Glenna C.L. Bett

Mouse model of Timothy syndrome recapitulates triad of autistic traits

Autism and autism spectrum disorder (ASD) typically arise from a mixture of environmental influences and multiple genetic alterations. In some rare cases, such as Timothy syndrome (TS), a specific mutation in a single gene can be sufficient to generate autism or ASD in most patients, potentially offering insights into the etiology of autism in general. Both variants of TS (the milder TS1 and the more severe TS2) arise from missense mutations in alternatively spliced exons that cause the same G406R replacement in the CaV1.2 L-type calcium channel. We generated a TS2-like mouse but found that heterozygous (and homozygous) animals were not viable. However, heterozygous TS2 mice that were allowed to keep an inverted neomycin cassette (TS2-neo) survived through adulthood. We attribute the survival to lowering of expression of the G406R L-type channel via transcriptional interference, blunting deleterious effects of mutant L-type channel overactivity, and addressed potential effects of altered gene dosage by studying CaV1.2 knockout heterozygotes. Here we present a thorough behavioral phenotyping of the TS2-neo mouse, capitalizing on this unique opportunity to use the TS mutation to model ASD in mice. Along with normal general health, activity, and anxiety level, TS2-neo mice showed markedly restricted, repetitive, and perseverative behavior, altered social behavior, altered ultrasonic vocalization, and enhanced tone-cued and contextual memory following fear conditioning. Our results suggest that when TS mutant channels are expressed at levels low enough to avoid fatality, they are sufficient to cause multiple, distinct behavioral abnormalities, in line with the core aspects of ASD.

Study familial hypertrophic cardiomyopathy using patient-specific induced pluripotent stem cells

Aims Familial hypertrophic cardiomyopathy (HCM) is one the most common heart disorders, with gene mutations in the cardiac sarcomere. Studying HCM with patient-specific induced pluripotent stem-cell (iPSC)-derived cardiomyocytes (CMs) would benefit the understanding of HCM mechanism, as well as the development of personalized therapeutic strategies. Methods and results To investigate the molecular mechanism underlying the abnormal CM functions in HCM, we derived iPSCs from an HCM patient with a single missense mutation (Arginine442Glycine) in the MYH7 gene. CMs were next enriched from HCM and healthy iPSCs, followed with whole transcriptome sequencing and pathway enrichment analysis. A widespread increase of genes responsible for ‘Cell Proliferation’ was observed in HCM iPSC-CMs when compared with control iPSC-CMs. Additionally, HCM iPSC-CMs exhibited disorganized sarcomeres and electrophysiological irregularities. Furthermore, disease phenotypes of HCM iPSC-CMs were attenuated with pharmaceutical treatments. Conclusion Overall, this study explored the possible patient-specific and mutation-specific disease mechanism of HCM, and demonstrates the potential of using HCM iPSC-CMs for future development of therapeutic strategies. Additionally, the whole methodology established in this study could be utilized to study mechanisms of other human-inherited heart diseases.

Electronic “expression” of the inward rectifier in cardiocytes derived from human-induced pluripotent stem cells

BACKGROUND Human-induced pluripotent stem cell (h-iPSC)-derived cardiac myocytes are a unique model in which human myocyte function and dysfunction are studied, especially those from patients with genetic disorders. They are also considered a major advance for drug safety testing. However, these cells have considerable unexplored potential limitations when applied to quantitative action potential (AP) analysis. One major factor is spontaneous activity and resulting variability and potentially anomalous behavior of AP parameters. OBJECTIVE To demonstrate the effect of using an in silico interface on electronically expressed I(K1), a major component lacking in h-iPSC-derived cardiac myocytes. METHODS An in silico interface was developed to express synthetic I(K1) in cells under whole-cell voltage clamp. RESULTS Electronic I(K1) expression established a physiological resting potential, eliminated spontaneous activity, reduced spontaneous early and delayed afterdepolarizations, and decreased AP variability. The initiated APs had the classic rapid upstroke and spike and dome morphology consistent with data obtained with freshly isolated human myocytes as well as the readily recognizable repolarization attributes of ventricular and atrial cells. The application of 1 µM of BayK-8644 resulted in anomalous AP shortening in h-iPSC-derived cardiac myocytes. When I(K1) was electronically expressed, BayK-8644 prolonged the AP, which is consistent with the existing results on native cardiac myocytes. CONCLUSIONS The electronic expression of I(K1) is a simple and robust method to significantly improve the physiological behavior of the AP and electrical profile of h-iPSC-derived cardiac myocytes. Increased stability enables the use of this preparation for a controlled quantitative analysis of AP parameters, for example, drug responsiveness, genetic disorders, and dynamic behavior restitution profiles.

The Toxoplasma Dense Granule Proteins GRA17 and GRA23 Mediate the Movement of Small Molecules between the Host and the Parasitophorous Vacuole

Toxoplasma gondii is a protozoan pathogen in the phylum Apicomplexa that resides within an intracellular parasitophorous vacuole (PV) that is selectively permeable to small molecules through unidentified mechanisms. We have identified GRA17 as a Toxoplasma-secreted protein that localizes to the parasitophorous vacuole membrane (PVM) and mediates passive transport of small molecules across the PVM. GRA17 is related to the putative Plasmodium translocon protein EXP2 and conserved across PV-residing Apicomplexa. The PVs of GRA17-deficient parasites have aberrant morphology, reduced permeability to small molecules, and structural instability. GRA17-deficient parasites proliferate slowly and are avirulent in mice. These GRA17-deficient phenotypes are rescued by complementation with Plasmodium EXP2. GRA17 functions synergistically with a related protein, GRA23. Exogenous expression of GRA17 or GRA23 alters the membrane conductance properties of Xenopus oocytes in a manner consistent with a large non-selective pore. Thus, GRA17 and GRA23 provide a molecular basis for PVM permeability and nutrient access.

Relaxin Suppresses Atrial Fibrillation by Reversing Fibrosis and Myocyte Hypertrophy, and Increasing Conduction Velocity and Sodium Current in Spontaneously Hypertensive Rat Hearts

Rationale: Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly and hypertensive patients and has been correlated to enhanced atrial fibrosis. Despite a lack of direct evidence that fibrosis causes AF, reversal of fibrosis is considered a plausible therapy. Objective: To evaluate the efficacy of the antifibrotic hormone relaxin (RLX) in suppressing AF in spontaneously hypertensive rats (SHR). Methods and Results: Normotensive Wistar-Kyoto (WKY) and SHR were treated for 2 weeks with vehicle (WKY+V and SHR+V) or RLX (0.4 mg/kg per day, SHR+RLX) using implantable mini-pumps. Hearts were perfused, mapped optically to analyze action potential durations, intracellular Ca2+ transients, and restitution kinetics, and tested for AF vulnerability. SHR hearts had slower conduction velocity (CV; P<0.01 versus WKY), steeper CV restitution kinetics, greater collagen deposition, higher levels of transcripts for transforming growth factor-&bgr;, metalloproteinase-2, metalloproteinase-9, collagen I/III, and reduced connexin 43 phosphorylation (P<0.05 versus WKY). Programmed stimulation triggered sustained AF in SHR (n=5/5) and SHR+V (n=4/4), but not in WKY (n=0/5) and SHR+RLX (n=1/8; P<0.01). RLX treatment reversed the transcripts for fibrosis, flattened CV restitution kinetics, reduced action potential duration at 90% recovery to baseline, increased CV (P<0.01), and reversed atrial hypertrophy (P<0.05). Independent of antifibrotic actions, RLX (0.1 µmol/L) increased Na+ current density, INa (≈2-fold in 48 hours) in human cardiomyocytes derived from inducible pluripotent stem cells (n=18/18; P<0.01). Conclusions: RLX treatment suppressed AF in SHR hearts by increasing CV from a combination of reversal of fibrosis and hypertrophy and by increasing INa. The study provides compelling evidence that RLX may provide a novel therapy to manage AF in humans by reversing fibrosis and hypertrophy and by modulating cardiac ionic currents.

Whole-Cell Mechanosensitive Currents in Rat Ventricular Myocytes Activated by Direct Stimulation

Abstract. Mechanosensitive channels may have a significant role in the development of cardiac arrhythmia following infarction, but the data on mechanical responses at the cellular level are limited. Mechanosensitivity is a ubiquitous property of cells, and although the structure of bacteriological mechanosensitive ion channels is becoming known by cloning, the structure and force transduction pathway in eukaryotes remains elusive.Isolated adult rat ventricular myocytes were voltage clamped and stimulated with a mechanical probe. The probe was set in sinusoidal motion (either in, or normal to, the plane of the cell membrane), and then slowly lowered onto the cell. The sinusoidal frequency was held constant at 1 Hz but the stimulation amplitude was increased and the probe gradually lowered until a mechanically sensitive whole cell current was seen, which usually followed several minutes of stimulation.The whole cell mechanosensitive current in rat cells had two components: (i) a brief large inward current spike current; (ii) a more sustained smaller inward current. The presence of the initial sharp inward current suggests that some structure within the cell either relaxes or is broken, exposing the mechanosensitive element(s) to stress. Metabolic changes induced by continued stress prior to the mechanosensitive response may weaken the elements that break producing the spike, or simple stress-induced fracture of the cytoskeleton itself may occur.

Models of HERG Gating

HERG (Kv11.1, KCNH2) is a voltage-gated potassium channel with unique gating characteristics. HERG has fast voltage-dependent inactivation, relatively slow deactivation, and fast recovery from inactivation. This combination of gating kinetics makes study of HERG difficult without using mathematical models. Several HERG models have been developed, with fundamentally different organization. HERG is the molecular basis of I(Kr), which plays a critical role in repolarization. We programmed and compared five distinct HERG models. HERG gating cannot be adequately replicated using Hodgkin-Huxley type formulation. Using Markov models, a five-state model is required with three closed, one open, and one inactivated state, and a voltage-independent step between some of the closed states. A fundamental difference between models is the presence/absence of a transition directly from the proximal closed state to the inactivated state. The only models that effectively reproduce HERG data have no direct closed-inactivated transition, or have a closed-inactivated transition that is effectively zero compared to the closed-open transition, rendering the closed-inactivation transition superfluous. Our single-channel model demonstrates that channels can inactivate without conducting with a flickering or bursting open-state. The various models have qualitative and quantitative differences that are critical to accurate predictions of HERG behavior during repolarization, tachycardia, and premature depolarizations.

Modeling and study of the mechanism of dilated cardiomyopathy using induced pluripotent stem cells derived from individuals with Duchenne muscular dystrophy

ABSTRACT Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), and is characterized by progressive weakness in skeletal and cardiac muscles. Currently, dilated cardiomyopathy due to cardiac muscle loss is one of the major causes of lethality in late-stage DMD patients. To study the molecular mechanisms underlying dilated cardiomyopathy in DMD heart, we generated cardiomyocytes (CMs) from DMD and healthy control induced pluripotent stem cells (iPSCs). DMD iPSC-derived CMs (iPSC-CMs) displayed dystrophin deficiency, as well as the elevated levels of resting Ca2+, mitochondrial damage and cell apoptosis. Additionally, we found an activated mitochondria-mediated signaling network underlying the enhanced apoptosis in DMD iPSC-CMs. Furthermore, when we treated DMD iPSC-CMs with the membrane sealant Poloxamer 188, it significantly decreased the resting cytosolic Ca2+ level, repressed caspase-3 (CASP3) activation and consequently suppressed apoptosis in DMD iPSC-CMs. Taken together, using DMD patient-derived iPSC-CMs, we established an in vitro model that manifests the major phenotypes of dilated cardiomyopathy in DMD patients, and uncovered a potential new disease mechanism. Our model could be used for the mechanistic study of human muscular dystrophy, as well as future preclinical testing of novel therapeutic compounds for dilated cardiomyopathy in DMD patients. Highlighted Article: Patient-derived induced pluripotent stem cells are used to establish an in vitro model of DMD-associated cardiomyopathy that could be used for future preclinical testing.

Kv1.4 channel block by quinidine: evidence for a drug‐induced allosteric effect

We studied quinidine block of Kv1.4ΔN, a K+ channel lacking N‐type inactivation, expressed in Xenopus ooctyes. Initially, quinidine intracellularly blocked the open channel so rapidly it overlapped with activation. This rapid open channel block was reduced (non‐additively) by interventions that slow C‐type inactivation: [K+]o elevation and an extracellular lysine to tyrosine mutation (K532Y). These manipulations reduced the affinity of rapid open channel block ≈10‐fold, but left the effective electrical distance unchanged at ≈0.15. Following rapid open channel block, there were time‐dependent quinidine effects: the rate of inactivation during a single depolarisation was increased, and repetitive pulsing showed use dependence. The rate of recovery from the time‐dependent aspect of quinidine block was similar to recovery from normal C‐type inactivation. Manipulations that prevented the channel from entering the C‐type inactivated state (i.e. high [K+]o or the K532Y mutation) prevented the development of the time‐dependent quinidine‐induced inactivation. The concentration dependence of the rapid block and the time‐dependent quinidine‐induced inactivation were similar, but the time‐dependent component was strongly voltage sensitive, with an effective electrical distance of 2. Clearly, this cannot reflect the permeation of quinidine through the electric field, but must be the result of some other voltage‐sensitive change in the channel. We propose that quinidine promotes the entry of the channel into a C‐type inactivated state in a time‐ and voltage‐dependent manner. We developed a mathematical model based on these results to test the hypothesis that, following rapid open channel block, quinidine promotes development of the C‐type inactivated state through a voltage‐dependent conformational change.

Ancillary subunits and stimulation frequency determine the potency of chromanol 293B block of the KCNQ1 potassium channel

KCNQ1 (Kv7.1 or KvLQT1) encodes the alpha‐subunit of a voltage‐gated potassium channel found in tissues including heart, brain, epithelia and smooth muscle. Tissue‐specific characteristics of KCNQ1 current are diverse, due to modification by ancillary subunits. In heart, KCNQ1 associates with KCNE1 (MinK), producing a slowly activating voltage‐dependent channel. In epithelia, KCNQ1 co‐assembles with KCNE3 (Mirp2) producing a constitutively open channel. Chromanol 293B is a selective KCNQ1 blocker. We studied drug binding and frequency dependence of 293B on KCNQ1 and ancillary subunits expressed in Xenopus oocytes. Ancillary subunits altered 293B potency up to 100‐fold (IC50 for KCNQ1 = 65.4 ± 1.7 μm; KCNQ1/KCNE1 = 15.1 ± 3.3 μm; KCNQ1/KCNE3 = 0.54 ± 0.18 μm). Block of KCNQ1 and KCNQ1/KCNE3 was time independent, but 293B altered KCNQ1/KCNE1 activation. We therefore studied frequency‐dependent block of KCNQ1/KCNE1. Repetitive rapid stimulation increased KCNQ1/KCNE1 current biphasically, and 293B abolished the slow component. KCNQ1/KCNE3[V72T] activates slowly with a KCNQ1/KCNE1‐like phenotype, but retains the high affinity binding of KCNQ1/KCNE3, demonstrating that subunit‐mediated changes in gating can be dissociated from subunit‐mediated changes in affinity. This study demonstrates the KCNQ1 pharmacology is significantly altered by ancillary subunits. The response of KCNQ1 to specific blockers will therefore be critically dependent on the electrical stimulation pattern of the target organ. Furthermore, the dissociation between gating and overall affinity suggests that mutations in ancillary subunits can potentially strongly alter drug sensitivity without obvious functional changes in gating behaviour, giving rise to unexpected side‐effects such as a predisposition to acquired long QT syndrome.