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The congenital long QT syndrome (LQTS) is a cardiac electrophysiological disorder that can cause sudden cardiac death. LQT1 is a subtype of LQTS caused by mutations in KCNQ1, affecting the slow delayed-rectifier potassium current ( I Ks ), which is essential for cardiac repolarization. Paradoxically, gain-of-function mutations in KCNQ1 have been reported to cause borderline QT prolongation, atrial fibrillation (AF), sinus bradycardia, and sudden death, however, the mechanisms are not well understood. The goal of the study is to investigate the ionic, cellular and tissue mechanisms underlying the complex phenotype of a gain-of-function mutation in KCNQ1, c.686G > A (p.G229D) using computer modeling and simulations informed by in vitro measurements. Previous studies have shown this mutation to cause AF and borderline QT prolongation. We report a clinical description of a family that carry this mutation and that a member of the family died suddenly during sleep at 21 years old. Using patch-clamp experiments, we confirm that KCNQ1-G229D causes a significant gain in channel function. We introduce the effect of the mutation in populations of atrial, ventricular and sinus node (SN) cell models to investigate mechanisms underlying phenotypic variability. In a population of human atrial and ventricular cell models and tissue, the presence of KCNQ1-G229D predominantly shortens atrial action potential duration (APD). However, in a subset of models, KCNQ1-G229D can act to prolong ventricular APD by up to 7% (19 ms) and underlie depolarization abnormalities, which could promote QT prolongation and conduction delays. Interestingly, APD prolongations were predominantly seen at slow pacing cycle lengths (CL > 1,000 ms), which suggests a greater arrhythmic risk during bradycardia, and is consistent with the observed sudden death during sleep. In a population of human SN cell models, the KCNQ1-G229D mutation results in slow/abnormal sinus rhythm, and we identify that a stronger L-type calcium current enables the SN to be more robust to the mutation. In conclusion, our computational modeling experiments provide novel mechanistic explanations for the observed borderline QT prolongation, and predict that KCNQ1-G229D could underlie SN dysfunction and conduction delays. The mechanisms revealed in the study can potentially inform management and treatment of KCNQ1 gain-of-function mutation carriers.

Cardiomyocytes from human pluripotent stem cells (hPSCs-CMs) could revolutionise biomedicine. Global burden of heart failure will soon reach USD $90bn, while unexpected cardiotoxicity underlies 28% of drug withdrawals. Advances in hPSC isolation, Cas9/CRISPR genome engineering and hPSC-CM differentiation have improved patient care, progressed drugs to clinic and opened a new era in safety pharmacology. Nevertheless, predictive cardiotoxicity using hPSC-CMs contrasts from failure to almost total success. Since this likely relates to cell immaturity, efforts are underway to use biochemical and biophysical cues to improve many of the ~30 structural and functional properties of hPSC-CMs towards those seen in adult CMs. Other developments needed for widespread hPSC-CM utility include subtype specification, cost reduction of large scale differentiation and elimination of the phenotyping bottleneck. This review will consider these factors in the evolution of hPSC-CM technologies, as well as their integration into high content industrial platforms that assess structure, mitochondrial function, electrophysiology, calcium transients and contractility. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Automated planar patch clamp systems are widely used in drug evaluation studies because of their ability to provide accurate, reliable, and reproducible data in a high-throughput manner. Typically, CHO and HEK tumorigenic cell lines overexpressing single ion channels are used since they can be harvested as high-density, homogenous, single-cell suspensions. While human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are physiologically more relevant, these cells are fragile, have complex culture requirements, are inherently heterogeneous, and are expensive to produce, which has restricted their use on automated patch clamp (APC) devices. Here, we used high efficiency differentiation protocols to produce cardiomyocytes from six different hPSC lines for analysis on the Patchliner (Nanion Technologies GmbH) APC platform. We developed a two-step cell preparation protocol that yielded cell catch rates and whole-cell breakthroughs of ∼80%, with ∼40% of these cells allowing electrical activity to be recorded. The protocol permitted formation of long-lasting (>15 min), high quality seals (>2 GΩ) in both voltage- and current-clamp modes. This enabled density of sodium, calcium, and potassium currents to be evaluated, along with dose-response curves to their respective channel inhibitors, tetrodotoxin, nifedipine, and E-4031. Thus, we show the feasibility of using the Patchliner platform for automated evaluation of the electrophysiology and pharmacology of hPSC-CMs, which will enable considerable increase in throughput for reliable and efficient drug evaluation.

Cardiomyocytes from human stem cells have applications in regenerative medicine and can provide models for heart disease and toxicity screening. Soluble components of the culture system such as growth factors within serum and insoluble components such as the substrate on which cells adhere to are important variables controlling the biological activity of cells. Using a combinatorial materials approach we develop a synthetic, chemically defined cellular niche for the support of functional cardiomyocytes derived from human embryonic stem cells (hESC-CMs) in a serum-free fully defined culture system. Almost 700 polymers were synthesized and evaluated for their utility as growth substrates. From this group, 20 polymers were identified that supported cardiomyocyte adhesion and spreading. The most promising 3 polymers were scaled up for extended culture of hESC-CMs for 15 days and were characterized using patch clamp electrophysiology and myofibril analysis to find that functional and structural phenotype was maintained on these synthetic substrates without the need for coating with extracellular matrix protein. In addition, we found that hESC-CMs cultured on a co-polymer of isobornyl methacrylate and tert-butylamino-ethyl methacrylate exhibited significantly longer sarcomeres relative to gelatin control. The potential utility of increased structural integrity was demonstrated in an in vitro toxicity assay that found an increase in detection sensitivity of myofibril disruption by the anti-cancer drug doxorubicin at a concentration of 0.05 μM in cardiomyocytes cultured on the co-polymer compared to 0.5 μM on gelatin. The chemical moieties identified in this large-scale screen provide chemically defined conditions for the culture and manipulation of hESC-CMs, as well as a framework for the rational design of superior biomaterials., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A scalable and cost-effective synthetic polymer substrate that supports robust expansion and subsequent multilineage differentiation of human pluripotent stem cells (hPSCs) with defined commercial media is presented. This substrate can be applied to common cultureware and used off-the-shelf after long-term storage. Expansion and differentiation of hPSCs are performed entirely on the polymeric surface, enabling the clinical potential of hPSC-derived cells to be realized., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost., (Copyright © 2013 WILEY Periodicals, Inc.)

Dinickel ditin zinc, Ni(2)Sn(2)Zn, crystallizes in the cubic space group Pm3m, with a lattice parameter of a = 8.845 (1) Å and with all atoms occupying special positions. The crystal structure exhibits pronounced similarities with that of the quaternary compound Ni(5.20)Sn(8.7)Zn(4.16)Cu(1.04). It shares structural features with other compounds in the Ni-Sn-Zn system, such as Ni(5)Sn(4)Zn and Ni(3)Sn(2).

In this work three complete isothermal sections of the Ni-Sn-Zn system at 700, 800 and 900 °C are presented. They were constructed based on experimental investigation of more than 60 alloy samples. Powder XRD, single crystal XRD, EPMA, and DTA measurements on selected samples were carried out. Two new ternary compounds, designated as τ2 (Ni 5 Sn 4 Zn) and τ3 (Ni 7 Sn 9 Zn 5 ), were identified and their homogeneity ranges and crystal structures could be described. Whereas τ3 is only present at 700 °C, the τ2-phase was found at both 700 and 800 °C. No truly ternary compound could be found in the isothermal section at 900 °C. A seemingly ternary compound at 20 at% Sn in the Ni-rich part of Ni-Sn-Zn was found at 800 and 900 °C. Our XRD results, however, indicate that this phase is a ternary solid solution of Ni 3 Sn-HT from constituent binary Ni-Sn. It is stabilized to lower temperatures by additions of Zn. These new experimental results will provide valuable information to the thermodynamic description of alloy systems relevant for high-temperature lead-free soldering.

The limited ability of the heart to regenerate has prompted development of new systems to produce cardiomyocytes for therapeutics. While differentiation of human embryonic stem cells (hESCs) into cardiomyocytes has been well documented, the process remains inefficient and/or expensive, and progress would be facilitated by better understanding the early genetic events that cause cardiac specification. By maintaining a transgenic cardiac-specific MYH6-monomeric red fluorescent protein (mRFP) reporter hESC line in conditions that promote pluripotency, we tested the ability of combinations of 15 genes to induce cardiac specification. Screening identified GATA4 plus TBX5 as the minimum requirement to activate the cardiac gene regulatory network and produce mRFP(+) cells, while a combination of GATA4, TBX5, NKX2.5, and BAF60c (GTNB) was necessary to generate beating cardiomyocytes positive for cTnI and α-actinin. Including the chemotherapeutic agent, Ara-C, from day 10 of induced differentiation enriched for cTnI/α-actinin double positive cells to 45%. Transient expression of GTNB for 5-7 days was necessary to activate the cardiogenesis through progenitor intermediates in a manner consistent with normal heart development. This system provides a route to test the effect of different factors on human cardiac differentiation and will be useful in understanding the network failures that underlie disease phenotypes.

Aims: Congenital long QT syndromes (LQTSs) are associated with prolonged ventricular repolarization and sudden cardiac death. Limitations to existing clinical therapeutic management strategies prompted us to develop a novel human in vitro drug-evaluation system for LQTS type 2 (LQT2) that will complement the existing in vitro and in vivo models., Methods and Results: Skin fibroblasts from a patient with a KCNH2 G1681A mutation (encodes I(Kr) potassium ion channel) were reprogrammed to human induced pluripotent stem cells (hiPSCs), which were subsequently differentiated to functional cardiomyocytes. Relative to controls (including the patient's mother), multi-electrode array and patch-clamp electrophysiology of LQT2-hiPSC cardiomyocytes showed prolonged field/action potential duration. When LQT2-hiPSC cardiomyocytes were exposed to E4031 (an I(Kr) blocker), arrhythmias developed and these presented as early after depolarizations (EADs) in the action potentials. In contrast to control cardiomyocytes, LQT2-hiPSC cardiomyocytes also developed EADs when challenged with the clinically used stressor, isoprenaline. This effect was reversed by β-blockers, propranolol, and nadolol, the latter being used for the patient's therapy. Treatment of cardiomyocytes with experimental potassium channel enhancers, nicorandil and PD118057, caused action potential shortening and in some cases could abolish EADs. Notably, combined treatment with isoprenaline (enhancers/isoprenaline) caused EADs, but this effect was reversed by nadolol., Conclusions: Findings from this paper demonstrate that patient LQT2-hiPSC cardiomyocytes respond appropriately to clinically relevant pharmacology and will be a valuable human in vitro model for testing experimental drug combinations.

Functional cardiomyocytes can now be derived routinely from hPSCs (human pluripotent stem cells), which collectively include embryonic and induced pluripotent stem cells. This technology presents new opportunities to develop pharmacologically relevant in vitro screens to detect cardiotoxicity, with a view to improving patient safety while reducing the economic burden to industry arising from high drug attrition rates. In the present article, we consider the need for human cardiomyocytes in drug-screening campaigns and review the strategies used to differentiate hPSCs towards the cardiac lineage. During early stages of differentiation, hPSC-cardiomyocytes display gene expression profiles, ultra-structures, ion channel functionality and pharmacological responses reminiscent of an embryonic phenotype, but maturation during extended time in culture has been demonstrated convincingly. Notably, hPSC-cardiomyocytes have been shown to respond in a highly predictable manner to over 40 compounds that have a known pharmacological effect on the human heart. This suggests that further development and validation of the hPSC-cardiomyocyte model as a tool for assessing cardiotoxicity is warranted.

The patho-physiologic process of restenosis and tissue growth may not be completely eliminated and is the primary concern of clinicians performing angioplasty and stent implantation procedures. Recent evidence suggests that the restenosis process is influenced by several factors: (1) geometry and size of vessel; (2) stent design; and (3) it's location that alter hemodynamic parameters, including local wall shear stress (WSS) distributions. The present three-dimensional (3D) analysis of pulsatile flow in a deployed coronary stent: (1) shows complex 3D variation of hemodynamic parameters; and (2) quantifies the changes in local WSS distributions for developed flow and compares with recently published WSS data for developing flow. Higher order of magnitude of WSS of 290 dyn/cm(2) is observed on the surface of cross-link intersections at the entrance of the stent for developed flow, which is about half of that for developing flow. Low WSS of 0.8 dyn/cm(2) and negative WSS of -8 dyn/cm(2) are seen at the immediate upstream and downstream regions of strut intersections. Persistent recirculation is observed at the downstream region of each strut cross-link and the regions of low and negative WSS may lead to patho-physiologic conditions near the stented region. The key finding of this study is that the location of stent in the coronary artery determines the developing or developed nature of the flow, which in turn, results in varied level of WSS.

A major consequence of stent implantation is restenosis that occurs due to neointimal formation. This patho-physiologic process of tissue growth may not be completely eliminated. Recent evidence suggests that there are several factors such as geometry and size of vessel, and stent design that alter hemodynamic parameters, including local wall shear stress distributions, all of which influence the restenosis process. The present three-dimensional analysis of developing pulsatile flow in a deployed coronary stent quantifies hemodynamic parameters and illustrates the changes in local wall shear stress distributions and their impact on restenosis. The present model evaluates the effect of entrance flow, where the stent is placed at the entrance region of a branched coronary artery. Stent geometry showed a complex three-dimensional variation of wall shear stress distributions within the stented region. Higher order of magnitude of wall shear stress of 530 dyn/cm2 is observed on the surface of cross-link intersections at the entrance of the stent. A low positive wall shear stress of 10 dyn/cm2 and a negative wall shear stress of -10 dyn/cm2 are seen at the immediate upstream and downstream regions of strut intersections, respectively. Modified oscillatory shear index is calculated which showed persistent recirculation at the downstream region of each strut intersection. The portions of the vessel where there is low and negative wall shear stress may represent locations of thrombus formation and platelet accumulation. The present results indicate that the immediate downstream regions of strut intersections are areas highly susceptible to restenosis, whereas a high shear stress at the strut intersection may cause platelet activation and free emboli formation.