One Ventricle, 3000 Proteins: Unraveling the Molecular Landscape of the Fontan Circulation

Remarkable achievements in surgical and medical management have improved outcomes for single ventricle heart disease, with >80% of patients surviving 20 years after the Fontan operation. ¹ , ² Despite improved early survival, aging with a Fontan circulation is associated with substantially increased morbidity and premature death. ³ , ⁴ , ⁵ In this issue of the Journal of the American Heart Association (JAHA), Kelly et al used high‐throughput proteomics to offer a glimpse into differences in the molecular biological processes between individuals with a Fontan circulation and healthy controls. ⁶ Proteomics offers complementary information to genomics and transcriptomics and helps generate a map of the complex, interconnected pathways and molecular systems involved in the Fontan circulation. Recent advancements in experimental technology have led to new proteomics methods, evolving from conventional methods, such as immunohistochemistry staining, western blot, and ELISA to more sophisticated high‐throughput methods such as mass spectrometry and, more recently, aptamer and antibody‐based proximity‐extension assays. A better understanding of proteomics may provide insight into potential therapeutic targets for the management of Fontan heart failure, protein‐losing enteropathy, plastic bronchitis, Fontan‐associated liver disease, and Fontan circulatory failure.

Kelly et al measured nearly 3000 proteins using the Olink platform in 58 individuals with Fontan circulation and 29 healthy controls and identified 424 upregulated proteins and 89 downregulated proteins unique to the Fontan circulation. Proteins involved in angiogenesis, bone and calcium homeostasis, metabolism, inflammation, and fibrosis were increased, while proteins involved in cholesterol synthesis and muscle structure and function were decreased. Other studies looking at a smaller number of plasma proteins have also reported increased angiogenesis, cell proliferation/turnover, chronic inflammation, and oxidative stress in adults with Fontan circulation. ⁷ , ⁸ , ⁹ , ¹⁰ Proteins involved in cholesterol metabolism have been reported to be represented in both up‐ and downregulated networks. ⁸ The article extends our understanding of the protein landscape in the Fontan cohort. By shedding light on the intricate interplay of proteins involved in various physiological processes, this work not only enhances our understanding of the Fontan physiology but also paves the way for potential therapeutic targets and interventions.

The results have face validity in the context of well described clinical and biochemical features of the Fontan circulation. For example, one would expect angiogenesis to be upregulated given the high incidence of development of abnormal blood vessels: venovenous collaterals, aortopulmonary collaterals, and pulmonary arteriovenous malformations. ¹¹ , ¹² For another, dyslipidemia associated with the Fontan circulation has been repeatedly described. ¹³ , ¹⁴ Inflammation is also known to play a critical role in the pathogenesis of cardiovascular disease, and inflammatory cytokines have been found to be elevated in individuals with Fontan circulation consistent with a state of chronic systemic dysregulation. ⁷ , ¹⁵ The end‐organ consequences of the Fontan circulation, including liver fibrosis, protein‐losing enteropathy, and plastic bronchitis, arise from a complex interplay of chronic venous congestion, inflammation, and lymphatic derangement. ¹⁶

The findings are intriguing and foretell of great potential for this class of technologies to provide new insights, although it must be admitted that these specific results from Kelly et al may not have a direct impact on our current understanding of the Fontan circulation. There are a number of study design considerations that constrain our interpretation of the data, and addressing these limitations will facilitate realization of the powerful future vision of Fontan proteomics promised by the current study (TableTable). First, the results must be interpreted with caution due to their preliminary nature and the inherent limitations in establishing causality. There are gaps and limitations on how to best translate proteomics to clinical use. The observed changes in protein levels may reflect a correlation rather than a direct cause‐and‐effect relationship. It is challenging to ascertain whether these protein changes are a consequence of the Fontan circulation or merely an associated phenomenon. Second, the statistical power was limited, with thousands of measurements made in <100 participants. The small sample size and cross‐sectional design of the study increases the probability of both false‐negative and false‐positive results. Third, and more importantly, only plasma protein levels were assessed. Fourth, there are few associated clinical data and no outcomes data on the cohort. Fifth, circulating protein concentrations can fluctuate for many reasons, including clinical status. The lack of longitudinal clinical data makes it hard to interpret the clinical relevance of these protein changes and what impact they have on the development of Fontan circulatory failure, need for cardiac transplantation, or death.

Table 1.

Study Design Considerations and a Future Vision for Fontan Proteomics Research

Design consideration	Implications	Future direction
Small sample size	Limited statistical power; increased risk of false positives/negatives	Multicenter consortia with larger, diverse cohorts
Single‐center experience	Results may not generalize to the broader Fontan population	Collaborative networks across institutions and countries
Cross‐sectional design, single measurement	No insights into protein dynamics or temporal relationship to disease progression, thereby limiting causal inference	Longitudinal studies assessing changes over time and response to clinical events
Plasma‐only proteomics	May not reflect tissue‐level pathophysiology in liver, myocardium, lymphatics; does not provide insight into reasons for altered protein levels	Multi‐tissue sampling (eg, liver biopsy, myocardial tissue, lymphatics), multiple omics approaches to provide more detailed understanding
Minimal clinical data or other phenotypic characterization	Difficult to correlate protein levels with outcomes or disease phenotypes	Combine with rich clinical phenotyping, imaging, and hemodynamics
Measurement of preselected proteins	Limited to those proteins that have been determined to be most important (or most readily measured), likely biasing results toward a subset of pathways and biological functions	Further expansion of number of proteins measured by aptamer or proximity‐extension assay methods and mass spectroscopy for unbiased protein measurement
Lack of validation of protein measurements	Aptamer‐based assays may have off‐target binding; platform‐specific limitations	Cross‐validation with orthogonal proteomic approaches (eg, mass spectrometry, proximity‐extension assay, ELISA)
Lack of functional validation	Proteins identified may not be causally involved in disease	Functional studies (in vitro, in vivo models) of top candidate proteins
No outcome association	Limits clinical interpretability and application	Association studies with prospective outcomes, including clinical end points

Taking one example of these challenges above, the study relies on plasma proteomics. The plasma proteome does not capture the full complexity of the biological processes at play, as it is influenced by multiple sources (eg, endothelium, liver, heart) and removal processes (eg, renal filtration). A more integrative approach integrating various omics approaches (eg, genomics, transcriptomics, metabolomics), across various tissue types (eg, plasma, liver, cardiac) over time and in response to measured exposures could provide a more complete understanding of the pathophysiology of the Fontan circulation. That, however, will require much greater resources and a much larger sample size than could be recruited from a single center. Such work can be catalyzed by established multicenter networks allowing data sharing, such as the Fontan Outcomes Network and newly established Single Ventricle Outcomes Network, to facilitate larger multicenter cohorts with longitudinal designs to better understand dynamic proteomic responses in the Fontan circulation. ¹⁷

This study represents a necessary and valuable initial step toward elucidating the proteomic impact of the Fontan physiology. The findings begin to lay the groundwork for future research aimed at exploring potential therapeutic targets and understanding the mechanisms behind the abnormal protein changes. As we move forward, a concerted effort to validate these findings and expand on our understanding of the Fontan proteome may prove a pivotal step toward improving long‐term outcomes for individuals with a Fontan circulation.

Disclosures

None.