Depicting gut dysbiosis and associated miRNA alteration in circulatory extracellular vesicles defines combined markers for pressure-overloaded remodeling in children

Heart failure caused by pressure-overloaded left-heart (poLV) diseases is especially difficult to manage in children. Long-term surveillance and early recognition of poLV remodeling are crucial, while current biomarkers remain inadequate for predicting remodeling in the pediatric population. This study aimed to investigate whether gut dysbiosis is associated with alterations in circulatory extracellular vesicles and their miRNA cargo, and to identify an exploratory combined microbial/miRNA signature associated with pediatric cardiac remodeling. For the animal experiments, a fecal microbiota transplantation strategy using germ-free (GF) mice was adopted. Circulatory extracellular vesicles (cEVs) were isolated and studied in primary mouse cardiomyocytes in vitro and in neonatal ascending aorta constriction (nAAC) mice in vivo. Metagenomics and miRNA sequencing were adopted to identify the characterized gut microbes in fecal samples and cEVs-miRNAs in mice. Next, we performed a clinical cohort study (poLV n = 52 vs. healthy controls n = 60). Fecal microbiota species and cEVs-miRNAs were further validated. Least absolute shrinkage and selection operator (LASSO) regression analysis was used to derive an exploratory combined panel in the clinical cohort. For experimental studies in vivo and in vitro, cEVs isolated from GF mice after transplantation of nAAC fecal microbiota (nAAC FMT-cEVs) significantly increased the cell surface area of primary mouse cardiomyocytes, compared with sham FMT-cEVs treatment. Supplementing nAAC FMT-cEVs to nAAC mice aggravated hypertrophy and fibrosis with worsened cardiac function. Metagenomic sequencing identified gut microbes that characterized nAAC and sham mice. miRNA sequencing identified the top differential miRNAs in FMT-cEVs. In the clinical cohort, four miRNAs and six gut microbes were validated as significant. LASSO analysis derived a four-feature exploratory combined panel including two cEV-miRNAs and two gut microbes, which showed good discriminative performance in the derivation cohort (AUC = 0.898). The panel value showed significant correlation to cardiac remodeling parameters and systolic function measured by echocardiography and computed tomography (CT). Gut dysbiosis was associated with altered cEV-associated miRNA composition and with functional effects on poLV remodeling in vitro and in vivo, offering an additional perspective on gut microbiota–cardiovascular interactions. In the clinical cohort, an exploratory combined panel integrating specific gut microbes and cEV-associated miRNAs showed good discriminative performance in the derivation cohort and was associated with cardiac remodeling parameters. These human findings should be interpreted as exploratory and require validation in independent pediatric cohorts.