The 8th International RASopathies Symposium: Expanding research and care practice through global collaboration and advocacy.

Germline pathogenic variants in the RAS/mitogen-activated protein kinase (MAPK) signaling pathway are the molecular cause of RASopathies, a group of clinically overlapping genetic syndromes. RASopathies constitute a wide clinical spectrum characterized by distinct facial features, short stature, predisposition to cancer, and variable anomalies in nearly all the major body systems. With increasing global recognition of these conditions, the 8th International RASopathies Symposium spotlighted global perspectives on clinical care and research, including strategies for building international collaborations and developing diverse patient cohorts in anticipation of interventional trials. This biannual meeting, organized by RASopathies Network, was held in a hybrid virtual/in-person format. The agenda featured emerging discoveries and case findings as well as progress in preclinical and therapeutic pipelines. Stakeholders including basic scientists, clinician-scientists, practitioners, industry representatives, patients, and family advocates gathered to discuss cutting edge science, recognize current gaps in knowledge, and hear from people with RASopathies about the experience of daily living. Presentations by RASopathy self-advocates and early-stage investigators were featured throughout the program to encourage a sustainable, diverse, long-term research and advocacy partnership focused on improving health and bringing treatments to people with RASopathies.

Cardio-rheumatology: the cardiovascular, pharmacological, and surgical risks associated with rheumatological diseases in women.

Cardiovascular disease (CVD) remains the number one cause of death worldwide. Women are at increased risk of death from CVD, but the mechanisms for how and why this occurs remain elusive. One subset of women who are exceptionally vulnerable to CVD are those with rheumatic diseases (RDs). Indeed, women account for 80% of all RDs, disorders that encompass a broad range of autoimmune and autoinflammatory diseases that lead to chronic inflammation and pathology. The clear association of increased CVD risk in women with RD is thought to be mediated by a number of factors, including RD pathology itself, pharmacological induction of CVD, and/or as yet unidentified mechanisms. As such, elucidation of the causes and treatments of these pathologies has given rise to a new subspecialty of cardiology: cardio-rheumatology. Here, we review and discuss the CVD risks in patients with RDs, the associated sex disparities in RD and CVD care, as well as the current therapeutic and interventional options available to specifically help women with RDs. We hope this discussion will provide guidance and support to patients, as well as to cardio-rheumatologists, as these groups are the most uniquely positioned to radically improve CVD care in these individuals. Moreover, we are hopeful this discussion may lead to better, more efficacious approaches to treating these disorders in women in the near future.

Myh6 promoter-driven Cre recombinase excises floxed DNA fragments in a subset of male germline cells.

Myh6-Cre transgenic mouse line was known to express Cre recombinase only in the heart. Nevertheless, during breeding Myh6-Cre to Rosa26fstdTom reporter (tdTom) mouse line, we observed that a significant part of their F2 tdTom/+ offspring had tdTom reporter gene universally activated. Our results show that Myh6-Cre transgenic mice have Cre recombinase activity in a subpopulation of the male germline cells, and that Myh6 gene transcripts are enriched in the interstitial Leydig cells and the undifferentiated spermatogonia stem cells. In summary, the current study confirms that the previously known "heart-specific" Myh6 promoter drives Cre expression in the testis.

The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

YAP/TEAD1 Complex Is a Default Repressor of Cardiac Toll-Like Receptor Genes.

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that modulate innate immune responses and play essential roles in the pathogenesis of heart diseases. Although important, the molecular mechanisms controlling cardiac TLR genes expression have not been clearly addressed. This study examined the expression pattern of Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, and Tlr9 in normal and disease-stressed mouse hearts. Our results demonstrated that the expression levels of cardiac Tlr3, Tlr7, Tlr8, and Tlr9 increased with age between neonatal and adult developmental stages, whereas the expression of Tlr5 decreased with age. Furthermore, pathological stress increased the expression levels of Tlr2, Tlr4, Tlr5, Tlr7, Tlr8, and Tlr9. Hippo-YAP signaling is essential for heart development and homeostasis maintenance, and YAP/TEAD1 complex is the terminal effector of this pathway. Here we found that TEAD1 directly bound genomic regions adjacent to Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, and Tlr9. In vitro, luciferase reporter data suggest that YAP/TEAD1 repression of Tlr4 depends on a conserved TEAD1 binding motif near Tlr4 transcription start site. In vivo, cardiomyocyte-specific YAP depletion increased the expression of most examined TLR genes, activated the synthesis of pro-inflammatory cytokines, and predisposed the heart to lipopolysaccharide stress. In conclusion, our data indicate that the expression of cardiac TLR genes is associated with age and activated by pathological stress and suggest that YAP/TEAD1 complex is a default repressor of cardiac TLR genes.

Inducible Pluripotent Stem Cell-Derived Cardiomyocytes Reveal Aberrant Extracellular Regulated Kinase 5 and Mitogen-Activated Protein Kinase Kinase 1/2 Signaling Concomitantly Promote Hypertrophic Cardiomyopathy in RAF1-Associated Noonan Syndrome.

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.

The sixth international RASopathies symposium: Precision medicine-From promise to practice.

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion.

The role of the protein tyrosine phosphatase SHP2 in cardiac development and disease.

Congenital heart disease is the most common human developmental disorder, affecting ∼1:100 newborns, and is the primary cause of birth-defect related deaths worldwide. As a major regulator of receptor tyrosine kinase (RTK), cytokine and G-protein coupled receptor signaling, the non-receptor protein tyrosine phosphatase SHP2 plays a critical role in normal cardiac development and function. Indeed, SHP2 participates in a wide variety of cellular functions, including proliferation, survival, differentiation, migration, and cell-cell communication. Moreover, human activating and inactivating mutations of SHP2 are responsible for two related developmental disorders called Noonan and LEOPARD Syndromes, respectively, which are both characterized, in part, by congenital heart defects. Structural, enzymologic, biochemical, and SHP2 mouse model studies have together greatly enriched our knowledge of SHP2 and, as such, have also uncovered the diverse roles for SHP2 in cardiac development, including its contribution to progenitor cell specification, cardiac morphogenesis, and maturation of cardiac valves and myocardial chambers. By delineating the precise mechanisms by which SHP2 is involved in regulating these processes, we can begin to better understand the pathogenesis of cardiac disease and find more strategic and effective therapies for treatment of patients with congenital heart disorders.

Rapidly progressive hypertrophic cardiomyopathy in an infant with Noonan syndrome with multiple lentigines: palliative treatment with a rapamycin analog.

Noonan syndrome with multiple lentigines (NSML) frequently manifests with hypertrophic cardiomyopathy (HCM). Recently, it was demonstrated that mTOR inhibition reverses HCM in NSML mice. We report for the first time on the effects of treatment with a rapamycin analog in an infant with LS and malignant HCM. In the boy, progressive HCM was diagnosed during the first week of life and a diagnosis of NSML was established at age 20 weeks by showing a heterozygous Q510E mutation in PTPN11. Immunoblotting with antibodies against pERK, pAkt, and pS6RP in fibroblasts demonstrated enhanced Akt/mTOR pathway activity. Because of the patient's critical condition, everolimus therapy was started at age 24 weeks and continued until heart transplantation at age 36 weeks. Prior to surgery, heart failure improved from NYHA stage IV to II and brain natriuretic peptide values decreased from 9,600 to <1,000 pg/ml, but no reversal of cardiac hypertrophy was observed. Examination of the explanted heart revealed severe hypertrophy and myofiber disarray with extensive perivascular fibrosis. These findings provide evidence that Akt/mTOR activity is enhanced in NSML with HCM and suggest that rapamycin treatment could principally be feasible for infantile NSML. The preliminary experiences made in this single patient indicate that therapy should start early to prevent irreversible cardiac remodelling.

Detection and Eradication of a Demodex Infestation in Specific Pathogen-free High-barrier Laboratory Mouse Facility Housing Immunocompromised Animals.

Demodex mites are a common ectoparasite in nonlaboratory Mus musculus (mouse) populations. While infrequently reported in laboratory research mice, the prevalence is thought to be as high as 35% of all colonies. Here, we discuss an outbreak of Demodex within an SPF high-barrier vivarium housing laboratory mice first identified through commercial sentinel-free PCR testing. Consequently, in-house PCR-mediated identification of individually infected cages was conducted, and a successful method for eradication of secondary reemergent infection was generated via recurrent testing and empirical 12-wk treatment with 3 mg/kg moxidectin and 13 mg/kg imidacloprid. While we were unable to determine the source of our primary outbreak, the secondary outbreak was traced to nongenetically modified C57B6/J immunocompetent mice, which were capable of harboring subclinical infection below our PCR threshold. Our eventual successful eradication of Demodex confirmed, first, that in-house PCR detection is a cost-effective means of monitoring an outbreak; second, that treatment with 3 mg/kg moxidectin and 13 mg/kg imidacloprid does kill Demodex mites in laboratory mice; and third, that treatment of only PCR-positive mice is an insufficient way to control an outbreak. Taken together, our methodological approach for infestations such as Demodex suggests it is possible to eradicate them but that it requires a thorough, systematic, and aggressive treatment regimen. Moreover, we recommend that all cages derived from infected animals be treated as positive, regardless of PCR positivity, to prevent recurrent and/or persistent infections within an animal colony.

Lineage-Specific Mesenchymal Stromal Cells Derived from Human iPSCs Showed Distinct Patterns in Transcriptomic Profile and Extracellular Vesicle Production.

Over the past decades, mesenchymal stromal cells (MSCs) have been extensively investigated as a potential therapeutic cell source for the treatment of various disorders. Differentiation of MSCs from human induced pluripotent stem cells (iMSCs) has provided a scalable approach for the biomanufacturing of MSCs and related biological products. Although iMSCs shared typical MSC markers and functions as primary MSCs (pMSCs), there is a lack of lineage specificity in many iMSC differentiation protocols. Here, a stepwise hiPSC-to-iMSC differentiation method is employed via intermediate cell stages of neural crest and cytotrophoblast to generate lineage-specific MSCs with varying differentiation efficiencies and gene expression. Through a comprehensive comparison between early developmental cell types (hiPSCs, neural crest, and cytotrophoblast), two lineage-specific iMSCs, and six source-specific pMSCs, are able to not only distinguish the transcriptomic differences between MSCs and early developmental cells, but also determine the transcriptomic similarities of iMSC subtypes to postnatal or perinatal pMSCs. Additionally, it is demonstrated that different iMSC subtypes and priming conditions affected EV production, exosomal protein expression, and cytokine cargo.

SHP2 as a primordial epigenetic enzyme expunges histone H3 pTyr-54 to amend androgen receptor homeostasis.

Mutations that decrease or increase the activity of the tyrosine phosphatase, SHP2 (encoded by PTPN11), promotes developmental disorders and several malignancies by varying phosphatase activity. We uncovered that SHP2 is a distinct class of an epigenetic enzyme; upon phosphorylation by the kinase ACK1/TNK2, pSHP2 was escorted by androgen receptor (AR) to chromatin, erasing hitherto unidentified pY54-H3 (phosphorylation of histones H3 at Tyr54) epigenetic marks to trigger a transcriptional program of AR. Noonan Syndrome with Multiple Lentigines (NSML) patients, SHP2 knock-in mice, and ACK1 knockout mice presented dramatic increase in pY54-H3, leading to loss of AR transcriptome. In contrast, prostate tumors with high pSHP2 and pACK1 activity exhibited progressive downregulation of pY54-H3 levels and higher AR expression that correlated with disease severity. Overall, pSHP2/pY54-H3 signaling acts as a sentinel of AR homeostasis, explaining not only growth retardation, genital abnormalities and infertility among NSML patients, but also significant AR upregulation in prostate cancer patients.

Biomimetic Nanomaterials for the Immunomodulation of the Cardiosplenic Axis Postmyocardial Infarction.

The spleen is an important mediator of both adaptive and innate immunity. As such, attempts to modulate the immune response provided by the spleen may be conducive to improved outcomes for numerous diseases throughout the body. Here, biomimicry is used to rationally design nanomaterials capable of splenic retention and immunomodulation for the treatment of disease in a distant organ, the postinfarct heart. Engineered senescent erythrocyte-derived nanotheranostic (eSENTs) are generated, demonstrating significant uptake by the immune cells of the spleen including T and B cells, as well as monocytes and macrophages. When loaded with suberoylanilide hydroxamic acid (SAHA), the nanoagents exhibit a potent therapeutic effect, reducing infarct size by 14% at 72 h postmyocardial infarction when given as a single intravenous dose 2 h after injury. These results are supportive of the hypothesis that RBC-derived biomimicry may provide new approaches for the targeted modulation of the pathological processes involved in myocardial infarction, thus further experiments to decisively confirm the mechanisms of action are currently underway. This novel concept may have far-reaching applicability for the treatment of a number of both acute and chronic conditions where the immune responses are either stimulated or suppressed by the splenic (auto)immune milieu.

Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation.

Fibrosis following myocardial infarction is associated with increases in arrhythmias and sudden cardiac death. Initial steps in the development of fibrosis are not clear; however, it is likely that cardiac fibroblasts play an important role. In immune cells, ATP release from pannexin 1 (Panx1) channels acts as a paracrine signal initiating activation of innate immunity. ATP has been shown in noncardiac systems to initiate fibroblast activation. Therefore, we propose that ATP release through Panx1 channels and subsequent fibroblast activation in the heart drives the development of fibrosis in the heart following myocardial infarction. We identified for the first time that Panx1 is localized within sarcolemmal membranes of canine cardiac myocytes where it directly interacts with the postsynaptic density 95/Drosophila disk large/zonula occludens-1-containing scaffolding protein synapse-associated protein 97 via its carboxyl terminal domain (amino acids 300-357). Induced ischemia rapidly increased glycosylation of Panx1, resulting in increased trafficking to the plasma membrane as well as increased interaction with synapse-associated protein 97. Cellular stress enhanced ATP release from myocyte Panx1 channels, which, in turn, causes fibroblast transformation to the activated myofibroblast phenotype via activation of the MAPK and p53 pathways, both of which are involved in the development of cardiac fibrosis. ATP release through Panx1 channels in cardiac myocytes during ischemia may be an early paracrine event leading to profibrotic responses to ischemic cardiac injury.

Mitochondria and the future of RASopathies: the emergence of bioenergetics.

RASopathies are a family of rare autosomal dominant disorders that affect the canonical Ras/MAPK signaling pathway and manifest as neurodevelopmental systemic syndromes, including Costello syndrome (CS). In this issue of the JCI, Dard et al. describe the molecular determinants of CS using a myriad of genetically modified models, including mice expressing HRAS p.G12S, patient-derived skin fibroblasts, hiPSC-derived human cardiomyocytes, an HRAS p.G12V zebrafish model, and human lentivirally induced fibroblasts overexpressing HRAS p.G12S or HRAS p.G12A. Mitochondrial proteostasis and oxidative phosphorylation were altered in CS, and inhibition of the AMPK signaling pathway mediated bioenergetic changes. Importantly, the pharmacological induction of this pathway restored cardiac function and reduced the developmental defects associated with CS. These findings identify a role for altered bioenergetics and provide insights into more effective treatment strategies for patients with RASopathies.

Distinct Stress-Dependent Signatures of Cellular and Extracellular tRNA-Derived Small RNAs.

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that transfer RNA-derived small RNAs (tDRs) play pivotal roles in stress responses. However, RNA modifications present on tDRs are barriers to accurately quantifying tDRs using traditional small RNA sequencing. Here, AlkB-facilitated methylation sequencing is used to generate a comprehensive landscape of cellular and extracellular tDR abundances in various cell types during different stress responses. Extracellular tDRs are found to have distinct fragmentation signatures from intracellular tDRs and these tDR signatures are better indicators of different stress responses than miRNAs. These distinct extracellular tDR fragmentation patterns and signatures are also observed in plasma from patients on cardiopulmonary bypass. It is additionally demonstrated that angiogenin and RNASE1 are themselves regulated by stressors and contribute to the stress-modulated abundance of sub-populations of cellular and extracellular tDRs. Finally, a sub-population of extracellular tDRs is identified for which AGO2 appears to be required for their expression. Together, these findings provide a detailed profile of stress-responsive tDRs and provide insight about tDR biogenesis and stability in response to cellular stressors.

Engineering spatial-organized cardiac organoids for developmental toxicity testing.

Emerging technologies in stem cell engineering have produced sophisticated organoid platforms by controlling stem cell fate via biomaterial instructive cues. By micropatterning and differentiating human induced pluripotent stem cells (hiPSCs), we have engineered spatially organized cardiac organoids with contracting cardiomyocytes in the center surrounded by stromal cells distributed along the pattern perimeter. We investigated how geometric confinement directed the structural morphology and contractile functions of the cardiac organoids and tailored the pattern geometry to optimize organoid production. Using modern data-mining techniques, we found that pattern sizes significantly affected contraction functions, particularly in the parameters related to contraction duration and diastolic functions. We applied cardiac organoids generated from 600 µm diameter circles as a developmental toxicity screening assay and quantified the embryotoxic potential of nine pharmaceutical compounds. These cardiac organoids have potential use as an in vitro platform for studying organoid structure-function relationships, developmental processes, and drug-induced cardiac developmental toxicity.

Deletion of Ptpn11 (Shp2) in cardiomyocytes causes dilated cardiomyopathy via effects on the extracellular signal-regulated kinase/mitogen-activated protein kinase and RhoA signaling pathways.

BACKGROUND: Heart failure is the leading cause of death in the United States. By delineating the pathways that regulate cardiomyocyte function, we can better understand the pathogenesis of cardiac disease. Many cardiomyocyte signaling pathways activate protein tyrosine kinases. However, the role of specific protein tyrosine phosphatases (PTPs) in these pathways is unknown. METHODS AND RESULTS: Here, we show that mice with muscle-specific deletion of Ptpn11, the gene encoding the SH2 domain-containing PTP Shp2, rapidly develop a compensated dilated cardiomyopathy without an intervening hypertrophic phase, with signs of cardiac dysfunction appearing by the second postnatal month. Shp2-deficient primary cardiomyocytes are defective in extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAPK) activation in response to a variety of soluble agonists and pressure overload but show hyperactivation of the RhoA signaling pathway. Treatment of primary cardiomyocytes with Erk1/2- and RhoA pathway-specific inhibitors suggests that both abnormal Erk/MAPK and RhoA activities contribute to the dilated phenotype of Shp2-deficient hearts. CONCLUSIONS: Our results identify Shp2 as the first PTP with a critical role in adult cardiac function, indicate that in the absence of Shp2 cardiac hypertrophy does not occur in response to pressure overload, and demonstrate that the cardioprotective role of Shp2 is mediated via control of both the Erk/MAPK and RhoA signaling pathways.

Generation of an induced pluripotent stem cell line (TRNDi003-A) from a Noonan syndrome with multiple lentigines (NSML) patient carrying a p.Q510P mutation in the PTPN11 gene.

Noonan syndrome with multiple lentigines (NSML), formerly known as LEOPARD Syndrome, is a rare autosomal dominant disorder. Approximately 90% of NSML cases are caused by missense mutations in the PTPN11 gene which encodes the protein tyrosine phosphatase SHP2. A human induced pluripotent stem cell (iPSC) line was generated using peripheral blood mononuclear cells (PBMCs) from a patient with NSML that carries a gene mutation of p.Q510P on the PTPN11 gene using non-integrating Sendai virus technique. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development to treat NSML.