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Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

Genetic cardiomyopathies involving intrinsic abnormalities of the heart, are frequently driven by gain-of-function mutations, producing toxic proteins or mRNAs that contribute to the disease pathogenesis. Treatment typically includes standard of care therapy for heart failure and arrhythmias rather than targeting the underlying genetic cause. Antibody-Oligonucleotide Conjugates (AOCs) offer a novel method to deliver small interfering RNA (siRNA) therapeutics directly to cardiac cells, holding a significant promise for precision medicine in cardiology, particularly for patients affected by gain-of-function mutations.. AOCs can be designed for precision cardiology therapies by reducing the expression of the targeted gene.. To evaluate the delivery and efficacy of AOCs to reduce the expression of targeted mRNA in heart.. We developed AOCs towards mice or non-human primates (NHPs) utilizing anti-TfR1 monoclonal antibodies conjugated to siRNA against the DMPK mRNA via a stable chemical linker. To evaluate the novel AOC platform to deliver siRNA and silence genes in the heart, mice were administered systemic intravenous AOC at 3 mg/kg (siRNA dose), and tissues were evaluated for siRNA concentration and Dmpk mRNA reduction on day 28. To evaluate the translation and tolerability of the AOC in larger species, NHPs were administered systemic intravenous AOC (2, 6 or 45 mg/kg siRNA dose) and tissues were evaluated for siRNA concentration and DMPK mRNA reduction.. A single dose of AOC at 3 mg/kg in mice resulted in approximately 75% reduction of Dmpk mRNA in both the heart and gastrocnemius muscle by day 28. However, no mRNA reduction was observed in the liver, despite similar siRNA concentrations in the liver and gastrocnemius tissues, with cardiac siRNA delivery being about 10 times greater. In NHPs, a single dose of AOC at 6 mg/kg led to significant DMPK mRNA reduction across various striated muscle tissues, including the heart, with no significant reduction observed in non-muscle tissues. A dose-response effect was also observed in cardiac DMPK mRNA reduction in NHPs following 9 months of chronic treatment (2 or 45 mg/kg). Additionally, AOC mediated reduction of DMPK mRNA in NHPs for 9 months was not associated with any adverse effects.. Our data suggests that AOC technology can efficiently deliver siRNA to the heart, significantly reducing the expression of targeted genes. This approach holds great promise as a therapeutic option for a subset of cardiomyopathies due to toxic gain-of-function mutations, particularly those with limited or nonexistent treatment options. [ABSTRACT FROM AUTHOR]

Background: Congenital heart disease (CHD) is the most common birth defect group and a significant contributor to neonatal and infant death. CHD with single ventricle anatomy, including hypoplastic left heart syndrome (HLHS), tricuspid atresia (TA), and various double‐inlet ventricle (DIV) malformations, is the most complex with the highest mortality. Prenatal risk factors associated with HLHS have been studied, but such data for DIV are lacking. Methods: We analyzed DIV cases and nonmalformed controls in the National Birth Defects Prevention Study, a case‐control, multicenter population‐based study of birth defects. Random forest analysis identified potential predictor variables for DIV, which were included in multivariable models to estimate effect magnitude and directionality. Results: Random forest analysis identified pre‐pregnancy diabetes, history of maternal insulin use, maternal total lipid intake, paternal race, and intake of several foods and nutrients as potential predictors of DIV. Logistic regression confirmed pre‐pregnancy diabetes, maternal insulin use, and paternal race as risk factors for having a child with DIV. Additionally, higher maternal total fat intake was associated with a reduced risk. Conclusions: Maternal pre‐pregnancy diabetes and history of insulin use were associated with an increased risk of having an infant with DIV, while maternal lipid intake had an inverse association. These novel data provide multiple metabolic pathways for investigation to identify better the developmental etiologies of DIV and suggest that public health interventions targeting diabetes prevention and management in women of childbearing age could reduce CHD risk. [ABSTRACT FROM AUTHOR]

Introduction: Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease associated with high morbidity and mortality. To improve disease treatments and prevention, a greater understanding of HLHS pathogenesis is necessary. While genetic risk factors and impaired blood flow during heart development have been implicated in the pathogenesis of HLHS, the role of intrinsic defects in ventricular cardiomyocyte formation and function remains unclear. Methods: To model ventricular hypoplasia seen in HLHS, we introduced, via CRISPR/Cas9, a tdTomato fluorescent reporter into the ventricular-specific myosin light chain 2 (MYL2) locus in healthy control and HLHS patient induced pluripotent stem cells (iPSCs). Cardiac directed differentiation was performed using small molecule modulation of the canonical Wnt pathway. MYL2-tdTomato positive cells were quantified and isolated, followed by gene expression, sarcomere alignment quantification, and contractility analysis. Results: We found that genome-edited control and HLHS iPSC lines expressed similar levels of pluripotency markers Nanog and Tra-1-81 prior to differentiation but a reduction in the fraction of MYL2-tdTomato positive iPSC-cardiomyocytes (iPSC-CMs) from HLHS lines compared to control at days 30 (control 69±13, HLHS-1 47±8, HLHS-2 51±14%, p<0.001), 45 (control 78±10 HLHS-1 45±9, HLHS-2 64±9%, p<0.001), and 60 (control 76±1, HLHS-1 52±14, HLHS-2 66±3%, p<0.001) of differentiation. Relative to control, day 30 ventricular HLHS iPSC-CMs showed a significant reduction in expression of multiple sarcomere protein genes (Myl2, Myl3, TnnT2, TnnI1, Myh7) and reduced sarcomere alignment (control 49.4±16.7, HLHS-1 13.5±11.4, HLHS-2 16.3±5.0%, p<0.01). Finally, ventricular HLHS iPSC-CMs displayed decreased contractile force, as measured by decreased maximal displacement (control 1.5±0.6, HLHS-1 1.0±0.4, HLHS-2 0.8±0.5μm, p<0.01) and velocity (control 10.0±4.3, HLHS-1 6.3±2.5, HLHS-2 6.0±3.4μm/s, p<0.01). Conclusions: Our findings support an intrinsic defect in ventricular cardiomyocyte formation and function in the pathogenesis of HLHS and the advantages of chamber-specific modeling of cardiomyogenesis for HLHS and perhaps other congenital anomalies. [ABSTRACT FROM AUTHOR]

Introduction: Congenital heart disease (CHD) is the most common birth defect and leading cause of neonatal and infant death. CHD with single ventricle anatomy, such as double-inlet left ventricle, also known as common ventricle (CV), are the most complex with the highest mortality. In contrast to other forms of CHD, prenatal risk factors associated with CV have not been previously studied. Methods: We analyzed 200 CV cases and 11,063 nonmalformed controls in the National Birth Defects Prevention Study, a case-control, multicenter population-based study of birth defects among 32,200 cases from 9 United States. Mothers completed surveys by trained interviewers 6 weeks to 24 months after the estimated date of delivery (October 1997 to December 2011). Random forest analysis identified potential predictor variables for CV among >200 variables. These potential predictor variables were included in multivariable models to estimate effects. Odds ratios (OR) and 95% confidence intervals were estimated using logistic regression for CV among all cases, cases with isolated CV, and non-isolated cases, defined as CV with at least 1 additional major non-cardiac malformation. Results: Random forest analysis identified pre-pregnancy diabetes, any history of maternal insulin use, maternal total lipid intake, and paternal race as potential predictors of CV. Table 1 reports risk factors identified via logistic regression. Higher maternal fat intake was associated with reduced risk of CV. Conclusions: Maternal pre-pregnancy diabetes and history of insulin use were associated with increased risk of having an infant with CV while maternal lipid intake had an inverse association. These novel data provide multiple metabolic pathways for investigation to better identify the developmental etiologies of CHD and suggest that public health interventions targeting balanced metabolism in women of childbearing age could reduce CHD risk. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have become critically important for the detailed study of cardiac development, disease modeling, and drug screening. However, directed differentiation of hiPSCs into cardiomyocytes often results in mixed populations of cardiomyocytes and other cell types, which may confound experiments that require pure populations of cardiomyocytes. Here, we detail the use of a CRISPR/Cas9 genome editing strategy to develop cardiomyocyte-specific reporters that allow for the isolation of hiPSC-derived cardiomyocytes and chamber-specific myocytes. Moreover, we describe a cardiac differentiation protocol to derive cardiomyocytes from hiPSCs, as well as a strategy to use fluorescence-activated cell sorting to isolate pure populations of fluorescently labeled cardiomyocytes for downstream applications.