Background:Pediatric dilated cardiomyopathy (DCM) is a rare, progressive heart disease with variable outcomes that range from recovery to heart transplantation. To date, there are no prognostic biomarkers for children with DCM. Identifying circulating biomarkers that are associated with clinical outcomes is critical for personalized management. Methods:miRNAs were identified by RNA-seq, whereas proteins were identified by SomaScan®. Machine learning methodologies were used to explore the predictive ability of circulating factors identified from serum samples collected at the time of presentation with acute heart failure. Results:Thirty patients experienced poor outcomes (cardiac transplantation, mechanical circulatory support, or death) and 19 patients recovered left ventricular function. Distinct miRNA and protein signatures differentiated outcomes groups. Top candidate proteins (COL2A1, CXCL12, and ADGRF5) and miRNAs (miR-874-3p, miR-335-3p, miR-323a-3p) demonstrated strong discriminatory performance within the study cohort (recovered vs poor outcomes; Area Under the Curve of 0.92). Ingenuity Pathway Analysis implicates cardiac remodeling, fibrosis, and inflammatory signaling as central pathways differentiating patient outcomes. Conclusions:Circulating miRNA and protein signatures at presentation identify a circulating molecular signature associated with divergent clinical trajectories in pediatric DCM. These findings support the potential utility of multi-omic biomarkers for early risk stratification and provide insight into mechanisms underlying divergent outcomes.

Fetal growth restriction (FGR) is a severe pregnancy complication often caused by placental insufficiency. Proper trophoblast invasion is essential for placental development and function, ensuring adequate nutrient and oxygen supply to the developing fetus. Dysregulation impairs placental perfusion, leading to FGR. This study uses a calorie-restricted mouse model to investigate genes/molecular mechanisms regulating trophoblast invasion across gestational timepoints. Pregnant mice received either a standard or 50 % calorie-restricted diet from E8.5. Placentas and invasion sites were analyzed at E10.5, E12.5, E14.5, E16.5, and E17.5. mRNA sequencing and RT/qPCR examined trophoblast invasion-related genes (Mmp2, Mmp9, Efna1, Rac1, Rras, Ascl2, Tfap2c, Prl7b1) and angiogenesis genes (Vegfa, Vegfb, Pdgf, Akt3). Immunohistochemistry of trophoblast cells (cytokeratin 8, CK8) and endothelial cell markers (endomucin, CD31, CCD105, VEGFR2) was performed. Statistical analysis used Student's t-test. Caloric restriction significantly reduced fetal/placental weights from E12.5, with persistent growth restriction at E16.5, and E17.5. IHC at E17.5 showed reduced decidual depth, trophoblast invasion distance, and trophoblast quantity within the decidua. This impaired growth was accompanied by reduced expression of trophoblast invasion genes (Mmp2, Mmp9, Efna1, Rac1, Rras, Ascl2, Tfap2c, Prl7b1) in FGR placentas, with a reduction in CK8 trophoblast staining. Angiogenesis reduction in FGR was demonstrated with reduced Vegfa, Vegfb, and Akt3 and supported by reduced CD31, CD105, and VEGF2 endothelial cell markers A caloric-restriction mouse model replicates key FGR pathophysiology, including reduced fetal/placental growth, downregulation of trophoblast invasion genes, impaired trophoblast invasion into the decidua, and reduced placenta angiogenesis. These findings offer molecular insights into placental insufficiency that merits further exploration regarding FGR pathogenesis.

Background:Current management of pediatric dilated cardiomyopathy (DCM) in children relies on guideline-directed medical therapy (GDMT) extrapolated from adult heart failure. However, due to small sample size, randomized trials of GDMT agents in children have failed to demonstrate efficacy and mortality benefits seen in adults, suggesting fundamental differences in disease mechanisms. We hypothesized that distinct age-dependent transcriptional programs underlie this therapeutic discordance. Methods:We performed comparative transcriptomic profiling using bulk RNA sequencing on explanted left ventricular tissue from pediatric (n=29) and adult (n=35) DCM patients (adult DCM from previously published data) compared with age-matched non-failing controls (n=22 pediatric, 14 adult). We analyzed differential gene expressions, pathway enrichment across disease etiologies, and the regulation of a conserved 430-gene β1-adrenergic receptor gene signaling network (β1-GSN) known to modulate remodeling in adult heart failure. Results:Transcriptional signatures were profoundly distinct, with only 7.4% of differentially expressed genes shared between adult and pediatric cohorts. Pediatric DCM was characterized by transcriptional reprogramming and the activation of developmental pathways, including WNT/β-catenin and Notch signaling. Conversely, adult DCM hearts were enriched for pathways associated with metabolic dysfunction, mitochondrial deficits, and inflammation. Crucially, while the β1-GSN was desensitized and extensively remodeled in adults, the pathway remained activated in children, with only 4 of 430 network genes showing antithetical regulation. Conclusion:The lack of pathological β-adrenergic remodeling in children could provide a molecular explanation for the lack of clear efficacy of β-blockers in this population. Collectively, these results suggest pediatric DCM represents a biologically distinct disease entity rather than an earlier manifestation of adult heart failure, and future therapeutic strategies must move beyond adult extrapolation to target pediatric-specific pathways.

Background Although operative and perioperative care continues to improve for patients with single ventricle (SV) congenital heart disease, long‐term morbidities and mortality remain high. Importantly, phosphodiesterase‐5 inhibitor (PDE5i) therapies are increasingly used, but their direct myocardial effects in SV congenital heart disease remain poorly understood. We previously demonstrated that the failing SV myocardium is characterized by increased PDE5 (phosphodiesterase‐5) activity and impaired mitochondrial bioenergetics. Here we sought to determine whether systemic circulating factors contribute to pathological metabolic remodeling in SV congenital heart disease, and whether PDE5i therapy mitigates these changes. Methods Using an established in vitro model whereby primary cardiomyocytes are treated with patient sera ± PDE5i, we assessed the impact of circulating factors on cardiomyocyte metabolism. Mass spectrometry‐based lipidomics and metabolomics assessed phospholipid and metabolite changes. Mitochondrial bioenergetics were evaluated using the Seahorse Bioanalyzer and a stable isotope‐based enzyme activity assay. Relative mitochondrial copy number was quantified using reverse transcriptase–quantitative polymerase chain reaction. Results Our data suggest that circulating factors contribute to fundamental changes in cardiomyocyte bioenergetics, including impaired mitochondrial function associated with decreased cardiolipin and other phospholipid species, impaired carnitine palmitoyltransferase activity, increased reactive oxygen species generation, and altered metabolite milieu. PDE5i treatment partially reversed these abnormalities by restoring phosphatidylglycerol levels, reducing reactive oxygen species, increasing carnitine palmitoyltransferase activity, improving energy production, and normalizing several metabolic intermediates. Conclusions Together, these data suggest circulating factors directly impair cardiomyocyte mitochondrial bioenergetics and may drive systemic metabolic dysfunction in SV congenital heart disease. PDE5i therapy partially attenuates these effects, supporting metabolic modulation as a potential therapeutic strategy for SV failure.

Background:Ventricular assist devices (VADs) are used as treatment for end-stage heart failure in children and adults. We previously demonstrated decreased mitochondrial function and changes in cardiolipin, a mitochondrial phospholipid, in explanted pediatric and adult failing hearts. In this study, we tested the hypothesis that VAD unloading of failing hearts leads to positive changes in myocardial cardiolipin in both pediatric and adult hearts. Methods:Ventricular tissue was collected from the same patient at time of VAD implantation and at transplant. Ejection fraction (EF), left ventricular internal diameter at end-diastole (LVIDd) and brain natriuretic peptide (BNP) were assessed pre- and post-VAD. Cardiolipin species from paired VAD core and explants were quantified using liquid chromatography mass spectrometry. Mitochondrial respiration was measured in ventricular tissue pre- and post-VAD in paired pediatric samples using the Oroboros Oxygraph-2k. Results:VAD support led to increased EF and decreased LVIDd and BNP. The predominant cardiolipin species in cardiac mitochondria, tetralinoleoylcardiolipin, was positively remodeled in pediatric post-VAD myocardium, while adult post-VAD myocardium demonstrated significantly increased total cardiolipin and decreased oxidized cardiolipin but did not demonstrate the tetralinoleoylcardiolipin remodeling seen in pediatric hearts. In pediatric patients, VAD support resulted in significant increases in Complex I+II activity, and a trend toward increases in Complex I activity. Conclusion:Our data demonstrate age-related differences in VAD-associated cardiolipin remodeling and suggest that improved mitochondrial function in pediatric VAD-supported hearts could be related to increased tetralinoleoylcardiolipin.

The Fragile-X (FraX) protein family regulates RNA metabolism, muscle development, and neuronal plasticity. These proteins are crucial for translation regulation, interacting with ribosomal subunits and RNA through specific domains. FXR1 has seven isoforms, including isoforms mostly expressed in skeletal and cardiac tissue, and plays a significant role in heart development and function. Additionally, FXR1 modulates microRNA function, impacting gene expression. Given FXR1's crucial role in cardiac differentiation, we evaluated whether expression of the muscle-specific isoforms of FXR1 was dysregulated in pediatric dilated cardiomyopathy (DCM) and sought to determine the impact of these isoforms on the function of miR-1, an important cardiac miRNA, and its regulation of the 3' untranslated region (3' UTR) of the gap junction protein connexin 43 (Cx43). Our results show that FXR1 protein levels are decreased in pediatric DCM left ventricular tissue compared to age-matched nonfailing controls. We investigated the function of muscle-specific isoforms FXR1-G and FXR1-E in an in vitro model of myocyte differentiation. H9c2 cells, differentiated to cardiomyocyte-like cells, show a significant increase in FXR1-G/E protein expression compared to H9c2 myoblasts. Furthermore, we show that FXR1G/E are essential for miR-1-mediated repression of Cx43 3' UTR, emphasizing the importance of miR binding proteins in myocyte homeostasis. Finally, we show that FXR1-G promotes interaction between miR-1 and the Cx43 3' UTR. Overall, we demonstrate that miR-1 regulation of the Cx43 3' UTR relies on muscle-specific isoforms of FXR1. Significantly, we are the first to report a reduction in the muscle-specific isoforms of FXR1 in pediatric DCM patients, underscoring an age-specific regulation of FXR1 expression.NEW & NOTEWORTHY The contribution of microRNAs to cardiovascular diseases has been extensively studied. However, the ability of microRNAs to regulate gene expression requires interactions with RNA-binding proteins (RBPs). Little is known about the contribution of RBPs to microRNA regulation in muscle. We now show that the muscle-specific isoforms of the RBP FXR1 are decreased in pediatric dilated cardiomyopathy hearts and are necessary for miR-1 repression of connexin 43 3' untranslated region (3' UTR), highlighting the importance of RBPs in miRNA function.

Background:Paediatric Idiopathic dilated cardiomyopathy (iDCM) is a life-threatening disease. The lack of disease-specific animal models limits our understanding of its mechanisms. We previously demonstrated that paediatric iDCM serum-circulating proteins promote pathologic remodeling in vitro, and that secreted frizzled related protein 1 (sFRP1) increases stiffness in cardiomyocytes. Here we investigated the mechanisms by which sFRP1 contributes to iDCM. Methods:The effect of sFRP1 in combination with isoproterenol (ISO) (to recapitulate the increase in circulating catecholamine observed in paediatric iDCM) was evaluated in neonatal rat ventricular myocytes (in vitro), and in neonatal rats through intraperitoneal injections (in vivo). Function and molecular mechanisms were investigated through echocardiography and next-generation-sequencing. Protein levels and localization were determined by Western blot. Tissue stiffness was measured by Atomic Force Microscopy. In vitro and in vivo data were compared to explanted human heart tissue. Results:We show that ISO+sFRP1 reactivates the fetal gene program in vitro, and promotes cardiac dysfunction, dilation and stiffness in vivo. Importantly, we show stiffness is also increased in paediatric iDCM hearts. We identified co-activation of Notch and WNT signaling in both ISO+sFRP1-treated rats and paediatric iDCM hearts. Mechanistically, in vitro inhibition of Notch or β-catenin prevented pathological remodeling, and Notch inhibition improved cardiac function, myocardial stiffness and ventricular dilation in ISO+sFRP1-treated rats. Conclusion:We identified alterations in Notch and WNT signaling in paediatric iDCM hearts and in our model. Notch inhibition abrogated pathologic changes in vitro and in vivo. These findings provide novel mechanistic insights and a potential therapeutic target for paediatric iDCM.

Background:While operative and perioperative care continues to improve for single ventricle congenital heart disease (SV), long-term morbidities and mortality remain high. Importantly, phosphodiesterase-5 inhibitor therapies (PDE5i) are increasingly used, however, little is known regarding the direct myocardial effects of PDE5i therapy in the SV population. Objectives:Our group has previously demonstrated that the failing SV myocardium is characterized by increased PDE5 activity and impaired mitochondrial bioenergetics. Here we sought to determine whether serum circulating factors contribute to pathological metabolic remodeling in SV, and whether PDE5i therapy abrogates these changes. Methods:Using an established in vitro model whereby primary cardiomyocytes are treated with patient sera +/- PDE5i, we assessed the impact of circulating factors on cardiomyocyte metabolism. Mass spectrometry-based lipidomics and metabolomics were performed to identify phospholipid and metabolite changes. Mitochondrial bioenergetics were assessed using the Seahorse Bioanalyzer and a stable isotope based mitochondrial enzyme activity assay. Relative mitochondrial copy number was quantified using RT-qPCR. Results:Our data suggest that serum circulating factors contribute to fundamental changes in cardiomyocyte bioenergetics, including impaired mitochondrial function associated with decreased cardiolipin and other phospholipid species, increased reactive oxygen species (ROS) generation, and altered metabolite milieu. Treatment with PDE5i therapy was sufficient to abrogate a number of these metabolic changes, including a rescue of phosphatidylglycerol levels, a reduction in ROS, improved energy production, and normalization of several key metabolic intermediates. Conclusions:Together, these data suggest PDE5i therapy has direct cardiomyocyte effects and contributes to beneficial cardiomyocyte metabolic remodeling in SV failure.

The heart is formed from diverse cell lineages that assemble into a functional unit, including the pericardium, a mesothelial sac that supports movement, homeostasis, and immune responses. However, its developmental origins remain unresolved. Here, we find the pericardium forms within the lateral plate mesoderm from dedicated mesothelial progenitors that are distinct from the classic heart field. Imaging of transgenic zebrafish reporters documents lateral plate mesoderm cells that emerge lateral of the heart field among a continuous mesothelial progenitor band. Single-cell transcriptomics and trajectories of hand2-expressing lateral plate mesoderm reveal distinct populations of mesothelial precursors, including pericardial precursors. Their mesothelial gene expression signature is conserved in mammals and carries over to post-natal development. Light sheet imaging and machine learning-supported cell tracking documents the migration of pericardial precursors from the edge of the heart field to form the pericardial cavity. Genetic perturbations reveal this process occurs independently of heart formation, with canonical Wnt/β-catenin signaling modulating pericardial cell number and tissue rigidity. We connect the pathological expression of secreted Wnt antagonists of the SFRP family found in pediatric dilated cardiomyopathy to increased pericardial stiffness in neonatal rats. Altogether, our data integrate pericardium formation as an independent process into heart morphogenesis.

Obesity in pregnant women increases offspring cardiovascular risk and causes fetal cardiac dysfunction. The underpinning mechanisms remain unclear. We hypothesized that circulating factors in serum from fetuses of women with obesity induce pathological cardiomyocyte hypertrophy. Pregnant women with obesity or healthy weight were recruited at term and provided umbilical cord serum and placentas, which were used for isolation of primary trophoblast cells. Primary cardiomyocytes were isolated from neonatal rats. Compared with cord serum from healthy weight women, cord serum from women with obesity upregulated cardiomyocyte mRNA expression of atrial natriuretic factor (Anf) and brain natriuretic peptide (Bnp) and increased the ratio of β-to α-myosin heavy chain expression (Myh7:Myh6), when it was supplemented into the culture medium. This effect was prevented by treating the cord serum with heat-freeze cycling and DNase or RNase digestion. Separately, conditioned medium from trophoblast cells from women with obesity increased cardiomyocyte Anf expression without altering Bnp or Myh7:Myh6. MicroRNAs miR-142 and miR-17, which are associated with cardiac function, were increased in abundance in extracellular vesicles isolated from cord serum from women with obesity. However, miR-142-3p, miR-142-5p, and miR-17-5p did not increase Anf, Bnp, or Myh7:Myh6 expression when they were transfected into cardiomyocytes. Neither cord serum nor the upregulated microRNAs from women with obesity altered cardiomyocyte size. The results show that human fetal circulating and placenta-derived factors induce gene expression hallmarks of pathological hypertrophy in cardiomyocytes and may mediate cardiac dysfunction in children of women with obesity.NEW & NOTEWORTHY Obesity in pregnant women increases risk for heart problems in their children. This study treated heart cells growing in a dish with blood plasma from the umbilical cords of newborn babies. Plasma from babies of women with obesity activated genes linked to heart failure. This means we could design treatments targeting plasma molecules, like microRNAs, or the way the placenta releases them. This could improve children's heart health if the mother has obesity.

Children with single ventricle heart disease (SVHD) demonstrate decreased arginine/NO metabolism following Stage 2 (Glenn) palliation, associated with poor postoperative outcomes. It is unknown if arginine dysregulation persists at Stage 3 (Fontan). The purpose of this study is to quantify circulating arginine metabolites in children undergoing Fontan palliation for SVHD to evaluate the relationship between metabolite concentrations and outcomes. Prospective cohort study of children undergoing Fontan operation (n = 82) and similar age healthy controls (n = 49). We measured circulating arginine metabolites pre- and post-op by tandem mass spectrometry. Postoperative outcomes included length of stay (LOS) and pleural drainage. Pre-op cases showed lower arginine, argininosuccinate, cysteine, NMMA, higher glutathione, and lower arginine/ADMA, arginine/citrulline, and arginine/ornithine ratios compared to controls. Post-op cases experienced progressively decreasing citrulline concentration and higher arginine/ADMA, arginine/citrulline, and arginine/ornithine ratios compared to pre-op. In uncorrected analysis, postoperative decreased citrulline level (31.4% and 33.9% longer LOS for 50% decrease in [citrulline] at 2 and 24 h, respectively) was associated with longer LOS. Decreased arginine/ADMA and arginine/ornithine ratios were significantly associated with longer LOS and greater pleural drainage. Arginine metabolism is altered in children with SVHD in both the pre- and post-Stage 3 period. Patients with greater postoperative derangements, including lower arginine/ADMA and arginine/ornithine ratios, experienced more morbidity. We speculate that alterations in arginine metabolism may be a modifiable risk factor for adverse post-Stage 3 outcomes in SVHD.

Aim Obesity in pregnant women increases offspring cardiovascular risk and causes fetal cardiac dysfunction. The underpinning mechanisms remain unclear. We hypothesised that circulating factors in serum from fetuses of women with obesity induce pathological cardiomyocyte hypertrophy. Methods Pregnant women with obesity or healthy weight were recruited at term and provided umbilical cord serum and placentas, which were used for isolation of primary trophoblast cells. Primary cardiomyocytes were isolated from neonatal rats. Results Compared to serum-free medium, supplementing cardiomyocytes with umbilical cord serum increased their mRNA expression of atrial natriuretic factor ( Anf ) and brain natriuretic peptide ( Bnp ), hallmarks of pathological hypertrophy, but did not alter their size. Cord serum from women with obesity further upregulated cardiomyocyte Anf and Bnp , and increased the ratio of beta- to alpha-myosin heavy chain expression ( Myh7:Myh6 ), compared to cord serum from healthy weight women. This effect was prevented by treating the cord serum with heat- freeze cycling and DNase or RNase digestion. Conditioned medium from trophoblast cells from women with obesity also increased cardiomyocyte Anf , Bnp and Myh7:Myh6 expression. MicroRNAs miR-142 and miR-17, which are associated with cardiac function, were increased in abundance in extracellular vesicles isolated from cord serum from women with obesity. However, miR-142-3p, miR-142-5p and miR-17-5p did not increase Anf , Bnp or Myh7:Myh6 expression when they were transfected into cardiomyocytes. Conclusion The results show that human fetal circulating and placenta-derived factors induce hallmarks of pathological hypertrophy in cardiomyocytes and may mediate cardiac dysfunction in children of women with obesity. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Endothelin-1 (ET1) is a potent vasoconstrictor and stimulator of pulmonary artery smooth muscle cell proliferation. We previously demonstrated that failure to suppress ET1 is associated with morbidity in infants with single ventricle heart disease (SVHD) undergoing stage 2 palliation. OBJECTIVES:The aim of this study is to evaluate whether persistent failure to suppress ET1 is associated with impaired recovery among children with SVHD undergoing the stage 3 (Fontan) operation. METHODS:A prospective cohort study that includes 84 children with SVHD undergoing stage 3 palliation and 50 controls. Samples for ET1 analysis were collected at preoperation (systemic and pulmonary vein), 2, 24, and 48 hours postoperation for SVHD cases and a single timepoint for controls. Primary outcomes were Fontan pressure and systemic oxygen saturation at 24 hours postoperation. RESULTS:SVHD cases showed higher ET1 in the systemic vein than pulmonary vein (1.0 vs 0.7 pg/mL, P < 0.001) and lower systemic vein levels than controls (1.0 vs 1.4 pg/mL, P = 0.001). Among cases, ET1 concentration peaked at 2 hours postoperation, decreased by 24 hours, and was stable but not back to baseline by 48 hours. Adjusting for clinical covariates, higher preoperative ET1 was associated with higher 24-hour Fontan pressure. Higher 24-hour postoperative ET1 was associated with lower systemic oxygen saturation at 24 hours postoperation, higher 24-hour Fontan pressure, more pleural drainage, and longer length of stay. CONCLUSIONS:SVHD children with higher peri-operative ET1 experience more post-stage 3 morbidity. Failure to suppress ET1 may be a modifiable risk factor for intolerance of SVHD palliation.

Mitochondria play a crucial role in multiple cellular processes such as energy metabolism, generation of reactive oxygen species, excitation–contraction coupling, cell survival and death. Dysfunction of mitochondria contributes to the development of cancer; neuromuscular, cardiovascular/congenital heart disease; and metabolic diseases, including diabetes. Mitochondrial dysfunction can result in excessive reactive oxygen species, a decrease in energy production, mitophagy and apoptosis. All these processes are known to be dysregulated in cardiovascular diseases. The focus of this review is to summarize our current knowledge of mitochondrial dysfunction, including mitophagy and apoptosis, in pediatric congenital heart disease due to maternal diabetes or due to structural cardiac defects, with a focus on single-ventricle congenital heart disease. We also discuss recent mitochondria-targeted therapies for cardiovascular diseases.