Cardiomyopathy (CM) are a heterogeneous group of pathologies characterized by structural and functional alterations of the heart. It is one of the most severe, potentially fatal electrical or muscle cardiac conditions. It may be heart-specific or a part of a more severe systemic disease, and it often resulting in cardiovascular death or progressive heart failure-related disability. ¹ Myocarditis is an inflammation of the myocardium that can occur from infections (e.g., viral, bacterial, fungal, parasitic) or non-infections (e.g., toxins, pharmacological sensitivities, immunological diseases) causes and may present as either acute or chronic. Myocarditis can impair cardiac function, potentially resulting in dilated cardiomyopathy (DCM) and heart failure. ² Dilated cardiomyopathy (DCM) is a clinical disorder distinguished by the enlargement of the left ventricle or both ventricles, as well as compromised contraction, which cannot be caused by abnormal loading circumstances such as hypertension, valvular heart disease, or coronary artery disease. Various genetic mutations have been identified as potential causes of dilated cardiomyopathy (DCM). ³ In spite of the significant body of work demonstrating the association of coxsackievirus with DCM the specific interaction between coxsackievirus and IL-12 remains underexplored. This study aims to explore the potential link between IL-12 and Coxsackievirus in the context of dilated cardiomyopathy (DCM). Specifically, it seeks to assess how these factors, along with lipid levels, troponin levels, interleukin-12 levels and coxsackievirus antibodies, may interact and contribute to the pathogenesis of DCM. The study will compare these markers between patients with DCM and a control group to identify potential correlations. T-helper 17 (Th17) cells are essential in causing cardiomyopathy Cytokines such as TNF, interferon alpha, interferon gamma, IL-6, and IL-1 alpha promote the production of IL-12 which activates T-helper 1 (Th1) cells. ⁴ IL-12, produced by single cells, promotes T lymphocytes and natural killer cells to generate interferon γ IFN-γ.IL-12 is considered to be pro-inflammatory factors that amplify down stream inflammatory signals. ⁵ Therefore these immunological and biochemical markers are important in early diagnosis. ⁶ By linking IL-12 and Coxsackieviruses, this study could present a novel in understanding the pathogenesis of DCM. Coxsackieviruses (CV) are prevalent single-stranded RNA viruses classified within the family Picornaviridae and the Enterovirus genus, which also encompasses poliovirus (PV), the most recognized of the enteroviruses (EV). They are mostly transmitted via the fecal–oral route and constitute a significant source of viral infections annually worldwide, particularly among children. ⁷ Coxsackieviruses are frequently linked to the etiology of chronic myocarditis, dilated cardiomyopathy, and autoimmune disorders, including type 1 diabetes (T1D). This study explores the contribution of viral persistence or chronic immune responses, particularly those initiated by viral infections, to DCM. Certain indications, including DCM, may arise as a delayed consequence of acute or chronic viral myocarditis, attributable to viral persistence or a chronic immune response predominantly instigated by a viral infection. ⁸

The control group showed significantly higher levels of cholesterol, triglycerides, LDL, and VLDL compared to DCM patients (p ≤ 0.001), indicating a healthy lipid profile. In contrast DCM patients showed lower lipid levels, suggesting potential lipid metabolism disorders associated. Although HDL levels were higher in DCM patients compared to controls, the difference was not statistically significant (p = 0.054). Regression analysis revealed that low HDL and LDL were negatively associated with DCM, while high cholesterol had a positive association. VLDL and triglycerides had no significant effect on DCM These findings highlight lipid metabolism disorders in DCM patients having lower levels of cholesterol, triglycerides, LDL cholesterol and VLDL. Myocardial dysfunction in DCM is known to impair lipid processing, contributing to these changes. Additionally, the use of statins in DCM patients which are prescribed to manage cardiovascular risk, can lower lipid levels and increase HDL levels. Statins are known to lower LDL and total cholesterol while promoting the synthesis of HDL thereby improving lipid profiles. ^{9, 10} However, a critical analysis of potential confounding factors is necessary. Factors such as medication use, viral persistence, immune variations, and inflammatory cytokines like IL-12 could influence lipid metabolism and cardiovascular outcomes in DCM patients. These uncontrolled variables, along with the presence of comorbidities like diabetes and hypertension, may contribute to the observed differences in lipid profiles and myocardial injury markers. Future studies should account for these factors for more accurate conclusions. Troponin levels were higher in patients with DCM than in controls p ≤ 0.001), which suggests that some patients may have had mild myocardial injury This shows that not all of the patients had serious damage to their heart muscle, even though troponin levels were positive in a small number of them. The difference in troponin levels shows that some people with DCM are still experiencing stress or injury to their heart muscle, which could be related to the condition getting worse or inflammation. Our results are consistent with those of Ref. 11. who reported that elevated troponin is a potential biomarker of poor prognosis in DCM patients. However, other studies such as have reported no differences between DCM patients and healthy people, suggesting that the role of troponin as a biomarker in DCM remains controversial. ¹² Troponin has a high specificity and sensitivity for detecting myocardial necrosis, making it a potent prognostic indicator. Regarding the lipid profile, our results did not show any significant association between troponin and cholesterol, triglycerides, HDL, LDL, and VLDL. the scatter panel analysis confirmed the absence of any clear clustering or trend, suggesting that the elevated troponin level in DCM reflect myocardial injury or stress independently of lipid metabolism. These results are consistent with a recent Ref. 13 study in dilated cardiomyopathy, which also did not mention any significant differences in lipid profile or troponin levels, supporting that high troponin in dilated cardiomyopathy reflects myocardial injury and not lipid dysregulation. However, some studies in patients with acute myocardial infarction have demonstrated significant association between troponin levels and lipid profile, suggesting may contribute to disease-specific mechanisms. ¹⁴ These results suggest that IL-12 plays an important role in differentiating DCM from healthy individuals, as its levels are significantly higher in DCM patients The Mann-Whitney U test (p < 0.001) confirmed a statistically significant difference between the two groups, supporting IL-12 as a potential biomarker for DCM differentiation. The substantial variation in IL-12 concentrations observed underscores its potential as a reliable marker for the identification of DCM patients. Upregulation of IL-12 may be associated with Th1 immune activation or the IFN-γ pathway, both of which are commonly involved in viral induced cardiac injury with Coxsackievirus being a known cause of Dilated Cardiomyopathy (DCM). The favorable Area Under the Curve (AUC) value in the ROC analysis suggests that IL-12 is linked to DCM especially in viral cases like those caused by Coxsackievirus. The ROC curve analysis yielded an AUC of 0.730 with a cut-off value of 8.80, Sensitivity was 0.8, and specificity was 0.482 demonstrating the biomarker’s ability to differentiate between DCM patients and healthy controls. While the AUC value is promising it is affected by the overlap in IL-12 concentrations between the two groups slightly reducing its discriminative power. Nonetheless, both the Mann-Whitney U test and the ROC analysis suggest that IL-12 may be a useful biomarker for DCM, aligning with previous studies on its role in viral-induced DCM These results are consistent with the concept proposed by Ref. 15 which highlights the use of multiple cytokines to differentiate DCM from healthy individuals. This approach supports improved diagnostic accuracy and patient management through the use of cytokine profiling to more accurately segment risk in cardiovascular diseases including dilated cardiomyopathy. The results showed a significant association between coxsackievirus infection and elevated IgG and IgM levels in DCM patients compared to health controls The Chi-Square test revealed a significant difference in IgG levels with 26.7% of DCM patients testing positive while none of the controls tested positive. This supports previous research suggesting that coxsackievirus infection may contribute to the development of DCM through immune responses. In addition 30% of DCM patients tested positive for IgM, an early marker of acute infection, supporting the hypothesis that viral infections may contribute to the pathogenesis of DCM. Presence of IgM in DCM patients without detection in controls highlights role of viral infections in exacerbation of heart disease. These findings align with the studies by Refs. 16, 17. which supports the role of Coxsackieviruses, in DCM disease. The association of IgG and IgM with Coxsackievirus infections could be instrumental in identifying high-risk patients and improving diagnostic strategies for DCM. However, There are no contradictory studies in the literature regarding the relationship between coxsackie virus infection and DCM. suggesting that the virus may contribute to its evolution. Further studies are needed to clarify the mechanisms involved. The Spearman’s rho correlation analysis revealed non-significant correlations between IL-12 and both IgM (r = 0.189, p = 0.075) and IgG (r = 0.099, p = 0.356). These results indicate that IL-12 does not have a meaningful relationship with either IgM or IgG in this dataset. The bootstrap confidence intervals for IL-12 and IgM were -0.015 to 0.386 and for IL-12 and IgG they were -0.083 to 0.278, which confirmed the mild associations. These findings may be attributed to various factors, such as the involvement of other immune factors, the possibility that IL-12 does not directly regulate IgM or IgG, and biological variability in immune responses. Overall, the results indicate that IL-12 does not have a substantial effect on IgM or IgG. Further studies with larger sample sizes or additional immunological markers may provide more insights. Additionally, IL-12 and its associated antibodies could be useful for screening, prognosis, and disease monitoring, offering valuable information on the progression of DCM and the impact of viral infections on heart function.

The study shows that there are significant correlations between immunological markers and DCM. The Mann-Whitney U test use to demonstrated the IL-12 levels were markedly increased in DCM patients compared to control group generates p ≤ 0.001. This confirms IL-12 as a possible biomarker to tell the difference between patients with DCM and control group. as a potential biomarker for differentiating DCM sufferers from healthy group. The ROC AUC curve analysis revealed a moderate value of 0.730, indicating that IL-12 is effective in distinguishing between the two groups. Coxsackievirus antibodies (IgG and IgM) demonstrated statistically significant differences between DCM patients and healthy controls, with p-values of 0.002 for IgG and <0.001 for IgM. Specifically, 26.7% of DCM patients exhibited positive IgG results, while 30% demonstrated positive IgM results, indicating that coxsackie virus infection may be a contributing factor to DCM. In conclusion IL-12 and Coxsackievirus antibodies (IgG and IgM) serve as possible biomarkers for the diagnosis and risk stratification in DCM. Further research is required to validate these findings and explore the mechanisms behind their contribution to the development of DCM.

This study was approved by the Ethics Committee of the College of Science, University of Baghdad, Department of Biotechnology, with approval reference number CSEC/0825/0098. Written informed consent was obtained from all participants (or their legal guardians for minors). The Ethics Approval letter is shown in Figure 8.