Patient-specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long-QT syndromes (LQTS). To study the LQT2-associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC-derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC-derived CMs. Further characterization of N996I-HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.

The Plasmadynamics and Electric Propulsion Laboratory High-speed Axial Reciprocating Probe system is used in conjunction with a floating emissive probe to measure plasma potential in the discharge chamber of the P5 Hall thruster. Plasma potential measurements are made at a constant voltage, 300 V, at two different discharge current conditions: 5.4 and 10 A. The plasma potential contours for the 5.4 A case indicate that the acceleration region begins several millimeters upstream of the exit plane, extends several centimeters downstream, and is uniform across the width of the discharge chamber. The 10 A case is similar to the 5.4 A case with the exception that the acceleration region is shifted downstream on centerline. Axial electric field profiles, computed from the measured potential, show a double peak structure in the 5.4 A case, indicating a zone of ion deceleration. Perturbations to the discharge current are shown to correspond spatially with the location of the peak electric field indicating that thruster perturbations may result from a disturbance to the Hall current, as opposed to ablation of probe material. This conclusion is supported by the lack of any observable material ablation.

During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1 1 precursors. Embryos deficient for Nkx2-5 in the Isl1 1 lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.

The use of electrostatic probes to measure local plasma parameters inside the discharge chamber of a Hall thruster presents significant difficulties. The high-temperature, dense plasma, and Hall current in the accelerating channel heat the probe rapidly causing ablation of probe material, which perturbs thruster operation and reduces probe lifetime. Results are presented which show the extent of perturbation to discharge current, cathode potential, and thrust for the case where probe material is ablated. A simple thermal model of probe material heating is developed and ablation times for a typical probe configuration are presented. Using the results of the thermal model, a high-speed axial reciprocating probe ͑HARP͒ system was developed to enable probe survival and reduce thruster perturbations during interrogation of the discharge chamber of a Hall thruster. Results using the HARP system are presented showing a significant reduction in thruster perturbation. The results also indicate that a mechanism other than material ablation is contributing to perturbation of the thruster. Based on emissive probe data, the tungsten conductor appears to provide a low impedance path between magnetic field lines, enhancing electron transport to the anode.