cfDNA methylation detection as potential liquid biopsy of multiple organ injury in myocarditis patients

Abstract

Myocarditis is inflammatory injury of the myocardium and causes approximately 30,000 deaths globally each year. Fulminant myocarditis is an extremely severe form of myocarditis. Currently, the clinical evaluation of myocarditis and fulminant myocarditis is primarily based on symptoms, ECG findings, and biochemical markers. Cardiac magnetic resonance and endomyocardial biopsy can provide further support for the diagnosis, but both have limitations in routine practice. Recent studies have shown that cell-free DNA (cfDNA) has distinct methylation patterns depending on the organ of origin, suggesting new possibilities for tracking specific types of organ damage. The core mechanism of fulminant myocarditis is a cytokine storm, leading to multiorgan damage, differing from clinically suspected myocarditis. We performed Genome-wide cfDNA methylation detection on plasma from 20 healthy donors and 22 patients (fulminant myocarditis: clinically suspected myocarditis = 9:13, COVID-19 positive: COVID-19 negative = 14:8) and found that cfDNA can be used to specifically identify early multiorgan damage caused by fulminant myocarditis, and its AUC is superior to traditional biochemical indicators such as troponin, high-sensitivity troponin, and lactate dehydrogenase. This is critically important for the timely clinical recognition and treatment of this condition. Furthermore, our study findings suggest that SARS-CoV-2 infection may exacerbate the severity of myocarditis and multiorgan damage. In summary, cfDNA shows great potential as a noninvasive, early, and sensitive biomarker for reflecting disease severity and systemic injury in fulminant myocarditis, which may help guide earlier risk stratification and intervention.

Keywords: Cell-free DNA; Cytokine storm; Early identification; Fulminant myocarditis; Multiorgan damage.

Fig. 1

Overall design of this study and analysis of DMRs. A Overall design of the study. B Global methylation levels in healthy controls (n = 20) and patients with fulminant myocarditis (n = 10) and clinically suspected myocarditis (n = 13). C Methylation status of DMRs in clinically suspected (n = 14) and fulminant myocarditis (n = 10). D The function proportion of DMR (clinically suspected: fulminant = 14:10). E Methylation status of promoter regions of genes associated with various immune pathways (in the heat map, red indicates high methylation, blue indicates low methylation, fulminant: clinically suspected myocarditis = 10:14, COVID positive: COVID negative = 14:10). F The heat map demonstrates the levels of IL6, IL8, and IFN-γ in the blood of patients (fulminant myocarditis: clinically suspected myocarditis = 8:11) as measured by ELISA. G Plasma levels of IL6, IL8, and IFN-γ in patients with fulminant (n = 8) and clinically suspected myocarditis (n = 11) measured by ELISA (Each sample was tested three times, with the average value taken as the final result). P values were calculated by two-sided Wilcoxon rank-sum test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

Analysis of cfDNA levels in donors and patients. A Total cfDNA concentration in healthy controls (n = 20) and patients with fulminant (n = 10) and clinically suspected myocarditis (n = 14). B Analysis of cfDNA released from healthy controls (n = 20) and patients with fulminant (n = 10) and clinically suspected myocarditis (n = 14). The Y-axis represents the proportion of cfDNA from different organ or tissue sources. C Fisher test results for cfDNA released from cardiac fibroblasts and renal tubular epithelial cells in patients with fulminant (n = 10) and clinically suspected myocarditis (n = 14). P values were calculated by two-sided Wilcoxon rank-sum test. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001

Fig. 3

The radar charts show the AUC of heart and kidney-derived cfDNA levels and traditional clinical indicators to distinguish fulminant myocarditis (n = 10) from clinically suspected myocarditis (n = 13) (A), clinical outcome survival (n = 19) from death (n = 4) (B), and ICU support required (n = 17) from ICU support not required (n = 6) (C)

Fig. 4

cfDNA traced damage can be used as a supplement to traditional clinical indicators. A Analysis of cfDNA derived from hearts, cardiomyocytes, and cardiac fibroblasts in patients with troponin I higher than the normal range on admission (n = 22). B Analysis of cfDNA derived from hearts, cardiomyocytes, and cardiac fibroblasts in patients with troponin T higher than the normal range on admission (n = 19). C Fisher test results of cardiac fibroblast-derived cfDNA levels and renal injury in patients with troponin I higher than the normal range on admission (n = 22). D Fisher test results of cardiac fibroblast-derived cfDNA levels and renal injury in patients with troponin T higher than the normal range on admission (n = 19). E Analysis of cardiac-derived cfDNA levels in patients with troponin and high-sensitivity troponin within or outside the normal range (troponin normal: troponin high = 9:15, high-sensitivity troponin normal: high-sensitivity troponin high = 6:18). F Results of continuous troponin I monitoring in selected patients with potential cardiac injury. The line graph represents the changes in troponin I levels over time, while circles, triangles, and squares represent the proportions of cfDNA from endothelial cells, the heart, and the kidneys, respectively. G Analysis of kidney-derived cfDNA levels in patients with serum creatinine within or outside the normal range (normal: high = 20:4). H Results of continuous monitoring of serum creatinine in some patients with potential renal impairment. The line graph represents the changes in serum creatinine levels over time, while circles, triangles, and squares represent the proportions of cfDNA from endothelial cells, the heart, and the kidneys, respectively