2.1. Experimental Animals and Grouping

In this study, a cohort of thirty-six adult male C57BL/6 mice, procured from Henan Skobes Biotechnology Co., Ltd, Henan, China, each weighing an average of 25 ± 2 g, were acclimatized to a controlled environment with a 12-hour light/dark cycle at a stable temperature of 25°C. They had unrestricted access to standard chow and water. The subjects were stratified into six distinct cohorts, each comprising six mice: the sham-operated group, the myocardial ischemia-reperfusion (MI/R) group, the sham-negative control (sham-NC)/reperfusion-negative control (R-NC) group, and the MI/R group treated with an H2AX inhibitor.

2.2. Mouse Model of Myocardial Ischemia/Reperfusion (MI/R)

For the experimental procedure, the mice received an intraperitoneal dose of 1% sodium pentobarbital (50 mg/kg, sourced from Sigma, St. Louis, USA) to induce anesthesia. Subsequent to tracheal intubation, the subjects were connected to a rodent ventilator (model ALC-V8, Shanghai, China) for mechanical respiration. Sterilization of the thoracic surface preceded a left-sided thoracotomy between the fourth and fifth ribs to reveal the cardiac structure. Ischemic conditions were simulated by the occlusion of the left anterior descending (LAD) coronary artery using a surgical suture for a duration of 30 minutes, succeeded by a 180 minute reperfusion phase initiated by suture release. The onset of ischemia was visually confirmed by a discernible discoloration in the myocardial tissue and the designated risk zone. Reperfusion was verified upon the removal of the occlusion and closure of the surgical site. In both the sham-operated group treated with H2AX inhibitor and the MI/R cohort receiving the same inhibitor, the compound was directly administered into the cardiac muscle tissue.

2.3. ELISA

Antigens for lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and creatine kinase (CK) were immobilized onto a microtiter plate, each well receiving 100 μL, and incubated at 37°C for a span of 4 hours. Following incubation, the wells were emptied. A 5% solution of fetal bovine serum was then introduced into each well, serving as a blocking agent against nonspecific interactions over a 40 minute incubation at 37°C. Subsequent to a triad of washes, the wells were filled with 100 μL of the appropriately diluted samples and incubated once more at 37°C for 40 to 60 minutes. After three additional washes, wells were treated with enzyme-conjugated antibodies and incubated for 60 minutes at 37°C. A final series of three washes preceded the addition of TMB substrate, ceasing the enzymatic reaction. The optical density (OD) of each well was quantified at a 450 nm wavelength using an ELISA microplate reader. The concentrations of LDH, AST, and CK were deduced by plotting the OD values against a pre-established standard curve.

2.4. 2,3,5-Triphenyltetrazolium Chloride (TTC) Staining

The cardiac specimens from each experimental subset underwent a perfusion process with 3 mL of 2% Evans blue dye (procured from Thermo Fisher) administered via the aortic conduit. Subsequently, they were methodically arranged on an icy metal surface and subjected to a −20°C environment for an overnight period. Post-freezing, the hearts were sectioned into 2 mm coronal segments utilizing a chilled, keen-edged instrument, in alignment with the longitudinal axis of the left ventricular chamber. These sections were then immersed in a 2% triphenyltetrazolium chloride (TTC) solution (sourced from Sigma Aldrich) maintained at 37°C for a quarter-hour, followed by a phosphate-buffered saline (PBS) rinse, and ultimately preserved in a 4% formalin solution.

2.5. Mouse Echocardiography for Cardiac Function Assessment

Upon the completion of the modeling phase and a fortnight of pharmacological intervention, the murine subjects were rendered unconscious using isoflurane. Subsequent to depilation of the thoracic region, the post-therapeutic cardiac architecture was scrutinized utilizing the Vevo 2100 imaging apparatus. Bidimensional echocardiographic snapshots were captured, delineating structural cardiac parameters such as left ventricular end-diastolic diameter (LVID; d), left ventricular end-systolic diameter (LVID; s), systolic thickness of the left ventricular anterior wall (LVAW; s), and diastolic thickness of the left ventricular anterior wall (LVAW; d). Functional cardiac indices were represented by the ejection fraction (EF) and fractional shortening (FS).

2.6. Masson’s Staining

Post-dissection, the cardiac tissues of the mice were preserved in 4% paraformaldehyde, subsequently encased within paraffin, and sectioned into 4 μm slices. These slices underwent a staining regimen with specific binding agents. Following a cleansing cycle, the sections were treated with Sirius Red solution (courtesy of Thermo Fisher) for a 3 minute duration, subjected to a differentiation process using a hydrochloric acid-alcohol solution, and then stained with Ponceau acid fuchsin for 10 minutes. This was succeeded by a 4 minute differentiation stage in 1% phosphomolybdic acid solution. The sections were further stained with a light green solution for 5 minutes and set in a neutral resin medium. The final step involved capturing the stained sections’ images through microscopic photography.

2.7. Transmission Electron Microscopy (TEM)

The specimens obtained were sectioned into trifurcated segments and subjected to stabilization using a 2.5% glutaraldehyde solution. Subsequently, they underwent a secondary fixation with a 1% osmium tetroxide preparation, followed by encapsulation within an epoxy resin matrix for an extended period. Post-embedding, the segments were sequentially treated with uranyl acetate for a duration of half an hour and lead citrate for a brief five-minute interval. The prepared samples were then examined utilizing a Philips CM120 transmission electron microscope to assess ultrastructural details.

2.8. Cell Treatment and Grouping

The HL-1 murine cardiomyocyte strain was procured from Procell Life Science&Technology Co.,Ltd.(Wuhan, China). These cells were propagated in MEM medium, enriched with 10% fetal bovine serum and 1% double antibody. The HL-1 culture was subjected to a pre-equilibration phase with a hypoxic gas mixture (95% nitrogen and 5% oxygen) for a minimum of two hours. This was succeeded by a four-hour hypoxic exposure within an incubator set to 95% nitrogen and 5% carbon dioxide at a controlled temperature of 37°C. Following this, the culture medium was refreshed, and the cells underwent a reoxygenation process in an atmosphere containing 95% air and 5% carbon dioxide for two hours. To delineate the negative control (NC) and the H2AX inhibitor-treated groups, HL-1 cells were administered an H2AX inhibitor. Furthermore, the cardiomyocytes within both the NC and H2AX inhibitor cohorts received treatment with the JNK pathway stimulant anisomycin and the P53 pathway activator MeOIstPyrd.

2.9. Western Blotting

Protein samples were isolated utilizing a dedicated extraction kit, and concentrations were quantified. A measure of 50 µg of the isolated protein underwent electrophoretic separation via SDS-PAGE, followed by the transference of the resolved proteins onto a nitrocellulose membrane through the blotting technique. To prevent nonspecific interactions, the membrane was saturated with 3.0% skim milk, then incubated with a suite of primary antibodies targeting H2AX (Abcam, ab26350, 1 : 1000), P53 (Abcam, ab307802, 1 : 1000), p-JNK (Abcam, ab32509, 1 : 1000), SHP2 (Abcam, ab62322, 1 : 1000), p-SHP2 (Abcam, ab32509, 1 : 1000), p-RAS (Cell Signaling, #3321, 1 : 1000), parkin (Abcam, ab77924, 1 : 2000), Drp1 (Abcam, ab184247, 1 : 1000), Cyt-C (Abcam, ab133504, 1 : 5000), Caspase-3 (Abcam, ab32351, 1 : 5000), and Caspase-8 (Abcam, ab32397, 1 : 1000), as well as GAPDH (Abcam, ab9485, 1 : 2500) at 4°C. Subsequent to thorough rinsing, the membrane was exposed to horseradish peroxidase-tagged goat anti-rabbit secondary antibody (Abcam, ab97080, 1 : 5000) at ambient temperature for a span of two hours. The final step involved capturing chemiluminescent signals and quantifying grayscale intensities via analytical software.

2.10. Statistical Analysis

The statistical evaluation and data manipulation were executed utilizing GraphPad Prism version 9.0. The quantitative data are articulated as mean ± standard deviation. For the assessment of differences across multiple cohorts, the chi-square test was employed, whereas the independent sample t-test facilitated the comparative analysis between two distinct groups. A p value falling below the threshold of 0.05 was deemed to denote statistical significance.

3.1. The Expression of p-H2AX in the Myocardial Tissue of Mice Subjected to Myocardial Ischemia-Reperfusion Significantly Increased

Initially, the study aimed to elucidate the function of phosphorylated H2AX in myocardial ischemia-reperfusion (I/R) events in murine models. To this end, a myocardial I/R model was established alongside a control cohort undergoing sham operations. Western blot analyses revealed a marked upregulation in the expression levels of phosphorylated H2AX in the myocardial I/R group when contrasted with the sham-operated counterparts, as depicted in Figures 1(a) and 1(b).

3.2. Inhibition of H2AX Can Improve Cardiac Function in Mice Subjected to Myocardial Ischemia-Reperfusion

Lactate dehydrogenase (LDH), a cytoplasmic enzyme, is pivotal in glycolysis, catalyzing the interconversion of lactate and pyruvate. Aspartate aminotransferase (AST), ubiquitous in cardiac, hepatic, muscular, and renal cells, serves as a biomarker for cardiac afflictions when assayed in serum. Creatine kinase (CK), integral to muscle function and energy homeostasis, is indicative of myocardial injury when elevated in the bloodstream. ELISA data indicated that LDH, AST, and CK levels in both sham-operated negative control (sham-NC) and sham-H2AX inhibitor groups remained low without notable variance. Contrastingly, these enzyme concentrations were elevated in the myocardial ischemia/reperfusion negative control (MI/R-NC) and MI/R-H2AX inhibitor groups, as shown in Figure 2(a). Notably, the MI/R-H2AX inhibitor group exhibited a substantial reduction in these enzymes compared to the MI/R-NC group, suggesting a mitigative effect of H2AX inhibition on enzymatic release post-injury.

Cardiac ultrasonographic evaluation of the mice revealed no significant disparities in EF, FS, LVAW; d, and LVAW; s, as well as LVID; d and LVID; s between the sham-NC and sham-H2AX inhibitor groups. However, in the MI/R-H2AX inhibitor group, there was a notable augmentation in EF, FS, LVAW; d, and LVAW; s, alongside a reduction in LVID; d and LVID; s when juxtaposed with the MI/R-NC group. These findings, depicted in Figure 2(b), underscore the potential of H2AX inhibition in enhancing myocardial function following I/R injury in mice.

3.3. Inhibition of H2AX Reduces Myocardial Infarct Size and Mitochondrial Injury in Myocardial Ischemia-Reperfusion Mice

Masson’s trichrome staining and TTC assay elucidated the myocardial infarction extent, revealing minimal infarct sizes in both the sham-NC and sham-H2AX inhibitor groups, with no discernible differences. Conversely, the myocardial infarction proportions were notably higher in the MI/R-NC and MI/R-H2AX inhibitor groups. A significant diminution in infarct size was observed in the MI/R-H2AX inhibitor group when juxtaposed with the MI/R-NC group, as illustrated in Figure 3(a) and 3(b). Further, the impact of H2AX inhibition on mitochondrial integrity was assessed in the myocardial I/R model via transmission electron microscopy, which offers detailed visualization of cellular architecture. In the sham cohorts, mitochondrial morphology remained intact. However, in the MI/R-NC group, mitochondria exhibited pronounced swelling and structural aberrations, including increased vacuolation, disrupted cristae, and compromised outer membranes. In contrast, the MI/R-H2AX inhibitor group displayed mitigated mitochondrial swelling and cristae deformation, with infrequent occurrences of membrane disruption, as shown in Figure 3(c). These observations suggest that H2AX inhibition may confer protection against mitochondrial damage in myocardial ischemia-reperfusion scenarios.

3.4. Inhibition of H2AX Can Suppress Mitochondrial Damage and the Apoptosis of Myocardial Cells by Regulating P53 and JNK Signaling Pathway

To delve deeper into the modulation of I/R injury by H2AX, we conducted western blot assays for p-H2AX, P53, p-JNK, SHP, p-SHP2, p-RAS, parkin, Drp1, Cyt-C, Caspase-3, and Caspase-8. Research indicates that augmented ROS levels may instigate the association of P53 with the SHP2 promoter, leading to a suppression of Src homology 2 domain-containing phosphatase-2 (SHP2) expression and a marginal reduction in P53 levels. RAS, a member of the small G protein family, orchestrates a variety of cellular signaling cascades, including those governing cell viability, proliferation, and differentiation. SHP2’s dephosphorylation of RAS-GTPase activating protein (RAS-GAP) attenuates its inhibitory effect on RAS, thereby potentiating RAS signaling and frequently culminating in the attenuation of P53 activity [18, 19]. JNK, or c-Jun N-terminal kinase, is a crucial kinase implicated in cellular stress responses [20], and it is postulated that H2AX may facilitate the phosphorylation of JNK [20]. Parkin is instrumental in mitochondrial maintenance, influencing cellular survival and metabolic equilibrium by modulating the interplay between mitochondria and apoptotic processes [21, 22]. Drp1, a mitochondrial fission regulator, dynamically shapes mitochondrial morphology. Cytochrome C (Cyt-C), a vital mitochondrial protein, is central to both the respiratory chain and the apoptotic pathway [23–25]. Additionally, Caspase-3 and Caspase-8 are pivotal in the execution phase of intrinsic apoptosis.

Through western blot analysis, it was observed that the suppression of H2AX led to a notable decrease in the levels of phosphorylated H2AX, P53, phosphorylated JNK, parkin, Drp1, cytochrome c, Caspase-3, and Caspase-8 within the H2AX inhibitor cohort relative to the normal control (NC) group. Conversely, the levels of SHP2, phosphorylated SHP2, and phosphorylated RAS were markedly elevated. Subsequent administration of JNK or P53 activators did not alter the levels of phosphorylated H2AX in either the NC or H2AX inhibitor groups; however, the expression of P53, phosphorylated JNK, parkin, Drp1, cytochrome c, Caspase-3, and Caspase-8 was augmented, while SHP2, phosphorylated SHP2, and phosphorylated RAS were diminished, with no significant disparities observed. Thus, the inhibition of H2AX is implicated in mitigating mitochondrial impairment and cardiomyocyte apoptosis through modulation of the P53 and JNK signaling cascades (Figure 4).