Alexander Blaha

The Fontan circulation is associated with progressive multisystem complications, yet its underlying molecular mechanisms remain incompletely understood. We aimed to characterize systemic proteomic alterations and identify biological pathways associated with Fontan physiology, long-term adaptation, and clinically unfavorable phenotypes. Peripheral vein serum proteomics was performed using mass spectrometry in 48 Fontan patients and matched controls. Differential protein expression and pathway enrichment analyses were applied across the overall cohort and clinically defined subgroups according to Fontan duration and composite clinical risk phenotyping. More than one third of quantified proteins were differentially expressed, demonstrating extensive systemic proteomic remodeling. Extracellular matrix remodeling emerged as the dominant signature, characterized by fibrosis-associated proteins and enrichment of TGF-β-related profibrotic pathways. Prolonged Fontan duration and clinically unfavorable status were not associated with substantially amplified extracellular matrix signatures. Instead, long-term Fontan patients demonstrated increasing immune, complement, and endothelial activation, whereas high-risk patients showed metabolic and redox-related alterations. These findings indicate that extracellular matrix remodeling and profibrotic signaling are dominant features of the Fontan circulation, while increasing Fontan duration and clinical deterioration are associated with immune activation, endothelial stress, and metabolic dysregulation with oxidative stress.

The total cavopulmonary anastomosis (Fontan procedure), a palliative procedure for single-ventricle congenital heart disease, improves survival but is associated with progressive multiorgan complications and high long-term morbidity. Prior blood-based proteomic studies in adults have been limited to targeted antibody-based panels or focused on methodological comparisons. Systemic molecular alterations in younger, clinically heterogeneous patients, particularly in untargeted pathways, remain incompletely characterized. Serum samples from 48 Fontan patients and 48 age- and sex-matched healthy controls were analyzed using mass spectrometry with TMT labeling. 2228 proteins were quantified, of which 124 were significantly differentially abundant (fold change > 1.5 or p < 0.05). Network analysis identified three major functional clusters: extracellular matrix (ECM) organization (predominantly increased), actin cytoskeleton organization, and platelet-related pathways (both predominantly decreased). Stratified analyses showed reduced ECM protein abundance in high-risk patients, suggesting a shift from active remodeling toward a more established fibrotic state, and uniquely elevated cytochrome b5 reductase 3 (CYB5R3), implicating altered redox homeostasis, nitric oxide metabolism, and cellular aging. Overall, our findings extend prior targeted analyses, reveal potential biomarkers such as CYB5R3 and underscore the complexity of the Fontan circulation, with implications for risk stratification and therapeutic targeting.