The role of myocardial energy metabolism perturbations in diabetic cardiomyopathy: from the perspective of novel protein post-translational modifications

Abstract

Diabetic cardiomyopathy (DbCM), a significant chronic complication of diabetes, manifests as myocardial hypertrophy, fibrosis, and other pathological alterations that substantially impact cardiac function and elevate the risk of cardiovascular diseases and patient mortality. Myocardial energy metabolism disturbances in DbCM, encompassing glucose, fatty acid, ketone body and lactate metabolism, are crucial factors that contribute to the progression of DbCM. In recent years, novel protein post-translational modifications (PTMs) such as lactylation, β-hydroxybutyrylation, and succinylation have been demonstrated to be intimately associated with the myocardial energy metabolism process, and in conjunction with acetylation, they participate in the regulation of protein activity and gene expression activity in cardiomyocytes. This review examines the epigenetic pathogenesis of DbCM, primarily focusing on myocardial energy metabolism perturbations and novel PTMs associated with them. It provides a detailed analysis of the mechanisms of these novel PTMs in DbCM to enhance the understanding of DbCM pathophysiology and establish a theoretical foundation for the development of new treatment strategies for DbCM.

Introduction

Diabetic cardiomyopathy (DbCM) is a significant chronic complication of diabetes, primarily characterized by myocardial hypertrophy [1], interstitial fibrosis [2], structural changes in sarcomeres [3], and coronary microangiopathy [4]. These alterations can lead to changes in cardiac cavity volume, reduced cardiac compliance, and impaired cardiac function [5]. Moreover, DbCM has a causal relationship with other cardiovascular diseases, such as atherosclerosis, hypertension, myocardial infarction, and heart failure [6]. This interaction not only complicates treatment strategies for DbCM but also greatly increases patient mortality and poses a serious threat to the overall health of patients.

The pathogenesis of DbCM is complex, and disturbance of myocardial energy substrate metabolism [7], involving glucose, fatty acids, and ketone bodies, is one of the main driving forces for the pathogenesis and development of DbCM [8]. First, disturbance of myocardial energy metabolism can directly affect the contractile and diastolic functions of cardiomyocytes. In addition, metabolic intermediates produced during metabolic disorders may act as downstream signaling molecules to activate or inhibit specific signaling pathways that influence cardiomyocyte proliferation, apoptosis, and fibrosis. Finally, metabolic disorders may also regulate the pathophysiological processes of DbCM through epigenetic mechanisms [9].

Epigenetics is a pivotal field in biology that investigates heritable alterations in biological phenotypes that occur without changes to the DNA sequence [10]. Among its various components, protein post-translational modifications (PTMs) are of particular significance. PTMs refer to the acylation modifications of proteins on amino acid residues, such as lysine, after translation. This process is dynamically regulated by specific writing enzymes and erasing enzymes. PTMs influence protein structure, stability, and function. On the one hand, PTMs on histones regulate the stability of histone binding to DNA, thereby affecting gene transcriptional activation in the relevant promoter region. In contrast, PTMs on non-histones regulate enzyme activity, functional localization, and protein–protein interactions [11].

The roles of previously discovered PTMs such as acetylation (Kac) [12] in DbCM have been extensively investigated. In recent years, the gradual application of high-precision mass spectrometry (MS) and the continuous development of specific antibodies have led to the identification and characterization of various novel PTMs [Fig. 1]. However, our understanding of their roles in biological regulation is limited. The formation of certain novel PTMs requires specific energy metabolic intermediates as acyl donors [13]. For instance, lactylation (Kla) uses lactate produced during glycolysis as a substrate [14], β-hydroxybutyrylation (Kbhb) employs the ketone body β-hydroxybutyrate (β-HB) as a substrate [15], and succinylation (Ksucc) relies on succinyl-CoA in the mitochondrial tricarboxylic acid (TCA) cycle as a substrate [16]. Recent studies have shown that these novel PTMs are involved in the metabolic regulation and expression of certain key genes in cardiomyocytes, thereby directly or indirectly influencing the occurrence and progression of DbCM [17].

Fig. 1

The discovery process and molecular structure of PTMs related to metabolic disturbance in DbCM. With the development of acyl antibodies and continuous improvement in the accuracy of MS detection, many PTMs related to metabolic disturbance of DbCM cardiomyocytes have been discovered in recent years, and the red part of their molecular structure is the corresponding acyl modification group

This review aimed to summarize the epigenetic effects of myocardial energy metabolism disorders in DbCM, focusing on the regulatory mechanism from the perspective of novel PTMs. First, we briefly analyzed the changes in the energy substrate metabolism observed in the DbCM myocardium. We then outline the relationship between DbCM and Kac. Finally, we describe in detail the formation process of new PTMs that are closely related to energy metabolism, as well as their roles and mechanisms in DbCM. This review provides a crucial theoretical basis for the elucidation of the epigenetic pathogenesis of DbCM and the search for new therapeutic targets, and has great significance for strengthening the prevention and treatment of DbCM.

Disturbance of energy metabolism in DbCM

The heart has the highest oxygen consumption in the body [18] and demonstrates remarkable flexibility in selecting substrates for energy metabolism in healthy adults [19]. Under normal circumstances, adenosine triphosphate (ATP) required by the myocardium is primarily derived from the β-oxidation of fatty acids and aerobic oxidation of glucose [20]. Approximately 60–90% of the energy supply is comes from fatty acids, while the remaining 10–40% is derived from glucose oxidation, with the contributions of amino acids and ketone bodies being relatively minor. However, in the context of DbCM, the metabolic processes of cardiomyocytes involving energy substrates such as glucose, fatty acids, and ketone bodies undergo significant alterations [21]. These changes adversely affect the overall function of the heart through complex mechanisms.

Glucose metabolism

Clinical studies have demonstrated that myocardial glucose metabolism is significantly impaired in patients with type 2 diabetes [22]. A study utilizing advanced hyperpolarized magnetic resonance spectroscopy revealed a marked reduction in the ability of pyruvate, produced through cytoplasmic glucose metabolism in cardiomyocytes to enter the mitochondria for TCA cycle metabolism [23]. Furthermore, diabetic patients frequently exhibit insulin resistance, leading to the downregulation of the glucose transporter GLUT4 on the surface of cardiomyocytes, consequently diminishing glucose uptake and utilization [24, 25]. In the context of diabetes, increased PPARα activity in cardiomyocytes may further inhibit oxidative glucose utilization by promoting Pdk4 transcription [26, 27]. This decline in glucose utilization not only compromises the energy supply efficiency of cardiomyocytes, but may also precipitate a series of metabolic abnormalities, such as heightened hyperglycemic toxicity, which can lead to further impairment of cardiomyocyte function.

Fatty acid metabolism

Cardiomyocytes of diabetic patients partially compensate for their impaired glucose metabolism by significantly enhancing fatty acid metabolism [22]. This compensatory mechanism operates effectively because of the markedly increased expression levels of membrane transporters, such as CD36, in cardiomyocytes, which facilitates the uptake of free fatty acids by the heart [28]. In the context of DbCM, abnormal activation of the transcription factor PPARα further induces the simultaneous upregulation of multiple downstream genes associated with fatty acid uptake, oxidation, and storage, thereby effectively coordinating the enhancement of myocardial fatty acid metabolism [26, 29]. Concurrently, elevated levels of fatty acid metabolic intermediates may directly inhibit the activity of glucose metabolic enzymes through allosteric regulation or covalent modification, resulting in decreased glucose utilization. This shift in the metabolic pattern triggers the gradual accumulation of fatty acids in cardiomyocytes, ultimately leading to increased lipid toxicity and further damage to the structure and function of cardiomyocytes [30]. Additionally, because the oxidation of fatty acids requires greater oxygen consumption, this process can increase the oxygen demand of cardiomyocytes, potentially resulting in a relatively hypoxic state of the myocardium.

Ketone body metabolism

Ketone bodies include acetoacetic acid, β-HB, and acetone, and the total amount of β-HB in human serum accounts for approximately 70% of all ketone bodies [31]. Ketone bodies exhibit greater efficiency in ATP production than glucose and fatty acid oxidation, while requiring relatively little oxygen. Thus, they are considered potential clean energy sources in the context of diabetes. However, research on ketone body metabolism in the diabetic heart is still insufficient, with only a limited number of conflicting experimental results. For instance, some clinical studies and animal experiments have indicated that the level of β-HB in the myocardium is elevated in cases of diabetic cardiomyopathy. Concurrently, the expression and activity of the regulatory enzyme BDH1, which catalyzes the production of β-HB, are significantly reduced [32]. Furthermore, in a study using a rat model of diabetic cardiomyopathy, an increased utilization of acetoacetic acid, another ketone body, was observed in the myocardium, a phenomenon closely linked to heightened activity of the acetoacetic acid-metabolizing enzyme SCOT [33, 34]. Recent studies have demonstrated that ketone bodies can inhibit the activation and release of the NLRP3 inflammasome in human monocytes, protect vascular endothelial function in mice with heart failure, and mitigate oxidative stress damage in sepsis-associated cardiomyopathy [35,36,37]. Therefore, ketone bodies may also contribute to delaying the progression of DbCM through these mechanisms.

Lactate metabolism

Lactate metabolism plays a crucial role in energy acquisition of cardiomyocytes. During the glycolysis of cardiomyocytes, glucose is transported to cells by glucose transporters to convert into two molecules of pyruvate. On the one hand, pyruvate in the cytoplasm can be transported to the mitochondria via mitochondrial pyruvate carriers, which are then converted to acetyl-CoA by pyruvate dehydrogenase and enter the TCA cycle. On the other hand, pyruvate can also be converted to lactate by lactate dehydrogenase (LDH) in the cytoplasm.

In diabetic hyperglycemia, the process of cardiomyocyte glycolysis is accelerated, but due to mitochondrial dysfunction, especially the activity of pyruvate carrier (MPC) is decreased. As a result, pyruvate cannot effectively enter mitochondria for oxidative phosphorylation [38], which promotes more conversion of pyruvate to lactate. In addition, lactate utilization by DbCM cardiomyocytes decreased. This change in substrate preference also resulted in increased lactate levels in cardiomyocytes [39]. In addition, the expression of monocarboxylate transporter 4 (MCT4) in cardiomyocytes is upregulated, which leads to increased lactate outflow, disrupts the lactate-pyruvate balance in cardiomyocytes, and triggers oxidative stress and inflammation, further aggravating myocardial damage [40].

In conclusion, DbCM is closely associated with multiple alterations in the metabolism of glucose, fatty acids, ketone bodies and lactate within cardiomyocytes, which collectively affect cardiac function through complex interactions. These metabolic changes not only diminish energy supply efficiency, but also initiate a series of pathological processes, including oxidative stress, inflammatory responses, and calcium homeostasis imbalances [41], ultimately resulting in cardiomyocyte damage and apoptosis. Long-term chronic hyperglycemia can induce oxidative stress, resulting in increased production of reactive oxygen species (ROS) that damage cardiomyocytes and promote myocardial fibrosis and heart failure [42]. Additionally, prolonged hyperglycemia can stimulate the immune system to generate a persistent inflammatory response, contributing to cardiomyocyte hypertrophy, fibrosis, and diastolic dysfunction. Furthermore, enhanced fatty acid oxidation may lead to mitochondrial dysfunction in cardiomyocytes, particularly through abnormal expression of the mitochondrial calcium homeostasis protein (MCU). This abnormality results in reduced mitochondrial calcium uptake, further exacerbating mitochondrial dysfunction in cardiomyocytes [43]. This cascade of events may lead to severe heart failure and other adverse outcomes. The disruption of cardiomyocyte energy metabolism in DbCM presents a potential therapeutic target. Research has demonstrated that empagliflozin can enhance the rate of glucose oxidation in the myocardium of db/db mice [44]. Additionally, other studies indicate that empagliflozin can decrease the expression of the fatty acid β-oxidation regulatory enzyme CPT1b in diabetic rat cardiomyocytes, consequently diminishing fatty acid utilization and ameliorating the energy metabolism disorder in cardiomyocytes [45].

Furthermore, in the context of DbCM, the protein PTMs formed by energy metabolism intermediates in cardiomyocytes may also exhibit corresponding changes, such as Kac, Kla, Kbhb, and Ksucc, which contribute to the emergence of epigenetic phenomena like “metabolic memory” among these modifications [46]. The relationship between Kac and DbCM, which was initially discovered, has been extensively studied. A systematic summary and in-depth exploration of the regulatory roles of other novel PTMs in the onset and progression of DbCM, along with the implementation of corresponding intervention strategies, holds significant practical importance for the prevention and treatment of DbCM.

Overview of the relationship between acetylation and DbCM

Acetyl-CoA, a key acyl group donor, plays an important role in the formation of Kac [47]. Acetyl-CoA is also a hub of energy metabolism in cardiomyocytes. In the oxidative metabolism of myocardial fatty acids, acetyl-CoA is mainly produced during the β-oxidation of fatty acids and then enters the TCA cycle [48]. In glucose metabolism, pyruvate, the end product of the glycolysis pathway, can be converted into acetyl-CoA after entering the mitochondria and further participates in the TCA cycle to provide energy for cardiomyocytes. In addition, in ketone body metabolism, acetoacetic acid can also be converted into acetyl-CoA, and then participate in the regulation of energy metabolism. Three types of enzymes are involved in the regulatory mechanism of Kac: writing enzymes (GCN5, etc.) [49, 50], erasing enzymes (HDAC1-5, SIRT1-7, etc.), and the corresponding reading enzymes (YEATS, etc.) [51, 52].

In the context of diabetes, the disruption of energy metabolism in cardiomyocytes results in an imbalance between the production and utilization of acetyl-CoA. This imbalance influences the pathogenesis of DbCM by regulating Kac levels of both histone and non-histone proteins. For instance, an experiment conducted in streptozotocin (STZ)-induced type 1 diabetic mice demonstrated that enhancing p53 deacetylation modifications can protect cardiomyocytes from damage caused by high glucose levels through a mechanism involving the inhibition of ferroptosis [53]. Another animal experiment demonstrated reduced Kac of heart histone H3 at lysine 9 and lysine 23 in db/db mice with renal failure, which contributes to cardiomyocyte hypertrophy in mice with type 2 diabetes [54]. Further findings revealed a significant role of specific Kac-modifying regulatory enzymes in DbCM. Lanfang et al. demonstrated through related experiments that jugular vein injection of an adenovirus overexpressing SIRT3 could markedly reduce the Kac level of p53 and the expression of TIGAR in the heart tissue of diabetic db/db mice, thereby effectively improving the pathological condition of DbCM [55]. Other researchers have reported that HDAC4 knockout induces heart failure in mouse models of type 1 and type 2 diabetes [56].

In summary, Kac modification and its regulatory enzymes play an indispensable role in the metabolic perturbation of cardiomyocytes in DbCM, and influence the occurrence and development of DbCM through a complex regulatory network. Future studies will further reveal the specific details of these regulatory mechanisms, and provide new ideas and targets for the prevention and treatment of DbCM.

Novel PTMs in DbCM

Lactylation

Kla was discovered in 2019, and lactate produced by glycolysis served as an acyl donor [57]. Lactate is an alternative energy source that sustains the energy supply of the heart in the DbCM state. During glycolysis, glucose is metabolized to pyruvate through a series of enzymatic reactions, resulting in the production of NADH and lactate [58]. Studies have demonstrated that the concentration of lactate in the peripheral blood of patients with DbCM is significantly elevated, which is closely associated with cardiac diastolic dysfunction [59, 60]. Under the influence of acyl-CoA synthetases such as ACSS2, lactate in the myocardium utilizes acetyl-CoA to form lactoyl-CoA. Subsequently, lactoyl-CoA undergoes Kla modification through the action of specific acyl-writing enzymes, thereby participating in the negative regulation of myocardial function.

Enzymes such as P300/CBP, GCN5, and HBO1 catalyze the covalent attachment of lactoyl-CoA to specific lysine residues on proteins, leading to Kla modifications [61,62,63]. In contrast, erasing enzymes, including HDAC1-3 and SIRT1-3, are responsible for removing Kla from proteins [64, 65], thereby restoring them to their original state and facilitating the precise regulation of the modification level. These writing and erasing enzymes not only contribute to the formation of Kla, but also exhibit the ability to catalyze other acylation modifications, underscoring their versatility in protein PTMs.

Kla plays a critical role as a bridge between glucose metabolism and epigenetic regulation. Recent studies have revealed the biological significance of Kla in DbCM. In DbCM mice, the abnormal upregulation of MCT4 induces excessive lactate excretion in cardiomyocytes, leading to lactoacylation of histone H4K12 in macrophages, which results in a series of inflammatory damages to cardiomyocytes. Thus, Kla may be associated with a detrimental microenvironment in cardiomyocytes [40]. In addition, the results of in vitro experiments indicated that exogenous lactate can promote pathological hypertrophy of the heart by enhancing the expression levels of histone H3K18 lactylation modification in cardiomyocytes. The use of glycolysis and LDH inhibitors to reduce histone lactoacylation modification levels in cardiomyocytes effectively inhibits the progression of cardiac hypertrophy. This finding may offer a novel targeted intervention strategy for reversing ventricular remodeling in DbCM [66]. Patients with DbCM experience a gradual deterioration of heart function, which may ultimately result in heart failure. A new study employed protein-modifying omics to characterize Kla in the hearts of mice suffering from heart failure. Under normal physiological conditions, the tail of the α-myosin heavy chain (α-MHC) binds to myosin, forming a thick muscle filament [67]. Stability of this thick muscle filament is crucial for maintaining the normal structure and contractile function of the heart. These findings indicate that in the context of heart failure, the level of Kla at the K1897 site of α-MHC was significantly reduced. This alteration disrupts the stability of thick muscle filaments and exacerbates the symptoms of heart failure [68]. The decreased level of α-MHC K1897 Kla was primarily attributed to the excessive outflow and depletion of lactate in cardiomyocytes during heart failure. However, the relationship between this phenomenon and the expression and localization of the corresponding lactate transporter receptors in the myocardial membrane requires further investigation. Consequently, it is essential to observe the dynamic changes in lactate levels and lactoacylation modification in cardiomyocytes throughout the progression of diabetic cardiomyopathy to heart failure, as this will enhance our understanding of the regulatory role of lactoacylation modification in the advancement of DbCM.

β-Hydroxybutyrylation

β-HB synthesis primarily occurs in the liver, although the kidneys and intestines also produce small amounts of β-HB [69]. However, it remains unclear whether the heart can synthesize ketone bodies. In the pathological state of diabetes, fatty acids are converted to acetyl-CoA through β-oxidation, which is further converted to β-HB by the liver ketone body synthetase system [70]. Subsequently, β-HB is transported to the cardiomyocytes via the bloodstream. Within cardiomyocytes, β-HB can be oxidized and phosphorylated through the TCA cycle, releasing energy to meet myocardial energy demands. Notably, β-HB not only serves as an additional energy source for cardiac muscle, but also functions as a potent antioxidant [71], mitigating damage to heart muscle cells caused by oxidative stress. In addition, β-HB has been implicated in PTMs of the DbCM heart, especially Kbhb, with the CoA-active form of β-HB as a precursor, which has recently attracted attention.

The regulatory mechanism of Kbhb is complex and intricate, and involves the synergistic action of multiple enzymes. Acyltransferase P300/CBP functions as a writing enzyme, catalyzing the covalent binding of β-hydroxybutyryl-CoA to lysine residues to form Kbhb [72]. This process is crucial for the regulation of various biological activities, including gene expression, cellular metabolism, and signal transduction. Concurrently, histone deacetylases HDAC1 and HDAC2 serve as erasing enzymes responsible for removing Kbhb modifications, thereby maintaining homeostasis of intracellular modification levels [73]. The identification of these regulatory enzymes associated with Kbhb modification offers new insights for a deeper understanding of its biological functions.

Research on Kbhb in DbCM is still in its early stages of development. A recent proteomic study employing liquid chromatography-tandem mass spectrometry (LC–MS/MS) successfully identified differences in the expression of Kbhb modifications in the heart tissue of DbCM rats induced by a high-glycemic, high-fat diet combined with STZ. Quantitative analysis revealed that 284 Kbhb modification sites were upregulated, whereas 52 were downregulated among 47 proteins in the hearts of DbCM rats. Bioinformatic analysis further suggested that Kbhb-modified proteins are involved in various energy metabolic pathways, including the TCA cycle, oxidative phosphorylation, and propionic acid metabolism. Thus, Kbhb may play a role in regulating the progression of DbCM by influencing myocardial energy metabolic status. Additionally, another proteomic analysis of Kbhb in the liver of fasting mice showed that Kbhb-modified proteins exhibited significant enrichment in fatty acid β-oxidation, TCA cycle, and ATP metabolism [74]. Accordingly, Kbhb may play a role in the regulation of ATP synthesis, thereby participating in the disturbance of energy metabolism in DCM cardiomyocytes. However, further studies are needed to explore the underlying mechanisms by which Kbhb regulates myocardial energy metabolism. Endothelial dysfunction is often regarded as an early pathological feature of diabetes-related vascular complications [75]. Diabetic hyperglycemia may impair cardiovascular endothelial function in patients with DbCM by activating oxidative stress, inflammatory responses, and formation of advanced glycation end products (AGEs). Wu et al. demonstrated that exogenous β-HB supplementation in STZ-induced SD rats with type 2 diabetes upregulated the modification level of histone H3K9bhb in the rat heart in a dose-dependent manner, resulting in the expression of vascular endothelial growth factor (VEGF) and subsequently improving aortic endothelial injury in DbCM hearts [76]. Interestingly, this study found that the repair effect of β-HB on the vascular endothelium was most obvious at moderate concentrations, suggesting that Kbhb modification induced by different concentrations of β-HB may have some unique effects or mechanisms on the improvement of vascular endothelial injury in DbCM.

In summary, the potential role of Kbhb in diabetic cardiomyopathy has not yet been fully explored. Future studies will further reveal the epigenetic regulatory mechanism of Kbhb and provide a scientific basis for the prevention and treatment of diabetic cardiomyopathy.

Succinylation

Succinyl-CoA plays a crucial role in the intricate network of myocardial energy metabolism, as a key intermediate in the TCA cycle [77]. It is formed when α-ketoglutarate undergoes decarboxylation facilitated by the α-ketoglutarate dehydrogenase complex, resulting in the binding of an acetyl group to CoA. Within the metabolic framework of the TCA cycle, succinyl-CoA is catalyzed by succinyl-thiokinase, which hydrolyzes its thioester bonds, thereby releasing free energy for the synthesis of GTP, it is the major pathway for succinyl-CoA consumption. With or without enzymatic action, succinyl-CoA can transfer succinyl groups to lysine residues in cellular proteins, thereby achieving Ksucc. Metabolomics studies have shown that succinyl-CoA is the most abundant short-chain acyl-CoA in the mouse heart and that Ksucc is particularly pronounced in this energy-consuming organ, showing its potential importance in the regulation of cardiac function [78].

The regulatory mechanism of Ksucc is a complex process that involves various types of enzymes. Among these, writing enzymes such as P300/CBP, GCN5, and KAT2 are primarily responsible for catalyzing Ksucc, a process that induces significant changes in protein function and structure [79, 80]. Concurrently, erasing enzymes, including SIRT1, SIRT2, and SIRT5, remove the succinyl group, thereby restoring the protein to its original state and regulating the balance between different biological processes within the cell [81]. However, current research on the direct reading of enzymes involved in the regulatory mechanism of Ksucc is relatively scarce, and the existing literature and experimental data are limited, which hinders a comprehensive understanding of this field.

In the diabetic state, abnormal metabolism of energy substrates within cardiomyocytes, such as glucose, can lead to the accumulation of TCA cycle intermediates, particularly succinyl-CoA. As a result, high levels of succinyl-CoA promote Ksucc modifications of proteins, which may influence the activity and localization of certain key metabolic enzymes and signaling molecules in cardiomyocytes. Recently, with the extensive study of Ksucc, its role in the occurrence and progression of DbCM has been acknowledged. For instance, a study that integrated broadly targeted metabolomics, 4D proteomics, and Ksucc omics revealed that in a mouse model of DbCM heart failure, SIRT5 expression was significantly reduced, resulting in a substantial increase of the Ksucc of carnitine palmitoyl transferase 2 (CPT2) [82]. This change leads to the accumulation of myocardial lipids and increased lipid toxicity. Consequently, SIRT5-mediated Ksucc may represent a potential target for the treatment of DbCM, it is of great clinical significance to investigate the therapeutic effects of SIRT5 activators, such as investin and resveratrol, on DbCM and other metabolic diseases associated with fat deposition, as well as to determine whether Ksucc serves as the underlying mechanism [83]. However, it is crucial to note that this study was based on the db/db mouse model, which exhibits severe metabolic disorders and genetic variants. Thus, further validation using additional animal models of DbCM is warranted.

In summary, Ksucc is a significant PTMs for cardiomyocytes; however, its specific mechanisms in the occurrence and development of DbCM have not been fully elucidated. Therefore, future studies are needed to further explore the enzymatic mechanism of Ksucc, substrate specificity, and its interaction with other modification modes.

Malonylation

Using specific malonylated antibodies and LC–MS/MS, researchers first identified malonylation (Kma) in proteins from E. coli and HeLa cells in 2011 [84]. Malonylated acyl donor malonyl-CoA is an indispensable intermediate metabolite in fatty acid synthesis and β-oxidation. In cardiomyocytes, malonyl-CoA is produced by acetyl-CoA carboxylase (ACC) [85, 86], which is regulated by various hormones and nutritional signals. For example, AMPK is dephosphorylated when blood glucose levels rise, thereby inhibiting its phosphorylation of ACC, which leads to ACC activation and promotes malonyl-CoA synthesis [87]. The resulting malonyl-CoA is then transported to different regions within the cell and is involved in various metabolic pathways. In the process of fatty acid β-oxidation, malonyl-CoA, as a negative regulator, can reduce the rate of fatty acids entering mitochondria for β-oxidation by inhibiting the activity of carnitine palmitoyl transferase-1 (CPT-1), thereby regulating the energy supply of cardiomyocytes [88].

Recently, numerous studies have demonstrated that Kma is closely associated with the occurrence and progression of DbCM. Researchers have observed significantly elevated serum malonyl-CoA levels in DbCM patients, which may be associated with myocardial insulin resistance, hyperglycemia, and abnormal fatty acid metabolism. SIRT5 is one of the few known Kma-demodifying enzymes [89]. In 2023, Can Wei et al. found that knocking out SIRT5 resulted in a significant increase of GSTP1 protein Kma in hyperglycemia-stimulated mouse primary cardiomyocytes. This increase promotes mitochondrial dysfunction, pyrodeath, and DNA damage in the cardiomyocytes. Conversely, the overexpression of SIRT5 can mitigate myocardial damage associated with DbCM [90]. However, the study had several limitations. For instance, only western blotting was employed for detection, and the changes in Kma levels of all proteins in cardiomyocytes were not comprehensively evaluated using MS or other methods. Additionally, the wild-type mice used in the experiment originated from a different source than the SIRT5 knockout mice, which somewhat undermines the persuasive strength of the conclusions of the study. Other studies have demonstrated that quercetin, a component found in various medicinal plants, promotes desuccinylation of IDH2 through SIRT5. This process helps maintain mitochondrial homeostasis in mouse cardiomyocytes, improves myocardial inflammation and fibrosis, and ultimately contributes to a reduction in the incidence of heart failure caused by DbCM and other related diseases [91]. Malonylated proteomic analysis of a mouse model of myocardial hypertrophy indicated that malonylated protein enrichment was significantly associated with cardiac structure, systolic function, cGMP-PKG signaling pathway, and various metabolic processes [92]. Given that the heart also exhibits hypertrophy during the progression of heart failure in the context of DbCM, it may represent a promising therapeutic target for enhancing cardiac remodeling in DbCM.

In summary, research on Kma not only elucidates the pathogenesis of DbCM, but also has the potential to offer new therapeutic avenues for other causes of heart failure, thereby expanding treatment strategies in this field.

Palmitoylation

The palmitoylation of proteins is a PTM in which palmitic acid or palmitoyl-CoA serves as an acyl group donor [93]. Palmitic acid, a 16-carbon saturated fatty acid, is a key product of lipid metabolism in cardiomyocytes and can be obtained through the diet or synthesized endogenously. During β-oxidation, palmitic acid is initially converted to palmitoyl-CoA within the mitochondria of cardiomyocytes [94]. Subsequently, palmitoyl-CoA undergoes a series of enzymatic reactions that gradually degrade into short-chain fatty acyl-CoA fragments. These fragments then enter the TCA cycle, ultimately producing acetyl-CoA, NADH, FADH2, and a small amount of ATP, which collectively provides energy for cardiac muscle cells.

The enzymes responsible for palmitoylation, particularly the DHHC family of proteins, help add palmitoyl groups to cysteine residues in proteins [95, 96]. Instead, erasing enzymes (such as APT1) removes palmitoyl groups from these protein molecules [97]. The expression and activities of these enzymes in cardiomyocytes are strictly regulated to maintain palmitoylation homeostasis.

Recently, an increasing number of studies have indicated that palmitoylation modifications may play a role in the pathological processes of DbCM by influencing lipid metabolism, insulin secretion, and related signal transduction pathways in cardiomyocytes. CD36 is a transmembrane protein responsible for approximately two-thirds of the fatty acid intake in the heart. In certain instances, functional migration of CD36 across the plasma membrane can enhance fatty acid absorption. A recent study demonstrated that the specific knockout of TGR5 in the myocardial cells of mice with DbCM led to increased activity of DHHC4, the enzyme responsible for catalyzing CD36 palmitoylation. This results in enhanced localization of CD36 to the cell membrane, thereby increasing the uptake of long-chain fatty acids by cardiomyocytes, which ultimately contributes to hypertrophy, fibrosis, and dysfunction of the heart [98]. Therefore, intervention in palmitoylation modification within cardiomyocytes may play a crucial role in preventing cardiac lipid toxicity and DbCM development. In addition to DHHC4, both DHHC5 and DHHC6 have also been implicated in the palmitoylation modification of CD36 [99], and future studies are needed to explore their epigenetic regulatory role in the development of DbCM. Research conducted by Professor Guofang Dong et al. has demonstrated that APT1 plays a significant role in the pathogenesis of diabetes by regulating the palmitoylation status of Scamp1, which in turn affects the function of islet β cells and insulin secretion [97]. A similar mechanism might be present in cardiomyocytes. The abnormal function of APT1 could lead to impaired cardiomyocyte function and cardiac remodeling by influencing palmitoylation of proteins associated with the energy metabolism of cardiomyocytes. Future studies will provide deeper insights into the specific mechanisms of action of palmitoylation and its regulatory enzymes in DbCM, which will encourage us to explore ways to optimize the treatment strategies for DbCM.

Conclusions and prospects

With the rapid development of modern techniques, such as protein modification omics, researchers have confirmed that various novel protein acylation reactions are associated with disturbances in myocardial energy metabolism in DbCM [Fig. 2]. In addition to the novel PTMs highlighted here, current research suggests that O-linked β-N-acetylglucosamine-modification (O-GlcNAcylation) may also be involved in mitochondrial dysfunction in DbCM [100]. However, our understanding of the relationship between novel protein acylation modifications and DbCM remains limited, and many potential molecular mechanisms require further exploration and study. For example, it is unclear whether these novel PTMs are more likely to significantly affect changes in cardiac morphology and function through non-histone or histone acylation. Based on the available evidence, histone PTMs appear to play a more significant and direct role in the epigenetic regulation of DbCM. By modifying chromatin structure, histone PTMs influence DNA accessibility, thereby regulating gene expression and transcriptional activity. Additionally, the combinations of histone PTMs are notably complex and diverse. In contrast, the functions of non-histone PTMs are more prominently associated with cell signaling and metabolic regulation, with their direct impact on gene expression being relatively limited. Consequently, there is an urgent need for comprehensive and in-depth pathophysiological studies to elucidate the specific details of these mechanisms.

Fig. 2

Metabolic perturbations in cardiomyocytes in DbCM and acyl donor formation of novel PTMs. Insulin resistance leads to reduced extracellular glucose uptake and glycolysis in diabetic cardiomyocytes. Additionally, aerobic oxidation of pyruvate within the mitochondria is inhibited, resulting in increased lactate levels in cardiomyocytes. Free fatty acids and ketone β-HB serve as alternative energy sources to glucose, enhancing their supply to cardiomyocytes and promoting the uptake of fatty acids and β-oxidation. However, the metabolism of ketone bodies in cardiomyocytes is bidirectional, with acyl-CoA being both formed and consumed during energy metabolism, thereby influencing the corresponding PTMs. DbCM, diabetic cardiomyopathy; β-HB, β-hydroxybutyrate; GLUT, glucose transporter; TCA cycle, tricarboxylic acid cycle; BDH1, β-hydroxybutyrate dehydrogenase 1; SCOT, succinyl-CoA:3-ketoacid CoA transferase; SCS, succinyl-CoA synthetase; AcAc, acetoacetic acid; FACS, fatty acyl-CoA synthetase; NADH, nicotinamide adenine dinucleotide; FADH2, flavin adenine dinucleotide hydrogen transmitter 2

Many protein acylation modifications are mediated by the same acyl-CoA synthetase, acyltransferase, and deacylase, which enhances the efficiency of identifying undiscovered regulatory enzymes and accelerates investigation of the biological functions of protein acylation modifications in DbCM [Table 1]. The researchers could further investigate whether the classic acyltransferase P300/CBP, along with the acyl-removing enzymes from the SIRT family, can catalyze the addition and removal of newly discovered acylated modifications. Additionally, they should continue to explore the roles of these regulatory enzymes in the modulation of these acylated modifications. Consequently, when exploring these reversible PTMs, it is crucial to thoroughly understand and closely monitor the dynamics of acyl donor levels under energy metabolic perturbations in DbCM as well as the dynamics of other modifications to gain a better understanding of the intricate regulatory network of protein acylation modifications in this pathological state.

Table 1 Summary of novel PTMs associated with myocardial metabolic perturbations in DbCM

Multiple PTMs can occur on the same protein, and these modifications can interact with one another, leading to either synergistic or antagonistic effects. Collectively, these interactions form the epigenetic background of DbCM. The interplay between PTMs may occur through mechanisms such as competition for the same modification site, structural alterations that render another site inaccessible to a different PTM modifier, or direct regulation of the activity of a secondary PTM modifier. Furthermore, whether amino acid residues within the same protein can undergo multiple modifications simultaneously in cardiomyocytes remains an open question. Consequently, it is essential to systematically investigate the functional effects of combinations of two or more novel PTMs within the same experiment as well as the potential interaction mechanisms between them.

Although PTMs have potential applications in the treatment of DbCM, several challenges and limitations remain. The high specificity and complexity of PTMs, along with the interactions and effects among different modifications, complicate precise control, leading to uncertainties regarding treatment efficacy and the potential for side effects. Furthermore, PTMs typically occur within cells, posing difficulties in directly targeting specific modifications through external administration. Even if a drug can be successfully delivered into the cell, ensuring that it specifically acts on the target PTMs presents a significant challenge. Additionally, it is likely that a drug targeting specific PTMs may also affect other modifications, resulting in off-target effects. This could reduce the treatment effectiveness for DbCM or increase the incidence of side effects.

In summary, novel PTMs associated with metabolic disorders are key to DbCM progression. Looking forward to the future, with the improvement of the precision of energy metabolity-related detection instruments, the significant improvement in the detection accuracy and application range of protein modification omics technology, and the development and marketing of more novel acylation modification site-specific antibodies, our understanding of energy metabolism disturbances in DbCM hearts will be more in-depth. Future studies should aim to establish a comprehensive network of regulatory relationships between PTMs and DbCM. Additionally, investigating PTMs in DbCM at the cellular level through techniques such as transcriptomics and modification omics will enhance our understanding of the functions and mechanisms underlying these associations. This knowledge will, in turn, facilitate the advancement and refinement of effective strategies for the prevention and treatment of DbCM.