Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.

Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity.

Conversion of human fibroblasts into functional cardiomyocytes by small molecules.

Reprogramming somatic fibroblasts into alternative lineages would provide a promising source of cells for regenerative therapy. However, transdifferentiating human cells into specific homogeneous, functional cell types is challenging. Here we show that cardiomyocyte-like cells can be generated by treating human fibroblasts with a combination of nine compounds that we term 9C.

Miniaturized iPS-Cell-Derived Cardiac Muscles for Physiologically Relevant Drug Response Analyses.

Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening.

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs.

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes.

Expandable Cardiovascular Progenitor Cells Reprogrammed from Fibroblasts.

Stem cell-based approaches to cardiac regeneration are increasingly viable strategies for treating heart failure. Generating abundant and functional autologous cells for transplantation in such a setting, however, remains a significant challenge. Here, we isolated a cell population with extensive proliferation capacity and restricted cardiovascular differentiation potentials during cardiac transdifferentiation of mouse fibroblasts.

KMT2D regulates specific programs in heart development via histone H3 lysine 4 di-methylation.

KMT2D, which encodes a histone H3K4 methyltransferase, has been implicated in human congenital heart disease in the context of Kabuki syndrome. However, its role in heart development is not understood. Here, we demonstrate a requirement for KMT2D in cardiac precursors and cardiomyocytes during cardiogenesis in mice.

'Therapeutic landscapes' and the importance of nostalgia, solastalgia, salvage and abandonment for psychiatric hospital design.

We examine emotional reactions to changes to medical spaces of care, linked with past experiences. In this paper we draw on findings from a qualitative study of the transfer of psychiatric inpatient care from an old to a newly built facility. We show how the meanings attributed to 'therapeutic landscapes' from one׳s past can evoke emotions and memories, manifesting in ideas about nostalgia, solastalgia, salvage and abandonment, which can impinge on one׳s present therapeutic experience.

Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales.

Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility.

Calcium transients closely reflect prolonged action potentials in iPSC models of inherited cardiac arrhythmia.

Long-QT syndrome mutations can cause syncope and sudden death by prolonging the cardiac action potential (AP). Ion channels affected by mutations are various, and the influences of cellular calcium cycling on LQTS cardiac events are unknown. To better understand LQTS arrhythmias, we performed current-clamp and intracellular calcium ([Ca(2+)]i) measurements on cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPS-CM).

Small molecules enable cardiac reprogramming of mouse fibroblasts with a single factor, Oct4.

It was recently shown that mouse fibroblasts could be reprogrammed into cells of a cardiac fate by forced expression of multiple transcription factors and microRNAs. For ultimate application of such a reprogramming strategy for cell-based therapy or in vivo cardiac regeneration, reducing or eliminating the genetic manipulations by small molecules would be highly desirable. Here, we report the identification of a defined small-molecule cocktail that enables the highly efficient conversion of mouse fibroblasts into cardiac cells with only one transcription factor, Oct4, without any evidence of entrance into the pluripotent state.

Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.

Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro.

Spaces for smoking in a psychiatric hospital: social capital, resistance to control, and significance for 'therapeutic landscapes'.

This paper reports on research framed by theories of therapeutic landscapes and the ways that the social, physical and symbolic dimensions of landscapes relate to wellbeing and healing. We focus especially on the question of how attributes of therapeutic landscapes are constructed in different ways according to the variable perspectives of individuals and groups. Through an ethnographic case study in a psychiatric hospital in the North of England we explore the perceived significance for wellbeing of 'smoking spaces' (where tobacco smoking is practiced in ways that may, or may not be officially sanctioned).

Compassionate containment? Balancing technical safety and therapy in the design of psychiatric wards.

This paper contributes to the international literature examining design of inpatient settings for mental health care. Theoretically, it elaborates the connections between conceptual frameworks from different strands of literature relating to therapeutic landscapes, social control and the social construction of risk. It does so through a discussion of the substantive example of research to evaluate the design of a purpose built inpatient psychiatric health care facility, opened in 2010 as part of the National Health Service (NHS) in England.

Creating 'therapeutic landscapes' for mental health carers in inpatient settings: a dynamic perspective on permeability and inclusivity.

Although there has been a shift toward treatment in the home and the community, in the UK, inpatient facilities are still important in modern mental health care. 'Informal carers', including family members, often play an essential role, not only in providing care in the community but also in care of patients during periods of hospitalisation. UK National Health Service policies increasingly consider the position of these carers as 'partners' in the care process, but relatively little attention has been paid to their position within the hospital settings where treatment is provided for inpatients.

Ion channel pharmacology under flow: automation via well-plate microfluidics.

Automated patch clamping addresses the need for high-throughput screening of chemical entities that alter ion channel function. As a result, there is considerable utility in the pharmaceutical screening arena for novel platforms that can produce relevant data both rapidly and consistently. Here we present results that were obtained with an innovative microfluidic automated patch clamp system utilizing a well-plate that eliminates the necessity of internal robotic liquid handling.

In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.

The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts,which represent 50%of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation.

Actions of ATX-II and other gating-modifiers on Na(+) currents in HEK-293 cells expressing WT and DeltaKPQ hNa(V) 1.5 Na(+) channels.

Voltage-gated Na(+) channels underlie the action potential upstroke in excitable cells, and both natural and synthetic inactivation inhibitors prolong the Na(+) current (I(Na)). The effects of Na(+) channel mutations on these pharmacological actions are incompletely investigated. Therefore, I compared the effects of inactivation inhibitors on I(Na) in WT or mutant (DeltaKPQ) human cardiac Na(+) channels expressed in HEK-293 cells, by measuring difference currents sensitive to 50muM tetrodotoxin.

Mechanisms underlying the effects of the pyrethroid tefluthrin on action potential duration in isolated rat ventricular myocytes.

Due to increased global use, acute exposures to pyrethroid insecticides in humans are of clinical concern. Pyrethroids have a primary mode of action that involves interference with the inactivation of Na+ currents (I(Na)) in excitable cells, which may include cardiac myocytes. To investigate the possible cardiac toxicity of these agents, we have examined the effects of a type-1 pyrethroid, tefluthrin, on isolated rat ventricular myocytes.

Differential effects of extracellular cesium on early afterdepolarizations in ventricular myocytes and arrhythmogenesis in isolated hearts of rats and guinea pigs.

CsCl has been shown to be arrhythmogenic in-vivo and to cause early afterdepolarizations (EADs) in isolated cardiac preparations, but the underlying electrophysiological mechanisms are ill-defined. To elucidate these actions further, the effects of extracellular solutions containing 3 mM CsCl and either 2 mM KCl (Cs2K solution) or 5 mM KCl (Cs5K solution) on membrane potential and ionic currents in rat and guinea-pig ventricular myocytes were compared. Cs2K solution rapidly and reversibly inhibited outward I(K1), and reduced other K(+) currents by about 20%.

Effects of Na+/Ca2+ exchange induced by SR Ca2+ release on action potentials and afterdepolarizations in guinea pig ventricular myocytes.

In cardiac cells, evoked Ca2+ releases or spontaneous Ca2+ waves activate the inward Na+/Ca2+ exchange current (INaCa), which may modulate membrane excitability and arrhythmogenesis. In this study, we examined changes in membrane potential due to INaCa elicited by sarcoplasmic reticulum (SR) Ca2+ release in guinea pig ventricular myocytes using whole cell current clamp, fluorescence, and confocal microscopy. Inhibition of INaCa by Na+-free, Li+-containing Tyrode solution reversibly abbreviated the action potential duration at 90% repolarization (APD90) by 50% and caused SR Ca2+ overload.

Inhibitory effects of the antiestrogen agent clomiphene on cardiac sarcolemmal anionic and cationic currents.

The aim of this study was to determine the effects of the antiestrogen agent clomiphene on cardiac anionic and cationic sarcolemmal ion channels. Whole-cell recordings were made from rat and guinea pig ventricular myocytes. Clomiphene inhibited the volume-regulated chloride current [I(Cl,vol), activated by cell swelling after hypotonic shock (approximately 145 mOsM)] with an IC(50) value of approximately 9.

Tefluthrin modulates a novel anionic background conductance (I(AB)) in guinea-pig ventricular myocytes.

This report describes for the first time a novel anionic background current (I(AB)) identified in guinea-pig isolated ventricular myocytes. It also shows that I(AB) has both novel and differential pharmacology from other (cardiac) chloride currents. Using the whole-cell patch-clamp technique and external anion substitution, I(AB) was found to be outwardly rectifying and highly permeable to NO(-)(3), with a relative permeability sequence of NO(-)(3) > I(-) > Cl(-).