Homocysteine (HCY) is a sulfhydryl-containing amino acid that occupies a pivotal role in one-carbon metabolism, functioning as a critical intermediate in two interconnected pathways: remethylation to methionine and transulfuration to cysteine (1, 2). Elevated plasma HCY levels, termed hyperhomocysteinemia, arise from a combination of genetic predispositions [e.g., mutations in methylenetetrahydrofolate reductase (MTHFR) or cystathionine β-synthase (CBS) genes], nutritional deficiencies (particularly folate, vitamin B6, or vitamin B12), and impaired renal clearance, which is a common contributor in chronic kidney disease (3). Accumulating evidence underscores hyperhomocysteinemia as a robust biomarker for cardiovascular pathology. Specifically, elevated HCY levels have been mechanistically linked to endothelial dysfunction via oxidative stress-mediated depletion of nitric oxide bioavailability, vascular inflammation through NF-κB activation and pro-inflammatory cytokine release, and thrombogenesis due to enhanced platelet aggregation and impaired anticoagulant pathways (4). These mechanisms collectively contribute to the pathogenesis of atherosclerosis, hypertension, and ischemic heart disease (5). Furthermore, clinical studies highlight a dose-dependent relationship between HCY levels and cardiovascular mortality, independent of traditional risk factors, solidifying its role as both a diagnostic and prognostic marker in cardiovascular medicine (6, 7).

Ejection fraction (EF) is a critical indicator for assessing cardiac systolic function, typically measured by echocardiography (8), with cardiac magnetic resonance (CMR) providing more accurate EF measurements (9, 10). Research indicates that when EF falls below 55%, patients experience significantly increased mortality and cardiovascular event risks (11). Furthermore, EF changes are closely related to prognosis, as an EF below 55% typically reflects substantial impairment of cardiac pumping capacity, potentially exacerbating heart failure symptoms and reducing quality of life (11). Consequently, a clinical threshold of 55% EF is commonly used to distinguish populations with normal EF from those with mildly abnormal cardiac function (12).

Myocarditis is clinically and pathologically defined as an inflammatory disease characterized by pathological changes predominantly confined to the myocardium, which can lead to cardiac dysfunction and ventricular remodeling (13, 14). Its etiology primarily includes infectious factors, such as viral and bacterial infections, and non-infectious causes, such as autoimmune diseases (15). In the pediatric population, myocarditis continues to exhibit high morbidity and significant disease burden. A nationwide epidemiological survey of hospitalized children in China revealed an increasing prevalence of myocarditis among pediatric inpatients, with a mortality rate of 4.13% (16).

Patients with severe myocarditis may develop ventricular remodeling, leading to a decrease in EF. The change in EF measured by the initial CMR performed early after acute myocarditis onset is an independent predictor of adverse outcomes in acute myocarditis (17, 18). Multiple studies have confirmed an inverse correlation between HCY levels and EF in patients with coronary artery disease, diabetes, and chronic heart failure (19–21). Nygard et al. (1997) observed in patients with coronary artery disease that elevated HCY levels were significantly associated with reduced LVEF, with a significantly higher mortality rate in the high HCY group (≥ 15 μmol/L), which suggests that HCY is not only a marker of cardiac insufficiency, but also an independent prognostic predictor (22). A negative correlation between HCY and LVEF was also identified in patients undergoing coronary artery bypass grafting (CABG) surgery (23). A study by El-Amrousy et al. (2017) reporting an association between HCY and EF in pediatric acute heart failure, no study to date has specifically examined the association between HCY levels and reduced EF in children diagnosed with myocarditis using CMR imaging (24). Given the unique pathophysiological features of myocarditis and the enhanced accuracy of CMR for evaluating left ventricular function, this study addresses a critical gap in the pediatric cardiovascular literature. This study aims to investigate the association between elevated HCY levels and reduced EF in pediatric patients with myocarditis, explore the dose-response relationship between HCY concentration and the risk of reduced EF, and examine whether this association varies across clinically relevant subgroups stratified by age, sex, and body mass index (BMI). We hypothesize that elevated plasma HCY levels are associated with an increased risk of reduced EF (EF < 55%) in pediatric patients with myocarditis.

Myocarditis affects individuals across all age groups, Compared to adults, the incidence of myocarditis in children exhibits a bimodal distribution, infants exhibit a higher incidence, followed by a secondary increase in young adults (26). Two-thirds have a history of preceding viral infections, and over 50% present with fever (10). Viral infection is the primary etiology, with pathogenesis involving direct damage from viral replication in host cells and inflammatory responses mediated by both viral activity and immune reactions (15). The pathophysiology of myocarditis can be divided into three stages: acute viral-induced injury; host innate and adaptive immune responses; and recovery or progression to scarring and dilated cardiomyopathy (15). Compared to adults, the pathophysiological course of myocarditis in children is more rapid, with fulminant myocarditis occurring more frequently than in adults (10). This may stem from the active immune systems of adolescents (27). Therefore, early recognition of myocarditis in pediatric patients is crucial for prognosis.

In this study, we investigated the association between elevated HCY levels and reduced EF in pediatric patients with myocarditis. we observed that elevated HCY levels were a risk factor for patients with EF <55%. This result remained statistically significant after correcting for confounders such as gender, age, and BMI. In previous studies, multiple investigations have extensively demonstrated a negative correlation between HCY and EF, and this association exhibits remarkable consistency across different clinical populations (20, 22–24, 28). This is consistent with the conclusions of the aforementioned studies. This finding demonstrates that elevated HCY levels in pediatric patients with myocarditis are associated with more severe cardiac function impairment. As this is a cross-sectional study, even if we can only establish an association between reduced EF and elevated HCY levels, it still holds significant clinical importance. In the 2024 ACC Expert Consensus Decision Pathway on Strategies and Criteria for the Diagnosis and Management of Myocarditis, reduced EF is recognized as a definitive poor prognostic factor (17). Given the correlation between HCY and reduced EF, early HCY assessment in blood biochemistry can serve as a prognostic reference during the initial treatment phase of myocarditis. This holds significant importance for determining pediatric treatment plans and subsequent follow-up care. Secondly, due to the uncertainty of causality, we cannot definitively determine whether elevated HCY levels during myocarditis result from the inflammatory response, thereby causing reduced EF, or whether individuals with high HCY levels inherently experience poorer outcomes when developing myocarditis. These questions require further clarification through cohort studies.

HCY is an independent risk factor for cardiovascular diseases (4). As a cross-sectional study, although this paper cannot establish a causal relationship between elevated HCY levels and reduced EF in children with myocarditis, we review the potential mechanisms underlying this association based on previous research. HCY has been demonstrated to possess cardiotoxicity, leading to myocardial remodeling and dysfunction (29). The underlying mechanisms involve multiple levels: at the genetic level, HCY decreases the S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio and induces DNA and histone hypomethylation, which alters gene expression and promotes cardiomyocyte hypertrophy (30). At the organelle level, HCY generates reactive oxygen species (ROS), triggers oxidative stress, and impairs mitochondrial protein content and function in cardiomyocytes. HCY also induces endoplasmic reticulum (ER) stress, which leads to Ca²⁺ accumulation, reduced ATP production, decreased membrane potential, and activation of mitosis and apoptosis (30). At the cellular level, HCY induces cardiomyocyte hypertrophy by up-regulating copper-transporting ATPase 1 (ATP7a) protein expression, decreasing copper content, and decreasing cytochrome c oxidase (COX) activity. Interactions between soluble epoxide hydrolase (sEH) and typical transient receptor potential 3 (TRPC3) channels, as well as the calreticulin-nuclear factor of activated T-cells (NFAT) pathway, may also contribute to HCY-mediated myocardial alterations (31–33). Additionally, elevated HCY levels may alter cell communication by regulating the expression of matrix metalloproteinases and connexins in myocardial tissue (34). In this study, patients with EF <55% exhibited higher HCY levels. These mechanisms provide a potential theoretical basis for the association between elevated HCY levels and more severe myocardial injury.

In our study, The RCS results showed an increased risk of EF < 55% in children with myocarditis with increasing plasma HCY. However, this relationship was not statistically significant (P > 0.05) due to the small sample size, but this trend still provides some indication of the relationship between HCY and EF. Several factors may explain this finding. First, the overall sample size, particularly the limited number of patients with significantly elevated HCY levels, constrained the statistical power of the RCS analysis. Second, HCY levels in this dataset primarily fell within the upper range of normal values (12.70 ± 0.84 μmol/L), exhibiting a relatively narrow distribution, which may have weakened the observable dose-response effect. Third, reduced EF in acute myocarditis is influenced by multiple interacting factors, including inflammatory activity, myocardial edema, viral infection. The presence of residual confounding factors may also obscure subtle nonlinear relationships. Therefore, the RCS results should be interpreted as hypothesis-generating findings, suggesting a potential risk pattern, requiring further validation through larger prospective studies.

Notably, in the subgroup analysis, the association between high HCY levels and EF < 55% risk showed greater significance in boys OR = 1.045 [95%CI, 1.008–1.083]; P = 0.016 and individuals with BMI ≥ 24 OR = 1.083 [95%CI, 1.019–1.151]; P = 0.010. In pediatric populations, HCY exhibits a weak correlation with overweight status (35). Previous studies have found elevated levels of total cholesterol and low-density lipoprotein in children with homocystineia. Homocysteinemia is significantly associated with LDL levels (36). The association between elevated HCY levels and overweight appears to be mediated through adipose tissue dysfunction, with the mechanism involving the inhibition of lipolysis through the activation of adenosine monophosphate-sensitive protein kinase (37). MTHFR gene polymorphisms are closely associated with HCY concentrations in obesity (38). However, evidence supporting this mechanistic link in pediatric populations remains insufficient, with only limited epidemiological data showing a conclusive association between hyperhomocysteinemia and excessive obesity in pediatric and adolescent populations (35). In previous studies, boys demonstrated higher HCY levels compared to girls (39), which may be attributed to sex-related differences in amino acid metabolism (40). HCY remethylation rates were found to be higher in females than in males, and this metabolic difference is consistent with sex differences in physiological requirements and utilization patterns of certain amino acids (41). Therefore, close monitoring of plasma HCY levels in overweight male children with myocarditis is essential to mitigate adverse outcomes.

This study has several limitations that should be acknowledged. First, it is a single-center study, which may limit the generalizability of the findings. Second, due to the cross-sectional design, causal inferences between HCY levels and cardiac function cannot be established. Third, the relatively small sample size may reduce the statistical power to detect certain associations. Finally, the absence of follow-up or longitudinal data precludes assessment of dynamic changes in HCY and cardiac function over time. Despite these limitations, our study provides novel insights into the association between HCY and left ventricular EF in pediatric patients with myocarditis. By identifying significant HCY–EF correlations and evaluating potential modifiers such as sex, age, and BMI, this work lays a foundation for future longitudinal studies.

The aim of this study was to investigate the relationship between HCY and EF in children with myocarditis. The results showed that elevated HCY levels were a risk factor for patients with EF < 55%. In addition, the association between high HCY levels and EF < 55% was more pronounced in two subgroups of patients with BMI ≥ 24 and in boys. HCY has great potential as a biomarker of mycarditis prognosis. Future studies should further investigate whether HCY lowering therapies improve cardiac function and establish a causal relationship between HCY and decreased EF in pediatric patients with myocarditis.