Protein acylation and heart failure: A review of novel lysine modifications in cardiac pathology

Abstract

Pathological cardiac hypertrophy is a primary contributor to heart failure (HF). Protein acylation, particularly acetylation, plays a crucial role in this process. Advances in mass spectrometry and proteomics have identified an increasing number of novel lysine acylation modifications, including crotonylation, lactylation, succinylation, β-hydroxybutyrylation, 2-hydroxyisobutyrylation, propionylation and malonylation. These novel acylations are implicated in key physiological processes and disease-related cellular functions, as they modify the physicochemical properties of proteins and closely link substrate metabolism to gene regulation. Research on these novel lysine acylations associated with cardiac hypertrophy and HF has been steadily increasing in recent years. This review aims to summarize the progress and significance of these novel lysine acylations in cardiac hypertrophy and HF, providing references for a deeper understanding of the pathogenesis of HF and development of innovative therapeutic strategies.

Key Words: Novel lysine acylations; Heart failure; Pathological cardiac hypertrophy; Protein post-translational modifications; Lysine acetylation

Core Tip: This review highlights novel lysine acylations that link substrate metabolism to gene regulation by altering protein properties and play key roles in pathological cardiac hypertrophy and heart failure. It summarizes their emerging roles and significance in these conditions, providing insights into the pathogenesis of heart failure and potential therapeutic strategies.

INTRODUCTION

Heart failure (HF) is the final stage of many cardiovascular diseases. It is a group of syndromes that result from insufficient blood perfusion in tissues and organs due to impaired cardiac filling or ejection function[1]. It has been defined as a global epidemic and it is a life-threatening syndrome. Due to the aging of the population and the earlier onset of cardiovascular diseases such as hypertension and ischemic heart disease, its morbidity and mortality rates remain high all year round. Studies have shown that > 64 million people worldwide suffer from HF, and that patients have a 1-year mortality rate of 15%-30% and a 5-year mortality rate of 50%-75%[2,3]. The resulting social and economic burden has become a major issue in global public health.

Pathological cardiac hypertrophy, resulting from cardiomyocyte hypertrophy, myocardial fibrosis, and energy metabolism disorders, is a primary factor that promotes progression of HF. In the early stages, the body maintains normal cardiac output through compensatory mechanisms such as the Frank-Starling law and neuroendocrine responses. However, during this compensatory phase, these mechanisms may disrupt energy metabolism or utilization, leading to cardiomyocyte hypertrophy or necrosis, alterations in the extracellular matrix, and changes in collagen networks. This cascade ultimately results in pathological remodeling and HF[4-7]. In recent years, a consensus has emerged regarding the management of HF, which encompasses both pharmacological and surgical interventions[1,4]. However, medical and surgical approaches have shown limited efficacy in enhancing quality of life and long-term outcomes. Therefore, there is an urgent need to develop novel therapies that can prevent or delay progression of HF.

Cardiac hypertrophy and HF are closely tied to a complex interplay of genetic[8], epigenetic[9,10], biological, and environmental[11] factors. Epigenetics serves as a bridge between environmental influences and heredity, and environmental changes frequently result in epigenetic modifications that ultimately disrupt gene expression and contribute to disease[12]. Protein post-translational modifications (PTMs) refer to the covalent alteration of amino acid residues within proteins and constitute a significant aspect of epigenetics. Lysine acylations represent one of the primary modes of PTM. Among the amino acids, lysine stands out due to its unique ε-amino group in the side chain, making it one of the most frequently modified residues[13].

Lysine acylations encompass both histone and nonhistone acylation. Histone acylation is crucial for the epigenetic regulation of gene transcription, as it influences gene expression by directly modifying chromatin and nucleosome structures, which subsequently affects the epigenome. While acylation is primarily associated with histones, it is also prevalent in nonhistone proteins. Nonhistone acylation predominantly regulates protein function, including stability, activity, localization, and protein-protein interactions, thus playing an essential role in the regulation of cellular activities[14]. Advances in mass spectrometry and proteomics have identified various forms of protein lysine acylations beyond the previously recognized lysine acetylation (Kac)[15]. These newly identified modifications include lysine crotonylation (Kcr)[16], lysine lactylation (Kla)[17], lysine butyrylation (Kbu)[18], lysine β-hydroxybutyrylation (Kbhb)[19], lysine 2-hydroxyisobutyrylation (Khib)[20], lysine succinylation (Ksucc)[21], lysine propionylation (Kpr)[22], and lysine malonylation (Kmal)[23]. Various protein acylation modifications, affecting both histones and nonhistones, have been implicated in the pathogenesis of cardiac hypertrophy and HF. This review summarizes the latest research concerning novel protein acylation modifications in cardiac hypertrophy and HF, and offers a new perspective for the development of targeted therapeutic agents.

OVERVIEW OF PREVIOUS RESEARCH ON CLASSICAL KAC AND PATHOLOGICAL CARDIAC HYPERTROPHY AND HF

Kac has been studied since its initial discovery in 1964. Table 1 presents the advances made in previous studies regarding Kac in cardiac hypertrophy and HF. It is a dynamic and reversible PTM characterized by transfer of acetyl groups from acetyl-CoA to the ε-amino side chains of lysine residues[24-37]. It was first identified in 1964 by Allfrey et al[38], who demonstrated its role in the regulation of transcription through acetylation of histones. Following this pivotal discovery, various components such as lysine acetyltransferases, lysine deacetylases, acetyl-lysine reader domains, and deacetylase inhibitors were identified. Advances in new technologies has significantly expedited the identification of nonhistone acetylated proteins, thereby enhancing our understanding of the associated regulatory processes[36,39].

Table 1 Summary of classical lysine acetylation in pathological cardiac hypertrophy and heart failure.

Typical lysine acylation	Models	Sites	Specific functions	Refs.
Acetylation	Mice	H3K4ac, H3K9ac, H3K27ac	H3K4ac, H3K9ac, and H3K27ac mediated by p300 are increased in cardiac hypertrophy and HF	[24,25]
Acetylation	Mice	H3K27ac	H3K27ac mediated by p300 exacerbates cardiac dysfunction	[26]
Acetylation	Mice	H3K9ac; H3K23ac	H3K23ac and H3K9ac are elevated in hearts of mice with diabetic cardiomyopathy	[27]
Acetylation	Mice	K3K14ac	H3K14ac is increased in mice induced by prenatal alcohol exposure, which contributed to abnormal fetal heart development	[28]
Acetylation	Rats	H3K122ac	H3K122ac is increased during the stages of HF	[29]
Acetylation	Mice, human	SERCA2a K492ac	Increased SERCA2a K492ac is associated with systolic dysfunction in HF	[30]
Acetylation	Mice	TFAM (K62ac, K76ac, K111ac, K118ac)	Acetylation of TFAM 4KR is diminished in response to low expression of GCN 5 L 1 in HF, which results in a reduction in mitochondrial DNA abundance and impaired cardiac energetics	[31]
Acetylation	Mice	P53 4KR (k98ac, K117ac, K161ac, K162ac)	Increased P53 4KR acetylation leads to coronary capillary sparsity, cardiac remodeling, and cardiac dysfunction	[32]
Acetylation	Mice	P53 K381ac	Lipopolysaccharide increases p53 K381ac and promotes the nuclear translocation of p53, contributing to cardiac dysfunction	[33]
Acetylation	Mice	Drp1 K642ac	High-fat diet leads to DRP1 K642ac increase, which activates DRP1 and results in cardiomyocyte dysfunction and death	[34]
Acetylation	Mice	SF3A2 K10ac	Ginsenoside Rb2 directly interacts with P300 to reduce SF3A2 K10ac, enhance mitochondrial respiration, and mitigate cardiac dysfunction	[35]

DRP1: Dynamin-related protein 1; HF: Heart failure; SERCA2a: Sarco/endoplasmic reticulum calcium ATPase 2a; TFAM: Mitochondrial transcription factor A.

Kac plays a crucial role in regulating chromatin remodeling and DNA transcription primarily through protein-nucleic acid and protein-protein interactions[39]. Over the past few decades, Kac has been extensively investigated in relation to cardiovascular diseases[40]. In spontaneously hypertensive rats, the inhibition of histone deacetylase (HDAC) by valproic acid reduces cardiac hypertrophy and fibrosis by increasing histone H3 acetylation at the promoter of the nuclear receptor subfamily 3 group C member 2 target gene, thereby suppressing gene expression[41]. HDAC inhibitor suberoylanilide hydroxamic acid prevents the prolongation of QT intervals in various arrhythmias, including those observed in malnourished mice[42]. The induction of interleukin-35 in plasma samples from ApoE knockout mice and patients with hypercholesterolemia was investigated. Interleukin-35 inhibited mitochondrial reactive oxygen species-mediated H3K14ac, thereby suppressing endothelial cell activation and preventing progression of arteriosclerosis[43].

Kac is a critical factor in the development of HF. The combination of elmisartan and thiorphan has been shown to significantly inhibit fibrosis in diabetic cardiomyopathy by targeting histone acetyltransferase, thereby reversing acetylation of histones H2AK5, H2BK5, H3K9, and H4K8[44]. VGLL4 K225ac inhibits the interaction between TEA domain transcription factor 1 and yes-associated protein, leading to degradation of TEA domain transcription factor 1. This process ultimately restricts the proliferation of nascent cardiomyocytes and contributes to cardiac growth defects[45]. Sirtuin (SIRT) 3 expression is downregulated in the HF of patients with obesity and metabolic syndrome, resulting in hyperacetylation of cyclophilin D, the opening of the mitochondrial permeability transition pore, and subsequent cardiac dysfunction[46]. Prenatal exposure to particulate matter < 2.5 mm is associated with significant reductions in birth weight and development of cardiac hypertrophy in adulthood, attributed to increased acetylation levels of CREB binding protein/P300 and H3K9[47]. Acetylation of H3K4, H3K9, H3K23, H3K122 and H3K27 is associated with cardiac hypertrophy and HF[24-29]. Among nonhistone acetylation modifications, sarco/endoplasmic reticulum calcium ATPase (SERCA) 2a K492ac is enhanced in HF, contributing to impairment of contractile function[30]. General control of amino acid synthesis 5-like 1 expression is reduced in the transverse aortic constriction (TAC) model of HF, leading to decreased acetylation of mitochondrial transcription factor A. However, mitochondrial transcription factor A acetylation is significantly upregulated in response to general control of amino acid synthesis 5-like 1 overexpression, which helps maintain mitochondrial DNA abundance and enhances cardiac energetics, thereby alleviating HF[48]. Increased acetylation of P53 4KR is associated with coronary capillary sparsity, and cardiac remodeling and dysfunction[32]. Lipopolysaccharide treatment elevates the acetylation level of p53 K381 and promotes its nuclear translocation, resulting in cardiac dysfunction[33]. High-fat diet leads to dynamin-related protein 1 K642ac increase, which activates dynamin-related protein 1 and results in cardiomyocyte dysfunction and death[34]. Ginsenoside Rb2 directly interacts with P300, leading to a reduction SF3A2 K10ac. This interaction enhances mitochondrial respiration and alleviates cardiac dysfunction[35].

In conclusion, cardiac hypertrophy and dysfunction influenced by factors such as stress overload, ischemia, metabolic changes, and drugs can mediate the development of HF through various pathways. Although the significance of these alterations in acetylation modifications is recognized, they do not entirely elucidate the complex pathogenesis of HF, highlighting the necessity for further research.

OVERVIEW OF NOVEL LYSINE ACYLATIONS IN RELATION TO PATHOLOGICAL CARDIAC HYPERTROPHY AND HF

Since the discovery of acetylation in the last century, Chen et al[18] reported two novel lysine acylations in 2007 - propionylation and butyrylation. This discovery paved the way for identifying a series of novel lysine acylations, including Kcr, Kla, Ksucc, Kmal, Kbhb and Khib. Many studies have concentrated on the regulatory mechanisms and pathophysiology related to novel lysine acylations, as well as their pathological associations. These novel acylations are known to play significant roles in many cellular biological processes, such as metabolic regulation, epigenetic regulation, and signal transduction. Furthermore, they are closely linked to a range of biological processes and diseases, including cardiovascular disease, metabolic disorders, cancer, neurodegeneration, and kidney disease, making them a focal point of research in the field of epigenetics[39,49,50].

Novel lysine acylations can also occur on both histones and nonhistones. Similar to acetylation, novel histone acetylation primarily influences the epigenome through transcriptional regulation of gene expression. In contrast, nonhistone acylation predominantly regulates protein functions, including stability, activity, localization, and protein-protein interactions. These novel lysine acylations are derived from three major metabolites: Glucose, amino acids, and fatty acids. Various metabolic intermediates, such as lactic acid, crotonate, β-hydroxybutyrate (BHB), succinyl-CoA, acetyl-CoA, and BHB, provide acyl groups that covalently modify proteins. The corresponding metabolites of Kcr, Kbu, Kpr, and Khib primarily originate from fatty acid oxidation and amino acid metabolism, while those of Kla, Kmal, Kbhb, and Ksucc are predominantly derived from glucose metabolism.

Further exploration of the intricate relationships between novel lysine acylations and HF will enhance the identification of additional targeted biomarkers and critical therapeutic targets. Table 2 summarizes the current knowledge regarding the regulatory mechanisms of novel acylations in the development of pathological cardiac hypertrophy and HF.

Table 2 Summary of novel lysine acylations and pathological cardiac hypertrophy and heart failure.

Novel lysine acylations	Models	Sites	Specific functions	Refs.
Crotonylation	Mice	TMP1 K28/29cr; IDH3α K199cr	TMP1 K28/29 and IDH3α K199cr are decreased in a model of cardiac dysfunction induced by ischemia-reperfusion	[53]
Crotonylation	Human; mice	H3K18cr, H2BK12cr	H3K18cr and H2BK12cr are elevated in hypertrophic cardiomyopathy	[54]
Crotonylation	Mice	NAE1 K238cr	NAE1 K238cr increases in the cardiac hypertrophy model	[55]
Crotonylation	Mice	SERCA2a K120cr	SERCA2a K120cr is significantly elevated in SIRT1 knockout mice, which impairs SERCA2a activity and leads to alterations in energy metabolism that precipitate cardiac hypertrophy and dysfunction	[56]
Lactylation	Mice	α-MHC K1897 La	α-MHC K1897 La reduction in heart failure weakens its interaction with titin, ultimately resulting in severe HF	[48]
Lactylation	Mice	H3K18 La	The elevation of H3K18 La is associated with pathological myocardial hypertrophy and cardiac dysfunction	[59]
Lactylation	Mice	H3K56 La	H3K56 La is reduced in the Mi/R model, and the inhibition of H3K56 La negated the cardioprotective effects of HSPA12A	[60]
Lactylation	Mice	H3K18 La	Exercise training enhances cardiac function by inhibiting YTHDF2, which reduces H3K18 lactylation and mitigates myocardial ischemia-reperfusion injury	[61]
Lactylation	Mice	Snail1	The lactylation of Snail1 was elevated following myocardial infarction, which contributed to myocardial fibrosis and worsened cardiac dysfunction	[62]
Succinylation	Mice	ECHA K351succ	SIRT5 mediates the desuccinylation of ECHA K351, thereby improving cardiac metabolism and function and reducing myocardial hypertrophy	[71]
Succinylation	Mice	IDH2	The inhibition of SIRT5 expression results in increased succinylation of IDH2, which disrupts mitochondrial homeostasis and contributes to myocardial fibrosis and HF	[72]
Succinylation	Human; mice	CPT2 K424succ	SIRT5 mediates the desuccinylation of CPT2 K424 in diabetic cardiomyopathy, thereby ameliorating lipid deposition, myocardial hypertrophy, and diastolic dysfunction	[73]
Succinylation	Mice	Txnrd1 K534succ	Txnrd1 K534succ increase in mice with a knockout of LDHA, leading to severe cardiac dysfunction in a pressure overload mouse model	[74]
β-hydroxybutyr-ylation	Mice	CS K395bhb	β-hydroxybutyrate mitigates heart failure with preserved ejection fraction by activating CS through the enhancement of CS K395bhb	[82]
Propionylation	Mice	H3K23pr	H3K23pr increase in cardiac hypertrophy and HF	[86]
Butyrylation	Mice	H3K9bu	H3K9bu is diminished due to the downregulation of β-oxidase in the heart following TAC surgery	[90]
Malonylation	Rat	VDAC2 K46mal	VDAC2 K46mal was elevated in mice with sepsis-induced cardiac dysfunction	[97]
Malonylation	Mice	GSTP1 K121mal	The abnormal decrease of SIRT5 increase of GSTP1 K121mal in DCM leads to oxidative stress and mitochondrial dysfunction	[98]
Malonylation	Mice	Ago2 K440mal	The increase of AGO2 K440mal enhances the production of ROS in mitochondria, subsequently impairing cardiac function in DCM	[99]
2-hydroxyisobu-tyrylation	Mice	DLD K440hib	DLD K440hib increase in the TAC-induced cardiac hypertrophy model	[105]

AGO2: Argonaute 2; CPT2: Carnitine palmitoyltransferase 2; CS: Citrate synthase; DCM: Diabetic cardiomyopathy; ECHA: E-CoA hydratase α subunit; GSTP1: Glutathione S-transferase 1; HF: Heart failure; HSPA12A: Heat shock protein family A member 12A; IDH: Isocitrate dehydrogenase; LDHA: Lactate dehydrogenase A; α-MHC: Α-myosin heavy chain; NAE1: NEDD8-activating enzyme E1 regulatory subunit; ROS: Reactive oxygen species; SERCA2a: Sarco/endoplasmic reticulum calcium ATPase 2a; SIRT: Sirtuin; Txnrd1: Thioredoxin reductase 1; VDAC2: Voltage-dependent anion-selective channel protein 2; YTHDF2: YT521-B homology domain family member 2; TAC: Transverse aortic constriction.

CURRENT ADVANCES OF NOVEL LYSINE ACYLATIONS IN PATHOLOGICAL CARDIAC HYPERTROPHY AND HF

Kcr

Kcr is a novel short-chain lysine acylation modification discovered in 2011[16]. It is a dynamic process involving the transfer of crotonyl groups to lysine residues, utilizing crotonyl-CoA as a substrate. This process is regulated bidirectionally by crotonyl acyltransferase and decrotonyl acylase. Kcr represents a novel PTM that occurs in both histone and nonhistone proteins. It plays a crucial role in various biological processes, including gene transcription, DNA repair, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, glycolysis, and regulation of protein activity and the cell cycle[51,52].

In a physiological context, Kcr is essential for normal energy metabolism and survival. crotonyl-CoA plays a crucial role in fatty acid metabolism, facilitating the breakdown of fatty acids within cells and generating energy for various physiological functions. Recent studies have concentrated on the role of Kcr in cardiac hypertrophy and HF. Myocardial ischemia-reperfusion injury has been shown to induce significant alterations in the Kcr levels of cardiac contractile proteins. These changes in Kcr are associated with the degradation of myocardial mitochondria, sarcomere structure, and gap junctions, as well as autophagy and apoptosis in cardiomyocytes[53]. It has been reported that a deficiency of enoyl-CoA hydratase 1 significantly increases levels of H3K18cr and H2BK12cr, which further drives gene expression in hypertrophic fetuses, ultimately promoting the hypertrophic growth of neonatal cardiomyocytes[54]. The levels of crotonylation at NEDD8-activating enzyme E1 regulatory subunit K238 are elevated in mice and in patients with cardiac hypertrophy[55]. Additionally, crotonylation of SERCA2a K120 was significantly elevated in SIRT1 knockout mice. The reduced activity of SERCA2a can be attributed to the diminished binding energy of crotonylated SERCA2a to ATP, which resulted in alterations in mitochondrial energy metabolism, and ultimately, cardiac hypertrophy and dysfunction[56]. These results establish a foundation for the future development of drugs aimed at targeting crotonylation, with the potential to treat cardiac hypertrophy and HF.

Kla

Lactate is the primary product of glycolytic metabolism in cells[57]. It serves as a critical molecule that connects cellular metabolism with the regulation of cellular activity, playing a significant role in energy supply and signal transduction[58]. The biological effects of lactate can be attributed to a novel PTM of Kla. This modification regulates gene transcription and protein function by altering histones and other functional proteins[17].

Kla is a significant factor in the regulation of cardiovascular diseases, particularly influencing the development of cardiac hypertrophy and HF. It has been reported that HKla is notably expressed in cardiac hypertrophy induced by TAC, as well as in neonatal mouse cardiomyocytes stimulated by angiotensin (Ang) II[59]. In the myocardial ischemia-reperfusion model, a decrease in lactylation of H3K56 was observed, and inhibiting H3K56 lactylation negated the cardioprotective effects of heat shock protein (HSP) family A member 12A[60]. Exercise training could inhibit YT521-B homology domain family member 2, thereby reducing H3K18 lactylation and alleviating myocardial ischemia-reperfusion injury[61]. Fan et al[62] demonstrated that Snail1 lactylation plays a critical role in the progression of cardiac fibrosis following myocardial infarction. In a model of Ang-induced HF, lactate efflux and depletion in cardiomyocytes led to decreased intracellular lactate levels and increased α-myosin heavy chain K1897 lactylation. This change significantly diminished the interaction between α-myosin heavy chain and Titin, resulting in severe HF[48]. Lactate metabolism and Kla represent novel therapeutic targets for cardiovascular diseases. Therefore, the future development of innovative drugs for HF, particularly those that focus on Kla, presents a promising avenue for exploration.

Ksucc

Succinylation is a novel acylation discovered in 2011. It is a dynamically reversible process that covalently attaches succinyl groups to lysine residues through enzymatic or nonenzymatic mechanisms[21,63]. The positive regulation of this modification involves the direct conversion of succinic acid into succinyl-CoA and its derived succinyl donors, facilitated by succinyl acyltransferases. In contrast, negative regulation primarily occurs through the action of desuccinylases[64,65]. Succinyl is an acidic group that, upon binding to lysine residues - typically characterized by a natural positive charge - induces a charge reversal in lysine. This alteration leads to changes in the spatial conformation, activity, stability, and function of the protein. This process is implicated in several biological mechanisms, including ketogenesis, fatty acid β-oxidation, the TCA cycle, and ATP synthesis[66-68].

Cardiomyocytes are abundant in mitochondria, which serve as the primary site for Ksucc. Consequently, alterations in Ksucc within the myocardium are readily apparent. Succinyl CoA levels and the extent of Ksucc in mitochondrial proteins are significantly reduced in the hearts of mice with HF[69]. Quantitative analysis of Ksucc revealed that 307 unique succinylated lysine sites were identified in the myofibrils of failing ischemic cardiomyopathy compared to nonfailing hearts. Specifically, 297 of these succinyl-lysine sites exhibited a decrease in Ksucc[70]. However, the succinylation levels of some mitochondrial proteins, such as TCA cycle enzymes and membrane proteins, showed a specific increase in chronic HF. Deletion of SIRT5 leads to the succinylation of E-CoA hydratase α subunit, which reduces its activity and negatively affects fatty acid oxidation, resulting in hypertrophic cardiomyopathy[71]. In a TAC mouse model, inhibition of SIRT5 expression resulted in increased Ksucc of isocitrate dehydrogenase 2, disrupting mitochondrial homeostasis and contributing to myocardial fibrosis and HF[72]. Elevation of carnitine palmitoyltransferase 2 K424succ in the myocardium of Sirt5-deficient mice has been shown to inactivate carnitine palmitoyltransferase 2 enzymatic activity, thereby impairing mitochondrial integrity and exacerbating cardiac lipotoxicity and dysfunction in diabetic cardiomyopathy[73]. Current research on Ksucc and HF has focused on proteins associated with mitochondrial metabolism[72-74]. However, the specific targets and regulators of histone Ksucc remain largely unexplored. Therefore, investigating the specific role and regulatory mechanisms of histone Ksucc in HF models is essential for facilitating drug development. In conclusion, Ksucc may represent a promising therapeutic target for HF.

Kbhb

During extended periods of exercise, starvation, fasting, or insufficient dietary carbohydrates, the liver can initiate the production of the endogenous ketone body BHB through the β-oxidation of fatty acids. Many researchers believe that BHB directly activates expression of a series of protective genes, playing a significant role in antioxidation, anti-inflammation, and neuroprotection[75,76]. In 2016, Xie et al[19] used mass spectrometry to demonstrate that BHB also promotes Kbhb. It refers to the covalent attachment of BHB to the ε-amino group of lysine, resulting in a protein adduct that is both metabolically sensitive and dependent on BHB concentration. This modification regulates the activity of histones and nonhistones and influences gene transcription in response to carbohydrate restriction or elevated BHB levels, both in vitro and in vivo[77]. Consequently, Kbhb is recognized as a significant component of ketone body regulation, which is intricately linked to chromatin remodeling, fatty acid oxidation, and energy metabolism[78].

The role of BHB and its associated Kbhb in cardiac function and the maintenance of cardiovascular health is multifaceted. These roles encompass direct myocardial protection, enhanced energy metabolic efficiency, and indirect protection through signal transduction. The decreased diastolic function in the hearts of 22-month-old mice may be associated with alterations in the 183 Kbhb site of 134 proteins when compared to 3-month-old mice[79]. A total of 244 Kbhb sites were identified in 142 proteins that exhibited significant alterations following the knockout of AMPKα2 in mice, leading to changes in myocardial mitochondrial ultrastructure and cardiac diastolic function. Kbhb underwent significant changes in rats with diabetic cardiomyopathy. Functional analyses indicated that Kbhb-modified proteins participate in various metabolic pathways[80]. Additionally, Chong et al[81] revealed that transient elevations of ketone bodies in the postnatal heart can enhance the Kbhb levels of mitochondrial proteins while inhibiting Kac. This modulation may be essential for mitochondrial maturation and the proper functioning of the heart in postnatal mice. BHB activates citrate synthase by directly increasing the Kbhb of citrate synthase at K395, while simultaneously inhibiting fatty acid uptake to reduce acetyl-CoA pools. This reduction, in turn, decreases protein acetylation in failing hearts with preserved ejection fraction, thereby preventing inflammation, fibrosis, and mitochondrial dysfunction[82]. Many studies have demonstrated that Kbhb plays a crucial role in regulating energy metabolism and alleviating mitochondrial dysfunction. However, the current body of research on the relationship between ketone bodies, particularly Kbhb, and cardiac hypertrophy, especially HF, remains limited. Future investigations should aim to elucidate the specific sites and regulatory mechanisms associated with Kbhb.

Kpr and Kbu

In 2007, Chen et al[18] identified two novel PTMs, Kpr and Kbu, on histones using mass spectrometry and other techniques. Kpr refers to the addition of a three-carbon propionyl group from propionyl-CoA to the ε-amino group of the lysine side chain; a reaction catalyzed by propionyltransferase or occurring via nonenzymatic means. This modification is dynamically regulated and can be reversed by lysine depropionylase. Propionic acid, a metabolite produced by intestinal bacteria, serves as a precursor in this process. Propionyl-CoA, an intermediate in propionic acid metabolism, acts as a substrate in the Kpr reaction, with its concentration directly influencing the extent of the reaction[83,84]. Elevated levels of propionyl-CoA, histone H3 propionylation, and genome-wide acetylation have been reported in the hearts of female mice exhibiting disrupted propionic acid metabolism. This altered gene expression subsequently affects cardiac function[85]. In mice with pressure-overload-induced cardiac hypertrophy, the levels of H3K23pr were increased at the promoters of genes with upregulated expression, while H3K23pr levels were decreased at the promoters of genes with downregulated expression. A reduction in branched-chain amino acid intake mitigates stress-overload-induced alterations in histone-acetylation-dependent gene expression in the heart, thereby delaying the progression of cardiomyopathy[86].

Kbu is a novel acylation modification characterized by the covalent attachment of a butyryl group to lysine residues, which occurs in histone and nonhistone proteins. This process is dynamically regulated by butyryl-CoA and its short-chain fatty acid derivative, butyric acid, through the actions of lysine acetyltransferases and deacetylases[87]. It has been reported that HSP90 K754bu reduces chemotherapy resistance in esophageal squamous cell carcinoma by maintaining the stability of HSP90[88]. Butyric acid-induced H3K9bu has been shown to mitigate damage associated with diabetic nephropathy[89]. In cardiovascular disease, accumulation of butyryl-CoA and reduced H3K9bu adversely affect the normal contractile function of the mouse heart[90]. Currently, there is limited research on Kbu in cardiovascular disease, underscoring the necessity for further studies on the epigenetic regulatory mechanisms involved in cardiac hypertrophy and HF.

Kmal

Kmal is a novel PTM that was identified in eukaryotic and prokaryotic cells in 2011[23]. This modification occurs when malonyl-CoA attaches malonyl groups to positively charged lysine residues, converting them into negatively charged entities. This alteration disrupts the electrostatic interactions between lysine and other amino acids, potentially leading to changes in protein conformation and affecting binding to target proteins[91]. Kmal has been demonstrated to occur in various metabolic pathways, including fatty acid synthesis and oxidation, mitochondrial respiration, and glycolysis. Furthermore, it plays a critical role in cancer, inflammation, type 2 diabetes, and angiogenesis[92-94].

Metabolic dysfunction is a fundamental issue in cardiovascular disease, where malonyl-CoA serves as a critical metabolic effector influencing obesity and fatty acid oxidation in the heart. Malonyl coenzyme A is particularly significant in the development of cardiovascular conditions, especially HF[95]. Kmal was found to decrease in H9C2 cardiomyocytes following 48 hours of Ang II stimulation. A total of 679 malonic sites were identified across 330 proteins in the hearts of Sham and TAC mice through liquid chromatography/tandem mass spectrometry analysis[96]. In a sepsis-induced myocardial dysfunction model, a significantly elevated voltage-dependent anion-selective channel protein 2 K46mal resulted in alterations to the N-terminal structure of voltage-dependent anion-selective channel protein 2 channels and mitochondrial dysfunction and ferroptosis in cardiomyocytes[97]. The loss of SIRT5 in diabetic cardiomyopathy significantly increases the Kmal of glutathione S-transferase p1, which reduces the stability of the glutathione S-transferase p1 protein. This reduction leads to oxidative stress and mitochondrial dysfunction, and contributes to cardiac dysfunction[98]. Similarly, the knockdown of SIRT3 elevated Argonaute 2 K440mal in diabetes-induced cardiac dysfunction, which resulted in decreased interaction between Argonaute 2 and TIMM17B on the transporter. This interaction could mitigate cardiac hypertrophy and enhance cardiac function[99]. Given the significant impact of Kmal on energy metabolism in the heart, further research is essential to elucidate the specific mechanisms and identify potential therapeutic drug targets related to Kmal in cardiac hypertrophy and HF.

Khib

Khib is a novel PTM first identified in 2014, which involves two substrates: 2-hydroxyisobutyryl and 2-hydroxyisobutyryl coenzyme A[20]. Notably, 2-hydroxybutyric acid, which is derived from the microbial degradation of dietary components, serves as a substrate for 2-hydroxyisobutyryl-CoA, thereby regulating Khib levels. Additionally, p300 and TIP60 function as substrates for 2-hydroxyisobutyryltransferase, which catalyzes the process of Khib. In contrast, HDAC1-3 and SIRT3 act as de-2-hydroxyisobutyrylases[100].

Khib occurs in a diverse array of histone and nonhistone proteins in animals and plants, playing a crucial role in protein synthesis, transcriptional regulation, and cellular metabolism. Reports indicate that the level of Khib modification in maize infected with Fusarium graminearum increases significantly. Khib modification is implicated in enhancing plant resistance to pathogen infection by regulating secondary metabolic processes[101]. In animal studies, Cheng et al[102] found that increased levels of Khib in mature sperm led to decreased sperm motility, resulting in asthenospermia. It has also been reported that aspirin reduces the activity of a-enolase, a crucial enzyme in glycolysis, by modulating the levels of a-enolase K2812hib[103]. There is a notable increase in glucose uptake and glycolytic rate in HF. P300 influences cellular processes by mediating the Khib of glycolytic enzymes, which subsequently affects glucose metabolism[104]. In a mouse model of transverse aortic coarctation-induced cardiac hypertrophy, the levels of dihydrolipoamide dehydrogenase (DLD) 2-hydroxy isobutyl were significantly increased, while pyruvate dehydrogenase activity was reduced. DLD has been identified as a direct substrate for P300. Ginsenoside Rg3 can reverse myocardial hypertrophy by inhibiting the acyltransferase activity of P300, thereby reducing the Khib level of DLD and restoring pyruvate dehydrogenase activity[105]. Unfortunately, the limited research on Khib has left its precise regulatory role in cardiac hypertrophy and HF unclear, necessitating additional clinical and basic research to elucidate this relationship.

In summary, by utilizing high-resolution mass spectrometry and modification omics technologies, researchers have systematically identified acylation modifications, including acetylation, crotonylation, succinylation, lactylation, malonylation, and β-hydroxybutyrylation, along with their substrate profiles and dynamic changes in HF (Figure 1). This has identified several acylation modifications within the context of HF. The discovery of novel acylation modifications highlights the direct regulatory role of metabolites as signaling molecules, operating through a multidimensional regulatory network of metabolism-modification-phenotype, thereby enhancing our understanding of the mechanisms underlying HF. The research focus has broadened from histone to nonhistone targets, significantly expanding the target lineage. This shift indicates that the impact of acylation modifications is no longer restricted to chromatin structure but extends directly to the functional level of proteins.

Figure 1 Novel protein lysine acylations in heart failure. 3-PG: 3-phosphoglycerate; Mal-CoA: Malonyl-CoA; Ac-CoA: Acetyl-CoA; Bu-CoA: Butyryl-CoA; Cr-CoA: Crotonyl-CoA; Pr-CoA: Propionyl-CoA; Hib-CoA: 2-hydroxyisobutyryl-CoA; Succ-CoA: Succinyl-CoA; α-KG: Α-ketoglutarate; Fum: Fumarate; BHB: Β-hydroxybutyrate; BCAA: Branched-chain amino acids; Lys: Lysine; Trp: Tryptophan.

However, it is important to note that current research on novel acylation modifications is limited to correlation studies at the modification level, with fewer in-depth explorations of causality. Additionally, in functional studies of modification sites, strategies such as lysine-arginine (K → R, mimicking demodification) and lysine-glutamine (K → Q, mimicking modification) point mutations are commonly used. However, these mutations may simultaneously alter the charge and structural stability of proteins, posing a risk of nonspecific effects. Interactions between these novel modifications in HF and the specific molecular logic of such interactions remain underexplored. Finally, various novel acylation modifications share the same “writers” (such as p300/CREB binding protein) and “erasers” (such as the SIRT family). These modifications lack specific modifying enzymes, which makes it difficult to distinguish the effects of acetylation, butyrylation, and succinylation.

CONCLUSION

Recent studies have demonstrated that PTMs in epigenetics, particularly lysine acylation, play a crucial role in the occurrence and development of cardiac hypertrophy and HF. We have reviewed various studies on histone PTMs associated with cardiac hypertrophy and HF, with a particular focus on novel modifications (Tables 1 and 2). This review aimed to provide a new perspective for future research into the pathophysiology of HF and the development of innovative drugs and diagnostic methods.

Lysine acylations are dynamic and reversible modifications mediated by regulatory enzymes. These modifications are influenced by regulatory factors and are dynamically altered in response to changes in cellular metabolic levels, such as lactic acid and pyruvate. Therefore, exogenous supplementation of lysine-modified precursors (e.g., crotonate, lactate, and pyruvate) or the regulation of modifying enzyme activity may represent a novel approach to treating cardiac hypertrophy and preventing HF.

The emergence of novel lysine acylations has prompted research in this area; however, most studies remain descriptive, lacking site-specific identification and mechanistic elucidation. Additionally, certain acylations, such as glutarylation and benzoylation, have not been explored in cardiovascular contexts, largely due to the scarcity of antibodies, high reagent costs, and technical limitations. Furthermore, current evidence regarding drugs targeting these modifications is primarily preclinical, with no clinical translation achieved to date. Therefore, a top priority is to validate their biological effects and clinical relevance in HF patient cohorts. This validation is essential for translation and will facilitate biomarker discovery, accurate efficacy assessment, and the eventual development and application of selective modulators of lysine acylation pathways.