under I/R stress conditions [157]. SIRT3 can inhibit reperfusion injury through the deacetylation of procyclin D, which prevents the opening of the mitochondrial permeability transition pore and subsequent cell death [158]. Sirtuin 5 (SIRT5) is a mitochondrial NAD⁺-dependent deacetylase that catalyzes the removal of succinyl groups from proteins. Proteomic analysis showed that the succinylation level of SIRT5 knockout mice was significantly higher than that of wild-type mice. The pretreatment of SIRT5 knockout mouse hearts with competitive succinate dehydrogenase inhibitors inhibited mitochondrial succinylation and mitigated I/R injury. This indicates that changes in succinate dehydrogenase activity significantly influence I/R damage, and succinylation may be a critical factor in the pathophysiology of I/R injury [159,160]. The loss of SIRT5 activity alters cardiac metabolic pathways, increases the succinylation of DHSA, promotes SDH activity, and exacerbates ischemia/reperfusion injury [161]. Charged multivesicular body protein 2B (CHMP2B), a subunit of the ESCRT-III complex, accumulates in the heart and impairs autophagic fluxes, while SIRT6 reduces the acetylation of FOXO1, promotes its transcriptional effect on the muscle-specific ubiquitin ligase Atrogin-1, and subsequently enhances the degradation of CHMP2B by Atrogin-1 to reduce I/R injury [162].

Among the related post-translational modifications, the most extensive studies related to I/R injury have been conducted on class III HDACs, as shown in Figure 5. HDACs may play an important role in mitigating myocardial I/R injury by modulating key biological processes, such as oxidative stress and apoptosis. However, the molecular mechanisms behind these processes remain to be explored in depth.

2.5. Other Diseases

It has been found that SIRT3 deacetylase can effectively reduce inflammation and the pro-fibrotic response in the human heart and neonatal rat cardiomyocytes [163]. K27 acetylation of histone H3 on the promoter DNA promotes FOS transcription through the FOS/AP-1 pathway, thereby driving myocardial cell fibrosis and inflammation. However, SIRT3 can inhibit cardiac hypertrophy by participating in the deacetylation of histone H3 [164,165]. Acetylation plays a crucial role in the occurrence and development of atrial fibrillation (AF). Both in vitro and in vivo studies have shown that the expression of SIRT3 is significantly downregulated in AF, accompanied by the abnormal expression of key downstream metabolic factors. AF increases the acetylation levels of long-chain acyl-CoA dehydrogenase, Acetyl-CoA synthetase 2 (AceCS2), and Glutamate dehydrogenase (GDH), attenuating their enzymatic activities. This results in abnormal metabolic changes and a reduction in ATP levels. Therefore, SIRT3 is a critical factor affecting the acetylation status of key metabolic enzymes in atrial muscle. Enhancing SIRT3 expression can reverse the metabolic remodeling of atrial muscle induced by atrial fibrillation [166]. In addition, SIRT7 inhibits apoptosis by deacetylating p53 and enhancing stress resistance of cardiomyocytes in vitro, whereas Sirt7^−/− mice exhibited increased apoptotic signaling and an inflammatory cardiomyopathy phenotype in the heart [85]. At the same time, SIRT1 inhibits mitochondrial division and reduces the angiotensin II-induced apoptosis of cultured cardiomyocytes by deacetylating p53 [167]. Hyperlipidemia promotes the development of vascular injury by mediating oxidative damage through the activation of the adaptor protein p66, which interacts with cytochrome c to promote mitochondrial ROS production [168], proved to be a key regulator in cardiovascular disease [169]. SIRT1 can promote H3 deacetylation to inhibit p66 expression [170] and inhibit p66 function through K81 deacetylation [171], thereby protecting cells. HDACs mediate various post-translational modifications that play a key role in cardiovascular disease. Nevertheless, these findings still need to be validated by clinical evidence. With the development of new technologies in recent years, we can better understand and study the mechanism of HDACs in PTM in CVD. However, more evidence is needed to fully understand the roles of HDACs in CVD, which will provide new ideas and targets for the prevention and treatment of cardiovascular diseases.

3. Regulation of Cardiovascular Disease by HDACis Targeting Post-Translational Protein Acetylation

At present, a total of five HDAC inhibitors have been approved worldwide. Vorinostat, Romidepsin, Belinostat, and Panobinostat have been approved by the US FDA for the clinical treatment of peripheral T-cell lymphoma (Vorinostat, Belinostat), cutaneous T-cell lymphoma (Romidepsin), and multiple myeloma (Panobinostat). In addition, Chidamide (trade name: Epidaza^®) has been approved by China’s NMPA for the treatment of peripheral T-cell lymphoma and breast cancer. Among these, Chidamide stands out as a selective inhibitor of HDAC Class I and the HDAC10 subtype, whereas the other four compounds act as pan-HDAC inhibitors. While inhibiting Class I HDACs and Class IIb HDACs, they also exhibit inhibitory activity against Class IIa HDACs (e.g., HDAC5, HDAC9) and Class IV HDACs (e.g., HDAC11), potentially leading to more toxic side effects mediated by the inhibition of Class IIa and Class IV HDAC enzymes.

Since the studies of post-translational modifications in drug development have mainly focused on the acetylation process, and the other modifications are less studied, the primary focus is on HDACis targeting protein acetylation. HDACs removes the acetyl group from lysine through a hydrolysis reaction. According to different catalytic mechanisms, the 18 types of HDACs can be divided into two categories, one comprising HDAC 1-11, which are zinc-dependent metalloenzymes, and the other comprising the seven sirtuins (SIRT1-7), which use NAD⁺ as a cofactor. The acyl group is transferred to the C2 position of the ribose sugar ring. Although these two enzyme families each perform specific biological functions in the process of hydrolyzing acyl lysine, HDACs are generally considered zinc-dependent enzymes. HDACis are currently one of the most widely studied types of drugs. Numerous studies have shown that HDACis can be used for cancer treatment. In addition to their role in tumor biology, HDACs play an important regulatory role in the pathological processes of multiple organs, including those affecting the nervous and cardiovascular systems [172]. In the cardiovascular field, it has been shown that some HDAC inhibitors, such as Trichostatin A (TSA), Vorinostat (SAHA), and valproic acid (VPA), have specific protective effects against cardiac hypertrophy, heart failure, atherosclerosis, and ischemia/reperfusion injury, as shown in

Table 2. Effects of HDAC inhibitors in acetylation on cardiovascular disease.

Biological Function	Drug	Target Protein	Modification Site	The Acetylation Level	Reference
Inhibition of cardiac hypertrophy	TSA	HDAC6	H3K9	↓	[173]
	VPA	HDACs		↑	[174]
	Fibroblast Growth Factor 21 (FGF21)	SIRT1		↓	[175]
Atherosclerosis	TSA	HDAC6	H3K9		[176]
Protecting the heart from ischemia/reperfusion injury	Entinostat	HDAC1/2/3		↑	[177]
	SAHA	HDAC1/2/3/6/7/11	H3K14	↑	[178]
	VPA	HDAC1/2	H3K4	↓	[179]
	Tubastatin A (TubA)	HDAC6		↓	[180]
	TSA	HDACs		↑	[181]
	Sodium valproate	HDACs		↑	[182]
	Remifentanil	HDAC3		↑	[183]
	TubA	HDAC6	Prdx1	↑	[150]
Slowing myocardial conduction and increasing susceptibility to refractory arrhythmias	Romidepsin	HDAC1/2/4/6		↓	[184]
	SAHA	HDACs		↑	[185]
Reduction in infarct size in rats with myocardial ischemia/reperfusion injury	Panobinostat	HDAC6		↑	[186]
Reduce myocardial infarction	TSA	HDAC6	H3K9		[187]
Apoptosis and cardiac fibrosis	β-hydroxybutyrate (β-OHB)	HDAC2		↑	[188]

. Protein acetylation has been known for over half a century, but its precise detection has only become available in the last two decades due to technical limitations. In addition, due to the technical and ethical issues of measuring acetylation levels in human patients receiving treatment, most studies have been conducted in animal models. Although these drugs have shown significant benefits in preclinical models, their clinical trials are still ongoing. Hence, these drugs require further study before they become viable options for clinical therapeutic interventions. To further improve the effectiveness and safety of HDACis and make up for the lack of specificity at this stage, more innovative strategies must be explored to optimize their design and application.

4. Directions for Future Research

Current studies on the roles of HDACs in the regulation of post-translational modifications mainly focus on acetylation and phosphorylation. Some studies have shown that HDAC1 undergoes SUMOylation at its C-terminus, which plays an important regulatory role in its transcriptional inhibition activity [189]. However, it is unclear whether SUMO directly alters the intrinsic activity of HDAC1 or whether it acts by regulating the formation of corepressor complexes. In addition, the Lys462 site of HDAC2 is also susceptible to SUMOylation, and SUMO-HDAC2 is catalytically active [190]. For example, Lys320 of p53 is targeted by SUMO-HDAC2, and deacetylated p53 cannot effectively regulate apoptosis of DNA-damaged cells [190]. S-nitrosylation is another important PTM of HDAC2, which was first discovered to play a role in neuronal development [191]. Nott et al. demonstrated that S-nitrosylation predominantly occurs at the Cys262 and Cys274 residues, it does not overlap with the phosphorylation site, and is a post-translational modification that directly acts on cysteine residues without relying on other modifications, which modulate HDAC2’s ability to bind to chromatin without altering its enzymatic activity [192]. Colussi et al. further investigated the therapeutic potential of HDAC2 S-nitrosation in a model of Duchenne muscular dystrophy. In this study, the intrinsic activity of HDAC2 was found to be significantly inhibited by S-nitrosylation, while endothelial nitric oxide synthase produced sufficient nitric oxide to inhibit HDAC2 activity. In addition, Malhotra et al. demonstrated HDAC2 S-nitrosylation and its functional relevance, which explains the inability to control chronic inflammation in chronic obstructive pulmonary disease (COPD) patients despite glucocorticoid therapy. S-nitrosylation can directly inhibit the activity of HDAC2, while de-nitrosylation can restore it. In summary, S-nitrosylation is an independent mechanism that regulates the intrinsic activity of HDAC2 and can reshape the transcription profile of its target genes. However, the functional role and biological significance of SUMOylation and S-nitrosylation of HDACs in CVD remain to be further elucidated. Therefore, in addition to studying the roles of HDACs in acetylation and phosphorylation, many other types of post-translational modifications are also relevant for this class of enzymes.

Post-translational modification is an indispensable mechanism of gene regulation that can rapidly respond to various cellular signals. Due to recent advances in high-resolution mass spectrometry and proteomics technologies, post-translational modifications have been extensively studied, revealing complex interactions between various modification types. When multiple PTMs target the same lysine residue, the underlying biological processes can be regulated through crosstalk or synergistic interactions [193]. For example, phosphorylation has been shown to affect protein acetylation [194,195,196]. Acetylation can, in turn, regulate protein phosphorylation, and HDACis targeting acetylation have also been found to affect protein phosphorylation. As an example, Class I HDACs inhibit phosphatase gene expression under stress conditions, whereas treatment with corresponding HDACis induces the expression of dual-specificity phosphatase 5 (dusp5) and further promotes dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) by altering histone acetylation levels. The same study found that Class I HDACis can alleviate stress-induced cardiac hypertrophy through a mechanism that partially relies on phosphatase-mediated intracellular ERK1/2 dephosphorylation [197]. Class IIa HDACs are highly sensitive to cellular signals because there are multiple phosphorylation sites within their important regulatory regions (including the NES and NLS). In addition to phosphorylation, modifying serine and threonine residues with an O-linked β-N-acetylglucosamine (O-GlcNAc) group represents another dynamic and irreversible PTM. Studies have shown that, in addition to targeting the identical residues, phosphorylation and O-GlcNAcylation can also undergo crosstalk through adjacent interactions [198]. Therefore, studying the effect of HDACis on the crosstalk between post-translational modifications, such as acetylation–crotonylation, may provide new ideas for treating cardiovascular diseases.

In recent years, the emergence of single-cell technologies, including genomics, has provided people with more accurate biomedical data and met the needs of precision medicine [199]. The two main technologies involved in single-cell genomics are single-cell whole-genome DNA sequencing and single-cell RNA sequencing (scRNA-seq) [199]. Other single-cell level technologies include single-cell epigenetic sequencing, proteomics, and metabolomics. Each technique has its specific features, involving different views of genetic variation and mutation, DNA methylation, histone modification, transcription, translation, and metabolism, all of which are conducive to CVD research. Using single-cell genomics technology, new therapeutic targets have been identified for various cardiovascular diseases. Previous studies have found that scRNA-seq technology has applications in cardiac hypertrophy [200], HF [201], Atherosclerosis [202], and I/R [203]. However, there are still some technical challenges, such as (1) the isolation of high-quality single cells, which is the most critical step; (2) the performance of clinical biopsy of cardiac samples, which is a difficult problem; (3) the obtaining of sufficient genomic material from single cells, which is critical; and (4) the depth of scRNA-seq transcriptome, which also determines the accuracy of the results. Therefore, single-cell genomics can provide new insights into CVD research, and in-depth research and improvement of this technology can promote new therapies for CVD. MS-based proteomic analyses are broadly grouped into three dominant approaches: bottom-up peptide-centric, top-down protein-centric, and targeted peptide/protein acquisitions. Cardiac proteomics can provide a broader and deeper understanding of the molecular mechanisms underlying cardiovascular diseases and suggest interventions for future treatment. However, nowadays, none of the techniques can be extended to analyze a large number of proteins simultaneously, and none of the techniques have been applied to cardiac problems, which requires further research.

The use of certain HDACi drugs has shown severe cardiac toxicity. This cardiotoxicity includes T-wave flattening, ST-segment depression, and QT interval prolongation, as detected by examining the ECG [204]. QT interval prolongation has been the most severe cardiac event in patients treated with HDACi to date due to its ability to lead to potentially fatal ventricular arrhythmia, known as torsades de pointes [205]. Therefore, it is of great significance to develop drugs that can provide higher efficacy at the site of action, while simultaneously reducing toxicity. Research has found that developing drugs that only act on a single HDAC subtype may provide significant clinical benefits, such as the development of selective inhibitors [206] or HDACi with low hERG affinity [207]. Moreover, among the HDACis currently approved by the FDA, none are specifically indicated for the treatment of CVD. Therefore, it is of great significance to develop drugs that specifically target CVD. The future outlook for HDACis therapy can be concisely encapsulated in three key areas: the enhancement of combination therapy approaches, the innovation of new drug formulations, and the identification of more precise biomarkers [208].

5. Summary and Outlook

Starting with cardiovascular diseases such as cardiac hypertrophy, heart failure, atherosclerosis, and myocardial ischemia/reperfusion injury, this paper summarizes the relationship between the roles of HDACs in various protein post-translational modifications, as well as HDACi in post-translational modifications that have been developed to date, aiming to provide new ideas for future research.

It is well known that cardiovascular diseases have a high incidence and morbidity, whereby their prevalence increases significantly with age. Therefore, it is particularly urgent to explore new therapeutic strategies for CVD in view of the rapid aging of the global population. In recent years, protein PTMs were shown to be closely related to the occurrence and development of CVD, which has become a focus of medical research. Most PTMs are reversible and can affect overall health by regulating cell states. PTMs are important in cardiovascular signaling pathways, regulating mitochondrial oxidative stress and inducing cardiomyocyte apoptosis. One of the key mechanisms regulating these modifications is mediated by HDACs. Since the discovery of HDACs and HDACis, researchers have gained a deeper understanding of their roles and potential therapeutic effects. HDACs are a major regulator that controls heart development and promotes stem cell-derived cardiogenesis. However, they also play a key role in mediating cardiac hypertrophy, heart remodeling, and functional recovery after cardiac injury [209]. In recent years, significant progress has been made in understanding the function of HDACs in physiological or pathological heart states as well as their regulatory modes. Although HDACis were initially highlighted as a new class of anticancer drugs, their application scope has gradually expanded to the treatment of various other diseases [210]. Accordingly, HDACs and HDACis critically regulate cardiovascular diseases via the modulation of post-translational modifications. Therefore, the in-depth study of HDACs and their inhibitors is critical. We anticipate that the continued study of HDACs and their inhibitors HDACis in various post-translational modifications will provide important theoretical support and potential targets for the clinical treatment of cardiovascular diseases.