Progress of Research on the Metabolic Regulation of Lactylation in Muscle Tissues and Its Disease Associations

Abstract

Lactylation serves as a vital link between cellular metabolism and epigenetic regulation and plays a pivotal role in muscle biology. Muscle tissue is the primary site of lactate production; its unique metabolic environment confers dynamism, specificity and functional diversity for lactylation. Under physiological conditions, lactylation regulates myocyte energy metabolism, proliferation, differentiation, and exercise adaptation through a dynamic “writer–eraser–reader” mechanism. In pathological states, lactate imbalance directly contributes to the progression of various muscular disorders. For instance, diminished histone lactylation during muscle aging suppresses the expression of genes critical for DNA repair and protein homeostasis. Aberrant lactylation is involved in the development of insulin resistance and diabetic cardiomyopathy. Furthermore, lactylation exerts dual effects in cardiovascular diseases; it provides protection by enhancing the transcription of repair genes and simultaneously aggravates injury by promoting processes such as fibrosis and ferroptosis. Collectively, these findings underscore the importance of lactylation in muscular pathologies and provide a theoretical foundation for the development of therapies that target this modification process. As the regulatory mechanisms of lactylation have become clearer, precise interventions targeting specific modification sites are expected to open new therapeutic avenues for muscular diseases.

1. Introduction

Lactate has long been recognized as both a metabolic byproduct and energy substrate, and recent studies have revealed that it serves as a substrate for lactylation, thereby contributing to epigenetic regulation [1]. This pivotal finding has established a new research frontier linking metabolic processes to epigenetic mechanisms [2].

Under physiological conditions, skeletal muscle is the site of approximately 25% of the total body lactate production, which increases to 70% during intense exercise [3,4]. Although cardiac muscle relies predominantly on aerobic metabolism, lactate production can increase significantly under hypoxic conditions. The production rate rises from 1.22 μmol·g dry weight⁻¹·min⁻¹ at rest to 18.5 μmol·g dry weight⁻¹·min⁻¹ [5]. Similarly, smooth muscle can produce lactate via anaerobic metabolism during hypoxia or vigorous intense contraction, with its content increasing from 29.83 ± 5.05 mg/100 g of wet weight to 65.36 ± 7.37 mg/100 g of wet tissue [6]. Lactate can serve as a substrate for protein lactylation, thereby participating in the regulation of cellular function.

Protein lactylation in muscle tissue exhibits four key characteristics: (1) Dynamic nature: lactylation levels change rapidly in response to muscle activity [7]; (2) Specificity: distinct modification patterns across different muscle fiber types [8]; (3) Functional significance: regulatory role in diverse physiological processes from energy metabolism to gene expression [9,10]; (4) Pathological relevance: dysregulated lactylation is associated with multiple muscular disorders, including muscle aging, metabolic myopathies, and cardiovascular diseases [11,12,13].

This review focuses on the metabolic regulatory mechanisms of lactylation in muscle tissues and their associations with disease, aiming to establish a foundation for further investigation into muscle pathophysiology and the development of therapeutic interventions.

2. Discovery and Characterization of Lactylation

The discovery of protein lactylation represents the progression of research from initial observations to developing a mechanistic understanding. In the late 1990s, lactyl-N-acetylneuraminic acid was identified in the mandibular glands of horses and human gastric aspirates, although its biological role remains unknown [14,15]. Pivotally, in 2019, Zhang [2] et al. used mass spectrometry to detect the lactylation of histones in macrophages, revealing its direct role in regulating inflammatory responses. Subsequent investigations established that lactylation occurs widely across various tissues and cell types. It is particularly prominent in metabolically active muscle tissues, where it exhibits dynamic, specific, functional, and pathogenic characteristics [7,8,9,10,11,12,13].

3. Chemical Basis and Mechanisms of Lactylation

3.1. Chemical Basis of Lactylation

Protein lysine lactylation is a post-translational modification involving a lactate molecule that forms an amide bond with the ε-amino group of a lysine residue, resulting in Nε-lactyllysine. This modification increases the mass of the residue by 72 Da and reduces its net charge by +1 [2]. Compared to acetyl, the lactyl group is bulkier and contains a hydroxyl group, which may introduce a more pronounced steric hindrance [16]. Under high glycolytic flux (e.g., in muscle ischemia or during intense exercise), glucose is metabolized in the cytosol via glycolysis to generate pyruvate, which is subsequently reduced to lactate by lactate dehydrogenase A (LDHA), thereby establishing a favorable environment for protein lactylation [17]. L-lactate-mediated lactylation represents an epigenetic regulatory mechanism by which cells respond to glycolysis. It mainly involves the covalent attachment of a polar hydroxyl-bearing l-lactoyl group to lysine residues [18,19]. This modification not only significantly enhances the polarity and solubility of lysine residues but also influences protein function by modulating the protein’s spatial conformation, receptor binding ability, stability, and interactions with other molecules [20]. Moreover, innate immune activation and the accumulation of intestinal bacteria can be regulated through D-lactate mediated lactylation [21,22].

3.2. Lactylation Mechanisms

L-lactate-mediated lactylation is an enzymatic post-translational modification governed by three core mechanisms: “writing”, “erasing”, and “reading”. During the “writing” phase, L-lactate is activated to lactoyl-CoA by lactoyl-CoA synthetases (e.g., acetyl-CoA synthetase 2 (ACSS2), succinyl-CoA synthetase (GTPSCS)) [23,24]. The lactyl group is then transferred to specific lysine residues on histone or non-histone proteins by lactyltransferases, including lysine acetyltransferases lysine acetyltransferase 2 (KAT2), E1A-binding protein p300 (p300), CREB-binding protein (CBP), general control non-depressible 5 (GCN5), and Tat-interactive protein 60 (TIP60) [20]. Additionally, an alternative ATP-dependent mechanism has been reported, wherein aminoacyl-tRNA synthetase 1 (AARS1) and AARS2 catalyze the formation of a lactyl-AMP intermediate, which subsequently modifies specific target proteins to alter their function [8,25]. The “erasing” mechanism is primarily mediated by Zn²⁺-dependent histone deacetylases 1–3 (HDAC1–3) and NAD⁺-dependent deacetylases Sirtuin1-3 (SIRT1-3), which remove lactyl modifications via hydrolysis [26]. The regulatory mechanisms governing the activity of key enzymes involved in lactylation are summarized in

Table 1. Regulatory information on lactylation-modifying enzymes.

Enzymes	Regulatory Factors	Response to Physiological /Pathological States	References
Lactyl-CoA synthetase
ACSS2	Signal initiation: The EGFR/ERK signaling pathway is activated, leading to the phosphorylation of ACSS2 at serine-267. Conformational change and translocation: PIN1 recognizes the phosphorylated site, induces a conformational change in ACSS2, and facilitates its nuclear translocation. Substrate requirements: The catalytic reaction requires the collective participation of lactate, CoA, and ATP.	High metabolic demand: In glycolytically active cells (tumor cells), they can respond to tumor microenvironmental changes such as growth factor signaling, hypoxia, and lactate accumulation.	[23]
GTPSCS	Nuclear localization signal (NLS): The NLS on the G1 subunit determines nuclear translocation. K73 acetylation on G2 subunit: This modification is a prerequisite for the interaction between GTPSCS and p300. Structural integrity: Substrate binding induces conformational rearrangement in the G2 subunit, affecting enzymatic activity. Substrate dependence: Lactate influences its catalytic activity.	High metabolic demand: In glycolysis-active tumor cells, lactate accumulation and increased levels of GTP/CoA lead to substantially enhanced activity.	[24]
Lysine lactyltransferase
P300/CBP	Post-translational modification: Phosphorylation can enhance its activity. Protein-protein interaction: Binding to specific transcription factors can guide it to target sites. Substrate dependence: Lactate concentration influences its catalytic activity.	Metabolic reprogramming: When cellular metabolism shifts from oxidative phosphorylation to glycolysis, the enzyme may show a preferential tendency to catalyze lactylation.	[30,31,32]
AARS1/2	Substrate concentration: Elevated lactate levels enhance its lactyltransferase activity. Competitive inhibition: β-alanine competes with lactate for binding. ATP dependency: The reaction is ATP-dependent.	Exercise/hypoxia: The decrease in oxygen partial pressure in muscles leads to an increase in AARS2 protein levels. Tumorigenesis: AARS1 acts as a lactate sensor, converting metabolic signals into protein modification signals.	[8,25]
Lysine delactylase
HDAC1-3	Constitutive activity: Its activity is regulated by subcellular localization, complex assembly, and phosphorylation. Zn2+-dependent catalytic activity: Maintained by Zn2+ and can be inhibited by chelators such as TSA (Trichostatin A). Direct inhibition by lactate: Evidence suggests that lactate itself may suppress its activity.	Constitutive regulation: As a fundamental delactylase, it likely maintains the baseline balance of lactylation modifications. Pathological State: In conditions such as atherosclerosis, its activity is altered.	[26,33,34]
SIRT1-3	NAD+/NADH ratio: The most critical regulatory factor. Elevated NAD+ activates the enzyme, while elevated NADH inhibits it. Direct inhibition by lactate: Evidence suggests that lactate itself may suppress its activity. Transcriptional and expression levels: Regulated by energy status (e.g., via AMPK) and other factors.	Energy sensing: During low-energy states (fasting or caloric restriction), the enzymatic activities of SIRT1-3 are enhanced. Pathological state: In heart failure, decreased NAD+ levels result in diminished delactylation capacity.	[35,36,37]

. The “reading” mechanism involves specific recognition of lactylated proteins by dedicated readers. The known readers include brahma-related gene 1 (Brg1), which recognizes H3K18 lactylation via its bromodomain to regulate stem cell reprogramming [27]; tripartite motif-containing protein 33 (TRIM33), which binds H3K18 lactylation through its PHD-bromodomain to modulate transcription in late-stage M1 macrophage activation [28]; and double PHD fingers 2 (PHD2), which interacts with H3K14 lactylation via its tandem PHD fingers to activate oncogenes [29]. However, the physiological and pathological mechanisms of lactylation in muscle tissues and other systems remain to be fully elucidated.

Recent studies have identified a non-enzymatic reaction in which an L-lactyl group is transferred from L-lactyl-CoA to lysine residues. This reaction is fundamentally chemical in nature, exhibiting clear time- and concentration-dependent characteristics. In vitro, it can modify a variety of proteins, including bovine serum albumin (BSA) in an enzyme-free system. Although this pathway is less efficient and less site-specific than its enzymatic counterparts, it represents a fundamental chemical mechanism that may regulate protein function in environments of locally high metabolite concentration. Furthermore, D-lactate-mediated lysine lactylation has been recognized as a significant, non-enzyme-dependent form of post-translational protein modification [38]. Furthermore, D-lactate-mediated lactylation is a significant non-enzymatic post-translational modification pathway. This process is initiated by methylglyoxal, a glycolytic intermediate primarily derived from the non-enzymatic degradation of phosphodihydroxyacetone and glyceraldehyde-3-phosphate [39]. During the catalysis of glyoxalase 1 (GLO1), methylglyoxal conjugates with glutathione to form lactoylglutathione. Lactoylglutathione serves as a lactyl donor, transferring the lactyl group to lysine residues on target proteins via a non-enzymatic mechanism, thereby facilitating lactylation. Subsequently, glyoxalase 2 (GLO2) catalyzes the hydrolysis of lactoylglutathione, regenerating glutathione for future catalytic cycles and releasing D-lactate [40]. Collectively, lactylation modifications constitute a crucial link between cellular metabolic states and epigenetic regulation (Figure 1).

4. Characteristics of Lactylation in Muscle Tissues

The muscles are a primary site for lactate production, particularly during high-intensity exercise or under hypoxic conditions, when anaerobic glycolysis is markedly activated, resulting in substantial lactate accumulation. This unique metabolic milieu makes muscle tissue an ideal model for studying lactylation. A locally high lactate concentration provides ample substrate for lysine lactylation, thereby driving a series of highly tissue-specific and dynamic modification processes. These modifications profoundly influence muscular energy homeostasis, cell fate, and disease progression, highlighting the direct role of the metabolic microenvironment in epigenetic regulation.

4.1. Dynamic Regulation of Lactylation in Muscle Tissue

The level of lactylation in muscle tissues is closely linked to lactate metabolism. Under resting conditions, skeletal muscle lactate concentration typically remains at 1 mM [41]. Conversely, during intense exercise, rapid glycolysis induces transient lactate accumulation, increasing concentrations to 5–10 mM [42]. This sharp increase promptly induces lactylation of both histone (e.g., H3K18) and non-histone proteins (e.g., pyruvate kinase M2) [43]. Notably, although blood lactate levels return to the baseline approximately 1 h after exercise cessation, protein lactylation in skeletal muscles (such as the soleus) peaks at 24 h post-exercise before gradually declining. After intense exercise, lactylation levels remain significantly elevated compared with resting controls, even at 48 and 72 h after exercise [7]. Zhang [2] et al. aptly termed this delayed response between lactylation dynamics and rapid blood lactate fluctuations as the “lactate clock”.

A similar time-delay phenomenon has been observed in postmortem muscles. Lactylation levels increased rapidly within the first two hours after death, which coincided with glycogen breakdown and lactate accumulation [44]. Although the lactate concentration and glycolytic potential peaked at four hours postmortem and subsequently declined, lactylation continued to increase, peaking at 8 h before decreasing significantly and stabilizing after 24 h, this pattern suggests delayed integration of metabolic signals [44]. This effect is likely linked to ATP availability, analogous to the mechanism of lactylation observed in tumor cells. The process is ATP-dependent at two key steps: first, ACSS2 consumes ATP to activate lactate into lactyl-CoA [23], and second, the lactyltransferase AARS1 utilizes ATP to form a lactyl-AMP intermediate, which subsequently transfers the lactyl group to specific target proteins [25]. As postmortem ATP depletes over time, reduced lactate dehydrogenase activity and insufficient lactyl-CoA availability lead to decreased lactylation levels; this effect is particularly evident in lactate-treated groups, indicating that ATP availability is crucial for maintaining the dynamic balance of protein lactylation [45].

4.2. Specificity of Lactylation in Muscle Tissue

Skeletal muscles consist of slow-twitch (type I) and fast-twitch (type II) fibers that exhibit distinct lactate metabolism and lactylation patterns due to their different metabolic characteristics and regulatory mechanisms. Type I fibers are rich in mitochondria and highly express aerobic metabolic enzymes that primarily rely on the oxidative metabolism of fat and glucose for energy, resulting in minimal lactate production [46]. However, they also express monocarboxylate transporter 1, which efficiently imports extracellular lactate for use as a substrate for oxidative phosphorylation [47]. This “consumer” characteristic enables type I fibers to display elevated lactylation levels following exercise. Research has demonstrated that after 30 min of running, although both fiber types exhibit lactate accumulation, type I fibers exhibit more pronounced lactylation at specific sites, including K336 of the pyruvate dehydrogenase E1 alpha 1 subunit (PDHA1) and K457/K458 of carnitine palmitoyltransferase 2 (CPT2) [8]. This pattern suggests that lactylation plays a crucial regulatory role in oxidative metabolism and endurance adaptation in type I muscle fibers.

In contrast, type II muscle fibers exhibit a characteristic glycolytic metabolic profile, characterized by high glycolytic enzyme activity, fewer mitochondria, and a primary dependence on anaerobic glycolysis for energy production [48]. These fibers can generate substantial lactate and rapidly export it via monocarboxylate transporter 4 (MCT4) [47]. As a result, type II fibers serve primarily as lactate “producers” and demonstrate relatively low levels of protein lactylation. Huang [7] et al. found that lactate levels were significantly elevated in the mouse soleus muscle, which is predominantly composed of oxidative fibers, at 24 h after an acute high-intensity interval training session. No comparable increase was detected in the gastrocnemius muscle, which is enriched with glycolytic fibers. Notably, other studies have indicated that neither acute nor chronic resistance or endurance training alter global lactylation levels in the human vastus lateralis muscle [49]. The potential influence of factors occurring outside the measured time points on skeletal muscle protein lactylation cannot be fully ruled out. Future human studies incorporating more frequent sampling are warranted to validate these findings. However, due to methodological limitations, it is challenging to assess the differences in fiber type-specific lactylation (type I vs. type II) in human muscle samples. Further investigations using animal models are required to address this. Information regarding key lactylation targets in muscle tissue can be found in

Table 2. Key lactylation site in muscle tissues.

Cell Type	Lactylation Site	Target Gene/ Pathway	Function	Reference
SKMC	PKM2, PKFM	/	Linked to glycolytic metabolism.	[9]
SKMC	PDHA1 K336, CPT2 K457/458	/	Inhibiting acetyl-CoA generation from both glucose and fatty acids.	[8]
SKMC	MT-ATP8, ATP5MG, ATP5PO	/	Suppressing the expression of related proteins to restrict oxidative phosphorylation.	[50]
SKMC	H3K9	Neu2	Driving myoblast differentiation into myotubes.	[51]
Macrophage	H3K18	/	Forecasting muscle regeneration potential based on injury biomarkers.	[52]
SKMC	VPS34 K356/K781	VPS34-BECNI, ATG14, UVRAG	Promoting autophagy and endo-lysosomal degradation.	[53]
SKMC	H3K9	Pds5b, Park7b, Eya1, Nedd4, Ubc, Wwp1	Activating DNA repair and proteostasis pathways to counteract muscle aging.	[11]
HSKMC	/	/	Linked to the development of insulin resistance.	[12]
PASMC	H3K18	Bmp5, Trpc5, Gbe1	Driving the proliferation of PASMCs and vascular remodeling.	[54]
PASMC	H3K18	Gbe1, Pgf, Mt2A, Ythdf2, Gys1	Driving the proliferation of PASMCs.	[55]
PASMC	TOP1, EMILIN-1	Yap/Taz, Akt-mTOR	Promoting PASMCs proliferation and survival.	[56]
VSMC	H3K18	CHI3L1	Inducing osteogenic transdifferentiation of VSMC.	[57]
VSMC	H3K18	Phospho1	Driving vascular calcification and regulating related gene expression.	[58]
VSMC	H4K12	SASP	Driving smooth muscle cell senescence and promoting disease development.	[59]
VSMC	Sox10	Cd74, C3, Lyz2	Inducing macrophage-like phenotypic switching in vascular smooth muscle cells	[60]
Macrophage	H3K18	PDHA, IL-10	Inducing M1 macrophage polarization.	[61]
PASMC	Histone	prelamin A	Elevating IL-6 secretion which drives proliferation of PASMC.	[62],
PASMC	H3K18	IL-1β, IL-6, TNF-α	Attenuating pro-inflammatory phenotypic switching in pulmonary artery smooth muscle cells.	[63]
Cardiomyocyte	PKM2, PKFM	/	Linked to glycolytic metabolism.	[64]
Cardiomyocyte	SCAD, ACAA2	/	Suppressing mitochondrial β-oxidation of fatty acids.	[65]
Cardiomyocyte	HADHA	/	Suppressing mitochondrial β-oxidation of fatty acids.	[66]
Cardiomyocyte	MDH2 K241	/	Participating in the regulation of mitochondrial function and cell death.	[67]
Cardiomyocyte	H4K12	Mex3b, E2f2, Rfc3	Promoting proliferative capacity of cardiomyocytes.	[68]
Cardiomyocyte	H3K18	CyclinD1, Cdk2 P21, P53	Activating the proliferation program in cardiomyocytes.	[69]
Macrophage	H4K12	HIF-1α	Reduced infiltration of HIF-1α-positive and IL-1β-positive inflammatory macrophages in the heart.	[70]
Cardiomyocyte	H3K18	Lrg1, Vegf-a, IL-10	Regulating monocyte–macrophage function; participating in the early and remote activation of genes.	[71]
Endothelial cell	Snail1	TGF-β/Smad2	Activating TGF-β/Smad2pathway.	[32]
Cardiomyocyte	H3K56	/	Supports cardiomyocyte survival under hypoxia/reoxygenation	[72]
Fibroblast	SA3K K351	RISK/SAFE, WNT	Enhancing the protein stability of SA3K and reducing cardiomyocyte apoptosis.	[73]
Cardiomyocyte	ACSL4 K83	/	Inducing lipid peroxidation-driven ferroptosis in cardiomyocytes.	[74]
Cardiomyocyte	GPX4 K21/K228	/	Sensitizing cardiomyocytes to ferroptosis through GPX4 inhibition.	[75]
Cardiomyocyte	NLRP3 K245	GSDMD	Promoting cardiomyocyte pyroptosis and aggravating injury. Mediating injury through the LDHA-NLRP3 pathway.	[76]
Cardiomyocyte	H3K18	YTHDF2	Participating in exercise-induced physiological cardiac hypertrophy and I/R injury repair.	[77]
Cardiomyocyte	α-MHC K1897	Titin	Maintaining sarcomere structure and function.	[78]
Cardiomyocyte	H3K18	/	Driving pathological cardiac hypertrophy.	[79]

.

4.3. Functional Diversity of Lactylation in Muscle Tissue

Lactylation plays a pivotal role in multifaceted and intricate regulatory networks governing energy metabolism in muscle cells. Its influence encompasses three major domains—glycolysis, lipid metabolism, and mitochondrial function—and it facilitates complex regulatory mechanisms under various physiological and pathological conditions.

4.3.1. Lactylation Regulates Glucose and Lipid Energy Metabolism in Muscle Cells

Lactylation provides multilayered, precise control of energy metabolism in muscle cells and regulates glucose metabolism, lipid metabolism, and mitochondrial function through context-dependent mechanisms.

For glucose metabolism, lactylation enhances the glycolytic pathway by modifying key glycolytic enzymes and inducing epigenetic changes. Mechanistically, lactylation at site 678 of phosphofructokinase-M (PFKM) markedly increases its enzymatic activity [80]. Similarly, the K62 site of pyruvate kinase M2 (PKM2) promotes the formation of highly active tetramers that work in concert to amplify glycolytic flux [81]. In muscle cells, lactylation targets both rate-limiting glycolytic enzymes (PFKM and PKM2) and non-rate-limiting enzymes (fructose-bisphosphate aldolase A and phosphoglycerate kinase 1) [9]. The lactylation levels of these enzymes are positively correlated with their protein expression levels, suggesting that lactylation may enhance the stability or activity of glycolytic enzymes [64]. Epigenetically, Research has confirmed that histone H4K12 lactylation can be enriched in the promoter region of the key glycolytic factor PKM2. This modification enhances glycolytic activity and lactate production in microglial cells, thereby establishing a positive feedback loop [82].

The regulation of lipid metabolism by lactylation demonstrates distinct dual-phase characteristics with opposing effects under physiological and pathological conditions. Under physiological conditions, an 8-week high-intensity interval training study revealed a novel metabolic pathway of lipid catabolism by lactylation. The study demonstrated that exercise-induced lactate accumulation triggers lactylation of both fatty acid synthase (FASN) and ATP-citrate lyase (ACLY) in adipose tissue. Fatty acid synthase lactylation directly inhibits its enzymatic activity, thereby curtailing the biosynthesis of its downstream products, including palmitate and triglycerides [83]. In contrast, under pathological conditions such as hypertrophic obstructive cardiomyopathy, lactylation induces metabolic disturbances. Lactylation of short-chain acyl-CoA dehydrogenase (ACADS) and acetyl-CoA acetyltransferase 2 (ACAA2) inhibits their activity, directly impairing myocardial fatty acid β-oxidation [65]. Furthermore, studies in septic heart tissue and lipopolysaccharide-induced cultured cells have shown that the deacetylases Sirtuin 1 and Sirtuin 3 regulate the lactylation of rifunctional enzyme subunit alph (HADHA) protein at lysine 166 and 728. This lactylation modification inhibits HADHA’s enzymatic activity, thereby impairing mitochondrial fatty acid β-oxidation [66].

Lactate modulates mitochondrial metabolism by regulating mitochondrial protein lactylation. At the metabolic entry point, lactate produced under hypoxic conditions is specifically recognized and catalyzed by the lactyltransferase AARS2 within the mitochondrial matrix. AARS2 mediates the lactylation of endogenous PDHA at lysine 336 and exogenously expresses CPT2 at lysines 457/458, this modification inhibits the activity of both enzymes, thereby limiting the substrate availability for oxidative phosphorylation (OXPHOS). The deacetylase SIRT3 can reverse this lactylation to restore OXPHOS activity [8]. Some phenolic acid compounds (e.g., chlorogenic acid and gallic acid) may restore OXPHOS substrate supply by reducing the lactylation levels of these enzymes [84,85]. Within the oxidative pathway, the mitochondrial enzyme malate dehydrogenase 2 (MDH2) catalyzes the reversible conversion of malate to oxaloacetate. Dysfunction of MDH2 disrupts cellular energy supply by causing an accumulation of tricarboxylic acid cycle intermediates [86]. Lactylation of MDH2 at lysine 241 is thought to downregulate its activity, thereby limiting OXPHOS [67]. At the terminal energy production stage, lactylation of multiple mitochondrial ATP synthase subunits (e.g., MT-ATP8, ATP5MG, and ATP5PO) decreases their protein expression; this impairment ultimately compromises the efficiency of OXPHOS [50] (Figure 2).

4.3.2. Lactylation Regulates Muscle Cell Proliferation and Differentiation

Lactylation serves as a key epigenetic mechanism in the regulation of muscle cell proliferation and differentiation.

In skeletal muscle, histone lactylation directly drives the differentiation of myoblasts. During differentiation, the promoter and enhancer regions of numerous genes associated with the terminal differentiation state acquire high levels of H3K18 lactylation. Treatment of myoblasts with lactate leads to upregulated expression of genes marked by specific H3K18 lactylation promoter peaks in myotubes. These genes are critically involved in skeletal muscle development and striated muscle cell differentiation [10]. These results suggest that lactate activates the expression of key genes by increasing histone lactylation in their promoter regions, thereby driving differentiation. Other investigations have shown that H3K9 lactylation accumulates in the enhancer region of Neuraminidase 2 (Neu2). This enrichment competitively inhibits HDAC3, enhances chromatin accessibility, and activates Neu2 transcription. Increased Neu2 expression promotes myoblast differentiation into myotubes and enhances the expression of the differentiation marker myosin heavy chain (MyHC) [51]. Furthermore, the H3K18 lactylation level in macrophages has emerged as a potential biomarker for predicting muscle regenerative capacity [52].

In cardiac muscles, lactylation is closely associated with metabolic transitions during heart development [64]. High glycolytic activity in neonatal cardiomyocytes generates substantial amounts of lactate, driving the lactylation of histones, including H3K18 and H4K12. These modifications promote chromatin accessibility and activate cell cycle and DNA replication genes (e.g., Mex3b, E2f2, and Rfc3), thereby supporting cardiomyocyte proliferation [68]. Specifically, H4K12 lactylation downregulates Vstm5 gene expression, thereby delaying the cell cycle progression. As the heart matures and metabolic patterns shift from glycolysis to fatty acid oxidation, lactylation levels decline along with the proliferative capacity [68]. In addition, studies show that exogenous 12-HEPE supplementation enhanced glycolysis via the Hippo-YAP pathway, subsequently promoting H3K18 lactylation; this modification downregulates the expression of cell cycle inhibition genes (e.g., P21 and P53) and upregulates the expression of pro-proliferative genes, including Cyclin D1 and Cdk2, thereby reactivating the proliferative program in cardiomyocytes [69].

In smooth muscle, lactylation drives pathological vascular remodeling by promoting the abnormal proliferation of smooth muscle cells. In pulmonary arterial hypertension, hypoxia enhances glycolysis through the HIF-1α/PDK signaling axis, leading to increased lactate production. The accumulated lactate induces H3K18 lactylation. This modification enriches the promoter regions of pro-proliferative genes, including Bmp5, Trpc5, and Gbe1, thereby significantly upregulating their transcription and directly driving the proliferation of pulmonary arterial smooth muscle cells (PASMCs) [54]. H3K18 lactylation is also highly enriched in the promoter regions of genes, such as Gbe1, Pgf, and Mt2A, which upregulates their expression and promotes the proliferation of PASMCs [55]. Widespread non-histone lactylation also contributes to this process, for example, hyper-lactylation induces over-accumulation of topoisomerase 1 (TOP1) and deficiency of elastin microfibril interface-located protein 1 (EMILIN-1) in PASMCs, leading to the upregulation of Yap/Taz, Akt-mTOR, TGFβ1, and increased proliferation [56] (Figure 2).

4.3.3. Lactylation Contributes the Adaptation of Skeletal Muscle to Exercise

Lactylation plays a key regulatory role in muscle adaptation to exercise by modulating multiple metabolic pathways and cellular functions.

Moderate-intensity exercise reduces lactate concentrations, thereby decreasing the lactylation of mitochondrial ATP synthase subunits (e.g., MT-ATP8, ATP5MG, and ATP5PO), enhancing oxidative phosphorylation efficiency, and ensuring adequate energy supply [50]. In contrast, during high-intensity exercise, lactate-mediated lactylation of mitochondrial PDHA and CPT2 inhibits their activity, thereby blocking acetyl-CoA production. This restriction of excessive oxidative phosphorylation prevents proton gradient collapse and excessive reactive oxygen species production, thereby protecting muscle cells from oxidative damage [8]. Furthermore, to mitigate post-vigorous-intensity exercise protein denaturation and mitochondrial damage, lactylation of the vacuolar protein sorting 34 (VPS34)-K356/K781 in skeletal muscle promotes autophagy and endolysosomal degradation, clearing damaged organelles and proteins to maintain cellular homeostasis [53].

Although lactate levels increase after exercise, some studies have reported no significant increase in protein lactylation in skeletal muscle after resistance training. For instance, despite a 3.5% increase in human vastus lateralis growth after 6 weeks of resistance training and 43.7% growth in mouse plantaris muscle following a 10-day overload-induced hypertrophy, these muscles showed no significant increase in nuclear or cytoplasmic protein lactylation but elevated blood and muscle lactate concentrations [49]. Potential explanations for this include: (1) exercise-induced lactate concentrations remain within physiological ranges, which are substantially lower than the levels required to induce lactylation in vitro; (2) limited availability of lactyl-CoA in muscle cells restricts its utility as a modification substrate; and (3) direct competition between lactylation and acetylation of lysine residues, as both modifications share regulatory enzymes (e.g., p300/CBP and HDAC1-3) and resistance training enhances acetylation, which may interfere with lactylation [20,49,87].

Collectively, these studies demonstrate that lactate functions not merely as a metabolic byproduct, but as a dynamic and precise signaling molecule. During exercise, it modulates muscle cells in real-time through lactylation. Under high-intensity conditions, lactate acts as a brake by inhibiting key enzymes to prevent energy system overload and oxidative damage, thereby protecting muscle tissue. At moderate intensity, it fine-tunes cellular metabolism by reducing its inhibitory effects on mitochondrial function, thereby enhancing energy production efficiency. Following strenuous exercise, lactate facilitates recovery by promoting autophagic clearance of damaged proteins and organelles. Interestingly, despite repeated elevations in lactate during long-term resistance training, widespread and persistent lactylation of muscle proteins has not been detected. This observation underscores the subtle nature of its regulation: rather than leaving permanent marks, lactylation likely constitutes a transient and precise mechanism that may cooperate or compete with other post-translational modifications, such as acetylation. Thus, the role of lactate in exercise adaptation is evolving from the outdated view of a “fatigue toxin” to a sophisticated signaling molecule central to the body’s intelligent adaptation to physical stress. Future research is needed to further elucidate how this small molecule orchestrates the processes that make muscle tissue stronger and more fatigue-resistant under diverse physiological conditions (Figure 2).

4.4. Central Mechanisms of Lactylation in Muscle Tissue Under Pathological Conditions

Lactylation not only regulates physiological processes such as muscle energy metabolism, exercise adaptation, and cell fate but is also closely linked to pathological conditions, including muscle aging, metabolic myopathies, and cardiovascular diseases.

4.4.1. Lactylation in Muscle Aging

Lactylation also plays a crucial regulatory role in muscle aging. Studies have demonstrated that histone lactylation levels (e.g., H3K9, H3K14, and H3K18) are significantly reduced in aged skeletal muscles (such as the gastrocnemius muscle), which is closely associated with diminished glycolytic activity, decreased lactate production, and reduced NAD⁺ levels. Mechanistically, this decline in lactylation induces epigenetic reprogramming, directly lowering lactylation levels at the promoter regions of genes involved in DNA repair (e.g., Pds5b, Park7, and Eya1) and protein homeostasis (e.g., Nedd4, Ubc, and Wwp1), thereby suppressing their expression; this dysregulation accelerates muscle aging [11]. Studies have demonstrated that both exogenous lactate treatment, which increases lactylation levels, and supplementation with the NAD precursor nicotinamide, a broad-spectrum competitive inhibitor of NAD⁺-dependent Sirtuins, can effectively delay muscle aging. The underlying mechanism involves the modulation of histone acetylation and succinylation, as well as the inhibition of delactylation [11,88,89].

Exercise is an effective strategy for counteracting muscle aging. Compared with sedentary controls, middle-aged mice subjected to a two-month treadmill running regimen (9 m/min, 30 min per session) significantly enhances glycolytic activity in skeletal muscles and increases histone lactylation. This exercise-induced “lactylation reprogramming” reactivates DNA repair and proteostasis genes, alleviating muscle dysfunction. Concurrently, running remodels muscle cellular composition by markedly increasing the proportion of IIb-type myonuclei and muscle stem cells while reducing adipocyte accumulation. This optimized cellular architecture restructures intercellular communication networks and coordinately activates pathways related to glycolysis, oxidative phosphorylation, and myofiber regeneration. Ultimately, these adaptations comprehensively improve muscle metabolic capacity, contractile function, and regenerative potential, thereby effectively counteracting the effects of aging [11,90] (Figure 3).

4.4.2. Lactylation in Metabolic Myopathies

Lactylation exhibits distinct physiological and pathological roles in metabolic regulation, which explains its apparent functional paradox. During acute stimuli, such as exercise, a transient surge in lactate levels enhances insulin sensitivity via the AMPK/PGC-1α/GLUT4 signaling axis; this is accompanied by rapid, short-term protein lactylation, which further promotes insulin sensitivity by augmenting glycolytic flux [43]. In contrast, chronic diseases such as obesity and diabetes induce persistent hyperlactatemia, leading to sustained protein lactylation. This chronic modification suppresses mitochondrial biogenesis genes, resulting in mitochondrial dysfunction and exacerbating insulin resistance [12,43,57]. Therefore, it is the abnormal and sustained elevation of lactate—rather than its transient physiological fluctuations—that serves as a common pathogenic driver of dysfunction in skeletal, cardiac, and vascular smooth muscle.

In skeletal muscles, lactylation is closely associated with insulin resistance and type 2 diabetes. A study involving 15 lean and 14 obese adults revealed significantly higher protein lactylation levels in the skeletal muscles of obese individuals, which positively correlated with the degree of insulin resistance [12]. Under insulin-resistant conditions, intracellular glucose metabolism is dysregulated, preferentially shifting toward lactate production [91]. This may persistently elevate lactylation levels and disrupt glucose metabolic homeostasis in skeletal muscle. Furthermore, the observed decrease in lactylation (e.g., H3K18 lactylation) following metformin treatment, which improves insulin resistance, corroborates its crucial role in this process [92].

The pathogenic effects of lactylation occur in other muscle tissues. In diabetic cardiomyopathy, elevated levels of free fatty acids stimulate cardiomyocytes to produce excess lactate, which is exported via MCT4 into the microenvironment. This promotes H4K12 lactylation in cardiac macrophages. MCT4 inhibition reverses cardiac macrophage H4K12 lactylation and alleviates cardiac inflammation [70]. Lactylation directly promotes pathological vascular calcification in diabetes. Under high-glucose conditions, global histone lactylation levels (e.g., H3K18 and H3K14) increase in muscle cells. Specifically, H3K18 lactylation upregulates chitinase-3-like protein 1 (CHI3L1) expression, activating the IL-13Rα2/JAK1/STAT3 signaling pathway and driving the transition of vascular smooth muscle cells (VSMCs) to an osteogenic phenotype [57]. Furthermore, H3K18 lactylation enrichment in the phosphatase 1 (Phospho1) promoter upregulates its expression and accelerates arterial calcification [58] (Figure 3).

4.4.3. Lactylation in Cardiovascular Diseases

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Atherosclerosis: The progression of atherosclerosis is closely linked to metabolic dysregulation and phenotypic changes in VSMCs and macrophages [93]. In VSMCs, cellular senescence is a key driver of atherosclerosis [94]. Senescent VSMCs undergo a metabolic shift from oxidative phosphorylation to aerobic glycolysis. The mitochondrial protein tumor necrosis factor receptor-associated protein 1 (TRAP1) is overexpressed in senescent VSMCs, which promotes glycolysis and suppresses the tricarboxylic acid cycle. Conversely, TRAP1 knockout reduces lactate levels and histone H4 lactylation. Further investigations have revealed that TRAP1 promotes H4K12 lactylation by downregulating HDAC3 via lactate, thereby activating senescence-associated secretory phenotype (SASP) transcription and accelerating VSMC senescence and atherosclerosis progression [59]. In advanced atherosclerotic plaques, chronic inflammation induces the transdifferentiation of some VSMCs into macrophage-like cells, a process associated with sex-determining region Y (SRY)-related HMG-box gene 10 (Sox10) lactylation, which exacerbates intimal inflammation and promotes vulnerable plaque formation [60].

However, lactylation can promote anti-inflammatory effects in macrophages. H3K18 lactylation activates the transcription of the reparative genes Interleukin-10 (IL-10) and pyruvate metabolic gene pyruvate dehydrogenase alpha (PDHA), promoting the transition from pro-inflammatory M1 to anti-inflammatory M2 macrophages, thereby attenuating atherosclerosis progression [61]. MCT4 plays a critical role in this process; its deficiency leads to intracellular lactate accumulation, which enhances H3K18 lactylation and facilitates macrophage polarization [61].

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Myocardial Infarction: Myocardial infarction (MI), as a severe clinical manifestation of atherosclerosis, is characterized by coronary artery occlusion that drastically reduces myocardial blood supply and impairs peripheral organ perfusion. This condition frequently induces hyperlactatemia, a metabolic disorder closely associated with progressive myocardial damage [95]. During the early phase of MI (day 1), lactate promotes H3K18 lactylation in monocytes and macrophages, thereby enhancing the transcription of repair-related genes (e.g., Lrg1, Vegf-α, and IL-10). Through their anti-inflammatory and pro-angiogenic activities, these genes foster a microenvironment conducive to tissue repair. Elevated H3K18 lactylation suppresses detrimental inflammation and improves cardiac function post-MI [71]. However, lactate is not entirely beneficial. In the later stage of MI (day 6), it may also promote Snail family transcriptional repressor 1 (Snail1) lactylation, activating the TGF-β/Smad2 signaling pathway and driving endothelial–mesenchymal transition (EndMT), which ultimately exacerbates myocardial fibrosis [32].

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Myocardial Ischemia-Reperfusion Injury: Myocardial ischemia-reperfusion injury (MIRI) is a secondary injury that occurs after blood flow restoration after post-myocardial infarction [96]. During MIRI, the cardiac energy metabolism shifts toward glycolysis, resulting in ATP depletion and lactate accumulation [97]. This subsequently induces protein lactylation, which exerts complex effects on cardiomyocyte survival. On the one hand, lactylation exhibits protective roles; heat shock protein A12A supports cardiomyocyte survival under hypoxia/reoxygenation by maintaining histone H3 lactylation and aerobic glycolytic homeostasis, and its deficiency exacerbates cardiac dysfunction [72]. Fibroblast-derived lactylated Serpina3k (SA3K) inhibits the WNT signaling pathway and activates the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) signaling pathways, reducing cardiomyocyte apoptosis [73].

In contrast, lactylation promotes injury progression. During MIRI, lactate induces the lactylation of the key ferroptosis regulators acyl-CoA synthetase long chain family member 4 (ACSL4)-K83 and glutathione peroxidase 4 (GPX4)-K21/K228, thereby enhancing and reducing their stability, respectively, which collectively exacerbate lipid peroxidation and ferroptosis [74,75]. MDH2-K24 lactylation impairs mitochondrial function, creating a favorable environment for cardiomyocyte ferroptosis [67]. Additionally, lactate promotes NLR family pyrin domain containing 3 (NLRP3)-K245 lactylation, facilitating inflammasome assembly and activation to induce cardiomyocyte death [76]. Furthermore, lactate can promote cardiomyocyte death by improving H3K18 lactylation levels and elevating intracellular N6-methyladenosine RNA-binding protein YTHDF2 [77].

In summary, lactylation plays a dual role in MIRI; under specific conditions, it confers cardioprotection by maintaining glycolytic homeostasis and activating protective signaling pathways, whereas in other contexts, it exacerbates injury by promoting ferroptosis and inflammatory responses. These contradictory roles likely stem from the differential modulation of distinct target proteins, coupled with their cell type-specific functions and molecular context, highlighting the complexity and context-dependent nature of MIRI pathogenesis.

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Heart Failure: Lactylation exerts dual regulatory effects in heart failure development, with its impact being highly target- and stage-specific. During heart failure progression, metabolic reprogramming increases lactate efflux and reduces intracellular lactate levels, consequently diminishing the lactylation of key proteins [98]. In both murine models and human patients, decreased lactylation at K1897 of α-myosin heavy chain (α-MHC) impairs its interaction with titin, compromising sarcomeric integrity and reducing contractility. Moreover, this modification deficiency upregulates the expression of the fibrotic markers alpha-smooth muscle actin and type I collagen, further deteriorating cardiac function. Experimentally, restoring α-MHC-K1897 lactylation by elevating lactate concentration or inhibiting lactate efflux effectively improves cardiac performance [78].

However, lactylation results in complex cardiac effects. In a mouse model of cardiac hypertrophy, elevated H3K18 lactylation levels in cardiomyocytes promoted pathological hypertrophy, whereas decreased H3K18 lactylation levels significantly attenuated this condition [79]. This indicates that the biological effects of lactylation vary substantially depending on the specific modification site and pathological stage. Collectively, these findings suggest that precise modulation of site-specific lactylation may offer novel metabolic intervention strategies for heart failure treatment.

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Pulmonary Hypertension: In pulmonary hypertension, lactylation contributes to pulmonary vascular remodeling by directly promoting smooth muscle proliferation and regulating cellular senescence and inflammatory phenotypes [54,55,56]. Lactylation modulates the senescence of PASMCs in pulmonary hypertension models. The accumulation of the senescence-associated prelamin A and the subsequent increase in Interleukin-6 (IL-6) secretion collectively create a microenvironment that promotes PASMC proliferation, and histone lactylation is implicated in driving this process. Lactylation plays a critical role in regulating inflammation [62]. The long non-coding RNA UNC5B-AS1 remodels cellular metabolism by suppressing glycolysis and enhancing oxidative phosphorylation, thereby reducing lactate levels and H3K18 lactylation enrichment at the pro-inflammatory genes, such as IL-1β, IL-6, and TNF-α. This mechanism attenuates the transition of PASMCs to a pro-inflammatory phenotype and inhibits the formation of a pro-inflammatory vascular microenvironment [63].

These findings expand our understanding of lactylation in PH, revealing its novel regulatory roles in cellular senescence and inflammation as well as highlighting potential therapeutic targets for PH treatment strategies focused on metabolic-epigenetic crosstalk (Figure 3).

5. Novel Strategies Targeting Lactylation for Muscular Disorders

Dysregulated lactylation is implicated in various pathological processes, including muscle aging, metabolic myopathies, and cardiovascular diseases, making the targeted modulation of lactylation a promising therapeutic strategy. The dynamic balance of lactylation in muscle tissue is maintained by specific “writer” and “eraser” enzymes: acetyltransferases (e.g., KAT2A, P300, and CBP) catalyze lactylation [20], while de-modifying enzymes (e.g., HDAC1-3 and SIRT1-3) remove these modifications [26].

To date, various small molecule compounds that selectively target these modifying enzymes have been identified. For instance, C646, A-485, and curcumin inhibit P300/CBP activity [78,99]; trichostatin A and suberoylanilide hydroxamic acid suppress HDAC1-3, and nicotinamide blocks the delactylation function of SIRT1-3 [13,71]. Therefore they serve as valuable molecular tools for modulating lactylation levels. Alternatively, targeting the upstream lactate production pathways has gained significant research attention. LDHA inhibitors (e.g., FX-11, GSK2837808A, and vitamin C) directly block lactate synthesis, whereas compounds such as stiripentol and gossypetin function as broad-spectrum dual LDH inhibitors [58,95]. Moreover, 2-deoxyglucose and lonidamine indirectly reduce lactate production by inhibiting hexokinase, whereas dichloroacetate reduces lactate levels by promoting pyruvate entry into the tricarboxylic acid cycle [12,100].

These interventions modulate lactylation through multiple mechanisms, thereby opening new therapeutic avenues for muscular disorders via metabolic regulation.

6. Conclusions and Future Perspectives

This review systematically examined the central role and regulatory mechanisms of lactylation in muscle physiology and pathology. As an emerging metabolic sensing mechanism, lactylation bridges glycolytic flux through gene transcription, protein function, and cell fate determination. Under physiological conditions, lactylation regulates the energy metabolism, proliferation, and adaptation of muscle cells, whereas dysregulated lactylation directly contributes to the pathogenesis of muscle aging, metabolic myopathies, and cardiovascular diseases.

Although significant progress has been achieved in this field, several challenges remain unresolved. First, lactylation exhibits dual—sometimes opposing—effects across different muscle types and disease stages, indicating high context-dependency. Future studies should employ single-cell sequencing and site-specific modification analyses to map the functional landscape of lactylation precisely. Second, the identification and functional understanding of lactylation “reader” proteins are still in their infancy and represent a key direction for mechanistic research. Finally, although existing targeting strategies (e.g., modifier enzyme inhibitors and lactate production inhibitors) have shown conceptual promise, their tissue specificity, temporal efficacy, and potential off-target effects require further evaluation using preclinical models.