Marlin Touma

Nkx2-5 Suppresses the Proliferation of Atrial Myocytes and Conduction System

Rationale: Tight control of cardiomyocyte proliferation is essential for the formation of four-chambered heart. Although human mutation of NKX2-5 is linked to septal defects and atrioventricular conduction abnormalities, early lethality and hemodynamic alteration in the mutant models have caused controversy as to whether Nkx2-5 regulates cardiomyocyte proliferation. Objective: In this study, we circumvented these limitations by atrial-restricted deletion of Nkx2-5. Method and Results: Atrial-specific Nkx2-5 mutants died shortly after birth with hyperplastic working myocytes and conduction system including two nodes and internodal tracts. Multicolor reporter analysis revealed that Nkx2-5–null cardiomyocytes displayed clonal proliferative activity throughout the atria, indicating the suppressive role of Nkx2-5 in cardiomyocyte proliferation after chamber ballooning stages. Transcriptome analysis revealed that aberrant activation of Notch signaling underlies hyperproliferation of mutant cardiomyocytes, and forced activation of Notch signaling recapitulates hyperproliferation of working myocytes but not the conduction system. Conclusions: Collectively, these data suggest that Nkx2-5 regulates the proliferation of atrial working and conduction myocardium in coordination with Notch pathway.

Repression of Sox9 by Jag1 Is Continuously Required to Suppress the Default Chondrogenic Fate of Vascular Smooth Muscle Cells

Acquisition and maintenance of vascular smooth muscle fate are essential for the morphogenesis and function of the circulatory system. Loss of contractile properties or changes in the identity of vascular smooth muscle cells (vSMCs) can result in structural alterations associated with aneurysms and vascular wall calcification. Here we report that maturation of sclerotome-derived vSMCs depends on a transcriptional switch between mouse embryonic days 13 and 14.5. At this time, Notch/Jag1-mediated repression of sclerotome transcription factors Pax1, Scx, and Sox9 is necessary to fully enable vSMC maturation. Specifically, Notch signaling in vSMCs antagonizes sclerotome and cartilage transcription factors and promotes upregulation of contractile genes. In the absence of the Notch ligand Jag1, vSMCs acquire a chondrocytic transcriptional repertoire that can lead to ossification. Importantly, our findings suggest that sustained Notch signaling is essential throughout vSMC life to maintain contractile function, prevent vSMC reprogramming, and promote vascular wall integrity.

Decoding the Long Noncoding RNA during Cardiac Maturation: A Roadmap for Functional Discovery

Background—Cardiac maturation during perinatal transition of heart is critical for functional adaptation to hemodynamic load and nutrient environment. Perturbation in this process has major implications in congenital heart defects. Transcriptome programming during perinatal stages is an important information but incomplete in current literature, particularly, the expression profiles of the long noncoding RNAs (lncRNAs) are not fully elucidated. Methods and Results—From comprehensive analysis of transcriptomes derived from neonatal mouse heart left and right ventricles, a total of 45 167 unique transcripts were identified, including 21 916 known and 2033 novel lncRNAs. Among these lncRNAs, 196 exhibited significant dynamic regulation along maturation process. By implementing parallel weighted gene co-expression network analysis of mRNA and lncRNA data sets, several lncRNA modules coordinately expressed in a developmental manner similar to protein coding genes, while few lncRNAs revealed chamber-specific patterns. Out of 2262 lncRNAs located within 50 kb of protein coding genes, 5% significantly correlate with the expression of their neighboring genes. The impact of Ppp1r1b-lncRNA on the corresponding partner gene Tcap was validated in cultured myoblasts. This concordant regulation was also conserved in human infantile hearts. Furthermore, the Ppp1r1b-lncRNA/Tcap expression ratio was identified as a molecular signature that differentiated congenital heart defect phenotypes. Conclusions—The study provides the first high-resolution landscape on neonatal cardiac lncRNAs and reveals their potential interaction with mRNA transcriptome during cardiac maturation. Ppp1r1b-lncRNA was identified as a regulator of Tcap expression, with dynamic interaction in postnatal cardiac development and congenital heart defects.

Decoding the Noncoding Transcripts in Human Heart Failure

Heart failure (HF) is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and for better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma BNP and Troponin, have been shown to have limited use in defining the etiology or prognosis of the disease1, 2. Consequently, current clinical identification and classification of heart failure remain descriptive, largely based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic vs. dilated or ischemic vs. non-ischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVAD) has assumed a growing role in the care of patients with end-stage HF 3. During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure and electrical properties4. This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVAD. However, the underlying mechanism for the reverse remodeling in the LVAD treated hearts is unclear and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.

Wnt11 regulates cardiac chamber development and disease during perinatal maturation

Ventricular chamber growth and development during perinatal circulatory transition is critical for functional adaptation of the heart. However, the chamber-specific programs of neonatal heart growth are poorly understood. We used integrated systems genomic and functional biology analyses of the perinatal chamber specific transcriptome and we identified Wnt11 as a prominent regulator of chamber-specific proliferation. Importantly, downregulation of Wnt11 expression was associated with cyanotic congenital heart defect (CHD) phenotypes and correlated with O2 saturation levels in hypoxemic infants with Tetralogy of Fallot (TOF). Perinatal hypoxia treatment in mice suppressed Wnt11 expression and induced myocyte proliferation more robustly in the right ventricle, modulating Rb1 protein activity. Wnt11 inactivation was sufficient to induce myocyte proliferation in perinatal mouse hearts and reduced Rb1 protein and phosphorylation in neonatal cardiomyocytes. Finally, downregulated Wnt11 in hypoxemic TOF infantile hearts was associated with Rb1 suppression and induction of proliferation markers. This study revealed a previously uncharacterized function of Wnt11-mediated signaling as an important player in programming the chamber-specific growth of the neonatal heart. This function influences the chamber-specific development and pathogenesis in response to hypoxia and cyanotic CHDs. Defining the underlying regulatory mechanism may yield chamber-specific therapies for infants born with CHDs.

Hematopoietic progenitors are required for proper development of coronary vasculature

RATIONALE During embryogenesis, hematopoietic cells appear in the myocardium prior to the initiation of coronary formation. However, their role is unknown. OBJECTIVE Here we investigate whether pre-existing hematopoietic cells are required for the formation of coronary vasculature. METHODS AND RESULTS As a model of for hematopoietic cell deficient animals, we used Runx1 knockout embryos and Vav1-cre; R26-DTA embryos, latter of which genetically ablates 2/3 of CD45(+) hematopoietic cells. Both Runx1 knockout embryos and Vav1-cre; R26-DTA embryos revealed disorganized, hypoplastic microvasculature of coronary vessels on section and whole-mount stainings. Furthermore, coronary explant experiments showed that the mouse heart explants from Runx1 and Vav1-cre; R26-DTA embryos exhibited impaired coronary formation ex vivo. Interestingly, in both models it appears that epicardial to mesenchymal transition is adversely affected in the absence of hematopoietic progenitors. CONCLUSION Hematopoietic cells are not merely passively transported via coronary vessel, but substantially involved in the induction of the coronary growth. Our findings suggest a novel mechanism of coronary growth.

Massive liver mass and parenteral nutrition extravasation secondary to umbilical venous catheter complications

JCN_124_13R6 C ASE R EPORT ››› Massive Liver Mass and Parenteral Nutrition Extravasation Secondary to Umbilical Venous Catheter Complication Joanna Yeh 1 , Jorge Vargas 1 , Laura J Wozniak 1 , Jeffrey B Smith 2 , Boechat M. Ines 3 , Marlin Touma 2 Departments of 1 Pediatric Gastroenterology, 2 Neonatology and Developmental Biology, 3 Pediatric Radiology, 10833 Le Conte Avenue, MDCC 12-383, Los Angeles, CA 90095-1752, USA ABSTRACT Umbilical vein catheters (UVC) are widely used in neonatal medicine. Serious complications from UVC placement are uncommon but do exist, including infection, thrombosis, arrhythmias, and hemorrhage. Although rare, hepatic complications, in particular, have been associated with signifi cant morbidity and mortality. Correct positioning of the catheter prior to starting infusion of hyperosmolar solutions and early recognition of UVC-related complication are crucial in minimizing iatrogenic injury. We report the case of a neonate who was found at 10 days of age to have large pleural and peritoneal effusions and a massive fl uid collection in the liver due to malposition of a UVC. Key words: Hepatic hematoma, intravenous lipids, liver mass, Neonate, total parenteral nutrition, umbilical venous catheter INTRODUCTION Umbilical vein cannulation was fi rst utilized in 1947 for early venous access in the sick neonate. Serious complications, including vascular, hepatic, and cardiac injuries are uncommon but do exist. TPN extravasation and hepatic injury from UVC malposition are rare but potentially life-threatening complications. CASE REPORT Th e patient was born at 36 weeks gestation via urgent cesarean section secondary to maternal status epilepticus. Because of perinatal asphyxia, the infant was intubated and transported to our Neonatal Intensive Care Unit within a few hours aft er birth. On admission, the infant was hemodynamically stable. No anomalies or hepatosplenomegaly were detected. A UVC and umbilical artery catheter (UAC) were placed shortly aft er admission. An initial radiograph of the chest and the abdomen showed the catheter tip at the level of T9 near the midline, just below the diaphragm [Figure 1a]. Total parental nutrition (TPN) and intravenous lipids were initiated via the UVC. On day of life (DOL) 10, the infant required increased ventilatory support. Examination showed abdominal distension. A chest X-ray revealed a right pleural eff usion [Figure 1b]. A limited abdominal ultrasound revealed ascites. Th e abdominal viscera were not completely shown on this study. Pleural and peritoneal drains were placed, both of which produced a large amount of milky white fl uid. Analysis revealed high triglyceride (TG) levels, 375 mg/dL in the pleural fl uid and 1446 mg/dL in the peritoneal fl uid (serum TG level was 174 mg/dL). Neither Journal of Clinical Neonatology | Vol. 3 | Issue 2 | April-June 2014 fl uid had evidence of blood contamination. Th e UVC line was promptly removed. An attempt to aspirate from the UVC prior to its removal did not yield blood or other fl uid. One day aft er the removal of the UVC, the white blood cell count increased to 33,000/mm 3 , and the ratio of immature to total neutrophils increased to 0.57. Th e patient was empirically treated with antimicrobial antibiotics for 7 days. Blood, peritoneal, and pleural cultures remained negative. On DOL 17, a follow up abdominal ultrasound revealed a complex hypoechoic lesion in the liver measuring 75 × 64 × 57 mm. A Doppler study showed normal hepatic vessels, with absence of Doppler fl ow in lesion. A CT scan [Figure 2] revealed a large lesion (80 × 72 × 38 mm) of heterogeneous intensity involving multiple hepatic segments. Th ree days later, given concern to exclude pre-existing tumor (i.e. mesenchymal hamartoma or hemangioendothelioma) and to track interval changes Address for correspondence: Dr. Joanna Yeh, Pediatric Hepatology and Gastroenterology, David Geffen School of Medicine at UCLA, Mattel Children’s Hospital UCLA, Department of Pediatrics, 10833 Le Conte Avenue, MDCC 12-383, Los Angeles, CA 90095-1752, USA. E-mail: jyeh@mednet.ucla.edu Access this article online Quick Response Code: Website: www.jcnonweb.com DOI:

A Path to Implement Precision Child Health Cardiovascular Medicine

Congenital heart defects (CHDs) affect approximately 1% of live births and are a major source of childhood morbidity and mortality even in countries with advanced healthcare systems. Along with phenotypic heterogeneity, the underlying etiology of CHDs is multifactorial, involving genetic, epigenetic, and/or environmental contributors. Clear dissection of the underlying mechanism is a powerful step to establish individualized therapies. However, the majority of CHDs are yet to be clearly diagnosed for the underlying genetic and environmental factors, and even less with effective therapies. Although the survival rate for CHDs is steadily improving, there is still a significant unmet need for refining diagnostic precision and establishing targeted therapies to optimize life quality and to minimize future complications. In particular, proper identification of disease associated genetic variants in humans has been challenging, and this greatly impedes our ability to delineate gene–environment interactions that contribute to the pathogenesis of CHDs. Implementing a systematic multileveled approach can establish a continuum from phenotypic characterization in the clinic to molecular dissection using combined next-generation sequencing platforms and validation studies in suitable models at the bench. Key elements necessary to advance the field are: first, proper delineation of the phenotypic spectrum of CHDs; second, defining the molecular genotype/phenotype by combining whole-exome sequencing and transcriptome analysis; third, integration of phenotypic, genotypic, and molecular datasets to identify molecular network contributing to CHDs; fourth, generation of relevant disease models and multileveled experimental investigations. In order to achieve all these goals, access to high-quality biological specimens from well-defined patient cohorts is a crucial step. Therefore, establishing a CHD BioCore is an essential infrastructure and a critical step on the path toward precision child health cardiovascular medicine.

Decoding the Long Noncoding RNA During Cardiac MaturationCLINICAL PERSPECTIVE

Background—Cardiac maturation during perinatal transition of heart is critical for functional adaptation to hemodynamic load and nutrient environment. Perturbation in this process has major implications in congenital heart defects. Transcriptome programming during perinatal stages is an important information but incomplete in current literature, particularly, the expression profiles of the long noncoding RNAs (lncRNAs) are not fully elucidated. Methods and Results—From comprehensive analysis of transcriptomes derived from neonatal mouse heart left and right ventricles, a total of 45 167 unique transcripts were identified, including 21 916 known and 2033 novel lncRNAs. Among these lncRNAs, 196 exhibited significant dynamic regulation along maturation process. By implementing parallel weighted gene co-expression network analysis of mRNA and lncRNA data sets, several lncRNA modules coordinately expressed in a developmental manner similar to protein coding genes, while few lncRNAs revealed chamber-specific patterns. Out of 2262 lncRNAs located within 50 kb of protein coding genes, 5% significantly correlate with the expression of their neighboring genes. The impact of Ppp1r1b-lncRNA on the corresponding partner gene Tcap was validated in cultured myoblasts. This concordant regulation was also conserved in human infantile hearts. Furthermore, the Ppp1r1b-lncRNA/Tcap expression ratio was identified as a molecular signature that differentiated congenital heart defect phenotypes. Conclusions—The study provides the first high-resolution landscape on neonatal cardiac lncRNAs and reveals their potential interaction with mRNA transcriptome during cardiac maturation. Ppp1r1b-lncRNA was identified as a regulator of Tcap expression, with dynamic interaction in postnatal cardiac development and congenital heart defects.

Decoding the Long Noncoding RNA During Cardiac MaturationCLINICAL PERSPECTIVE: A Roadmap for Functional Discovery

Background—Cardiac maturation during perinatal transition of heart is critical for functional adaptation to hemodynamic load and nutrient environment. Perturbation in this process has major implications in congenital heart defects. Transcriptome programming during perinatal stages is an important information but incomplete in current literature, particularly, the expression profiles of the long noncoding RNAs (lncRNAs) are not fully elucidated. Methods and Results—From comprehensive analysis of transcriptomes derived from neonatal mouse heart left and right ventricles, a total of 45 167 unique transcripts were identified, including 21 916 known and 2033 novel lncRNAs. Among these lncRNAs, 196 exhibited significant dynamic regulation along maturation process. By implementing parallel weighted gene co-expression network analysis of mRNA and lncRNA data sets, several lncRNA modules coordinately expressed in a developmental manner similar to protein coding genes, while few lncRNAs revealed chamber-specific patterns. Out of 2262 lncRNAs located within 50 kb of protein coding genes, 5% significantly correlate with the expression of their neighboring genes. The impact of Ppp1r1b-lncRNA on the corresponding partner gene Tcap was validated in cultured myoblasts. This concordant regulation was also conserved in human infantile hearts. Furthermore, the Ppp1r1b-lncRNA/Tcap expression ratio was identified as a molecular signature that differentiated congenital heart defect phenotypes. Conclusions—The study provides the first high-resolution landscape on neonatal cardiac lncRNAs and reveals their potential interaction with mRNA transcriptome during cardiac maturation. Ppp1r1b-lncRNA was identified as a regulator of Tcap expression, with dynamic interaction in postnatal cardiac development and congenital heart defects.