Tests of a Laser Ion Source at the Heidelberg Electron Beam Ion Trap

A laser ion source (LIS) has been designed and successfully tested for loading the Heidelberg electron beam ion trap (H-EBIT) with ions of practically all solid-state elements. A pulsed YAG:Nd3þ laser (30 mJ, 8 ns) is used to produce plasma from a solid target. Lowly charged ions are extracted from the plasma and accelerated by a short high-voltage pulse, generating a pulsed ion beam with energy of up to 6 keV per charge. The ion beam is transported into the EBIT, decelerated and captured within it by switching the trapping electrode potentials. The electron beam further ionizes the trapped ions to the desired highly charged states. The experimental setup is described together with the source parameters measured for different target materials. X-ray spectra of up to He-like highly charged ions (Cu, Mo, Pb, Al and Ge) produced in the EBIT after injection from the LIS have been measured. The reliability and convenience of this method allows the use of the LIS to produce highly charged ions of both insulating and conducting solid elements with an EBIT as needed for different experiments

Fine Mapping of the 1p36 Deletion Syndrome Identifies Mutation of PRDM16 as a Cause of Cardiomyopathy

Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM

Monitoring Voltage-Dependent Charge Displacement of Shaker B-IR K+ Ion Channels Using Radio Frequency Interrogation

Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K+ ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ∼120 µM Cu2+ addition to the external bath. Cu2+ is known to bind to the ShB-IR ion channel and inhibit Shaker K+ conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu2+-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains — capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug–protein interactions

Chamber identity programs drive early functional partitioning of the heart

The vertebrate heart muscle (myocardium) develops from the first heart field (FHF) and expands by adding second heart field (SHF) cells. While both lineages exist already in teleosts, the primordial contributions of FHF and SHF to heart structure and function remain incompletely understood. Here we delineate the functional contribution of the FHF and SHF to the zebrafish heart using the cis-regulatory elements of the draculin (drl) gene. The drl reporters initially delineate the lateral plate mesoderm, including heart progenitors. Subsequent myocardial drl reporter expression restricts to FHF descendants. We harnessed this unique feature to uncover that loss of tbx5a and pitx2 affect relative FHF versus SHF contributions to the heart. High-resolution physiology reveals distinctive electrical properties of each heart field territory that define a functional boundary within the single zebrafish ventricle. Our data establish that the transcriptional program driving cardiac septation regulates physiologic ventricle partitioning, which successively provides mechanical advantages of sequential contraction

Metastable Atrial State Underlies the Primary Genetic Substrate for MYL4 Mutation-Associated Atrial Fibrillation

Background:Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models.Methods:Using an MYH6:mCherry knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell–derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled human MYL4 mutants, one of the few definitive genetic causes of AF. To explore non–cell-autonomous components of AF substrate, we also created a zebrafish Myl4 knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations.Results:There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)–related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in MYL4 mutants, as well as in atrial biopsies from unselected forms of human AF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models.Conclusions:These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.</p

Human cardiomyopathy mutations induce myocyte hyperplasia and activate hypertrophic pathways during cardiogenesis in zebrafish

To assess the effects during cardiac development of mutations that cause human cardiomyopathy, we modeled a sarcomeric gene mutation in the embryonic zebrafish. We designed morpholino antisense oligonucleotides targeting the exon 13 splice donor site in the zebrafish cardiac troponin T (tnnt2) gene, in order to precisely recapitulate a human TNNT2 mutation that causes hypertrophic cardiomyopathy (HCM). HCM is a disease characterized by myocardial hypertrophy, myocyte and myofibrillar disarray, as well as an increased risk of sudden death. Similar to humans with HCM, the morphant zebrafish embryos displayed sarcomere disarray and there was a robust induction of myocardial hypertrophic pathways. Microarray analysis uncovered a number of shared transcriptional responses between this zebrafish model and a well-characterized mouse model of HCM. However, in contrast to adult hearts, these embryonic hearts developed cardiomyocyte hyperplasia in response to this genetic perturbation. The re-creation of a human disease-causing TNNT2 splice variant demonstrates that sarcomeric mutations can alter cardiomyocyte biology at the earliest stages of heart development with distinct effects from those observed in adult hearts despite shared transcriptional responses

An infrared optical pacing system for screening cardiac electrophysiology in human cardiomyocytes

Human cardiac myocytes derived from pluripotent stem cells (hCM) have invigorated interest in genetic disease mechanisms and cardiac safety testing; however, the technology to fully assess electrophysiological function in an assay that is amenable to high throughput screening has lagged. We describe a fully contactless system using optical pacing with an infrared (IR) laser and multi-site high fidelity fluorescence imaging to assess multiple electrophysiological parameters from hCM monolayers in a standard 96-well plate. Simultaneous multi-site action potentials (FluoVolt) or Ca2+ transients (Fluo4-AM) were measured, from which high resolution maps of conduction velocity and action potential duration (APD) were obtained in a single well. Energy thresholds for optical pacing were determined for cell plating density, laser spot size, pulse width, and wavelength and found to be within ranges reported previously for reliable pacing. Action potentials measured using FluoVolt and a microelectrode exhibited the same morphology and rate of depolarization. Importantly, we show that this can be achieved accurately with minimal damage to hCM due to optical pacing or fluorescence excitation. Finally, using this assay we demonstrate that hCM exhibit reproducible changes in repolarization and impulse conduction velocity for Flecainide and Quinidine, two well described reference compounds. In conclusion, we demonstrate a high fidelity electrophysiological screening assay that incorporates optical pacing with IR light to control beating rate of hCM monolayers