Double outlet right ventricle - the 50% rule has always been about the conus.

PURPOSE OF REVIEW: There has been much variability in the definition of double outlet right ventricle (DORV) spanning the last century. Historically, emphasis has been placed on the assignment of the great arteries to the right ventricle as a definition of DORV. In this review, we aim to underscore the importance of conal muscle, rather than rules surrounding assignment of great arteries to ventricles. We will be outlining the variability in patient anatomy that results from variations in conal muscle development in DORV, which may not fit perfectly into predefined constructs. This anatomic variability directly determines physiology and surgical repair options. RECENT FINDINGS: There is a growing appreciation of the utility of cross-sectional imaging in complex DORV, and the generation of patient-specific 3D models with virtual reality simulations for surgical planning. These models improve the prediction of candidacy for biventricular repair and allow the mapping of complex baffle pathways preoperatively. SUMMARY: DORV is not a disease entity in itself, but rather a vast spectrum of disorders associated with maldevelopment of conal muscle and often abnormal expansion of one the great vessels. Patient-specific 3D models will be crucial for improved surgical planning and patient outcomes.

HRAS-Mutant Cardiomyocyte Model of Multifocal Atrial Tachycardia.

BACKGROUND: Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. METHODS: HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. RESULTS: ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CONCLUSIONS: CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

The Placenta in Congenital Heart Disease: Form, Function and Outcomes.

The maternal-fetal environment, controlled and modulated by the placenta, plays a critical role in the development and well-being of the fetus, with long-term impact through programming of lifelong health. The fetal cardiovascular system and placenta emerge at the same time embryologically, and thus placental form and function are altered in the presence of congenital heart disease (CHD). In this review, we report on what is known about the placenta from a structural and functional perspective when there is CHD. We describe the various unique pathologic findings as well as the diagnostic imaging tools used to characterize placental function in utero. With growing interest in the placenta, a standardized approach to characterizing placental pathology has emerged. Furthermore, application of ultrasonography techniques and magnetic resonance imaging now allow for insights into placental blood flow and functionality in vivo. An improved understanding of the intriguing relationship between the placenta and the fetal cardiovascular system will provide opportunities to develop novel ways to optimize outcomes. Once better understood, therapeutic modulation of placental function offered during the vulnerable period of fetal plasticity may be one of the most impactful ways to alter the course of CHD and its complications.

Mid-gestational fetal placental blood flow is diminished in the fetus with congenital heart disease.

OBJECTIVE: Data suggest fetuses with congenital heart disease (CHD) have placental abnormalities. Their abnormal placental vasculature may affect fetal placental blood flow, which has not previously been explored. METHOD: We performed a retrospective cross-sectional study comparing umbilical venous volume flow (UVVF) of single ventricle, D-transposition of the great arteries, and tetralogy of Fallot fetuses with fetuses without CHD. UVVF and combined cardiac output (CCO) were calculated from fetal echocardiography and compared using t tests, χ2 and Fisher's exact tests. RESULTS: Mean gestational age and fetal weight were greater in CHD fetuses (26.5 weeks, 1119.4 g; n = 81, P < .001) compared to controls (23.1 weeks, 675 g; n = 170, P < .001). UVVF/fetal weight was nevertheless decreased among cases (99.8 vs 115.3 mL/min/kg, P < .001). Subgroup analysis of 20- to 25-week fetuses demonstrated no significant differences in case and control baseline characteristics. In CHD fetuses (n = 31) compared to controls (n = 144), absolute UVVF (50.8 vs 62.1 mL/min, P = .006), and UVVF/fetal weight (98.8 vs 118.5 mL/min/kg, P < .001) were decreased. Findings were similar in single ventricle (n = 24) and hypoplastic left heart syndrome (n = 14). CONCLUSION: Mid-gestational placental blood flow in CHD fetuses is decreased compared to controls. Further study is needed to explore the relationship between UVVF and placental pathology, and impact on outcomes.

Autosomal Recessive Cardiomyopathy Presenting as Acute Myocarditis.

BACKGROUND: Myocarditis is inflammation of the heart muscle that can follow various viral infections. Why children only rarely develop life-threatening acute viral myocarditis (AVM), given that the causal viral infections are common, is unknown. Genetic lesions might underlie such susceptibilities. Mouse genetic studies demonstrated that interferon (IFN)-α/ß immunity defects increased susceptibility to virus-induced myocarditis. Moreover, variations in human TLR3, a potent inducer of IFNs, were proposed to underlie AVM. OBJECTIVES: This study sought to evaluate the hypothesis that human genetic factors may underlie AVM in previously healthy children. METHODS: We tested the role of TLR3-IFN immunity using human induced pluripotent stem cell-derived cardiomyocytes. We then performed whole-exome sequencing of 42 unrelated children with acute myocarditis (AM), some with proven viral causes. RESULTS: We found that TLR3- and STAT1-deficient cardiomyocytes were not more susceptible to Coxsackie virus B3 (CVB3) infection than control cells. Moreover, CVB3 did not induce IFN-α/ß and IFN-α/ß-stimulated genes in control cardiomyocytes. Finally, exogenous IFN-α did not substantially protect cardiomyocytes against CVB3. We did not observe a significant enrichment of rare variations in TLR3- or IFN-α/ß-related genes. Surprisingly, we found that homozygous but not heterozygous rare variants in genes associated with inherited cardiomyopathies were significantly enriched in AM-AVM patients compared with healthy individuals (p = 2.22E-03) or patients with other diseases (p = 1.08E-04). Seven of 42 patients (16.7%) carried rare biallelic (homozygous or compound heterozygous) nonsynonymous or splice-site variations in 6 cardiomyopathy-associated genes (BAG3, DSP, PKP2, RYR2, SCN5A, or TNNI3). CONCLUSIONS: Previously silent recessive defects of the myocardium may predispose to acute heart failure presenting as AM, notably after common viral infections in children.

Autonomous and Non-autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes.

Germline mutations in BRAF cause cardio-facio-cutaneous syndrome (CFCS), whereby 40% of patients develop hypertrophic cardiomyopathy (HCM). As the role of the RAS/MAPK pathway in HCM pathogenesis is unclear, we generated a human induced pluripotent stem cell (hiPSC) model for CFCS from three patients with activating BRAF mutations. By cell sorting for SIRPα and CD90, we generated a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM. BRAF-mutant SIRPα(+)/CD90(-) cardiomyocytes displayed cellular hypertrophy, pro-hypertrophic gene expression, and intrinsic calcium-handling defects. BRAF-mutant SIRPα(-)/CD90(+) cells, which were fibroblast-like, exhibited a pro-fibrotic phenotype and partially modulated cardiomyocyte hypertrophy through transforming growth factor ß (TGFß) paracrine signaling. Inhibition of TGFß or RAS/MAPK signaling rescued the hypertrophic phenotype. Thus, cell autonomous and non-autonomous defects underlie HCM due to BRAF mutations. TGFß inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies.

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRPα and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition. Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

Human Engineered Cardiac Tissues Created Using Induced Pluripotent Stem Cells Reveal Functional Characteristics of BRAF-Mediated Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden cardiac death that often goes undetected in the general population. HCM is also prevalent in patients with cardio-facio-cutaneous syndrome (CFCS), which is a genetic disorder characterized by aberrant signaling in the RAS/MAPK signaling cascade. Understanding the mechanisms of HCM development in such RASopathies may lead to novel therapeutic strategies, but relevant experimental models of the human condition are lacking. Therefore, the objective of this study was to develop the first 3D human engineered cardiac tissue (hECT) model of HCM. The hECTs were created using human cardiomyocytes obtained by directed differentiation of induced pluripotent stem cells derived from a patient with CFCS due to an activating BRAF mutation. The mutant myocytes were directly conjugated at a 3:1 ratio with a stromal cell population to create a tissue of defined composition. Compared to healthy patient control hECTs, BRAF-hECTs displayed a hypertrophic phenotype by culture day 6, with significantly increased tissue size, twitch force, and atrial natriuretic peptide (ANP) gene expression. Twitch characteristics reflected increased contraction and relaxation rates and shorter twitch duration in BRAF-hECTs, which also had a significantly higher maximum capture rate and lower excitation threshold during electrical pacing, consistent with a more arrhythmogenic substrate. By culture day 11, twitch force was no longer different between BRAF and wild-type hECTs, revealing a temporal aspect of disease modeling with tissue engineering. Principal component analysis identified diastolic force as a key factor that changed from day 6 to day 11, supported by a higher passive stiffness in day 11 BRAF-hECTs. In summary, human engineered cardiac tissues created from BRAF mutant cells recapitulated, for the first time, key aspects of the HCM phenotype, offering a new in vitro model for studying intrinsic mechanisms and screening new therapeutic approaches for this lethal form of heart disease.

Identification and purification of human induced pluripotent stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression.

The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC) reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN). When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.

Induced pluripotent stem cell-derived cardiomyocytes as models for genetic cardiovascular disorders.

PURPOSE OF REVIEW: The development of induced pluripotent stem cell (iPSC) technology has led to many advances in the areas of directed cell differentiation and characterization. New methods for generating iPSC-derived cardiomyocytes provide an invaluable resource for the study of certain cardiovascular disorders. This review highlights the current technology in this field, its application thus far to the study of genetic disorders of the RAS/MAPK pathway and long-QT syndrome (LQTS), and future directions for the field. RECENT FINDINGS: Enhanced methods increase the efficiency of generating and stringently purifying iPSC-derived cardiomyocytes. The use of cardiomyocytes derived from patients with LEOPARD syndrome and LQTS has shed light on the molecular mechanisms of disease and validated their use as reliable human disease models. SUMMARY: The use of iPSC-derived cardiomyocytes to study genetic cardiovascular disorders will enable a deeper and more applicable understanding of the molecular mechanisms of human disease, as well as improving our ability to achieve successful cell-based therapies. Methods to efficiently generate these cells are improving and provide promise for future applications of this technology.

UNC-51/ATG1 kinase regulates axonal transport by mediating motor-cargo assembly.

Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor-cargo assembly are not well understood. Here we show that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser(143), and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor-cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser(143) phosphorylation in axonal transport. Thus, our data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins.

Common SNPs in HMGCR in micronesians and whites associated with LDL-cholesterol levels affect alternative splicing of exon13.

UNLABELLED: Background- Variation in LDL-cholesterol (LDL-C) among individuals is a complex genetic trait involving multiple genes and gene-environment interactions. METHODS AND RESULTS: In a genome-wide association study (GWAS) to identify genetic variants influencing LDL-C in an isolated population from Kosrae, we observed associations for SNPs in the gene encoding 3hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase (HMGCR). Three of these SNPs (rs7703051, rs12654264, and rs3846663) met the statistical threshold of genome-wide significance when combined with data from the Diabetes Genetics Initiative GWAS. We followed up the association results and identified a functional SNP in intron13 (rs3846662), which was in linkage disequilibrium with the SNPs of genome-wide significance and affected alternative splicing of HMGCR mRNA. In vitro studies in human lymphoblastoid cells demonstrated that homozygosity for the rs3846662 minor allele was associated with up to 2.2-fold lower expression of alternatively spliced HMGCR mRNA lacking exon13, and minigene transfection assays confirmed that allele status at rs3846662 directly modulated alternative splicing of HMGCR exon13 (42.9+/-3.9 versus 63.7+/-1.0%Deltaexon13/total HMGCR mRNA, P=0.02). Further, the alternative splice variant could not restore HMGCR activity when expressed in HMGCR deficient UT-2 cells. CONCLUSIONS: We identified variants in HMGCR that are associated with LDL-C across populations and affect alternative splicing of HMGCR exon13.