Defining Cardiomyocyte Repolarization Response to Pharmacotherapy in Long-QT Syndrome Type 3.

BACKGROUND: Long-QT syndrome is a primary cardiac ion channelopathy predisposing a patient to ventricular arrhythmia through delayed repolarization on the resting ECG. We aimed to establish a patient-specific, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes model of long-QT syndrome type 3 (LQT3) using clustered regularly interspaced palindromic repeats (CRISPR/Cas9), for disease modeling and drug challenge. METHODS AND RESULTS: HiPSCs were generated from a patient with LQT3 harboring an SCN5A pathogenic variant (c.1231G>A; p.Val411Met), and an unrelated healthy control. The same SCN5A pathogenic variant was engineered into the background healthy control hiPSCs via CRISPR/Cas9 gene editing to generate a second disease model of LQT3 for comparison with an isogenic control. All 3 hiPSC lines were differentiated into cardiomyocytes. Both the patient-derived LQT3 (SCN5A+/-) and genetically engineered LQT3 (SCN5A+/-) hiPSC-derived cardiomyocytes showed significantly prolonged cardiomyocyte repolarization compared with the healthy control. Mexiletine, a cardiac voltage-gated sodium channel (NaV1.5) blocker, shortened repolarization in both patient-derived LQT3 and genetically engineered LQT3 hiPSC-derived cardiomyocytes, but had no effect in the control. Notably, calcium channel blockers nifedipine and verapamil showed a dose-dependent shortening of repolarization, rescuing the phenotype. Additionally, therapeutic drugs known to prolong the corrected QT in humans (ondansetron, clarithromycin, and sotalol) demonstrated this effect in vitro, but the LQT3 clones were not more disproportionately affected compared with the control. CONCLUSIONS: We demonstrated that patient-derived and genetically engineered LQT3 hiPSC-derived cardiomyocytes faithfully recapitulate pathologic characteristics of LQT3. The clinical significance of such an in vitro model is in the exploration of novel therapeutic strategies, stratifying drug adverse reaction risk and potentially facilitating a more targeted, patient-specific approach in high-risk patients with LQT3.

Using Ribonucleoprotein-based CRISPR/Cas9 to Edit Single Nucleotide on Human Induced Pluripotent Stem Cells to Model Type 3 Long QT Syndrome (SCN5A±).

Human induced pluripotent stem cells (hiPSCs) have been widely used in cardiac disease modelling, drug discovery, and regenerative medicine as they can be differentiated into patient-specific cardiomyocytes. Long QT syndrome type 3 (LQT3) is one of the more malignant congenital long QT syndrome (LQTS) variants with an SCN5A gain-of-function effect on the gated sodium channel. Moreover, the predominant pathogenic variants in LQTS genes are single nucleotide substitutions (missense) and small insertion/deletions (INDEL). CRISPR/Cas9 genome editing has been utilised to create isogenic hiPSCs to control for an identical genetic background and to isolate the pathogenicity of a single nucleotide change. In this study, we described an optimized and rapid protocol to introduce a heterozygous LQT3-specific variant into healthy control hiPSCs using ribonucleoprotein (RNP) and single-stranded oligonucleotide (ssODN). Based on this protocol, we successfully screened hiPSCs carrying a heterozygous LQT3 pathogenic variant (SCN5A±) with high efficiency (6 out of 69) and confirmed no off-target effect, normal karyotype, high alkaline phosphatase activity, unaffected pluripotency, and in vitro embryonic body formation capacity within 2 weeks. In addition, we also provide protocols to robustly differentiate hiPSCs into cardiomyocytes and evaluate the electrophysiological characteristics using Multi-electrode Array. This protocol is also applicable to introduce and/or correct other disease-specific variants into hiPSCs for future pharmacological screening and gene therapeutic development.

EED orchestration of heart maturation through interaction with HDACs is H3K27me3-independent.

In proliferating cells, where most Polycomb repressive complex 2 (PRC2) studies have been performed, gene repression is associated with PRC2 trimethylation of H3K27 (H3K27me3). However, it is uncertain whether PRC2 writing of H3K27me3 is mechanistically required for gene silencing. Here, we studied PRC2 function in postnatal mouse cardiomyocytes, where the paucity of cell division obviates bulk H3K27me3 rewriting after each cell cycle. EED (embryonic ectoderm development) inactivation in the postnatal heart (EedCKO) caused lethal dilated cardiomyopathy. Surprisingly, gene upregulation in EedCKO was not coupled with loss of H3K27me3. Rather, the activating histone mark H3K27ac increased. EED interacted with histone deacetylases (HDACs) and enhanced their catalytic activity. HDAC overexpression normalized EedCKO heart function and expression of derepressed genes. Our results uncovered a non-canonical, H3K27me3-independent EED repressive mechanism that is essential for normal heart function. Our results further illustrate that organ dysfunction due to epigenetic dysregulation can be corrected by epigenetic rewiring.

Novel Roles of GATA4/6 in the Postnatal Heart Identified through Temporally Controlled, Cardiomyocyte-Specific Gene Inactivation by Adeno-Associated Virus Delivery of Cre Recombinase.

GATA4 and GATA6 are central cardiac transcriptional regulators. The postnatal, stage-specific function of the cardiac transcription factors GATA4 and GATA6 have not been evaluated. In part, this is because current Cre-loxP approaches to cardiac gene inactivation require time consuming and costly breeding of Cre-expressing and "floxed" mouse lines, often with limited control of the extent or timing of gene inactivation. We investigated the stage-specific functions of GATA4 and GATA6 in the postnatal heart by using adeno-associated virus serotype 9 to control the timing and extent of gene inactivation by Cre. Systemic delivery of recombinant, adeno-associated virus 9 (AAV9) expressing Cre from the cardiac specific Tnnt2 promoter was well tolerated and selectively and efficiently recombined floxed target genes in cardiomyocytes. AAV9:Tnnt2-Cre efficiently inactivated Gata4 and Gata6. Neonatal Gata4/6 inactivation caused severe, rapidly lethal systolic heart failure. In contrast, Gata4/6 inactivation in adult heart caused only mild systolic dysfunction but severe diastolic dysfunction. Reducing the dose of AAV9:Tnnt2-Cre generated mosaics in which scattered cardiomyocytes lacked Gata4/6. This mosaic knockout revealed that Gata4/6 are required cell autonomously for physiological cardiomyocyte growth. Our results define novel roles of GATA4 and GATA6 in the neonatal and adult heart. Furthermore, our data demonstrate that evaluation of gene function hinges on controlling the timing and extent of gene inactivation. AAV9:Tnnt2-Cre is a powerful tool for controlling these parameters.

Ultrasound-guided transthoracic intramyocardial injection in mice.

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Cardiovascular disease in Noonan syndrome.

BACKGROUND: Noonan syndrome (NS), a relatively common autosomal dominant disorder with an incidence of 1 in 1000 to 2500 live births, is the most common syndromic cause of congenital heart disease after Trisomy 21. OBJECTIVE: To comprehensively define the spectrum of cardiac morphology and specific clinical course of a large cohort of NS patients. DESIGN: Retrospective, descriptive case series study. PATIENTS: An international Harvard-based NS registry was combined with clinical data from NS patients followed at Boston Children's Hospital, Massachusetts, USA. RESULTS: We identified 293 patients with NS. Cardiovascular disease was seen in 81% (n=237) including pulmonary stenosis in 57%, secundum atrial septal defects in 32% and hypertrophic cardiomyopathy in 16%. A genetic mutation of the RAS-MAPK signalling pathway was identified in 62% (n=136). Genotype-phenotype associations were noted between PTPN11 mutations and atrial septal defects (p=0.001), and pulmonary stenosis (p<0.001). RAF1 mutations were associated with hypertrophic cardiomyopathy (p<0.001). Cardiovascular outcomes that differed specifically in a NS cohort included high re-intervention rates (65%) after percutaneous balloon pulmonary valvuloplasty for valvar pulmonary stenosis. Additionally, in NS patients with hypertrophic cardiomyopathy, a clinically significant regression of hypertrophy (17%) was observed as was a markedly higher incidence of concomitant congenital heart defects (70%). CONCLUSIONS: Patients with NS have a distinct spectrum of cardiac phenotypes that may have a natural history and response to therapy atypical to that normally seen in non-syndromic heart disease. A diagnosis of NS in a patient with pulmonary stenosis or infant-onset hypertrophic cardiomyopathy would facilitate condition-specific counselling on outcome and prognosis.

Heterotaxy syndrome: defining contemporary disease trends.

The purpose of this study was to define a population of visceral heterotaxy and to investigate the incidence of bacterial sepsis in the current era of universal pediatric pneumococcal immunization. Pediatric echocardiography and radiology databases, along with electronic medical records, were searched for patients followed-up since birth between 1999 and 2009 with either asplenia or polysplenia and cardiac anatomy consistent with heterotaxy syndrome. A total of 29 patients were identified. Seven patients (24%) had a total of 8 sepsis events, and 6 patients (86%) developed sepsis while taking appropriately prescribed antibiotic prophylaxis. Of the patients with sepsis, 5 had polysplenia and 2 had asplenia. Sixty-two percent of sepsis events were nosocomially acquired. No cases of pneumococcal sepsis occurred after the introduction of the conjugated pneumococcal vaccination to the pediatric vaccination schedule. Bacterial sepsis was associated with a 44% mortality rate. An unexpected finding in 3 patients with visceral heterotaxy, asplenia, and an interrupted inferior vena cava (IVC) as the only anomaly on echocardiography was associated intestinal malrotation. Children with visceral heterotaxy remain at significant risk of life-threatening bacterial infection. In addition, the finding of interrupted IVC on echocardiography should prompt screening for intestinal malrotation, even in the absence of additional structural heart disease.