Abstract Myocardial ischemia‐reperfusion injury (MIRI) significantly worsens the outcomes of patients with cardiovascular diseases. Dexmedetomidine (Dex) is recognized for its cardioprotective properties, but the related mechanisms, especially regarding metabolic reprogramming, have not been fully clarified. A total of 60 patients with heart valve disease are randomly assigned to Dex or control group. Blood samples are collected to analyze cardiac injury biomarkers and metabolomics. In vivo and vitro rat models of MIRI are utilized to assess the effects of Dex on cardiac function, lactate production, and mitochondrial function. It is found that postoperative CK‐MB and cTNT levels are significantly lower in the Dex group. Metabolomics reveals that Dex regulates metabolic reprogramming and reduces lactate level. In Dex‐treated rats, the myocardial infarction area is reduced, and myocardial contractility is improved. Dex inhibits glycolysis, reduces lactate, and improves mitochondrial function following MIRI. Lactylation proteomics identifies that Dex reduces the lactylation of Malate Dehydrogenase 2（MDH2), thus alleviating myocardial injury. Further studies reveal that MDH2 lactylation induces ferroptosis, leading to MIRI by impairing mitochondrial function. Mechanistic analyses reveal that Dex upregulates Nuclear Receptor Subfamily 3 Group C Member 1（NR3C1) phosphorylation, downregulates Pyruvate Dehydrogenase Kinase 4 (PDK4), and reduces lactate production and MDH2 lactylation. These findings provide new therapeutic targets and mechanisms for the treatment for MIRI. Keywords: dexmedetomidine, ferroptosis, lactylation, metabolic reprogramming, myocardial ischemia‐reperfusion injury __________________________________________________________________ Dex reduces lactate levels and downregulates MDH2 lactylation to enhance mitochondrial function and prevent ferroptosis, ultimately alleviating myocardial ischemia‐reperfusion injury. This mechanism involves through Dex facilitating the phosphorylation and nuclear export of NR3C1, leading to the suppression of PDK4 and influencing metabolic reprogramming. graphic file with name ADVS-11-2409499-g008.jpg 1. Introduction Ischemic heart disease is a leading cause of death worldwide, accounting for 16% of total mortality. The number of deaths caused by this disease increased from 2 million in 2000 to 8.9 million in 2019.^[ [56]^1 ^] While reperfusion is essential for preventing cardiac function deterioration and improving clinical outcomes, restoring blood flow through thrombolysis or revascularization may result in severe myocardial ischemia/reperfusion injury (MIRI),^[ [57]^1 ^] which accounts for up to 50% of the final myocardial infarction area.^[ [58]^2 ^] Additionally, MIRI is a common complication in cardiopulmonary bypass and a critical factor leading to perioperative myocardial injury.^[ [59]^3 ^] The pathophysiology of MIRI is complex, involving oxidative stress, inflammatory responses, and various forms of cell death.^[ [60]^4 , [61]^5 , [62]^6 ^] Although significant theoretical breakthroughs have emerged in treating MIRI^[ [63]^7 ^]over the past few decades, practical challenges remain in the applicability, safety, and efficacy of these treatments. Therefore, further research to identify measures for the prevention and treatment of MIRI is of significant clinical importance. The heart is a critical organ with high energy consumption.^[ [64]^8 ^] Under normal physiological conditions, the heart relies on oxidative phosphorylation to produce ATP to meet its high energy demands.^[ [65]^9 ^] During myocardial ischemia‐reperfusion (I/R), the regular oxidative phosphorylation is inhibited, and glycolysis becomes the primary energy source. This metabolic shift leads to a rapid decrease in ATP production and an accumulation of lactate, which causes cellular damage.^[ [66]^10 ^] Studies have shown that lactate is not only a metabolic byproduct, but it also regulates gene expression through protein lactylation.^[ [67]^11 ^] Recent research indicated that lactate can limit mitochondrial oxidative phosphorylation via protein lactylation.^[ [68]^12 ^] However, the role and related mechanism of lactate as well as lactylation in the progression of MIRI remain unclear. Dexmedetomidine (Dex) is a novel, highly selective α2‐adrenergic receptor agonist that has been widely used in clinical anesthesia and intensive care in recent years.^[ [69]^13 , [70]^14 ^] Its unique pharmacological properties extend beyond sedation, analgesia, and anxiolysis to provide significant cardiovascular protection.^[ [71]^15 , [72]^16 ^] For instance, perioperative administration of Dex has been shown to effectively reduce postoperative mortality and shorten hospital stay of patients after coronary artery bypass graft surgery.^[ [73]^17 ^] A high‐quality meta‐analysis involving 48 trials and 6273 participants also reported that perioperative use of Dex reduced short‐term mortality after cardiac surgery.^[ [74]^18 ^] Additionally, basic research indicates that Dex plays a crucial role in mitigating ischemia‐reperfusion injury, exhibiting antioxidative and anti‐inflammatory effects.^[ [75]^15 , [76]^19 ^] Our previous study has found that Dex can alleviate vascular leakage by inhibiting ferroptosis in vascular endothelial cells through metabolic reprogramming.^[ [77]^20 ^] However, it remains unclear whether Dex confers myocardial protection against I/R injury through metabolic remodeling. In this study, we employed multi‐omics techniques to explore the role and mechanism of Dex in regulating myocardial metabolic reprogramming and alleviating MIRI. This research specifically focused on the role of lactate and lactylation, in hope to provide new insights for the prevention and treatment of MIRI. 2. Results 2.1. Dex reduced the Lactate Level of Cardiopulmonary Bypass Surgery Patients by Metabolic Reprogramming The temporarily interrupted blood supply for heart in cardiopulmonary bypass (CPB) surgery, could cause potential myocardial ischemia‐reperfusion injury (MIRI). Therefore, we included 60 patients diagnosed with heart valve disease and who underwent valve replacement surgery under CPB. The 60 patients were randomly divided into the Dex group and the Con group in a 1:1 ratio (The baseline information and the status of regular medications for the two groups of patients were shown in Table [78]S2, Supporting Information). We collected venous blood both preoperatively (pre) and postoperatively (post) to measure the cardiac injury biomarkers CK‐MB and C‐TNT, as well as metabolomics (Figure [79]1A). Results showed that the level of CK‐MB and C‐TNT in both Con and Dex group was low preoperatively (with no statistical difference between groups) but increased significantly postoperatively (Figure [80]1B,C). However, postoperative levels of CK‐MB and C‐TNT were significantly lower in the Dex group compared to the Con group (Figure [81]1B,C), suggesting that Dex effectively reduced MIRI. Figure 1. Figure 1 [82]Open in a new tab Dex regulated the lactate level of patients undergoing cardiopulmonary bypass surgery by metabolic reprogramming. A) Flowchart for collecting blood samples to measure cardiac injury biomarkers and metabolomics. B) Comparison of CK‐MB in the Con group and Dex group during the preoperative stage (pre) and the post‐operative stage (post) (n = 30 samples each group). C) Comparison of C‐TNT in the Con group and Dex group during the preoperative stage (pre) and the post‐operative stage (post) (n = 30 samples each group). D) Principal Component Analysis (PCA) of the difference in metabolic patterns between the Con‐pre and Con‐post groups. E) PCA of the difference in metabolic patterns between the Dex‐post and Con‐post groups. F,G) KEGG pathway enrichment analysis for differentially expressed (DE) metabolites. H,I) Volcano plot of DE‐metabolites, each dot represented a metabolite, blue indicated down‐regulated DE‐metabolites, red indicated up‐regulated, and gray indicated no significant difference. J,K) Box plots showing changes in levels of L‐lactic acid in different groups. L,M) Box plots showing changes in levels of D‐lactic acid in different groups. N,O) ROC curves evaluating the diagnostic capacity of L‐lactic acid and D‐lactic acid in different groups. a: p < 0.05, as compared with the Con‐pre group; b: p < 0.05, as compared with the Dex‐pre group; c: p < 0.05, as compared with the Con‐post group. Con‐pre: Con‐pre group; Con‐post: Con‐post group; Dex‐pre: Dex‐pre group; Dex‐post: Dex‐post group. Metabolomics was conducted to explore whether Dex conferred cardioprotective effects by modulating metabolic reprogramming. Principal Component Analysis (PCA) results indicated significant metabolic differences before and after surgery in both Con and Dex group (Figure [83]1D,E). We identified 232 differentially expressed (DE) metabolites between Con‐pre and Con‐post groups, enriched in Glycolysis/Gluconeogenesis, TCA cycle, Pyruvate metabolism, Ferroptosis, and Glutathione metabolism pathways. Similarly, there were 247 DE‐metabolites between Con‐post and Dex‐post groups, enriched in the same metabolic pathways (criteria for screening DE‐metabolites: VIP > 1 and P‐value < 0.05) (Figure [84]1F,G), suggesting that glycolysis activation and ferroptosis might be critical causes of MIRI, and the mechanism of Dex reducing MIRI might be related to regulating glycolysis and TCA cycle reprogramming and inhibiting ferroptosis. The metabolomics showed that the level of lactic acid significantly increased on the third day after surgery, while the postoperative lactic acid level in the Dex group was significantly lower than that in the Con group (Figure [85]1H,M). This finding indicated that Dex might influence lactic acid levels through metabolic reprogramming. In addition, the ROC curve results showed that lactic acid had a good discriminatory ability for preoperative and postoperative, as well as for Dex‐post and Con‐post. These results suggested that lactic acid might be a potential biomarker for MIRI (Figure [86]1N,O). 2.2. Dex Improved Mitochondrial Function and Reduced MIRI by Downregulating Lactate Level We constructed a rat model of MIRI and observed the protective effects of Dex. The results showed that compared to the Con group, I/R injury significantly impaired cardiac function, manifesting as a significant increase in myocardial infarction area (Figure [87]2A,B), a marked decrease in myocardial contractility, and a reduction in LVEF (Figure [88]2C–E). However, compared to the I/R group, Dex treatment effectively alleviated MIRI, as evidenced by a reduction in infarction area, increased myocardial contractility, and elevated LVEF (Figure [89]2A–E). Additionally, we used OGD/R to establish an I/R model at the cellular level and analyzed the effects of OGD/R and Dex treatment on metabolic reprogramming in cardiomyocytes. The results showed that OGD/R led to significant increases in glycolysis rate and reductions in mitochondrial oxygen consumption rate (OCR), which were all relieved by Dex treatment (Figure [90]2F,G). Additionally, we found that MIRI increased the lactate level in cardiomyocytes, whereas Dex effectively inhibited lactate production (Figure [91]2H). Figure 2. Figure 2 [92]Open in a new tab Dex enhanced mitochondrial function and mitigated MIRI by reducing lactate level. A,B) Evans Blue‐TTC staining was used to detect the impact of I/R and Dex on the myocardial infarction area in rats (n = 6 rats each group). The left ventricular area (LV), area at risk (AAR), and infarct area (IA) were calculated. AAR/LV (%) indicated ischemic region size, while IA/AAR (%) indicated infarcted region size. C) Cardiomyocyte contraction curves were used to reflect the impact of Dex on myocardial contractility in rats (n = 6 rats each group). D,E) Echocardiography was used to detect rats LVEF (n = 6 rats each group). F) ECAR was used to reflect the glycolysis rate (n = 3 independent experiments). G) Seahorse was used to detect mitochondrial OCR (n = 3 independent experiments). H) Lactate levels were measured in rats (n = 6 rats each group) and cells (n = 3 independent experiments). I) Transmission electron microscopy was used to observe the structure of myocardial mitochondria (bar = 1µm) (n = 6 rats each group). J) Confocal microscopy was used to observe mitochondrial morphology of H9c2 cells (each group randomly selected 30 cells and the mitochondrial morphology was blindly scored and classified into two categories: Long (>6µm), Short (≤3µm) (bar = 15µm) (n = 3 independent experiments)). K,L) JC‐1 and DCFH‐DA were used to detect mitochondrial membrane potential (bar = 15µm) and ROS (bar = 50µm) respectively (n = 3 independent experiments). M) The effect of Dex on ATP levels in OGD/R‐treated cells (n = 3 independent experiments). N,O) Evans Blue‐TTC staining was used to detect the effect of exogenous lactate on myocardial infarction area (n = 6 rats each group). The effect of exogenous lactate on P) cardiomyocytes contractility and Q,R) LVEF (n = 6 rats each group). S,T) The effect of exogenous lactate on mitochondrial morphology (bar = 15µm) (n = 3 independent experiments). U,V) The effect of exogenous lactate on ROS (bar = 50µm) and mitochondrial membrane potential of H9c2 cells (bar = 15µm) (n = 3 independent experiments). The effect of exogenous lactate on W) mitochondrial OCR and X) ATP levels (n = 3 independent experiments) of H9c2 cells. a: p < 0.05, as compared with the Con or Nor group; b: p < 0.05, as compared with the I/R or OGD/R group; c: p < 0.05, as compared with the Dex group. Con: control group; Nor: normal group; I/R: I/R group; OGD/R: OGD/R group; Dex: Dex‐treated I/R or OGD/R group; Dex + Lac: Dex + Lac treated I/R or OGD/R group. Furthermore, we examined the effects of OGD/R and Dex treatment on the structure and function of myocardial mitochondria. Transmission electron microscopy and confocal microscopy results showed that compared to the Con (Nor) group, I/R (OGD/R) caused abnormal mitochondrial structures, characterized by increased mitochondrial vacuolization, cristae disruption, and mitochondrial fragmentation, whereas Dex effectively improved mitochondrial morphology and reduced mitochondrial fragmentation (Figure [93]2I,J). Moreover, OGD/R induced mitochondrial dysfunction in cardiomyocytes, manifested by decreased mitochondrial membrane potential, increased reactive oxygen species (ROS) production, and reduced ATP levels, which were all improved by Dex treatment (Figure [94]2K–M). These results suggested that the protective effects of Dex after MIRI might be related to regulating metabolic reprogramming, inhibiting lactate production, and improving mitochondrial function. To further verify the role of lactate in this process, we pretreated cells and rats by adding exogenous lactate (sodium lactate, Lac) to directly upregulate lactate levels.^[ [95]^21 ^] The results showed that lactate concentrations ≥10 mm significantly reduced the viability of cardiomyocytes (Figure [96]S1, Supporting Information). Additionally, we found that lactate administration effectively counteracted the protective effects of Dex on myocardial injury in I/R rats. Compared to the Dex group, the Dex + Lac showed a significant increase in infarct size, reduced myocardial contractility, and lower LVEF (Figure [97]2N–R). Similarly, lactate administration counteracted the protective effects of Dex on the structure and function of mitochondria in OGD/R‐treated cells. Compared to the Dex group, the Dex + Lac increased mitochondrial fragmentation, decreased mitochondrial membrane potential, elevated ROS production, inhibited mitochondrial OCR, and reduced ATP production (Figure [98]2S–X). These results indicated Dex could improve mitochondrial function and reduce MIRI by downregulating lactate level. 2.3. Dex Downregulated the Lactylation of MDH2 Lactate regulated cell functions by affecting protein lysine lactylation (Kla). Therefore, we collected myocardial tissues from I/R and Dex rats and conducted lactylation proteomics (Figure [99]3A). A total of 1026 lactylation sites across 238 proteins were identified, among them, 731 lactylation sites from 167 proteins were quantified. The number of Kla sites per protein is shown in Figure [100]3B. Next, we used the iceLogo tool to analyze the amino acids surrounding the identified Kla sites against all human background sequences. Significant enrichment Alanine and Aspartate were found at the −1 and +1 positions of the Kla sites (Figure [101]3C). A volcano plot of the differential analysis showed that, compared to the I/R group, 446 lactylation‐modified peptides were significantly upregulated, but 554 lactylation‐modified peptides were significantly downregulated in the Dex group (Figure [102]3D). GSEA pathway enrichment analysis of the differential proteins revealed that the TCA cycle pathway was significantly enriched (Figure [103]3E), suggesting that Dex might regulate the lactylation of TCA cycle‐related proteins. Using Protein‐Protein Interaction (PPI) interaction analysis to score and screen key proteins, we found that malate dehydrogenase 2 (MDH2) was the hub protein among the identified Kla proteins (Figure [104]3F). MDH2 is a critical enzyme located in the mitochondrial matrix and plays an essential role in the TCA cycle.^[ [105]^22 ^] Immunofluorescence and WB analyses were used to confirm that MDH2 is localized in the mitochondria (Figure [106]S2A,B, Supporting Information). Lactylation proteomics analysis revealed that the Lys241 (K241) site of MDH2 was significantly downregulated in the Dex group compared to the I/R group (Figure [107]3G–I). MDH2 K241 was found to be highly conserved among different species (Figure [108]3J). These results indicated that Dex could downregulate the lactylation of MDH2 in MIRI. Figure 3. Figure 3 [109]Open in a new tab Lactylation proteomics indicated that Dex reduced the lactylation of MDH2. A) Workflow of the strategy for lactylation proteomics analysis. B) Statistical distribution of lactylation sites. C) Icelogo representation displaying flanking sequence preferences for all Kla