Since the discovery of human induced pluripotent stem cells (hiPSCs), numerous strategies have been established to efficiently derive cardiomyocytes from hiPSCs (hiPSC-CMs). Here, we describe a cost-effective strategy for the subsequent massive expansion (>250-fold) of high-purity hiPSC-CMs relying on two aspects: removal of cell-cell contacts and small-molecule inhibition with CHIR99021. The protocol maintains CM functionality, allows cryopreservation, and the cells can be used in downstream assays such as disease modeling, drug and toxicity screening, and cell therapy.

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against the CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects on CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo, providing a proof of principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging, and arrhythmia management.

Background: With the advent of more intensive rhythm monitoring strategies, ventricular arrhythmias (VAs) are increasingly detected in Fontan patients. However, the prognostic implications of VA are poorly understood. We assessed the incidence of VA in Fontan patients and the implications on transplant-free survival. Methods: Medical records of Fontan patients seen at a single center between 2002 and 2019 were reviewed to identify post-Fontan VA (nonsustained ventricular tachycardia >4 beats or sustained >30 seconds). Patients with preFontan VA were excluded. Hemodynamically unstable VA was defined as malignant VA. The primary outcome was death and heart transplantation. Death with censoring at transplant was a secondary outcome. Results: Of 431 Fontan patients, transplant-free survival was 82% at 15 years post-Fontan with 64 (15%) meeting primary outcome of either death (n=16, 3.7%), at a median 4.6 (0.4–10.2) years post-Fontan, or transplant (n=48, 11%), at a median of 11.1 (5.9–16.2) years post-Fontan. Forty-eight (11%) patients were diagnosed with VA (90% nonsustained ventricular tachycardia, 10% sustained ventricular tachycardia). Malignant VA (n=9, 2.0%) was associated with younger age, worse systolic function, and valvular regurgitation. Risk for VA increased with time from Fontan, 2.4% at 10 years to 19% at 20 years. History of Stage 1 surgery with right ventricular to pulmonary artery conduit and older age at Fontan were significant risk factors for VA. VA was strongly associated with an increased risk of transplant or death (HR, 9.2 [95% CI, 4.5–18.7]; P <0.001), with a transplant-free survival of 48% at 5-year post-VA diagnosis. Conclusions: Ventricular arrhythmias occurred in 11% of Fontan patients and was highly associated with transplant or death, with a transplant-free survival of <50% at 5-year post-VA diagnosis. Risk factors for VA included older age at Fontan and history of right ventricular to pulmonary artery conduit. A diagnosis of VA in Fontan patients should prompt increased clinical surveillance.

BACKGROUND: DSP cardiomyopathy is a distinct subset of arrhythmogenic cardiomyopathy, reported primarily in adults, that has predominantly left ventricular involvement and features of myocarditis. Clinical characteristics, risk stratification, and management of pediatric patients with DSP variants are not well known. We sought to identify phenotypic features and prognosis of pediatric patients with DSP pathogenic or likely pathogenic variants. METHODS: Multicenter, retrospective study of patients <21 years of age with DSP variants from 6 tertiary pediatric hospitals. RESULTS: Thirty-four patients, including 10 probands with clinical disease and 24 genotype-positive phenotype-negative patients, were included in the study. The majority of probands were initially diagnosed with myocarditis (50%) and had biventricular (60%) or left ventricular predominant (40%) disease. Chest pain was the most common symptom at presentation (30%), and all had troponin elevation. Probands with homozygous or compound heterozygous DSP variants were likely to present at an early age (<13 years) with symptoms of heart failure, severe biventricular involvement, and dermatologic abnormalities. Low-voltage QRS was the most prominent ECG abnormality. Of those who underwent implantable cardioverter defibrillator implantation, 50% received appropriate implantable cardioverter defibrillator therapy and were found to have significant biventricular involvement in addition to severe left ventricular dysfunction with an ejection fraction <35%. CONCLUSIONS: DSP cardiomyopathy in children and adolescents has varied phenotypic manifestations based on age and genotype and often can be diagnosed as myocarditis. Severe left ventricular dysfunction and biventricular involvement may be associated with a higher likelihood of malignant ventricular tachyarrhythmia.