AMI refers to injury of the coronary artery caused by acute and/or chronic ischemia and hypoxia [1]. Currently, AMI is becoming one of the leading causes of mortality among people with cardiovascular disease worldwide. Despite the fact that thrombolytic and interventional treatments have considerably improved the prognosis of AMI patients, mortality rates remain high [2]. Cardiac troponin I (cTnI) and cTnT are the recommended gold biomarkers for the diagnosis of AMI. However, the levels of cTn may be elevated not just after AMI but also as a consequence of other nonvascular myocardial injuries [31]. This impact limited the specificity of cTn for AMI and made the diagnosis complicated. As a result, a significant number of patients do not receive timely treatment, resulting in irreversible myocardial injury and a poor prognosis. Rapid and accurate diagnosis of AMI helps increase therapy efficiency and improve patient prognosis. Consequently, it is necessary to identify effective diagnostic biomarkers for precisely predicting AMI.
Previous studies screening for diagnostic markers of AMI have mostly focused on ferroptosis- and immune-related genes [16, 32]. However, the pathogenesis of AMI is a particularly complicated process involving multiple biological pathways. A recent study has shown that cuproptosis is associated with the development and prognosis of AMI, suggesting a potential target for AMI treatment [18]. Intracellular copper accumulation is a significant cause of cuproptosis, which often occurs in cells with high metabolic activities, like mitochondria [19]. Therefore, we hypothesize that the cuproptosis involved in the development of AMI may be dependent on mitochondrial dysfunction [33]. Moreover, investigations have demonstrated that copper accumulation in the mitochondria is also connected to ferroptosis [20, 34]. This indicates a potentially complex relationship between ferroptosis and cuproptosis. There are currently no studies examining the link between cuproptosis and ferroptosis in AMI. We used bioinformatics tools to investigate the role of CFRGs in AMI as well as their interaction with immune infiltration, attempting to identify more precise AMI diagnostic markers.
In this study, we systematically screened 19 differential CFRGs in the peripheral blood of 74 healthy controls and 83 AMI patients. Functional enrichment analysis revealed that the differential CFRGs were enriched in the regulation of reactive oxygen species metabolic processes, inflammatory response-related pathways, and lipid and atherosclerosis, implying that differential CFRGs might participate in the initiation and progression of AMI through these biological processes. Most studies have demonstrated that inflammatory responses play an important role in the initiation and subsequent repair of myocardial infarction [35, 36]. This is in line with the results of our study. Then, we constructed the PPI network and screened 10 hub CFRGs (JUN, STAT3, MAPK14, TLR4, CDKN1A, DUSP1, DDIT3, ATM, CXCL2, SOCS1), all of which are potential candidate biomarkers closely related to AMI.
Immune cells are critical for maintaining cardiac homeostasis and repairing post-ischemic injury [37–38]. Innate immune cells, such as macrophages and neutrophils, have been recruited into the heart after myocardial infarction, facilitating the clearance of necrotic cardiomyocytes and participating in the inflammatory response [39–40]. Adaptive immune cells also participate in the immune response following AMI, but the underlying processes are still poorly understood. An observational study [41] indicated that after early coronary reperfusion, the number of CD8 cells in the blood was drastically reduced in STEMI cases, probably due to cell recruitment into the heart ischemic tissue. Our analysis shows that macrophages, neutrophils, and Tfh cells are highly expressed in the MI group, while CD8 + T cells, NK cells, Th1 cells, and TIL cells are lower. It was consistent with the previous studies. CCR has been demonstrated to modulate leukocyte infiltration and migration to inflammatory sites [42]. The CCL2-CCR2 axis is one of the classical CCR signaling pathways, which has contributed to regulating monocyte recruitment in the infarcted myocardium [43]. Several studies have shown that higher levels of CCL2 increase the risk of coronary artery disease and myocardial infarction [44]. In our study, we also demonstrated that the immune function of CCR was elevated in the AMI group. In the follow-up analysis, we identified the 6 hub CFRGs (STAT3, TLR4, CDKN1A, DUSP1, DDIT3, and CXCL2) most associated with immune infiltration as diagnostic feature genes through the Spearman correlation test. But the expression levels of all 6 feature genes were strongly positively linked with the CCR. This finding suggests that the 6 feature genes participate in the immunological and inflammatory responses of AMI, probably through CCR signaling pathways.
Then, we constructed a nomogram for predicting AMI using the 6 feature genes listed above. The ROC curve can be used to evaluate the diagnostic efficacy of the nomogram, and the AUC areas of the training and validation datasets are 0.805 and 0.94, respectively. This suggests that the 6-gene signature may have a good predictive value for AMI. Similarly, after validation using the AMI mouse model and peripheral blood of AMI patients, we found that 6 feature genes were significantly elevated in the AMI group, consistent with the results of bioinformatics analysis and previous studies. DDIT3, also known as pro-apoptotic transcriptional factor C/EBP homologous protein (CHOP), is one of the endoplasmic reticulum (ER) stress features. During AMI, the ER stress pathway-associated apoptotic responses were activated, and CHOP was upregulated, leading to increased apoptosis of myocardial cells [45–46]. In addition, it has been demonstrated that CHOP has a close relationship with STAT3 in AMI. Li Y et al, [47] revealed that hypoxia-induced mitogenic factor (HIMF) can activate the CHOP-STAT3 signaling pathway and inhibit M2 macrophage polarization, adversely regulating myocardial repair. But another study indicated that upregulated P-STAT3 promotes anti-inflammatory M2 macrophage polarization and protects against AMI [48]. CXCL2, a neutrophil recruiting chemokine, elevated after AMI and involved in the process of inflammation-mediated myocardial injury [49–51]. DUSP is known as MAPK phosphatases (MKPs), which can dephosphorylate MAPKs. It has been shown that DUSP1-mediated JNK dephosphorylation has an antiapoptotic effect in myocardial ischemia-reperfusion (I/R) injury [52–53]. And Zhang N et al, [32] have identified DUSP1 as an immune-related diagnostic biomarker for AMI, which is consistent with our findings. CDKN1A, also called p21, has been shown to inhibit cardiomyocyte proliferation by acetylation modification in MI [54]. Meanwhile, Zheng Y et al, [55] found CDKN1A may be potential therapeutic targets for patients with MI. TLR4 is a key molecular pattern recognition receptor that is expressed on the cell membrane surface of macrophages and cardiomyocytes. Elevated TLR4 expression in MI patients aggravates the ischemia injury to cardiomyocytes [56], and could participate in multiple inflammatory signaling pathways of MI [57–60]. These investigations provide valuable insight into the potential that 6 hub CFRGs might be diagnostic and therapeutic targets for AMI.
Finally, we predicted the miRNAs and candidate drugs that regulate the 6 feature genes. MiR-648 is predicted to regulate 3 feature genes (DDIT3, CXCL2, and STAT3) and has been linked to inhibiting tumor development and overcoming chemotherapy resistance [61]. But there are no studies about miR-648 in cardiac disease. MiR-4798-3p was indicated to modulate TLR4 and STAT3, and it was closely linked with the prevalence of atrial fibrillation in males and also could regulate genes involved in the pathogenesis of AF [62]. MiR-483-5p was found to be elevated in people with AF, which suggests that it could be used to predict AF [63], and it can exert neuroprotective effects after cardiopulmonary resuscitation by blocking ROS production and reducing MDA activity to attenuate oxidative stress injury [64]. In the current study, curcumin was shown to affect all 6 feature genes, and it has been discovered to have multiple effects, such as anti-inflammatory, anti-fibrosis, and so on. Liu Y et al, [65] found that curcumin reduces cardiomyocyte injury generated by LPS by inhibiting the inflammatory response. Mahmoudi A et al, [66] demonstrated that curcumin affects the process of liver fibrosis by impacting genes involved in extracellular matrix communication and oxidative stress. Although the function of the above-mentioned medicines and miRNAs in AMI is unclear, the particular pathways need further investigation. It also provides inspiration for the research and development of diagnosis and treatment related to AMI.