Ethyl Acetylsalicylate (E-ASA) Protects Against Myocardial Ischemia-Reperfusion Injury

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This study aimed to evaluate the cardioprotective effects of aspirin (ASA) and to identify aspirin ester derivatives with improved efficacy and safety profiles. Methods The cardioprotective potential of ASA was validated in established animal models of myocardial IRI. Multiple aspirin ester derivatives were comparatively analyzed and screened based on their structural and physicochemical characteristics. Among these compounds, ethyl acetylsalicylate (E‑ASA) was identified as exhibiting higher cell membrane permeability and a lower bleeding risk than ASA. The cardioprotective mechanisms of E‑ASA were further investigated using acetylation proteomics and metabolic pathway analysis in myocardial tissue. Results E‑ASA treatment significantly reduced myocardial infarct size and preserved cardiac function following IRI compared with ASA. Proteomic analyses revealed that E‑ASA induced hyperacetylation of key metabolic enzymes involved in cardiac energy metabolism, suggesting that its cardioprotective effects may be mediated through modulation of metabolic remodeling. Acute high‑dose administration of E‑ASA also provided stronger cardioprotection than equimolar doses of ASA in both cellular and in vivo models. Conclusion E‑ASA demonstrated improved cardioprotective effects and a lower bleeding tendency compared with aspirin in preclinical models. These findings suggest that E‑ASA may represent a potential therapeutic lead requiring further validation for the prevention and treatment of ischemic heart disease. myocardial ischemia-reperfusion aspirin derivative ethyl acetylsalicylate cardiac metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Ischemic heart disease is one of the leading causes of death global(Vos et al. 2020 ). For patients with myocardial ischemia, restoring blood flow through thrombolysis or percutaneous coronary intervention is the standard therapy recommended by current treatment guidelines(Ibanez et al. 2018 ). However, the restoration of blood flow can exacerbate myocardial cell damage and death, leading to ischemia-reperfusion (I/R) injury (Yellon and Hausenloy 2007 ). As a common complication, myocardial ischemia-reperfusion injury is characterized by high morbidity and mortality rates. Its physiological mechanism is complex, involving multiple processes including oxidative stress, calcium overload, and inflammatory responses, and there is still a lack of effective therapeutic approaches(Hausenloy et al. 2019 ). Aspirin serves as a secondary prevention medication for cardiovascular and cerebrovascular diseases. It works by irreversibly acetylating cyclooxygenase-1 (COX-1) at serine 530, inhibiting platelet aggregation and thus exerting antithrombotic effects(Loll et al. 1995 ). Research has shown that aspirin has certain effects in inhibiting myocardial inflammation(Dong et al. 2022 ), reducing myocardial fibrosis(Liu et al. 2017 ), and alleviating myocardial ischemia-reperfusion injury(Seth et al. 1994 ).However, the mechanism of aspirin's cardioprotective effects has not yet been clearly elucidated. As a secondary prevention medication, long-term use of aspirin significantly increases patients' risk of bleeding(Antithrombotic Trialists et al. 2009) and damage to the gastrointestinal tract(Lanas et al. 2011 ), liver, and kidneys(Zheng and Roddick 2019). Therefore, many studies have attempted to develop aspirin analogues or prodrugs(Willetts and Foley 2020 ) to reduce its adverse drug reactions or find alternatives or adjuvants with better therapeutic effects(Wood et al. 1962 ). In animal models, we first confirmed the cardioprotective effects of aspirin in myocardial ischemia-reperfusion models, and we explored an aspirin derivative - ethyl acetylsalicylate (E-ASA), which is esterified at the carboxyl group of aspirin. Related studies have shown that the carboxyl group of aspirin may be associated with its bleeding risk(Killackey et al. 1982 ). Drug esterification is a common derivatization strategy, with advantages typically including improved drug absorption, reduced toxicity, enhanced sustained activation capability, and facilitation of targeted drug delivery(Zhou 2024 ). Based on clinical application needs, aspirin still has certain limitations in terms of drug safety and efficacy. Therefore, we also explored the therapeutic potential of E-ASA in myocardial ischemia-reperfusion models and conducted preliminary investigations into its safety, time effectiveness, and dosage. Result Chronic feeding mice with aspirin protects myocardium from I/R injury. Aspirin, with its century-long history, has been extensively utilized in clinical practice for the prevention of ischemic heart disease (Patrono et al. 2005 ). However, the effects of aspirin on the myocardial I/R injury are less well understood. Toward this aim, we established a mouse model of myocardial I/R injury (45 minutes of ischemia followed by reperfusion) according to established protocols (Lindsey et al. 2018 ). The mice were fed with 0.15 mg/ml aspirin-containing drinking water for 3 weeks, followed by I/R, and then the myocardial I/R injury was assessed 24 h after reperfusion (Fig. 1 a). The dosage of chronic aspirin treatment in mice was close to the daily dose of 100 mg aspirin for secondary prophylaxis of myocardial infarction (MI) in adult humans (Reagan-Shaw et al. 2008 ) (also see Methods on dosage conversion between human and mice). Strikingly, chronic feeding of aspirin significantly reduced the myocardial infarction areas in mice indicated by the Evans blue and TTC staining (Fig. 1 b-d). Plasma creatine kinase - myocardial band isoenzyme (CK-MB) and aspartate aminotransferase (AST) are commonly used diagnostic markers for myocardial damage, and their levels are correlated with the extent of myocardial death (Nigam 2007 ). Likewise, we found that the plasma levels of CK-MB and AST were significantly increased in the I/R mice 24 h after reperfusion compared with the sham group (Fig. 1 e, f). However, aspirin-treated I/R mice showed lower plasma levels of CK-MB and AST than saline-treated I/R mice (vehicle) 24 h after reperfusion (Fig. 1 e, f). Next, we assessed the cardiac function of the I/R mice 24 h after reperfusion through echocardiographic (ECG) recording. The I/R mice showed a significant reduction in the ejection fraction (EF) and fractional shortening (FS) compared with the sham group (Fig. 1 g-i). Intriguingly, aspirin-treated I/R mice showed significant improvements in both EF and FS% 24 h after reperfusion, compared with saline-treated I/R mice (Fig. 1 g-i). Taken together, these results demonstrate that chronic treatment with aspirin before MI may protect myocardial I/R injury. Acute treatment with aspirin after MI shows minor protection against myocardial I/R injury in mice. In the following study, we sought to determine whether acute treatment with aspirin after MI can protect myocardial from I/R injury in mice. To this end, we performed intraperitoneal (i.p.) injection of 50 mg/kg aspirin 30 min after MI (i.e., 15 min before reperfusion), and then the myocardial I/R injury was assessed 24 h after reperfusion (Fig. 2 a). The dosage of acute aspirin treatment in mice was close to the high dose of 300 mg aspirin administrated right after MI in adult patients (Ganjehei and Becker 2015 ) (also see Methods on dosage conversion between human and mice). Different from the results of chronic feeding with aspirin (Fig. 1 ), acute treatment with aspirin 30 min after MI did not reduce the myocardial infarction areas (Fig. 2 g-i) or decrease the plasma levels of CK-MB and AST (Fig. 2 e and f) in the I/R mice 24 h after reperfusion. In contrast, acute treatment with aspirin 30 min after MI led to significant increases in both EF and FS 24 h after reperfusion (Fig. 2 b-d), indicating an improvement on cardiac functions. Taken together, these results demonstrate that acute treatment with aspirin after MI has minor protection against myocardial I/R injury in mice. E-ASA shows improved cell membrane permeability than aspirin Aspirin has a poor cell membrane permeability due to the presence of a free carboxyl group (Menichetti et al. 2019 ), which may lower the intracellular concentrations of aspirin and thus restrict the protective effects of acute aspirin treatment against myocardial I/R injury. Esterification of the carboxyl group can neutralize its negative charge under physiological pH conditions and thus enhance the cell membrane permeability of aspirin. Conversion of the carboxyl group into an ester typically increases membrane permeability, thereby enhancing pharmacological efficacy(Stella et al. 1985 ; Rautio et al. 2008 ). Accordingly, esterification of aspirin yields its alkyl esters—methyl (M‑ASA), ethyl (E‑ASA), and propyl acetylsalicylate (P‑ASA) (Fig. 3 a). To test this hypothesis, we analyzed the permeability across cell membranes of ASA, M-ASA, E-ASA and P-ASA with a web-based tool called PreADMET ( https://preadmet.qsarhub.com/ ). The prediction results demonstrated significantly improved cell membrane permeability for all three esterified aspirin derivatives compared to aspirin (Fig. 3 b). However, ASA, M‑ASA, and E‑ASA exhibit higher flux values than P‑ASA(Gerber et al. 2006 ), indicating greater membrane permeability and absorption rates, which can be attributed to differences in lipophilicity, molecular weight, ionization, and membrane interactions. Considering bioavailability, we excluded P-ASA from further consideration. A compound safety assessment was conducted via the PubChem database ( https://pubchem.ncbi.nlm.nih.gov/compound/68484 ). M-ASA (CAS No. 580-02-9) was excluded due to its documented skin irritation, eye and respiratory tract irritation, and specific target organ toxicity. Following the preliminary evaluations, we further verified the in vivo safety of E‑ASA in comparison with ASA (Fig. S1 a). After three weeks of treatment with the same concentration (0.15 mg/ml) of ASA or E‑ASA, there were no significant differences in body weight gain or daily water consumption among the three groups (vehicle, ASA, and E‑ASA) (Fig. S1 b, c). However, analysis of plasma biochemical markers related to hepatic and renal function—including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine (CREA), and uric acid (UA)—revealed that plasma AST, LDH, BUN, and CREA levels did not differ significantly among the three groups (Fig. S1 e-h). In contrast, ALT and UA levels were lower in the E‑ASA group than in the ASA group, suggesting that E‑ASA causes less hepatic and renal injury (Fig. S1 d, i). Given the well‑known bleeding risk associated with ASA in clinical settings, we also assessed tail‑bleeding time to compare the hemostatic safety of ASA and E‑ASA in mice. The ASA‑treated mice exhibited a significantly prolonged bleeding time, whereas the E‑ASA group showed no significant difference from the vehicle group, indicating that E‑ASA confers a markedly lower bleeding risk in vivo (Fig. S1 j). Building on the above findings, we compared the membrane permeability of ASA and E‑ASA in human cardiomyocyte cells (AC16 cell line). In vitro experiments demonstrated that E‑ASA exhibited significantly higher membrane permeability than ASA (Fig. 3 c). Acute treatment with E‑ASA after MI was associated with reduced myocardial injury and improved cardiac function in mice. Given the improved membrane permeability of E‑ASA relative to aspirin, we next examined whether post‑treatment could provide additional cardiac protection. To this aim, we performed intraperitoneal (i.p.) injection of 48 mg/kg aspirin and E-ASA 30 min after MI (i.e., 15 min before reperfusion), and then the myocardial I/R injury was assessed 24 h after reperfusion (Fig. 2 a). While posttreatment with aspirin did not reduce the myocardial infarction areas, posttreatment with E-ASA significantly attenuate the myocardial infarction in the I/R mice 24 h after reperfusion (Fig. 2 g-i). Likewise, posttreatment with E-ASA rather than aspirin significantly decreased the serum levels of AST and CK-MB in the I/R mice 24 h after reperfusion (Fig. 2 e and f). Through echocardiography, we found that the cardiac function (EF and FS) of the I/R mice was significantly improved after acute treatment with ASA 24 h after reperfusion, but the improvement was more pronounced in the E-ASA group (Fig. 2 b-d). Taken together, these results indicate that posttreatment with E-ASA confers better cardioprotection against I/R injury than aspirin in mice. E-ASA increased acetylation of proteins involved in the myocardial metabolism and I/R injury Aspirin has an acetyl group and thus has been shown to acetylate a variety of intracellular proteins (Alfonso et al. 2009 ; Guo et al. 2020 ). Since E-ASA also has an acetyl group (Fig. 3 a), we hypothesized that E-ASA would be able to acetylate intracellular proteins in the heart. To identify the intracellular proteins that can be acetylated by E-ASA, we performed label-free proteomic assay in heart tissues from saline (vehicle) and E-ASA-treated mice. The mice were i.p. injected with saline or 50 mg/kg E-ASA, and then the homogenates of heart tissues were collected and digested with trypsin 24 h after E-ASA treatment. The acetylated peptides were purified with the anti-acetyl-lysine (anti-Ac-K) antibodies and then were analyzed by the liquid chromatography plus tandem mass spectrometry (LC-MS/MS) (Fig. 4 a). From three independent experiments, we identified 1341 Ac-K sites that matched to 494 acetylated proteins from vehicle mice, and 1532 Ac-K sites that matched to 559 acetylated proteins from E-ASA-treated mice (Fig. 4 b and Table S1 ). The majority of the Ac-K sites and acetylated proteins are conserved between control and E-ASA groups (Fig. 4 b). Comparative analysis of the proteomics data from two groups of mice identified 296 upregulated acetylation sites (fold change > 1.2, P value < 0.05) which matched to 216 acetylated proteins (Fig. 4 c). In contrast, only 50 acetylation sites were found to be downregulated (fold change < 0.83, P value < 0.05) in mouse heart after E-ASA treatment (Fig. 4 c). Analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that acetylated proteins upregulated by E-ASA were significantly enriched in metabolic pathways (Fig. 4 d). The acetylation of several key enzymes in the processes of glycolysis, tricarboxylic acid cycle (TCA), and fatty acid oxidation was increased in the heart tissues after E-ASA treatment (Fig. 4 d and Table S1 ). On the other hand, acetylated proteins upregulated by E-ASA were also significantly overrepresented in the myocardial injury-related pathways (Fig. 4 e). The acetylation of several proteins involved in the processes of HIF-1 signaling pathway, PPAR signaling pathway, and hypertrophic cardiomyopathy was increased in the heart tissues after E-ASA treatment (Fig. 4 e and Table S1 ). E‑ASA treatment appeared to enhance the acetylation of key enzymes involved in cardiac energy metabolism. E-ASA increased the acetylation of K179 in SDHA (succinate dehydrogenase isoform α, a key enzyme involved in TCA and respiratory complex II) (Fig. 5 ) which has been shown to reduce its activity(Horton et al. 2016 ). E-ASA increased the acetylation of K305 in Pkm (Pyruvate Kinase, Muscle type) is a key enzyme in the glycolysis pathway (Fig. 5 ) which has been shown to reduce its activity and inhibits the enzymatic function of PKM and promotes its degradation, redirecting cellular metabolism toward a branching pathway for glucose metabolism. (Lv et al. 2011 ). Although our mass spectrometry results show that there are many site-specific acetylation-upregulated proteins, no studies have yet reported the effects of acetylation on protein activity. However, many proteins have been reported to be associated with myocardial injury and cardioprotection. The study found that inhibiting Carnitine palmitoyltransferase-1b(CPT1b)-mediated fatty acid oxidation can change the metabolic state of the myocardium in the form of metabolic reprogramming and promote myocardial regeneration(Li et al. 2023 ). Also it has been suggested that inhibiting CPT1 activity by specific CPT1 inhibitors exerts protective effects against cardiac hypertrophy and heart failure(He et al. 2012 ). In addition, key metabolic enzymes such as Pfkm (Zhou et al. 2022 ), Aldoa (Luo et al. 2021 ), Gapdh (Yao et al. 2012 ), Pgk1(Lu et al. 2023 ), Pgam2(Okuda et al. 2013 ), and Pdha1(Lewandowski and White 1995 ) have also been reported to play an important role in myocardial function and cardioprotection. In the results of mass spectrometry analysis, there are relatively few studies on the effects of acetylation on the activity of E-ASA differentially upregulated proteins, and the correlation between some differentially upregulated proteins and cardiac function remains largely unknown. We annotated key proteins upregulated by acetylation in the major differentially enriched metabolic pathways, including glycolysis, fatty acid oxidation, and ATP synthesis, including sites upregulated by E-ASA-mediated differential acetylation (Fig. 5 ). Blue indicates sites with literature reported on the effects of acetylation on protein activity, while red indicates sites with unknown effects on protein activity. Disussion Our preliminary experiments in animal models indicated that E‑ASA showed a more pronounced cardioprotective effect compared with classical aspirin. This enhanced efficacy may be related to the modification of ASA's carboxyl group through esterification, which potentially increases the drug's cell membrane permeability while maintaining a lower bleeding risk profile. The bleeding risk assessment was conducted through in vivo tail bleeding experiments. In subsequent studies, we plan to further investigate E-ASA's affinity for COX-1 and its effects on COX-1 activity to elucidate the mechanisms underlying its reduced bleeding risk. Long-term E-ASA administration resulted in altered cardiac acetylation levels, with increased acetylation potentially stemming from E-ASA's inherent acetyl group structure. Biological acetylation occurs through two primary mechanisms: enzyme-dependent and enzyme-independent pathways(Weinert et al. 2018 ). The enzyme-dependent acetylation is primarily catalyzed by histone acetyltransferases (HATs), which transfer acetyl groups from acetyl-CoA to lysine residues on target proteins. The enzyme-independent form relies mainly on acetyl group donors (such as acetyl-CoA) concentration and the chemical environment. Therefore, E-ASA may exert its biological functions through modulating the complex internal environment, coordinating both enzyme-dependent and enzyme-independent pathways. Acetylproteomic analysis revealed that E‑ASA administration was associated with increased acetylation levels in several cardiac energy metabolism-related proteins. Although these modifications encompass acetylation changes in various rate-limiting enzymes across multiple metabolic pathways, which may affect protein structural properties or biological activities, we cannot fully investigate each individual mechanism in detail. Previous studies on metabolic regulators such as Cpt1(Li et al. 2023 ) and the SDH (Zhang and Lang 2023 ; Yu et al. 2025 ) family have demonstrated cardioprotective roles, which may provide mechanistic context for the effects observed with E‑ASA. We also explored the differences in E-ASA's effects across various dosages and time points, which may be related to its in vivo pharmacokinetics and metabolism - aspects we plan to investigate further in subsequent experiments. Given the abundance of esterases in biological systems, E-ASA's in vivo stability presents certain challenges that warrant consideration. Materials and Methods Animals Male C57BL/6J mice aged 8–12 weeks were obtained from Shanghai Jihui Laboratory Animal Care Co., Ltd. Male mice were selected for all experimental procedures to avoid confounding effects associated with estrous cycles. Animals were maintained in a specific pathogen-free (SPF) facility under standard laboratory conditions with a 12:12-hour light-dark cycle and provided with food and water ad libitum. In the three‑week chronic feeding experiment, 30 male C57BL/6 mice (8–12 weeks old) were randomly divided into three groups: Sham (normal drinking water, sham surgery), Vehicle (normal drinking water, I/R), and E‑ASA (E‑ASA‑containing drinking water, I/R), n = 10 mice per group. In the acute administration experiment, 32 male C57BL/6 mice (8–12 weeks old) were randomly divided into four groups: Sham (saline, sham surgery), Vehicle (saline, I/R), ASA (aspirin, I/R), and E‑ASA (E‑ASA, I/R), n = 8 mice per group. For acetylation proteomics, six mice (12 weeks old) were used, with three in the Vehicle group and three in the E‑ASA group. In the three‑week chronic feeding safety evaluation, 33 mice (8–12 weeks old) were randomly divided into three groups: Vehicle (normal drinking water), ASA (aspirin‑containing water), and E‑ASA (E‑ASA‑containing water), n = 11 mice per group. Minor discrepancies in n-values among experiments may result from quality‑control exclusions during data acquisition. Cellular Uptake of E‑ASA in AC16 Cardiomyocytes Human AC16 cardiomyocytes, kindly provided by Professor Tao Zhong (East China Normal University, China), were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in 5% CO₂. Cells at approximately 80% confluence were incubated with E‑ASA at a final concentration of 0.1 mM for 40, 80, 120, and 160 min. After incubation, cells were washed twice with ice‑cold PBS to remove residual compound and lysed on ice using (lysis buffer) for quantitative analysis. Cell lysates were centrifuged, and the supernatants were subjected to LC–MS/MS determination of E‑ASA levels using standard calibration curves. Intracellular E‑ASA concentrations were normalized to total protein quantified by the BCA assay. Tail Bleeding Time Assay Under sustained anesthesia with 2% isoflurane, mice from each experimental group underwent tail transection at a diameter of 2 mm with a single, rapid cut. The transected tail was immediately immersed in warm saline, and the bleeding time (duration from initial bleeding to hemostasis) was recorded. In vivo model of myocardial ischemia and reperfusion Male C57BL/6 mice aged 8–12 weeks were used to establish the I/R animal model. The mice were fixed on a heating pad maintained at 37°C using medical tape and anesthetized with 2% isoflurane via inhalation. After removing the chest and abdominal hair, a sterile scissor was used to make an incision at the position of strongest heartbeat, and a 4 − 0 suture was prepared for pre-suturing. Using curved forceps, the thoracic cavity above the heart was bluntly dissected through the third and fourth intercostal space. The mouse position was adjusted to gently compress the thoracic cavity, extruding the heart. An 8 − 0 suture was used to create a slipknot around the left anterior descending coronary artery, with one end of the suture exposed outside the thoracic cavity. The heart was then returned to the thoracic cavity, excess air was evacuated, and the skin was sutured. The mouse was gently placed in a warming box. After 45 minutes, the retained slipknot was released, and the mouse was returned to its housing cage for continued maintenance. Echocardiography Echocardiographic measurements were performed using the Vevo 2100 system (MS400C probe) in all groups of mice. The sonographers was blinded to group allocation. Left ventricular end-diastolic and end-systolic dimensions were derived from M-mode images. Fractional shortening was calculated as a percentage using the following formula: (left ventricular end-diastolic dimension - left ventricular end-systolic dimension)/left ventricular end-diastolic dimension. All measurements were conducted at the papillary muscle level following standardized protocols. Infarct size measurement At 24 hours after myocardial ischemia-reperfusion, the chest cavity was reopened to expose the mouse heart. The left anterior descending artery was re-ligated at the same location, followed by immediate injection of 1 mL 2% Evans blue staining solution (Sigma-Aldrich, St. Louis, MO, United States, dissolved in phosphate buffer) into the aorta. The heart was then excised and rapidly frozen at -80°C for 10 minutes. The frozen heart was evenly cut into 5 slices from the ligation site to the apex. The heart slices were incubated in 2% TTC solution (2,3,5-Triphenyltetrazolium Chloride, Sigma-Aldrich, St. Louis, MO, United States, dissolved in phosphate buffer pH 7.4, 3 mL per heart) at 37°C for 30 minutes. After staining, the heart slices were fixed in 4% paraformaldehyde for 15 minutes before digital scanning and photography. The non-ischemic area of the left ventricle was stained dark blue, viable tissue within the area at risk appeared bright red, and infarcted tissue appeared white or pale yellow. Image analysis was performed using ImageJ software. The infarct size as a percentage of the area at risk was calculated using standard methods as previously described(Xie et al. 2014 ). Biochemical assessment After 24 hours of myocardial ischemia-reperfusion, blood samples were collected from each group of mice using 3.8% (w/v) sodium citrate as an anticoagulant. After standing at room temperature for 30 minutes, the collected blood was centrifuged at 3000 rpm at 4°C for 15 minutes. The supernatant was collected as plasma and stored at -80°C for subsequent analysis, avoiding repeated freeze-thaw cycles. The indicators for assessing myocardial injury included aspartate aminotransferase (AST) and creatine kinase isoenzyme. For the three groups of mice (Control, ASA, and E-ASA) that underwent three weeks of drug administration, plasma samples were collected following the aforementioned method upon completion of treatment and stored at -80°C for subsequent analysis. To evaluate potential hepatic and renal injury during the three-week treatment period, the following parameters were measured: aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) for comprehensive assessment of liver function; blood urea nitrogen (BUN), urinary creatinine, and uric acid for comprehensive assessment of renal function.All plasma parameter measurements were conducted by Wuhan Servicebio Technology Co., Ltd. 4D-Label Free Acetylation Mass Spectrometry Analysis Heart tissues from control and E-ASA-treated mice were homogenized in urea-based lysis buffer supplemented with protease and deacetylase inhibitors. The extracted proteins underwent reduction with dithiothreitol (DTT), alkylation with iodoacetamide, and overnight tryptic digestion. Following desalting, acetylated peptides were enriched using anti-acetyl-lysine antibody-conjugated agarose beads. The samples were then loaded onto a 25 cm C18 analytical column (25 cm × 75 µm inner diameter, 1.6 µm C18 particles, IonOpticks) at a flow rate of 300 nL/min and subjected to gradient elution. The resulting experimental data were processed using MaxQuant software (Version 1.6.17.0) for database searching. Statistical analysis All the data were shown as mean ± SEM unless otherwise mentioned. The sample size justification was based on the previous studies and met the requirement of statistics power. The N number of mice could be different for distinctive assays. All the statistical analyses were performed by the software of GraphPad Prism 8.0.2. Statistically significant difference was indicated as follows: **** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Authors and Affiliations Department of Ultrasound, Changning Maternity and Infant Health Hospital, East China Normal University, Shanghai, 200050, China. Bi-Han Wang, Hai-Huan Gao and Bi-Yuan He Key Laboratory of Brain Functional Genomics, Ministry of Education and Shanghai, School of Life Science, East China Normal University, Shanghai, 200062, China. Bi-Han Wang, Wen-Liang Jiang and Dong-Min Yin Corresponding author Correspondence to Bi-Yuan He. Ethics approval Mouse care and experiments were performed according to the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the Animal Care and Use Committee of East China Normal University (m20230704). Clinical trial number: not applicable. Funding The study was supported by PI research team program of health commission foundation of Changning district, Shanghai, China(PI202431). Author Contribution Bi-Han Wang: Methodology, Validation, Formal analysis, Investigation, Data Curation and Project Administration. Hai-Huan Gao: Review of original draft and Visualization. Wen-Liang Jiang: Supervision, Data Curation. Dong-Min Yin, Bi-yuan He: Conceptualization, Supervision, Funding acquisition, Writing – Review & Editing. The final version of the manuscript has been reviewed and approved by all authors. The authors declare that all data were generated in-house and that no paper mill was used. References Vos T, Lim SS, Abbafati C, Abbas KM, Abbasi M, Abbasifard M, et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. The lancet. 2020;396(10258):1204–22 Ibanez B, James S, Agewall S, Antunes MJ, Bucciarelli-Ducci C, Bueno H, et al. 2017 ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC). European heart journal. 2018;39(2):119–77 Yellon DM, Hausenloy DJ. Myocardial reperfusion injury. N Engl J Med. 2007;357(11):1121–35.https://doi.org/[10.1056/NEJMra071667 Hausenloy DJ, Chilian W, Crea F, Davidson SM, Ferdinandy P, Garcia-Dorado D, et al. The coronary circulation in acute myocardial ischaemia/reperfusion injury: a target for cardioprotection. Cardiovasc Res. 2019;115(7):1143–55.https://doi.org/[10.1093/cvr/cvy286 Loll PJ, Picot D, Garavito RM. The structural basis of aspirin activity inferred from the crystal structure of inactivated prostaglandin H2 synthase. Nat Struct Biol. 1995;2(8):637–43.https://doi.org/[10.1038/nsb0895-637 Dong Z, Yang L, Jiao J, Jiang Y, Li H, Yin G, et al. Aspirin in combination with gastrodin protects cardiac function and mitigates gastric mucosal injury in response to myocardial ischemia/reperfusion. Front Pharmacol. 2022;13:995102.https://doi.org/[10.3389/fphar.2022.995102 Liu PP, Liu HH, Sun SH, Shi XX, Yang WC, Su GH, et al. Aspirin alleviates cardiac fibrosis in mice by inhibiting autophagy. Acta Pharmacol Sin. 2017;38(4):488–97.https://doi.org/[10.1038/aps.2016.143 Seth SD, Maulik M, Manchanda SC, Maulik SK. Role of aspirin in modulating myocardial ischemic reperfusion injury. Agents Actions. 1994;41(3-4):151–5.https://doi.org/[10.1007/BF02001909 Antithrombotic Trialists C, Baigent C, Blackwell L, Collins R, Emberson J, Godwin J, et al. Aspirin in the primary and secondary prevention of vascular disease: collaborative meta-analysis of individual participant data from randomised trials. Lancet. 2009;373(9678):1849–60.https://doi.org/[10.1016/S0140-6736(09)60503-1 Lanas A, Wu P, Medin J, Mills EJ. Low doses of acetylsalicylic acid increase risk of gastrointestinal bleeding in a meta-analysis. Clin Gastroenterol Hepatol. 2011;9(9):762–8 e6.https://doi.org/[10.1016/j.cgh.2011.05.020 Zheng SL, Roddick AJ. Association of aspirin use for primary prevention with cardiovascular events and bleeding events: a systematic review and meta-analysis. Jama. 2019;321(3):277–87 Willetts S, Foley DW. True or false? Challenges and recent highlights in the development of aspirin prodrugs. Eur J Med Chem. 2020;192:112200.https://doi.org/[10.1016/j.ejmech.2020.112200 Wood PH, Harvey-Smith E, Dixon ASJ. Salicylates and gastro-intestinal bleeding. British Medical Journal. 1962;1(5279):669 Killackey JJ, Killackey BA, Philp RB. Structure-activity studies of aspirin and related compounds on platelet aggregation, arachidonic acid metabolism in platelets and artery, and arterial prostacyclin activity. Prostaglandins Leukot Med. 1982;9(1):9–23.https://doi.org/[10.1016/0262-1746(82)90068-3 Zhou G. Exploring Ester Prodrugs: A Comprehensive Review of Approaches, Applications, and Methods. Pharmacology & Pharmacy. 2024;15(8):269–84 Patrono C, Garcia Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med. 2005;353(22):2373–83.https://doi.org/[10.1056/NEJMra052717 Lindsey ML, Bolli R, Canty JM, Jr., Du XJ, Frangogiannis NG, Frantz S, et al. Guidelines for experimental models of myocardial ischemia and infarction. Am J Physiol Heart Circ Physiol. 2018;314(4):H812–H38.https://doi.org/[10.1152/ajpheart.00335.2017 Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J. 2008;22(3):659–61.https://doi.org/[10.1096/fj.07-9574LSF Nigam PK. Biochemical markers of myocardial injury. Indian J Clin Biochem. 2007;22(1):10–7.https://doi.org/[10.1007/BF02912874 Ganjehei L, Becker RC. Aspirin dosing in cardiovascular disease prevention and management: an update. J Thromb Thrombolysis. 2015;40(4):499–511.https://doi.org/[10.1007/s11239-015-1267-6 Menichetti R, Kanekal KH, Bereau T. Drug-Membrane Permeability across Chemical Space. ACS Cent Sci. 2019;5(2):290–8.https://doi.org/[10.1021/acscentsci.8b00718 Stella VJ, Charman WN, Naringrekar VH. Prodrugs. Do they have advantages in clinical practice? Drugs. 1985;29(5):455–73.https://doi.org/[10.2165/00003495-198529050-00002 Rautio J, Kumpulainen H, Heimbach T, Oliyai R, Oh D, Jarvinen T, et al. Prodrugs: design and clinical applications. Nat Rev Drug Discov. 2008;7(3):255–70.https://doi.org/[10.1038/nrd2468 Gerber M, Breytenbach JC, Hadgraft J, du Plessis J. Synthesis and transdermal properties of acetylsalicylic acid and selected esters. Int J Pharm. 2006;310(1-2):31–6.https://doi.org/[10.1016/j.ijpharm.2005.11.018 Alfonso LF, Srivenugopal KS, Bhat GJ. Does aspirin acetylate multiple cellular proteins? (Review). Mol Med Rep. 2009;2(4):533–7.https://doi.org/[10.3892/mmr_00000132 Guo L, Gao J, Gao Y, Zhu Z, Zhang Y. Aspirin Reshapes Acetylomes in Inflammatory and Cancer Cells via CoA-Dependent and CoA-Independent Pathways. J Proteome Res. 2020;19(2):962–72.https://doi.org/[10.1021/acs.jproteome.9b00853 Horton JL, Martin OJ, Lai L, Riley NM, Richards AL, Vega RB, et al. Mitochondrial protein hyperacetylation in the failing heart. JCI insight. 2016;1(2):e84897 Lv L, Li D, Zhao D, Lin R, Chu Y, Zhang H, et al. Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth. Mol Cell. 2011;42(6):719–30.https://doi.org/[10.1016/j.molcel.2011.04.025 Li X, Wu F, Günther S, Looso M, Kuenne C, Zhang T, et al. Inhibition of fatty acid oxidation enables heart regeneration in adult mice. Nature. 2023;622(7983):619–26 He L, Kim T, Long Q, Liu J, Wang P, Zhou Y, et al. Carnitine palmitoyltransferase-1b deficiency aggravates pressure overload-induced cardiac hypertrophy caused by lipotoxicity. Circulation. 2012;126(14):1705–16.https://doi.org/[10.1161/CIRCULATIONAHA.111.075978 Zhou M, Sun X, Wang C, Wang F, Fang C, Hu Z. PFKM inhibits doxorubicin-induced cardiotoxicity by enhancing oxidative phosphorylation and glycolysis. Sci Rep. 2022;12(1):11684.https://doi.org/[10.1038/s41598-022-15743-0 Luo G, Wang R, Zhou H, Liu X. ALDOA protects cardiomyocytes against H/R-induced apoptosis and oxidative stress by regulating the VEGF/Notch 1/Jagged 1 pathway. Mol Cell Biochem. 2021;476(2):775–83.https://doi.org/[10.1007/s11010-020-03943-z Yao LL, Wang YG, Liu XJ, Zhou Y, Li N, Liu J, et al. Phenylephrine protects cardiomyocytes from starvation-induced apoptosis by increasing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. J Cell Physiol. 2012;227(10):3518–27.https://doi.org/[10.1002/jcp.24053 Lu Y, Zhao N, Wu Y, Yang S, Wu Q, Dong Q, et al. Inhibition of phosphoglycerate kinase 1 attenuates autoimmune myocarditis by reprogramming CD4+ T cell metabolism. Cardiovasc Res. 2023;119(6):1377–89.https://doi.org/[10.1093/cvr/cvad029 Okuda J, Niizuma S, Shioi T, Kato T, Inuzuka Y, Kawashima T, et al. Persistent overexpression of phosphoglycerate mutase, a glycolytic enzyme, modifies energy metabolism and reduces stress resistance of heart in mice. PLoS One. 2013;8(8):e72173.https://doi.org/[10.1371/journal.pone.0072173 Yuan S, Xie R, Zhang X, Tang Y, Jin K, Fan J, et al. Double face of glycolytic enzyme ENO1 in heart failure. medRxiv. 2025:2025.03. 31.25324995 Lewandowski ED, White LT. Pyruvate dehydrogenase influences postischemic heart function. Circulation. 1995;91(7):2071–9.https://doi.org/[10.1161/01.cir.91.7.2071 Weinert BT, Narita T, Satpathy S, Srinivasan B, Hansen BK, Scholz C, et al. Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome. Cell. 2018;174(1):231–44 e12.https://doi.org/[10.1016/j.cell.2018.04.033 Zhang W, Lang R. Succinate metabolism: a promising therapeutic target for inflammation, ischemia/reperfusion injury and cancer. Front Cell Dev Biol. 2023;11:1266973.https://doi.org/[10.3389/fcell.2023.1266973 Yu J, Yao Q, Hu T, Zhang Y, Liu Y, Xiao Y, et al. The Role of HINT3 in Myocardial Ischemia-Reperfusion Injury in Male Mice: Mechanisms Involving SDHA and its Acetylation. Adv Sci (Weinh). 2025;12(33):e03109.https://doi.org/[10.1002/advs.202503109 Xie M, Kong Y, Tan W, May H, Battiprolu PK, Pedrozo Z, et al. Histone deacetylase inhibition blunts ischemia/reperfusion injury by inducing cardiomyocyte autophagy. Circulation. 2014;129(10):1139–51 Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx FigS1.jpg Fig.S1 Effects of three-week oral administration of ASA and E-ASA on the safety of mice. a, Experimental design. 8 to 12 week-old C57BL/6 mice were randomly divided into three groups: one group received regular drinking water, one group received drinking water containing 0.15 mg/mL ASA, and one group received drinking water containing 0.15 mg/mL E‑ASA. All groups were fed a standard chow diet, and safety analysis was performed after three weeks. b, Changes in body weight of mice following three weeks of administration with ASA or E-ASA. Two-way-ANOVA followed by Tukey's multiple comparisons test, n=10 mice each group. c, Daily water intake of mice during the three-week treatment with ASA or E-ASA. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=21 mice each group. d,Plasma alanine aminotransferase (ALT) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. e, Plasma aspartate aminotransferase (AST) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. f, Plasma lactate dehydrogenase (LDH) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. g, Plasma blood urea nitrogen (BUN) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. h, Plasma creatinine (CREA) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9. i, Plasma uric acid (UA) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. j, ASA prolonged bleeding time in mice, whereas E‑ASA significantly reduced tail bleeding time compared with ASA. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. ALT: alanine aminotransferase, AST: aspartate aminotransferase, LDH: lactate dehydrogenase, BUN: blood urea nitrogen, CREA: creatinine, UA: uric acid. Data are presented as the mean ±SEM. *P < 0.05, **P < 0.01, ns means not significant. Cite Share Download PDF Status: Published Journal Publication published 13 Apr, 2026 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted Editorial decision: Revision requested 07 Nov, 2025 Editor assigned by journal 07 Nov, 2025 Submission checks completed at journal 07 Nov, 2025 First submitted to journal 03 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8019780","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":541652003,"identity":"12e646dd-4c3d-45b2-9f20-ef2f0d3b897b","order_by":0,"name":"Bi-Han Wang","email":"","orcid":"","institution":"Department of Ultrasound, Changning Maternity and Infant Health Hospital, East China Normal University, Shanghai, 200050, China","correspondingAuthor":false,"prefix":"","firstName":"Bi-Han","middleName":"","lastName":"Wang","suffix":""},{"id":541652004,"identity":"6cb17f25-91f9-42c5-81a3-d6f2ed1a245d","order_by":1,"name":"Hai-Huan Gao","email":"","orcid":"","institution":"Department of Ultrasound, Changning Maternity and Infant Health Hospital, East China Normal University, Shanghai, 200050, China","correspondingAuthor":false,"prefix":"","firstName":"Hai-Huan","middleName":"","lastName":"Gao","suffix":""},{"id":541652005,"identity":"a3e806b2-f529-4e70-9cd8-2a1bc29be0c9","order_by":2,"name":"Wen-Liang Jiang","email":"","orcid":"","institution":"Department of Ultrasound, Changning Maternity and Infant Health Hospital, East China Normal University, Shanghai, 200050, China","correspondingAuthor":false,"prefix":"","firstName":"Wen-Liang","middleName":"","lastName":"Jiang","suffix":""},{"id":541652006,"identity":"06283dcd-6d2c-4544-808d-119319da08cb","order_by":3,"name":"Dong-min Yin","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Dong-min","middleName":"","lastName":"Yin","suffix":""},{"id":541652007,"identity":"d76dce04-a242-463b-8da9-48e11516a9c5","order_by":4,"name":"Biyuan He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYBACAwkkNoiQY2NvPkCMFgMokcBgzMdzLIE0LYnzJHIU8Goxl+4xe/Dhzx+77e2Ht0n8/GGT3saQw8Dwo2IbTi2Wc86YG85sM0iecyatTLInIS23jeHsAcaeM7dxO+xGjpk0b4NBsgRDjpkET8Lh3DbGvgRmxjYCWnj+ALXwvzGT/JPwP52NmceACC1sBnYSEiBGwoEENjZCWu4cKwf6xThBQuJZsbVMWrJhGw9bwkG8frndvA0YYnL2EvzJG2++sbGTl5//+OCDHxW4tTAwcJiByMQGZLEDeNQDAfszEGmPX9EoGAWjYBSMaAAAHHVVFvI7wyMAAAAASUVORK5CYII=","orcid":"","institution":"Department of Ultrasound, Changning Maternity and Infant Health Hospital, East China Normal University, Shanghai, 200050, China","correspondingAuthor":true,"prefix":"","firstName":"Biyuan","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2025-11-03 13:38:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8019780/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8019780/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00210-026-05308-7","type":"published","date":"2026-04-13T15:56:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":96610548,"identity":"ee0f31e6-dd90-44f3-8028-b30afde4820c","added_by":"auto","created_at":"2025-11-24 09:31:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3446111,"visible":true,"origin":"","legend":"\u003cp\u003eChronic feeding of ASA exerts cardioprotective effects in a mouse model of myocardial ischemia‑reperfusion (I/R) injury. \u003cstrong\u003ea,\u003c/strong\u003eExperimental design. Adult mice were fed with 0.1 mg/ml ASA or E-ASA-containing drinking water for three weeks, followed by myocardial ischemia (MI) for 45 min. The myocardial function, infarction area, and I/R-related enzymes were analyzed 24 h after reperfusion. \u003cstrong\u003eb-d,\u003c/strong\u003e ASA improved myocardial function in the I/R mice. b, The representative echocardiography images from three groups of mice: Sham, I/R + Veh, I/R + ASA. Scale bar, 1 mm, 100 ms. c, d, ASA and E-ASA increased the myocardial ejection fraction (EF) (c) and fractional shortening (FS) (d) in the I/R mice. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n = 10 mice each group.\u003cstrong\u003e e,\u003c/strong\u003e E-ASA reduced the serum levels of AST in the I/R mice. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n = 10 mice each group. \u003cstrong\u003ef,\u003c/strong\u003e E-ASA reduced the serum levels of CK-MB in the I/R mice, Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n = 8 mice each group. \u003cstrong\u003eg, \u003c/strong\u003eRepresentative images of Evans blue plus TTC (2,3,5-triphenyltetrazolium chloride) staining of heart tissue from two groups of mice: I/R + Veh, I/R + ASA. Scale bar, 1 mm. h, ASA did not affect the areas at risk (AAR) in the I/R mice. P= 0.9560, Unpaired t test, n =8 mice each group. i, ASA alleviated the myocardial infarction in the I/R mice, Unpaired t test, n =8 mice each group. I/R: ischemia‑reperfusion, Veh: vehicle, ASA: aspirin, E-ASA: ethyl acetylsalicylate, EF: ejection fraction, FS: fractional shortening, AAR: area at risk, LV: left ventricle, AST: aspartate aminotransferase, CK-MB: creatine kinase - myocardial band isoenzyme. Data are shown as the mean ± SEM for each group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8019780/v1/e3fcc2a15eced228fab87d85.jpg"},{"id":96611053,"identity":"509d6048-f511-4325-a20a-b8f9b73a5f7c","added_by":"auto","created_at":"2025-11-24 09:32:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3882350,"visible":true,"origin":"","legend":"\u003cp\u003eAcute administration of E‑ASA after MI confers markedly greater protection against myocardial ischemia/reperfusion (I/R) injury in mice.\u003cstrong\u003e a,\u003c/strong\u003eExperimental design. ASA or E‑ASA was administered intraperitoneally 30 minutes after the onset of myocardial ischemia. After 45 minutes of ischemia, reperfusion was initiated, and cardiac function was evaluated 24 hours post‑reperfusion. \u003cstrong\u003eb,\u003c/strong\u003eThe representative echocardiography images from four groups of mice: Sham, I/R + Veh, I/R + ASA, I/R + E-ASA. Scale bar, x axis = 100 ms, y axis = 1 mm. \u003cstrong\u003ec, d,\u003c/strong\u003e ASA and E-ASA increased the myocardial ejection fraction (EF) (c) and fractional shortening (FS) (d) in the I/R mice., Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, Sham n = 5, I/R + Veh, I/R + ASA, I/R + E-ASA, n = 8 each group. \u003cstrong\u003ee,\u003c/strong\u003e Serum AST levels were significantly decreased in myocardial I/R mice treated with E‑ASA, while no significant change was observed with ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n = 8 mice each group. \u003cstrong\u003ef, \u003c/strong\u003eSerum CK-MB levels were significantly decreased in myocardial I/R mice treated with E‑ASA, while no significant change was observed with ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n = 8 mice each group. \u003cstrong\u003eg, \u003c/strong\u003eRepresentative images of Evans blue plus TTC (2,3,5-triphenyltetrazolium chloride) staining of heart tissue from four groups of mice: Sham, I/R + Veh, I/R + ASA, I/R + E-ASA. Scale bar, 1 mm. \u003cstrong\u003eh,\u003c/strong\u003e Saline, ASA, E-ASA did not affect the areas at risk (AAR) in the I/R mice, Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n =8 mice each group.\u003cstrong\u003e i,\u003c/strong\u003eE-ASA alleviated the myocardial infarction in the I/R mice, while no significant change was observed with ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n =8 mice each group. I/R: ischemia‑reperfusion, Veh: vehicle, ASA: aspirin, E-ASA: ethyl acetylsalicylate, EF: ejection fraction, FS: fractional shortening, AAR: area at risk, LV: left ventricle, AST: aspartate aminotransferase, CK-MB: creatine kinase - myocardial band isoenzyme. Data are shown as the mean ± SEM for each group. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8019780/v1/131c77d1b2150d0dc717de21.jpg"},{"id":96610960,"identity":"ce3a5c1d-ef47-4ea0-8dc3-133a94299d6f","added_by":"auto","created_at":"2025-11-24 09:32:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2598154,"visible":true,"origin":"","legend":"\u003cp\u003eE-ASA shows improved cell membrane permeability than aspirin\u003cstrong\u003e. a,\u003c/strong\u003e Chemical structures of ASA, M‑ASA, E‑ASA, and P‑ASA. \u003cstrong\u003eb,\u003c/strong\u003ePredicted cell membrane permeability of ASA, M‑ASA, E‑ASA, and P‑ASA obtained using \u003cem\u003ePreADMET\u003c/em\u003e. \u003cstrong\u003ec,\u003c/strong\u003eAfter incubation with AC16 cardiomyocyte cells, the intracellular concentration of E‑ASA was significantly higher than that of ASA. **** P \u0026lt; 0.0001, Two-way-ANOVA followed by Sidak's multiple comparisons test, n =3 mice each group.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8019780/v1/4c363228c53b30c311c41cd3.jpg"},{"id":96610931,"identity":"3ac43b55-5760-4bef-8376-37ee086d054b","added_by":"auto","created_at":"2025-11-24 09:32:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3421865,"visible":true,"origin":"","legend":"\u003cp\u003eAcetylation proteomics detected major changes in energy metabolism in the mouse heart after E-ASA administration. \u003cstrong\u003ea,\u003c/strong\u003e Schematic diagram of the mass spectrometry detection workflow. \u003cstrong\u003eb,\u003c/strong\u003eThe acetylated proteomic analysis: (left) number of upregulated acetylated proteins between the vehicle and E‑ASA groups, and (right) number of upregulated acetylation sites between the two groups. \u003cstrong\u003ec,\u003c/strong\u003e Volcano plot showing differentially upregulated acetylation sites in the E‑ASA group compared with the vehicle group. \u003cstrong\u003ed,\u003c/strong\u003eKEGG pathway analysis of differentially upregulated acetylated proteins in the E‑ASA group compared with the vehicle group, highlighting metabolism-related pathways. \u003cstrong\u003ee,\u003c/strong\u003e KEGG pathway analysis of differentially upregulated acetylated proteins in the E‑ASA group compared with the vehicle group, highlighting myocardial injury–related pathways.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8019780/v1/b39acb79f8c23e7cceda0347.jpg"},{"id":96610801,"identity":"46a541c2-4a81-4505-a7bd-208790546e3b","added_by":"auto","created_at":"2025-11-24 09:32:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3079060,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential regulation of acetylation levels of key enzymes in cardiac energy metabolism pathways by E-ASA. Blue indicates sites with literature reported on the effects of acetylation on protein activity, while red indicates sites with unknown effects on protein activity. TCA: tricarboxylic acid cycle, ETC: electron transport chain.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8019780/v1/098e5d3b886bf13712d94dba.jpg"},{"id":107350695,"identity":"e92032bd-1a8f-41e7-b201-e06d7cc8a0a0","added_by":"auto","created_at":"2026-04-20 16:00:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16896555,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8019780/v1/700d6f3e-c822-4d42-9a69-28e6b7f80c65.pdf"},{"id":96611061,"identity":"8909f3a6-048d-45f0-b6b5-a18a185ef0b4","added_by":"auto","created_at":"2025-11-24 09:32:29","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":902124,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8019780/v1/61f92a0e9bbf055a19e8bc61.xlsx"},{"id":96610958,"identity":"7fce4afb-56d5-4d9a-843f-388eba6e619f","added_by":"auto","created_at":"2025-11-24 09:32:16","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3237044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.S1 \u003c/strong\u003eEffects of three-week oral administration of ASA and E-ASA on the safety of mice. \u003cstrong\u003ea, \u003c/strong\u003eExperimental design. 8 to 12 week-old C57BL/6 mice were randomly divided into three groups: one group received regular drinking water, one group received drinking water containing 0.15 mg/mL ASA, and one group received drinking water containing 0.15 mg/mL E‑ASA. All groups were fed a standard chow diet, and safety analysis was performed after three weeks. \u003cstrong\u003eb,\u003c/strong\u003e Changes in body weight of mice following three weeks of administration with ASA or E-ASA. Two-way-ANOVA followed by Tukey's multiple comparisons test, n=10 mice each group. \u003cstrong\u003ec,\u003c/strong\u003e Daily water intake of mice during the three-week treatment with ASA or E-ASA. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=21 mice each group. \u003cstrong\u003ed,\u003c/strong\u003ePlasma alanine aminotransferase (ALT) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. \u003cstrong\u003ee, \u003c/strong\u003ePlasma aspartate aminotransferase (AST) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. \u003cstrong\u003ef,\u003c/strong\u003e Plasma lactate dehydrogenase (LDH) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. \u003cstrong\u003eg,\u003c/strong\u003e Plasma blood urea nitrogen (BUN) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. \u003cstrong\u003eh,\u003c/strong\u003e Plasma creatinine (CREA) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9. \u003cstrong\u003ei,\u003c/strong\u003e Plasma uric acid (UA) levels after three weeks of ASA or E-ASA treatment. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. j, ASA prolonged bleeding time in mice, whereas E‑ASA significantly reduced tail bleeding time compared with ASA. Ordinary one-way-ANOVA followed by Turkey’s multiple-comparison test, n=9 mice each group. ALT: alanine aminotransferase, AST: aspartate aminotransferase, LDH: lactate dehydrogenase, BUN: blood urea nitrogen, CREA: creatinine, UA: uric acid. Data are presented as the mean ±SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ns means not significant.\u003c/p\u003e","description":"","filename":"FigS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8019780/v1/6458742bd3243187a7828db8.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ethyl Acetylsalicylate (E-ASA) Protects Against Myocardial Ischemia-Reperfusion Injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIschemic heart disease is one of the leading causes of death global(Vos et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For patients with myocardial ischemia, restoring blood flow through thrombolysis or percutaneous coronary intervention is the standard therapy recommended by current treatment guidelines(Ibanez et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the restoration of blood flow can exacerbate myocardial cell damage and death, leading to ischemia-reperfusion (I/R) injury (Yellon and Hausenloy \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). As a common complication, myocardial ischemia-reperfusion injury is characterized by high morbidity and mortality rates. Its physiological mechanism is complex, involving multiple processes including oxidative stress, calcium overload, and inflammatory responses, and there is still a lack of effective therapeutic approaches(Hausenloy et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAspirin serves as a secondary prevention medication for cardiovascular and cerebrovascular diseases. It works by irreversibly acetylating cyclooxygenase-1 (COX-1) at serine 530, inhibiting platelet aggregation and thus exerting antithrombotic effects(Loll et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Research has shown that aspirin has certain effects in inhibiting myocardial inflammation(Dong et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), reducing myocardial fibrosis(Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and alleviating myocardial ischemia-reperfusion injury(Seth et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).However, the mechanism of aspirin's cardioprotective effects has not yet been clearly elucidated. As a secondary prevention medication, long-term use of aspirin significantly increases patients' risk of bleeding(Antithrombotic Trialists et al. 2009) and damage to the gastrointestinal tract(Lanas et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), liver, and kidneys(Zheng and Roddick 2019).\u003c/p\u003e\u003cp\u003eTherefore, many studies have attempted to develop aspirin analogues or prodrugs(Willetts and Foley \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) to reduce its adverse drug reactions or find alternatives or adjuvants with better therapeutic effects(Wood et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). In animal models, we first confirmed the cardioprotective effects of aspirin in myocardial ischemia-reperfusion models, and we explored an aspirin derivative - ethyl acetylsalicylate (E-ASA), which is esterified at the carboxyl group of aspirin. Related studies have shown that the carboxyl group of aspirin may be associated with its bleeding risk(Killackey et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Drug esterification is a common derivatization strategy, with advantages typically including improved drug absorption, reduced toxicity, enhanced sustained activation capability, and facilitation of targeted drug delivery(Zhou \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Based on clinical application needs, aspirin still has certain limitations in terms of drug safety and efficacy. Therefore, we also explored the therapeutic potential of E-ASA in myocardial ischemia-reperfusion models and conducted preliminary investigations into its safety, time effectiveness, and dosage.\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cb\u003eChronic feeding mice with aspirin protects myocardium from I/R injury.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAspirin, with its century-long history, has been extensively utilized in clinical practice for the prevention of ischemic heart disease (Patrono et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). However, the effects of aspirin on the myocardial I/R injury are less well understood. Toward this aim, we established a mouse model of myocardial I/R injury (45 minutes of ischemia followed by reperfusion) according to established protocols (Lindsey et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The mice were fed with 0.15 mg/ml aspirin-containing drinking water for 3 weeks, followed by I/R, and then the myocardial I/R injury was assessed 24 h after reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The dosage of chronic aspirin treatment in mice was close to the daily dose of 100 mg aspirin for secondary prophylaxis of myocardial infarction (MI) in adult humans (Reagan-Shaw et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) (also see Methods on dosage conversion between human and mice).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eStrikingly, chronic feeding of aspirin significantly reduced the myocardial infarction areas in mice indicated by the Evans blue and TTC staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-d). Plasma creatine kinase - myocardial band isoenzyme (CK-MB) and aspartate aminotransferase (AST) are commonly used diagnostic markers for myocardial damage, and their levels are correlated with the extent of myocardial death (Nigam \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Likewise, we found that the plasma levels of CK-MB and AST were significantly increased in the I/R mice 24 h after reperfusion compared with the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). However, aspirin-treated I/R mice showed lower plasma levels of CK-MB and AST than saline-treated I/R mice (vehicle) 24 h after reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). Next, we assessed the cardiac function of the I/R mice 24 h after reperfusion through echocardiographic (ECG) recording. The I/R mice showed a significant reduction in the ejection fraction (EF) and fractional shortening (FS) compared with the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-i). Intriguingly, aspirin-treated I/R mice showed significant improvements in both EF and FS% 24 h after reperfusion, compared with saline-treated I/R mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-i). Taken together, these results demonstrate that chronic treatment with aspirin before MI may protect myocardial I/R injury.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAcute treatment with aspirin after MI shows minor protection against myocardial I/R injury in mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the following study, we sought to determine whether acute treatment with aspirin after MI can protect myocardial from I/R injury in mice. To this end, we performed intraperitoneal (i.p.) injection of 50 mg/kg aspirin 30 min after MI (i.e., 15 min before reperfusion), and then the myocardial I/R injury was assessed 24 h after reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The dosage of acute aspirin treatment in mice was close to the high dose of 300 mg aspirin administrated right after MI in adult patients (Ganjehei and Becker \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) (also see Methods on dosage conversion between human and mice).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDifferent from the results of chronic feeding with aspirin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), acute treatment with aspirin 30 min after MI did not reduce the myocardial infarction areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-i) or decrease the plasma levels of CK-MB and AST (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and f) in the I/R mice 24 h after reperfusion. In contrast, acute treatment with aspirin 30 min after MI led to significant increases in both EF and FS 24 h after reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d), indicating an improvement on cardiac functions. Taken together, these results demonstrate that acute treatment with aspirin after MI has minor protection against myocardial I/R injury in mice.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eE-ASA shows improved cell membrane permeability than aspirin\u003c/h2\u003e\u003cp\u003eAspirin has a poor cell membrane permeability due to the presence of a free carboxyl group (Menichetti et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which may lower the intracellular concentrations of aspirin and thus restrict the protective effects of acute aspirin treatment against myocardial I/R injury. Esterification of the carboxyl group can neutralize its negative charge under physiological pH conditions and thus enhance the cell membrane permeability of aspirin. Conversion of the carboxyl group into an ester typically increases membrane permeability, thereby enhancing pharmacological efficacy(Stella et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Rautio et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAccordingly, esterification of aspirin yields its alkyl esters\u0026mdash;methyl (M‑ASA), ethyl (E‑ASA), and propyl acetylsalicylate (P‑ASA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). To test this hypothesis, we analyzed the permeability across cell membranes of ASA, M-ASA, E-ASA and P-ASA with a web-based tool called PreADMET (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://preadmet.qsarhub.com/\u003c/span\u003e\u003cspan address=\"https://preadmet.qsarhub.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The prediction results demonstrated significantly improved cell membrane permeability for all three esterified aspirin derivatives compared to aspirin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHowever, ASA, M‑ASA, and E‑ASA exhibit higher flux values than P‑ASA(Gerber et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), indicating greater membrane permeability and absorption rates, which can be attributed to differences in lipophilicity, molecular weight, ionization, and membrane interactions. Considering bioavailability, we excluded P-ASA from further consideration. A compound safety assessment was conducted via the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/compound/68484\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/compound/68484\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). M-ASA (CAS No. 580-02-9) was excluded due to its documented skin irritation, eye and respiratory tract irritation, and specific target organ toxicity.\u003c/p\u003e\u003cp\u003eFollowing the preliminary evaluations, we further verified the in vivo safety of E‑ASA in comparison with ASA (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). After three weeks of treatment with the same concentration (0.15 mg/ml) of ASA or E‑ASA, there were no significant differences in body weight gain or daily water consumption among the three groups (vehicle, ASA, and E‑ASA) (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb, c). However, analysis of plasma biochemical markers related to hepatic and renal function\u0026mdash;including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine (CREA), and uric acid (UA)\u0026mdash;revealed that plasma AST, LDH, BUN, and CREA levels did not differ significantly among the three groups (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee-h). In contrast, ALT and UA levels were lower in the E‑ASA group than in the ASA group, suggesting that E‑ASA causes less hepatic and renal injury (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed, i).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven the well‑known bleeding risk associated with ASA in clinical settings, we also assessed tail‑bleeding time to compare the hemostatic safety of ASA and E‑ASA in mice. The ASA‑treated mice exhibited a significantly prolonged bleeding time, whereas the E‑ASA group showed no significant difference from the vehicle group, indicating that E‑ASA confers a markedly lower bleeding risk in vivo (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ej).\u003c/p\u003e\u003cp\u003eBuilding on the above findings, we compared the membrane permeability of ASA and E‑ASA in human cardiomyocyte cells (AC16 cell line). In vitro experiments demonstrated that E‑ASA exhibited significantly higher membrane permeability than ASA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAcute treatment with E‑ASA after MI was associated with reduced myocardial injury and improved cardiac function in mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the improved membrane permeability of E‑ASA relative to aspirin, we next examined whether post‑treatment could provide additional cardiac protection. To this aim, we performed intraperitoneal (i.p.) injection of 48 mg/kg aspirin and E-ASA 30 min after MI (i.e., 15 min before reperfusion), and then the myocardial I/R injury was assessed 24 h after reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). While posttreatment with aspirin did not reduce the myocardial infarction areas, posttreatment with E-ASA significantly attenuate the myocardial infarction in the I/R mice 24 h after reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-i). Likewise, posttreatment with E-ASA rather than aspirin significantly decreased the serum levels of AST and CK-MB in the I/R mice 24 h after reperfusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and f). Through echocardiography, we found that the cardiac function (EF and FS) of the I/R mice was significantly improved after acute treatment with ASA 24 h after reperfusion, but the improvement was more pronounced in the E-ASA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). Taken together, these results indicate that posttreatment with E-ASA confers better cardioprotection against I/R injury than aspirin in mice.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eE-ASA increased acetylation of proteins involved in the myocardial metabolism and I/R injury\u003c/h3\u003e\n\u003cp\u003eAspirin has an acetyl group and thus has been shown to acetylate a variety of intracellular proteins (Alfonso et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Since E-ASA also has an acetyl group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), we hypothesized that E-ASA would be able to acetylate intracellular proteins in the heart. To identify the intracellular proteins that can be acetylated by E-ASA, we performed label-free proteomic assay in heart tissues from saline (vehicle) and E-ASA-treated mice. The mice were i.p. injected with saline or 50 mg/kg E-ASA, and then the homogenates of heart tissues were collected and digested with trypsin 24 h after E-ASA treatment. The acetylated peptides were purified with the anti-acetyl-lysine (anti-Ac-K) antibodies and then were analyzed by the liquid chromatography plus tandem mass spectrometry (LC-MS/MS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFrom three independent experiments, we identified 1341 Ac-K sites that matched to 494 acetylated proteins from vehicle mice, and 1532 Ac-K sites that matched to 559 acetylated proteins from E-ASA-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The majority of the Ac-K sites and acetylated proteins are conserved between control and E-ASA groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Comparative analysis of the proteomics data from two groups of mice identified 296 upregulated acetylation sites (fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.2, P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) which matched to 216 acetylated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In contrast, only 50 acetylation sites were found to be downregulated (fold change\u0026thinsp;\u0026lt;\u0026thinsp;0.83, P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in mouse heart after E-ASA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eAnalysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that acetylated proteins upregulated by E-ASA were significantly enriched in metabolic pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The acetylation of several key enzymes in the processes of glycolysis, tricarboxylic acid cycle (TCA), and fatty acid oxidation was increased in the heart tissues after E-ASA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). On the other hand, acetylated proteins upregulated by E-ASA were also significantly overrepresented in the myocardial injury-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). The acetylation of several proteins involved in the processes of HIF-1 signaling pathway, PPAR signaling pathway, and hypertrophic cardiomyopathy was increased in the heart tissues after E-ASA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eE‑ASA treatment appeared to enhance the acetylation of key enzymes involved in cardiac energy metabolism.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eE-ASA increased the acetylation of K179 in SDHA (succinate dehydrogenase isoform α, a key enzyme involved in TCA and respiratory complex II) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e) which has been shown to reduce its activity(Horton et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). E-ASA increased the acetylation of K305 in Pkm (Pyruvate Kinase, Muscle type) is a key enzyme in the glycolysis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e) which has been shown to reduce its activity and inhibits the enzymatic function of PKM and promotes its degradation, redirecting cellular metabolism toward a branching pathway for glucose metabolism. (Lv et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough our mass spectrometry results show that there are many site-specific acetylation-upregulated proteins, no studies have yet reported the effects of acetylation on protein activity. However, many proteins have been reported to be associated with myocardial injury and cardioprotection. The study found that inhibiting Carnitine palmitoyltransferase-1b(CPT1b)-mediated fatty acid oxidation can change the metabolic state of the myocardium in the form of metabolic reprogramming and promote myocardial regeneration(Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Also it has been suggested that inhibiting CPT1 activity by specific CPT1 inhibitors exerts protective effects against cardiac hypertrophy and heart failure(He et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In addition, key metabolic enzymes such as Pfkm (Zhou et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), Aldoa (Luo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), Gapdh (Yao et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), Pgk1(Lu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Pgam2(Okuda et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and Pdha1(Lewandowski and White \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) have also been reported to play an important role in myocardial function and cardioprotection.\u003c/p\u003e\u003cp\u003eIn the results of mass spectrometry analysis, there are relatively few studies on the effects of acetylation on the activity of E-ASA differentially upregulated proteins, and the correlation between some differentially upregulated proteins and cardiac function remains largely unknown. We annotated key proteins upregulated by acetylation in the major differentially enriched metabolic pathways, including glycolysis, fatty acid oxidation, and ATP synthesis, including sites upregulated by E-ASA-mediated differential acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Blue indicates sites with literature reported on the effects of acetylation on protein activity, while red indicates sites with unknown effects on protein activity.\u003c/p\u003e"},{"header":"Disussion","content":"\u003cp\u003eOur preliminary experiments in animal models indicated that E‑ASA showed a more pronounced cardioprotective effect compared with classical aspirin. This enhanced efficacy may be related to the modification of ASA's carboxyl group through esterification, which potentially increases the drug's cell membrane permeability while maintaining a lower bleeding risk profile. The bleeding risk assessment was conducted through in vivo tail bleeding experiments. In subsequent studies, we plan to further investigate E-ASA's affinity for COX-1 and its effects on COX-1 activity to elucidate the mechanisms underlying its reduced bleeding risk.\u003c/p\u003e\u003cp\u003eLong-term E-ASA administration resulted in altered cardiac acetylation levels, with increased acetylation potentially stemming from E-ASA's inherent acetyl group structure. Biological acetylation occurs through two primary mechanisms: enzyme-dependent and enzyme-independent pathways(Weinert et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The enzyme-dependent acetylation is primarily catalyzed by histone acetyltransferases (HATs), which transfer acetyl groups from acetyl-CoA to lysine residues on target proteins. The enzyme-independent form relies mainly on acetyl group donors (such as acetyl-CoA) concentration and the chemical environment. Therefore, E-ASA may exert its biological functions through modulating the complex internal environment, coordinating both enzyme-dependent and enzyme-independent pathways.\u003c/p\u003e\u003cp\u003eAcetylproteomic analysis revealed that E‑ASA administration was associated with increased acetylation levels in several cardiac energy metabolism-related proteins. Although these modifications encompass acetylation changes in various rate-limiting enzymes across multiple metabolic pathways, which may affect protein structural properties or biological activities, we cannot fully investigate each individual mechanism in detail. Previous studies on metabolic regulators such as Cpt1(Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and the SDH (Zhang and Lang \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) family have demonstrated cardioprotective roles, which may provide mechanistic context for the effects observed with E‑ASA.\u003c/p\u003e\u003cp\u003eWe also explored the differences in E-ASA's effects across various dosages and time points, which may be related to its in vivo pharmacokinetics and metabolism - aspects we plan to investigate further in subsequent experiments. Given the abundance of esterases in biological systems, E-ASA's in vivo stability presents certain challenges that warrant consideration.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e Male C57BL/6J mice aged 8\u0026ndash;12 weeks were obtained from Shanghai Jihui Laboratory Animal Care Co., Ltd. Male mice were selected for all experimental procedures to avoid confounding effects associated with estrous cycles. Animals were maintained in a specific pathogen-free (SPF) facility under standard laboratory conditions with a 12:12-hour light-dark cycle and provided with food and water ad libitum.\u003c/p\u003e\u003cp\u003eIn the three‑week chronic feeding experiment, 30 male C57BL/6 mice (8\u0026ndash;12 weeks old) were randomly divided into three groups: Sham (normal drinking water, sham surgery), Vehicle (normal drinking water, I/R), and E‑ASA (E‑ASA‑containing drinking water, I/R), n\u0026thinsp;=\u0026thinsp;10 mice per group. In the acute administration experiment, 32 male C57BL/6 mice (8\u0026ndash;12 weeks old) were randomly divided into four groups: Sham (saline, sham surgery), Vehicle (saline, I/R), ASA (aspirin, I/R), and E‑ASA (E‑ASA, I/R), n\u0026thinsp;=\u0026thinsp;8 mice per group. For acetylation proteomics, six mice (12 weeks old) were used, with three in the Vehicle group and three in the E‑ASA group. In the three‑week chronic feeding safety evaluation, 33 mice (8\u0026ndash;12 weeks old) were randomly divided into three groups: Vehicle (normal drinking water), ASA (aspirin‑containing water), and E‑ASA (E‑ASA‑containing water), n\u0026thinsp;=\u0026thinsp;11 mice per group. Minor discrepancies in n-values among experiments may result from quality‑control exclusions during data acquisition.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCellular Uptake of E‑ASA in AC16 Cardiomyocytes\u003c/h2\u003e\u003cp\u003eHuman AC16 cardiomyocytes, kindly provided by Professor Tao Zhong (East China Normal University, China), were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37\u0026deg;C in 5% CO₂. Cells at approximately 80% confluence were incubated with E‑ASA at a final concentration of 0.1 mM for 40, 80, 120, and 160 min. After incubation, cells were washed twice with ice‑cold PBS to remove residual compound and lysed on ice using (lysis buffer) for quantitative analysis. Cell lysates were centrifuged, and the supernatants were subjected to LC\u0026ndash;MS/MS determination of E‑ASA levels using standard calibration curves. Intracellular E‑ASA concentrations were normalized to total protein quantified by the BCA assay.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTail Bleeding Time Assay\u003c/h3\u003e\n\u003cp\u003eUnder sustained anesthesia with 2% isoflurane, mice from each experimental group underwent tail transection at a diameter of 2 mm with a single, rapid cut. The transected tail was immediately immersed in warm saline, and the bleeding time (duration from initial bleeding to hemostasis) was recorded.\u003c/p\u003e\n\u003ch3\u003eIn vivo model of myocardial ischemia and reperfusion\u003c/h3\u003e\n\u003cp\u003eMale C57BL/6 mice aged 8\u0026ndash;12 weeks were used to establish the I/R animal model. The mice were fixed on a heating pad maintained at 37\u0026deg;C using medical tape and anesthetized with 2% isoflurane via inhalation. After removing the chest and abdominal hair, a sterile scissor was used to make an incision at the position of strongest heartbeat, and a 4\u0026thinsp;\u0026minus;\u0026thinsp;0 suture was prepared for pre-suturing. Using curved forceps, the thoracic cavity above the heart was bluntly dissected through the third and fourth intercostal space. The mouse position was adjusted to gently compress the thoracic cavity, extruding the heart. An 8\u0026thinsp;\u0026minus;\u0026thinsp;0 suture was used to create a slipknot around the left anterior descending coronary artery, with one end of the suture exposed outside the thoracic cavity. The heart was then returned to the thoracic cavity, excess air was evacuated, and the skin was sutured. The mouse was gently placed in a warming box. After 45 minutes, the retained slipknot was released, and the mouse was returned to its housing cage for continued maintenance.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEchocardiography\u003c/h2\u003e\u003cp\u003eEchocardiographic measurements were performed using the Vevo 2100 system (MS400C probe) in all groups of mice. The sonographers was blinded to group allocation. Left ventricular end-diastolic and end-systolic dimensions were derived from M-mode images. Fractional shortening was calculated as a percentage using the following formula: (left ventricular end-diastolic dimension - left ventricular end-systolic dimension)/left ventricular end-diastolic dimension. All measurements were conducted at the papillary muscle level following standardized protocols.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eInfarct size measurement\u003c/h2\u003e\u003cp\u003eAt 24 hours after myocardial ischemia-reperfusion, the chest cavity was reopened to expose the mouse heart. The left anterior descending artery was re-ligated at the same location, followed by immediate injection of 1 mL 2% Evans blue staining solution (Sigma-Aldrich, St. Louis, MO, United States, dissolved in phosphate buffer) into the aorta. The heart was then excised and rapidly frozen at -80\u0026deg;C for 10 minutes. The frozen heart was evenly cut into 5 slices from the ligation site to the apex. The heart slices were incubated in 2% TTC solution (2,3,5-Triphenyltetrazolium Chloride, Sigma-Aldrich, St. Louis, MO, United States, dissolved in phosphate buffer pH 7.4, 3 mL per heart) at 37\u0026deg;C for 30 minutes. After staining, the heart slices were fixed in 4% paraformaldehyde for 15 minutes before digital scanning and photography. The non-ischemic area of the left ventricle was stained dark blue, viable tissue within the area at risk appeared bright red, and infarcted tissue appeared white or pale yellow. Image analysis was performed using ImageJ software. The infarct size as a percentage of the area at risk was calculated using standard methods as previously described(Xie et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eBiochemical assessment\u003c/h2\u003e\u003cp\u003eAfter 24 hours of myocardial ischemia-reperfusion, blood samples were collected from each group of mice using 3.8% (w/v) sodium citrate as an anticoagulant. After standing at room temperature for 30 minutes, the collected blood was centrifuged at 3000 rpm at 4\u0026deg;C for 15 minutes. The supernatant was collected as plasma and stored at -80\u0026deg;C for subsequent analysis, avoiding repeated freeze-thaw cycles. The indicators for assessing myocardial injury included aspartate aminotransferase (AST) and creatine kinase isoenzyme.\u003c/p\u003e\u003cp\u003eFor the three groups of mice (Control, ASA, and E-ASA) that underwent three weeks of drug administration, plasma samples were collected following the aforementioned method upon completion of treatment and stored at -80\u0026deg;C for subsequent analysis. To evaluate potential hepatic and renal injury during the three-week treatment period, the following parameters were measured: aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) for comprehensive assessment of liver function; blood urea nitrogen (BUN), urinary creatinine, and uric acid for comprehensive assessment of renal function.All plasma parameter measurements were conducted by Wuhan Servicebio Technology Co., Ltd.\u003c/p\u003e\u003cp\u003e\u003cb\u003e4D-Label Free Acetylation Mass Spectrometry Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHeart tissues from control and E-ASA-treated mice were homogenized in urea-based lysis buffer supplemented with protease and deacetylase inhibitors. The extracted proteins underwent reduction with dithiothreitol (DTT), alkylation with iodoacetamide, and overnight tryptic digestion. Following desalting, acetylated peptides were enriched using anti-acetyl-lysine antibody-conjugated agarose beads. The samples were then loaded onto a 25 cm C18 analytical column (25 cm \u0026times; 75 \u0026micro;m inner diameter, 1.6 \u0026micro;m C18 particles, IonOpticks) at a flow rate of 300 nL/min and subjected to gradient elution. The resulting experimental data were processed using MaxQuant software (Version 1.6.17.0) for database searching.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll the data were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM unless otherwise mentioned. The sample size justification was based on the previous studies and met the requirement of statistics power. The N number of mice could be different for distinctive assays. All the statistical analyses were performed by the software of GraphPad Prism 8.0.2. Statistically significant difference was indicated as follows: **** P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, *** P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eAuthors and Affiliations\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Ultrasound, Changning Maternity and Infant Health Hospital, East China Normal University, Shanghai, 200050, China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBi-Han Wang, Hai-Huan Gao and Bi-Yuan He\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKey Laboratory of Brain Functional Genomics, Ministry of Education and Shanghai, School of Life Science, East China Normal University, Shanghai, 200062, China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBi-Han Wang, Wen-Liang Jiang and Dong-Min Yin\u003c/p\u003e\n\u003ch2\u003eCorresponding author\u003c/h2\u003e\n\u003cp\u003eCorrespondence to Bi-Yuan He.\u003c/p\u003e\n\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eMouse care and experiments were performed according to the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the Animal Care and Use Committee of East China Normal University (m20230704). Clinical trial number: not applicable.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe study was supported by PI research team program of health commission foundation of Changning district, Shanghai, China(PI202431).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eBi-Han Wang: Methodology, Validation, Formal analysis, Investigation, Data Curation and Project Administration. Hai-Huan Gao: Review of original draft and Visualization. Wen-Liang Jiang: Supervision, Data Curation. Dong-Min Yin, Bi-yuan He: Conceptualization, Supervision, Funding acquisition, Writing \u0026ndash; Review \u0026amp; Editing. The final version of the manuscript has been reviewed and approved by all authors. The authors declare that all data were generated in-house and that no paper mill was used.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVos T, Lim SS, Abbafati C, Abbas KM, Abbasi M, Abbasifard M, et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1990\u0026ndash;2019: a systematic analysis for the Global Burden of Disease Study 2019. The lancet. 2020;396(10258):1204\u0026ndash;22 \u003c/li\u003e\n\u003cli\u003eIbanez B, James S, Agewall S, Antunes MJ, Bucciarelli-Ducci C, Bueno H, et al. 2017 ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force for the management of acute myocardial infarction in patients presenting with ST-segment elevation of the European Society of Cardiology (ESC). European heart journal. 2018;39(2):119\u0026ndash;77 \u003c/li\u003e\n\u003cli\u003eYellon DM, Hausenloy DJ. Myocardial reperfusion injury. N Engl J Med. 2007;357(11):1121\u0026ndash;35.https://doi.org/[10.1056/NEJMra071667\u003c/li\u003e\n\u003cli\u003eHausenloy DJ, Chilian W, Crea F, Davidson SM, Ferdinandy P, Garcia-Dorado D, et al. The coronary circulation in acute myocardial ischaemia/reperfusion injury: a target for cardioprotection. Cardiovasc Res. 2019;115(7):1143\u0026ndash;55.https://doi.org/[10.1093/cvr/cvy286\u003c/li\u003e\n\u003cli\u003eLoll PJ, Picot D, Garavito RM. The structural basis of aspirin activity inferred from the crystal structure of inactivated prostaglandin H2 synthase. Nat Struct Biol. 1995;2(8):637\u0026ndash;43.https://doi.org/[10.1038/nsb0895-637\u003c/li\u003e\n\u003cli\u003eDong Z, Yang L, Jiao J, Jiang Y, Li H, Yin G, et al. Aspirin in combination with gastrodin protects cardiac function and mitigates gastric mucosal injury in response to myocardial ischemia/reperfusion. Front Pharmacol. 2022;13:995102.https://doi.org/[10.3389/fphar.2022.995102\u003c/li\u003e\n\u003cli\u003eLiu PP, Liu HH, Sun SH, Shi XX, Yang WC, Su GH, et al. Aspirin alleviates cardiac fibrosis in mice by inhibiting autophagy. Acta Pharmacol Sin. 2017;38(4):488\u0026ndash;97.https://doi.org/[10.1038/aps.2016.143\u003c/li\u003e\n\u003cli\u003eSeth SD, Maulik M, Manchanda SC, Maulik SK. Role of aspirin in modulating myocardial ischemic reperfusion injury. Agents Actions. 1994;41(3-4):151\u0026ndash;5.https://doi.org/[10.1007/BF02001909\u003c/li\u003e\n\u003cli\u003eAntithrombotic Trialists C, Baigent C, Blackwell L, Collins R, Emberson J, Godwin J, et al. Aspirin in the primary and secondary prevention of vascular disease: collaborative meta-analysis of individual participant data from randomised trials. Lancet. 2009;373(9678):1849\u0026ndash;60.https://doi.org/[10.1016/S0140-6736(09)60503-1\u003c/li\u003e\n\u003cli\u003eLanas A, Wu P, Medin J, Mills EJ. Low doses of acetylsalicylic acid increase risk of gastrointestinal bleeding in a meta-analysis. Clin Gastroenterol Hepatol. 2011;9(9):762\u0026ndash;8 e6.https://doi.org/[10.1016/j.cgh.2011.05.020\u003c/li\u003e\n\u003cli\u003eZheng SL, Roddick AJ. Association of aspirin use for primary prevention with cardiovascular events and bleeding events: a systematic review and meta-analysis. Jama. 2019;321(3):277\u0026ndash;87 \u003c/li\u003e\n\u003cli\u003eWilletts S, Foley DW. True or false? Challenges and recent highlights in the development of aspirin prodrugs. Eur J Med Chem. 2020;192:112200.https://doi.org/[10.1016/j.ejmech.2020.112200\u003c/li\u003e\n\u003cli\u003eWood PH, Harvey-Smith E, Dixon ASJ. Salicylates and gastro-intestinal bleeding. British Medical Journal. 1962;1(5279):669 \u003c/li\u003e\n\u003cli\u003eKillackey JJ, Killackey BA, Philp RB. Structure-activity studies of aspirin and related compounds on platelet aggregation, arachidonic acid metabolism in platelets and artery, and arterial prostacyclin activity. Prostaglandins Leukot Med. 1982;9(1):9\u0026ndash;23.https://doi.org/[10.1016/0262-1746(82)90068-3\u003c/li\u003e\n\u003cli\u003eZhou G. Exploring Ester Prodrugs: A Comprehensive Review of Approaches, Applications, and Methods. Pharmacology \u0026amp; Pharmacy. 2024;15(8):269\u0026ndash;84 \u003c/li\u003e\n\u003cli\u003ePatrono C, Garcia Rodriguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med. 2005;353(22):2373\u0026ndash;83.https://doi.org/[10.1056/NEJMra052717\u003c/li\u003e\n\u003cli\u003eLindsey ML, Bolli R, Canty JM, Jr., Du XJ, Frangogiannis NG, Frantz S, et al. Guidelines for experimental models of myocardial ischemia and infarction. Am J Physiol Heart Circ Physiol. 2018;314(4):H812\u0026ndash;H38.https://doi.org/[10.1152/ajpheart.00335.2017\u003c/li\u003e\n\u003cli\u003eReagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J. 2008;22(3):659\u0026ndash;61.https://doi.org/[10.1096/fj.07-9574LSF\u003c/li\u003e\n\u003cli\u003eNigam PK. Biochemical markers of myocardial injury. Indian J Clin Biochem. 2007;22(1):10\u0026ndash;7.https://doi.org/[10.1007/BF02912874\u003c/li\u003e\n\u003cli\u003eGanjehei L, Becker RC. Aspirin dosing in cardiovascular disease prevention and management: an update. J Thromb Thrombolysis. 2015;40(4):499\u0026ndash;511.https://doi.org/[10.1007/s11239-015-1267-6\u003c/li\u003e\n\u003cli\u003eMenichetti R, Kanekal KH, Bereau T. Drug-Membrane Permeability across Chemical Space. ACS Cent Sci. 2019;5(2):290\u0026ndash;8.https://doi.org/[10.1021/acscentsci.8b00718\u003c/li\u003e\n\u003cli\u003eStella VJ, Charman WN, Naringrekar VH. Prodrugs. Do they have advantages in clinical practice? Drugs. 1985;29(5):455\u0026ndash;73.https://doi.org/[10.2165/00003495-198529050-00002\u003c/li\u003e\n\u003cli\u003eRautio J, Kumpulainen H, Heimbach T, Oliyai R, Oh D, Jarvinen T, et al. Prodrugs: design and clinical applications. Nat Rev Drug Discov. 2008;7(3):255\u0026ndash;70.https://doi.org/[10.1038/nrd2468\u003c/li\u003e\n\u003cli\u003eGerber M, Breytenbach JC, Hadgraft J, du Plessis J. Synthesis and transdermal properties of acetylsalicylic acid and selected esters. Int J Pharm. 2006;310(1-2):31\u0026ndash;6.https://doi.org/[10.1016/j.ijpharm.2005.11.018\u003c/li\u003e\n\u003cli\u003eAlfonso LF, Srivenugopal KS, Bhat GJ. Does aspirin acetylate multiple cellular proteins? (Review). Mol Med Rep. 2009;2(4):533\u0026ndash;7.https://doi.org/[10.3892/mmr_00000132\u003c/li\u003e\n\u003cli\u003eGuo L, Gao J, Gao Y, Zhu Z, Zhang Y. Aspirin Reshapes Acetylomes in Inflammatory and Cancer Cells via CoA-Dependent and CoA-Independent Pathways. J Proteome Res. 2020;19(2):962\u0026ndash;72.https://doi.org/[10.1021/acs.jproteome.9b00853\u003c/li\u003e\n\u003cli\u003eHorton JL, Martin OJ, Lai L, Riley NM, Richards AL, Vega RB, et al. Mitochondrial protein hyperacetylation in the failing heart. JCI insight. 2016;1(2):e84897 \u003c/li\u003e\n\u003cli\u003eLv L, Li D, Zhao D, Lin R, Chu Y, Zhang H, et al. Acetylation targets the M2 isoform of pyruvate kinase for degradation through chaperone-mediated autophagy and promotes tumor growth. Mol Cell. 2011;42(6):719\u0026ndash;30.https://doi.org/[10.1016/j.molcel.2011.04.025\u003c/li\u003e\n\u003cli\u003eLi X, Wu F, G\u0026uuml;nther S, Looso M, Kuenne C, Zhang T, et al. Inhibition of fatty acid oxidation enables heart regeneration in adult mice. Nature. 2023;622(7983):619\u0026ndash;26 \u003c/li\u003e\n\u003cli\u003eHe L, Kim T, Long Q, Liu J, Wang P, Zhou Y, et al. Carnitine palmitoyltransferase-1b deficiency aggravates pressure overload-induced cardiac hypertrophy caused by lipotoxicity. Circulation. 2012;126(14):1705\u0026ndash;16.https://doi.org/[10.1161/CIRCULATIONAHA.111.075978\u003c/li\u003e\n\u003cli\u003eZhou M, Sun X, Wang C, Wang F, Fang C, Hu Z. PFKM inhibits doxorubicin-induced cardiotoxicity by enhancing oxidative phosphorylation and glycolysis. Sci Rep. 2022;12(1):11684.https://doi.org/[10.1038/s41598-022-15743-0\u003c/li\u003e\n\u003cli\u003eLuo G, Wang R, Zhou H, Liu X. ALDOA protects cardiomyocytes against H/R-induced apoptosis and oxidative stress by regulating the VEGF/Notch 1/Jagged 1 pathway. Mol Cell Biochem. 2021;476(2):775\u0026ndash;83.https://doi.org/[10.1007/s11010-020-03943-z\u003c/li\u003e\n\u003cli\u003eYao LL, Wang YG, Liu XJ, Zhou Y, Li N, Liu J, et al. Phenylephrine protects cardiomyocytes from starvation-induced apoptosis by increasing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. J Cell Physiol. 2012;227(10):3518\u0026ndash;27.https://doi.org/[10.1002/jcp.24053\u003c/li\u003e\n\u003cli\u003eLu Y, Zhao N, Wu Y, Yang S, Wu Q, Dong Q, et al. Inhibition of phosphoglycerate kinase 1 attenuates autoimmune myocarditis by reprogramming CD4+ T cell metabolism. Cardiovasc Res. 2023;119(6):1377\u0026ndash;89.https://doi.org/[10.1093/cvr/cvad029\u003c/li\u003e\n\u003cli\u003eOkuda J, Niizuma S, Shioi T, Kato T, Inuzuka Y, Kawashima T, et al. Persistent overexpression of phosphoglycerate mutase, a glycolytic enzyme, modifies energy metabolism and reduces stress resistance of heart in mice. PLoS One. 2013;8(8):e72173.https://doi.org/[10.1371/journal.pone.0072173\u003c/li\u003e\n\u003cli\u003eYuan S, Xie R, Zhang X, Tang Y, Jin K, Fan J, et al. Double face of glycolytic enzyme ENO1 in heart failure. medRxiv. 2025:2025.03. 31.25324995 \u003c/li\u003e\n\u003cli\u003eLewandowski ED, White LT. Pyruvate dehydrogenase influences postischemic heart function. Circulation. 1995;91(7):2071\u0026ndash;9.https://doi.org/[10.1161/01.cir.91.7.2071\u003c/li\u003e\n\u003cli\u003eWeinert BT, Narita T, Satpathy S, Srinivasan B, Hansen BK, Scholz C, et al. Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome. Cell. 2018;174(1):231\u0026ndash;44 e12.https://doi.org/[10.1016/j.cell.2018.04.033\u003c/li\u003e\n\u003cli\u003eZhang W, Lang R. Succinate metabolism: a promising therapeutic target for inflammation, ischemia/reperfusion injury and cancer. Front Cell Dev Biol. 2023;11:1266973.https://doi.org/[10.3389/fcell.2023.1266973\u003c/li\u003e\n\u003cli\u003eYu J, Yao Q, Hu T, Zhang Y, Liu Y, Xiao Y, et al. The Role of HINT3 in Myocardial Ischemia-Reperfusion Injury in Male Mice: Mechanisms Involving SDHA and its Acetylation. Adv Sci (Weinh). 2025;12(33):e03109.https://doi.org/[10.1002/advs.202503109\u003c/li\u003e\n\u003cli\u003eXie M, Kong Y, Tan W, May H, Battiprolu PK, Pedrozo Z, et al. Histone deacetylase inhibition blunts ischemia/reperfusion injury by inducing cardiomyocyte autophagy. Circulation. 2014;129(10):1139\u0026ndash;51 \u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"myocardial ischemia-reperfusion, aspirin derivative, ethyl acetylsalicylate, cardiac metabolism","lastPublishedDoi":"10.21203/rs.3.rs-8019780/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8019780/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTimely reperfusion remains the standard therapy for myocardial ischemia, but it inevitably causes myocardial ischemia‑reperfusion injury (IRI), which remains a major clinical challenge. This study aimed to evaluate the cardioprotective effects of aspirin (ASA) and to identify aspirin ester derivatives with improved efficacy and safety profiles.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe cardioprotective potential of ASA was validated in established animal models of myocardial IRI. Multiple aspirin ester derivatives were comparatively analyzed and screened based on their structural and physicochemical characteristics. Among these compounds, ethyl acetylsalicylate (E‑ASA) was identified as exhibiting higher cell membrane permeability and a lower bleeding risk than ASA. The cardioprotective mechanisms of E‑ASA were further investigated using acetylation proteomics and metabolic pathway analysis in myocardial tissue.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eE‑ASA treatment significantly reduced myocardial infarct size and preserved cardiac function following IRI compared with ASA. Proteomic analyses revealed that E‑ASA induced hyperacetylation of key metabolic enzymes involved in cardiac energy metabolism, suggesting that its cardioprotective effects may be mediated through modulation of metabolic remodeling. Acute high‑dose administration of E‑ASA also provided stronger cardioprotection than equimolar doses of ASA in both cellular and in vivo models.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eE‑ASA demonstrated improved cardioprotective effects and a lower bleeding tendency compared with aspirin in preclinical models. These findings suggest that E‑ASA may represent a potential therapeutic lead requiring further validation for the prevention and treatment of ischemic heart disease.\u003c/p\u003e","manuscriptTitle":"Ethyl Acetylsalicylate (E-ASA) Protects Against Myocardial Ischemia-Reperfusion Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 09:25:57","doi":"10.21203/rs.3.rs-8019780/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-07T15:48:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-07T06:09:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-07T06:08:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2025-11-03T13:33:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"840cc514-fb9d-4c54-8b50-61154d3c48b7","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T15:59:59+00:00","versionOfRecord":{"articleIdentity":"rs-8019780","link":"https://doi.org/10.1007/s00210-026-05308-7","journal":{"identity":"naunyn-schmiedebergs-archives-of-pharmacology","isVorOnly":false,"title":"Naunyn-Schmiedeberg's Archives of Pharmacology"},"publishedOn":"2026-04-13 15:56:49","publishedOnDateReadable":"April 13th, 2026"},"versionCreatedAt":"2025-11-24 09:25:57","video":"","vorDoi":"10.1007/s00210-026-05308-7","vorDoiUrl":"https://doi.org/10.1007/s00210-026-05308-7","workflowStages":[]},"version":"v1","identity":"rs-8019780","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8019780","identity":"rs-8019780","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}