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Background: Dilated cardiomyopathy (DCM) is a leading cause of death in children with heart failure. The outcome of pediatric heart failure treatment is inconsistent, and large cohort studies are lacking. Progress may be achieved through personalized therapy that takes age- and disease-related pathophysiology, pathology, and molecular fingerprints into account. We present single nuclei RNA sequencing from pediatric patients with DCM as the next step in identifying cellular signatures.Methods: We performed single nuclei RNA sequencing with heart tissues from 6 children with DCM with an age of 0.5, 0.75, 5, 6, 12, and 13 years. Unsupervised clustering of 18 211 nuclei led to the identification of 14 distinct clusters with 6 major cell types.Results: The number of nuclei in fibroblast clusters increased with age in patients with DCM, a finding that was confirmed by histological analysis and was consistent with an age-related increase in cardiac fibrosis quantified by cardiac magnetic resonance imaging. Fibroblasts of patients with DCM >6 years of age showed a profoundly altered gene expression pattern with enrichment of genes encoding fibrillary collagens, modulation of proteoglycans, switch in thrombospondin isoforms, and signatures of fibroblast activation. In addition, a population of cardiomyocytes with a high proregenerative profile was identified in infant patients with DCM but was absent in children >6 years of age. This cluster showed high expression of cell cycle activators such as cyclin D family members, increased glycolytic metabolism and antioxidative genes, and alterations in ß-adrenergic signaling genes.Conclusions: Novel insights into the cellular transcriptomes of hearts from pediatric patients with DCM provide remarkable age-dependent changes in the expression patterns of fibroblast and cardiomyocyte genes with less fibrotic but enriched proregenerative signatures in infants. [ABSTRACT FROM AUTHOR]

Background: More than 90% of individuals with Noonan syndrome (NS) with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated HCM.Methods: We used patient-derived RAF1S257L/+ and CRISPR-Cas9-generated isogenic control inducible pluripotent stem cell (iPSC)-derived cardiomyocytes to model NS RAF1-associated HCM and to further delineate the molecular mechanisms underlying the disease.Results: We show that mutant iPSC-derived cardiomyocytes phenocopy the pathology seen in hearts of patients with NS by exhibiting hypertrophy and structural defects. Through pharmacological and genetic targeting, we identify 2 perturbed concomitant pathways that, together, mediate HCM in RAF1 mutant iPSC-derived cardiomyocytes. Hyperactivation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), but not extracellular regulated kinase 1/2, causes myofibrillar disarray, whereas the enlarged cardiomyocyte phenotype is a direct consequence of increased extracellular regulated kinase 5 (ERK5) signaling, a pathway not previously known to be involved in NS. RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iPSC-derived cardiomyocytes and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways that could contribute to the NS-associated HCM.Conclusions: Taken together, the results of our study identify the molecular mechanisms by which NS RAF1 mutations cause HCM and reveal downstream effectors that could serve as therapeutic targets for treatment of NS and perhaps other, more common, congenital HCM disorders. [ABSTRACT FROM AUTHOR]

Introduction: Noonan Syndrome (NS) is caused by mutations that affect the RAS/ERK1/2-MAPK pathway. More than 90% of NS individuals with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated-HCM. Methods: Here, we used patient-derived RAF1S257L/+ and CRISPR-Cas9-generated isogenic control iPSC-derived cardiomyocytes (iCMs) to delineate the molecular mechanisms underlying HCM in NS-associated RAF1 mutations. Results: We found that mutant iCMs phenocopy the pathology seen in NS by exhibiting hypertrophy and structural defects (n=5). At the molecular level, while RAF1S257L/+ iCMs had elevated RAF1 and MEK1/2 activities, ERK1/2 itself was modestly enhanced. To test whether ERK1/2 activation was responsible for increased size of RAF1S257L/+ iCMs, we inhibited MEK1/2 activity with three different inhibitors (U0126, PD or Tram) and found that, while neither PD nor Tram reduced mutant iCMs area, both rescued myofibrillar disarray (n=3), suggesting that MEK1/2 and/or ERK1/2 regulate myofilament organization but not cell size. Since, PD and Tram block MEK1/2 activation by RAF, whereas U0126 prevents activation of ERK1/2 by active MEK1/2, we hypothesized that MEK1/2 rather than ERK1/2 regulates myofilament organization. Accordingly, we found that specific inhibition or ERK1/2 activity by ectopic expression of MKP3 did not rescue myofibrillar disarray in mutant RAF1 iCMs. Strikingly, U0126 was able to reduce iCMs surface area, suggesting that an ERK1/2-independent pathway was responsible for hypertrophy. Since U0126 also targets MEK5, we treated RAF1S257L/+ iCMs with a specific MEK5/ERK5 inhibitor and found that hypertrophy was drastically reduced (n=3), indicating that the ERK5 pathway triggered hypertrophy in RAF1S257L/+ iCMs. Finally, RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iCMs and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways and known as critical regulators of cardiac development, as well as extracellular matrix, cytoskeletal and actin assembly homeostasis that could contribute to the NS-associated HCM. Conclusion: Taken together, our study identifies the molecular mechanisms by which NS RAF1 mutations cause HCM and reveals downstream effectors that could serve as therapeutic targets for treatment of HCM in NS patients. [ABSTRACT FROM AUTHOR]