Baseline azithromycin resistance in the gut microbiota of preterm born infants.

BACKGROUND: Macrolides, including azithromycin, are increasingly used in preterm-born infants to treat Ureaplasma infections. The baseline carriage of macrolide resistance genes in the preterm stool microbiota is unknown. OBJECTIVES: Identify carriage of azithromycin resistant bacteria and the incidence of macrolide resistant genes. METHODS: Azithromycin resistant bacteria were isolated from serial stool samples obtained from preterm infants (≤32 weeks' gestation) by culturing aerobically/anaerobically, in the presence/absence of azithromycin. Using quantitative PCR, we targeted 6 common macrolide resistance genes (erm(A), erm(B), erm(C), erm(F), mef(A/E), msr(A)) in DNA extracted from selected bacteria resistant to azithromycin. RESULTS: From 89 stool samples from 37 preterm-born infants, 93.3% showed bacterial growth in aerobic or anaerobic conditions. From the 280 azithromycin resistant isolates that were identified, Staphylococcus (75%) and Enterococcus (15%) species dominated. Macrolide resistance genes were identified in 91% of resistant isolates: commonest were erm(C) (46% of isolates) and msr(A) (40%). Multiple macrolide resistance genes were identified in 18% of isolates. CONCLUSION: Macrolide resistance is common in the gut microbiota of preterm-born infants early in life, most likely acquired from exposure to the maternal microbiota. It will be important to assess modulation of macrolide resistance, if macrolide treatment becomes routine in the management of preterm infants. IMPACT STATEMENT: Azithromycin resistance is present in the stool microbiota in the first month of life in preterm infants 91% of azithromycin resistant bacteria carried at least one of 6 common macrolide resistant genes Increasing use of macrolides in the preterm population makes this an important area of study.

Prognostic value of electrocardiographic time intervals and QT rate dependence in hypertrophic cardiomyopathy.

INTRODUCTION: Preventing sudden cardiac death (SCD) is one of the main goals in hypertrophic cardiomyopathy (HCM). Many variables have been proposed, however the European and American guidelines do not incorporate any ECG or Holter monitoring derived variables other than the presence of ventricular arrhythmia in their risk stratification models. In the present study we evaluated electrocardiographic parameters in risk stratification of HCM. METHODS AND RESULTS: Novel electrocardiographic parameters including the index of cardio-electrophysiological balance (iCEB), individualized QT correction (QTi) and QT rate dependence were evaluated along with established risk factors. A composite endpoint of SCD was defined as out of hospital cardiac arrest, appropriate ICD shock and sustained ventricular tachycardia. Cox regression analysis was used to evaluate predictors of SCD. Out of the 466 HCM patients, 31 reached the composite endpoint during a follow up of 75 ±â€¯86 months. In a multivariate model, nor iCEB, QTi or QT rate dependence were predictors of SCD. Only male gender (p < 0.01; OR 13.1; CI 1.74-98.83), negative T waves in the inferior leads (p = 0.04; OR 2.51; CI 1.03-6.13) and familial sudden death (p < 0.01; OR 3.03; CI 1.39-6.59) were significant predictors. On top of either the ESC risk score or the 3 traditional 'American risk factors', only male gender was a significant predictor of SCD. CONCLUSION: No ECG or Holter monitoring parameters added in risk stratification for SCD in HCM. However, male gender and negative T waves in the inferior leads are promising novel markers to evaluate in larger cohorts.

The respiratory consequences of early-term birth and delivery by caesarean sections.

In England and Wales, 19% of live births in 2012 were at 37-38 weeks' gestation, equating to nearly 140 000 early-term births each year. Since caesarean sections (CS) are often performed at early-term gestations, this accounts for some of the increased proportion of the early-term births. Infants born early-term are at an increased risk of neonatal respiratory morbidity particularly if they are delivered by caesarean section. The long term lung function data are limited but available data suggest that early-term delivery is associated with respiratory morbidity in childhood. CS also appears to be associated with increased neonatal morbidity and future development of respiratory symptoms. However, future studies need to confirm the independent effects of caesarean sections and early-term deliveries particularly for long term outcomes as both are likely to affect the respiratory system differently.

Evaluation of Index of Cardio-Electrophysiological Balance (iCEB) as a New Biomarker for the Identification of Patients at Increased Arrhythmic Risk.

BACKGROUND: Recently a new risk marker for drug-induced arrhythmias called index of cardio-electrophysiological balance (iCEB), measured as QT interval divided by QRS duration, was evaluated in an animal model. It was hypothesized that iCEB is equivalent to the cardiac wavelength λ (λ = effective refractory period (ERP) x conduction velocity) and that an increased or decreased value of iCEB would potentially predict an increased susceptibility to TdP or non-TdP mediated VT/VF, respectively. METHODS: First, the correlation between QT interval and ERP was evaluated by invasively measuring ERP during a ventricular stimulation protocol in humans (N = 40). Then the effect of administration of sotalol and flecainide on iCEB was measured in 40 patients with supraventricular tachycardias. Finally iCEB was assessed in carriers of a long QT syndrome (LQTS, N = 70) or Brugada syndrome (BrS, N = 57) mutation and compared them with genotype negative family members (N = 65). RESULTS: The correlation between QT interval and ERP was established (Pearson R(2) = 0.25) which suggests that iCEB≈ERPxCV≈QT/QRS. Sotalol administration increased iCEB (+ 0.23; P = 0.01), while it decreased with the administration of flecainide (-0.21, P = 0.03). In the LQTS group iCEB was increased (5.22 ± 0.93, P < 0.0001) compared to genotype negative family members (4.24 ± 0.5), while it was decreased in the BrS group (3.52 ± 0.43, P < 0.0001). CONCLUSIONS: Our data suggest that iCEB (QT/QRS) is a simple but effective ECG surrogate of cardiac wavelength. iCEB is increased in situations that predispose to TdP and is decreased in situations that predispose to non-TdP mediated VT/VF. Therefore, iCEB might serve as a noninvasive and readily measurable marker to detect increased arrhythmic risk.

Experimentally calibrated population of models predicts and explains intersubject variability in cardiac cellular electrophysiology.

Cellular and ionic causes of variability in the electrophysiological activity of hearts from individuals of the same species are unknown. However, improved understanding of this variability is key to enable prediction of the response of specific hearts to disease and therapies. Limitations of current mathematical modeling and experimental techniques hamper our ability to provide insight into variability. Here, we describe a methodology to unravel the ionic determinants of intersubject variability exhibited in experimental recordings, based on the construction and calibration of populations of models. We illustrate the methodology through its application to rabbit Purkinje preparations, because of their importance in arrhythmias and safety pharmacology assessment. We consider a set of equations describing the biophysical processes underlying rabbit Purkinje electrophysiology, and we construct a population of over 10,000 models by randomly assigning specific parameter values corresponding to ionic current conductances and kinetics. We calibrate the model population by closely comparing simulation output and experimental recordings at three pacing frequencies. We show that 213 of the 10,000 candidate models are fully consistent with the experimental dataset. Ionic properties in the 213 models cover a wide range of values, including differences up to ±100% in several conductances. Partial correlation analysis shows that particular combinations of ionic properties determine the precise shape, amplitude, and rate dependence of specific action potentials. Finally, we demonstrate that the population of models calibrated using data obtained under physiological conditions quantitatively predicts the action potential duration prolongation caused by exposure to four concentrations of the potassium channel blocker dofetilide.

The Role of the Airway and Gut Microbiome in the Development of Chronic Lung Disease of Prematurity.

Chronic lung disease (CLD) of prematurity, a common cause of morbidity and mortality in preterm-born infants, has a multifactorial aetiology. This review summarizes the current evidence for the effect of the gut and airway microbiota on the development of CLD, highlighting the differences in the early colonisation patterns in preterm-born infants compared to term-born infants. Stool samples from preterm-born infants who develop CLD have less diversity than those who do not develop CLD. Pulmonary inflammation, which is a hallmark in the development of CLD, may potentially be influenced by gut bacteria. The respiratory microbiota is less abundant than the stool microbiota in preterm-born infants. There is a lack of clear evidence for the role of the respiratory microbiota in the development of CLD, with results from individual studies not replicated. A common finding is the presence of a single predominant bacterial genus in the lungs of preterm-born infants who develop CLD. Probiotic preparations have been proposed as a potential therapeutic strategy to modify the gut or lung microbiota with the aim of reducing rates of CLD but additional robust evidence is required before this treatment is introduced into routine clinical practice.

Development of a new hazard scoring system in primary neuronal cell cultures for drug-induced acute neuronal toxicity identification in early drug discovery.

We investigated drug-induced acute neuronal electrophysiological changes using Micro-Electrode arrays (MEA) to rat primary neuronal cell cultures. Data based on 6-key MEA parameters were analyzed for plate-to-plate vehicle variability, effects of positive and negative controls, as well as data from over 100 reference drugs, mostly known to have pharmacological phenotypic and clinical outcomes. A Least Absolute Shrinkage and Selection Operator (LASSO) regression, coupled with expert evaluation helped to identify the 6-key parameters from many other MEA parameters to evaluate the drug-induced acute neuronal changes. Calculating the statistical tolerance intervals for negative-positive control effects on those 4-key parameters helped us to develop a new weighted hazard scoring system on drug-induced potential central nervous system (CNS) adverse effects (AEs). The weighted total score, integrating the effects of a drug candidate on the identified six-pivotal parameters, simply determines if the testing compound/concentration induces potential CNS AEs. Hereto, it uses four different categories of hazard scores: non-neuroactive, neuroactive, hazard, or high hazard categories. This new scoring system was successfully applied to differentiate the new compounds with or without CNS AEs, and the results were correlated with the outcome of in vivo studies in mice for one internal program. Furthermore, the Random Forest classification method was used to obtain the probability that the effect of a compound is either inhibitory or excitatory. In conclusion, this new neuronal scoring system on the cell assay is actively applied in the early de-risking of drug development and reduces the use of animals and associated costs.

The Potential Mechanisms behind Loperamide-Induced Cardiac Arrhythmias Associated with Human Abuse and Extreme Overdose.

Loperamide has been a safe and effective treatment for diarrhea for many years. However, many cases of cardiotoxicity with intentional abuse of loperamide ingestion have recently been reported. We evaluated loperamide in in vitro and in vivo cardiac safety models to understand the mechanisms for this cardiotoxicity. Loperamide slowed conduction (QRS-duration) starting at 0.3 µM [~1200-fold (×) its human Free Therapeutic Plasma Concentration; FTPC] and reduced the QT-interval and caused cardiac arrhythmias starting at 3 µM (~12,000× FTPC) in an isolated rabbit ventricular-wedge model. Loperamide also slowed conduction and elicited Type II/III A-V block in anesthetized guinea pigs at overdose exposures of 879× and 3802× FTPC. In ion-channel studies, loperamide inhibited hERG (IKr), INa, and ICa currents with IC50 values of 0.390 µM, 0.526 µM, and 4.091 µM, respectively (i.e., >1560× FTPC). Additionally, in silico trials in human ventricular action potential models based on these IC50s confirmed that loperamide has large safety margins at therapeutic exposures (≤600× FTPC) and confirmed repolarization abnormalities in the case of extreme doses of loperamide. The studies confirmed the large safety margin for the therapeutic use of loperamide but revealed that at the extreme exposure levels observed in human overdose, loperamide can cause a combination of conduction slowing and alterations in repolarization time, resulting in cardiac proarrhythmia. Loperamide's inhibition of the INa channel and hERG-mediated IKr are the most likely basis for this cardiac electrophysiological toxicity at overdose exposures. The cardiac toxic effects of loperamide at the overdoses could be aggravated by co-medication with other drug(s) causing ion channel inhibition.

Does Enhanced Structural Maturity of hiPSC-Cardiomyocytes Better for the Detection of Drug-Induced Cardiotoxicity?

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are currently used following the Comprehensive in vitro Proarrhythmic Assay (CiPA) initiative and subsequent recommendations in the International Council for Harmonization (ICH) guidelines S7B and E14 Q&A, to detect drug-induced cardiotoxicity. Monocultures of hiPSC-CMs are immature compared to adult ventricular cardiomyocytes and might lack the native heterogeneous nature. We investigated whether hiPSC-CMs, treated to enhance structural maturity, are superior in detecting drug-induced changes in electrophysiology and contraction. This was achieved by comparing hiPSC-CMs cultured in 2D monolayers on the current standard (fibronectin matrix, FM), to monolayers on a coating known to promote structural maturity (CELLvo™ Matrix Plus, MM). Functional assessment of electrophysiology and contractility was made using a high-throughput screening approach involving the use of both voltage-sensitive fluorescent dyes for electrophysiology and video technology for contractility. Using 11 reference drugs, the response of the monolayer of hiPSC-CMs was comparable in the two experimental settings (FM and MM). The data showed no functionally relevant differences in electrophysiology between hiPSC-CMs in standard FM and MM, while contractility read-outs indicated an altered amplitude of contraction but not changes in time course. RNA profiling for cardiac proteins shows similarity of the RNA expression across the two forms of 2D culture, suggesting that cell-to-matrix adhesion differences may explain account for differences in contraction amplitude. The results support the view that hiPSC-CMs in both 2D monolayer FM and MM that promote structural maturity are equally effective in detecting drug-induced electrophysiological effects in functional safety studies.

Storage on Maternal Plants Affects Temperature Requirements during Germination in Rumex obtusifolius.

Aerial seed banks facilitate population persistence by extending the temporal range of seed dispersal. Knowing the temporal range of germination will improve our understanding of the relationship between seed germination dynamics and aerial seed bank storage duration. We tested the effects of temperature (12/12 h of 5/10, 10/20, 20/30 and 25/35 °C) and light variation (12 h light/12 h darkness and 24 h darkness per day) on germination of Rumex obtusifolius L. seeds retained in an aerial seed bank for 0, 2, 4, 6, 8 and 10 months. Freshly harvested R. obtusifolius were non-dormant and exhibited germination rates of up to 92%. Overall, seeds of R. obtusifolius germinated reliably at all but the lowest temperature (5/10 °C). Seeds maintained high viability throughout the collection period, indicating that fluctuating weather conditions had little influence on seed germination. Thus, the species can maintain viable seeds in aerial storage for up to 10 months and contribute viable seeds to the soil seed bank year-round. This ability to maintain a renewed soil seed bank contributes to the species' strong resilience in colonizing disturbed areas and makes it a difficult weed to control.

High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures.

Drug-induced seizure liability is a significant safety issue and the basis for attrition in drug development. Occurrence in late development results in increased costs, human risk, and delayed market availability of novel therapeutics. Therefore, there is an urgent need for biologically relevant, in vitro high-throughput screening assays (HTS) to predict potential risks for drug-induced seizure early in drug discovery. We investigated drug-induced changes in neural Ca2+ oscillations, using fluorescent dyes as a potential indicator of seizure risk, in hiPSC-derived neurons co-cultured with human primary astrocytes in both 2D and 3D forms. The dynamics of synchronized neuronal calcium oscillations were measured with an FDSS kinetics reader. Drug responses in synchronized Ca2+ oscillations were recorded in both 2D and 3D hiPSC-derived neuron/primary astrocyte co-cultures using positive controls (4-aminopyridine and kainic acid) and negative control (acetaminophen). Subsequently, blinded tests were carried out for 25 drugs with known clinical seizure incidence. Positive predictive value (accuracy) based on significant changes in the peak number of Ca2+ oscillations among 25 reference drugs was 91% in 2D vs. 45% in 3D hiPSC-neuron/primary astrocyte co-cultures. These data suggest that drugs that alter neuronal activity and may have potential risk for seizures can be identified with high accuracy using an HTS approach using the measurements of Ca2+ oscillations in hiPSC-derived neurons co-cultured with primary astrocytes in 2D.

Water Stress Inhibits Germination While Maintaining Embryo Viability of Subtropical Wetland Seeds: A Functional Approach With Phylogenetic Contrasts.

Wetland species commonly exhibit a range of strategies to cope with water stress, either through drought tolerance or through avoidance of the period of limited water availability. Natural populations provide a genetic resource for ecological remediation and may also have direct economic value. We investigated the effects of drought stress on the seed germination of wetland species. Nineteen species were germinated in four concentrations of polyethylene glycol 6000 (PEG) and were evaluated daily (12-h light photoperiod) or after 35 days (continuous darkness) to determine seed germination under water stress. Germination percentage decreased with an increase in polyethylene glycol 6000 (PEG) concentration, but species' germination response to PEG concentration varied significantly. Seeds recovered their germinability after the alleviation of water stress, but the extent of recovery was species-dependent.

Is the forming of neuronal network activity in human-induced pluripotent stem cells important for the detection of drug-induced seizure risks?

BACKGROUND: Functional network activity is a characteristic for neuronal cells, and the complexity of the network activity represents the necessary substrate to support complex brain functions. Drugs that drastically increase the neuronal network activity may have a potential higher risk for seizures in human. Although there has been some recent considerable progress made using cultures from different types of human-induced pluripotent stem cell (hiPSC) derived neurons, one of the primary limitations is the lack of - or very low - network activity. METHOD: In the present study, we investigated whether the limited neuronal network activity in commercial hiPSC-neurons (CNS.4U®) is capable of detecting drug-induced potential seizure risks. Therefore, we compared the hiPSC-results to those in rat primary neurons with known high neuronal network activity in vitro. RESULTS: Gene expression and electrical activity from in vitro developing neuronal networks were assessed at multiple time-points. Transcriptomes of 7, 28, and 50 days in vitro were analyzed and compared to those from human brain tissues. Data from measurements of electrical activity using multielectrode arrays (MEAs) indicate that neuronal networks matured gradually over time, albeit in hiPSC this developed slower than rat primary cultures. The response of neuronal networks to neuronal active reference drugs modulating glutamatergic, acetylcholinergic and GABAergic pathways could be detected in both hiPSC-neurons and rat primary neurons. However, in comparison, GABAergic responses were limited in hiPSC-neurons. CONCLUSION: Overall, despite a slower network development and lower network activity, CNS.4U® hiPSC-neurons can be used to detect drug induced changes in neuronal network activity, as shown by well-known seizurogenic drugs (affecting e.g., the Glycine receptor and Na+ channel). However, lower sensitivity to GABA antagonists has been observed.

Identifying Acute Cardiac Hazard in Early Drug Discovery Using a Calcium Transient High-Throughput Assay in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Introduction: Early identification of cardiac risk is essential for reducing late-stage attrition in drug development. We adapted the previously published cardiac hazard risk-scoring system using a calcium transient assay in human stem cell-derived CMs for the identification of cardiac risks recorded from the new hiPSC-CM line and investigated its predictivity and translational value based on the screening of a large number of reference and proprietary compounds. Methods: Evaluation of 55 reference drugs provided the translation of various pharmacological effects into a single hazard label (no, low, high, or very high hazard) using a Ca2+-sensitive fluorescent dye assay recorded by -by FDSS/µCell Functional Drug Screening System (Hamamatsu on hiPSC-CM line (FCDI iCell Cardiomyocytes2). Results: Application of the adapted hazard scoring system in the Ca2+ transient assay, using a second hiPS-CM line, provided comparable scoring results and predictivity of hazard, to the previously published scoring approach, with different pharmacological drug classes, as well as screening new chemical entities (NCE's) using a single hazard label from four different scoring levels (no, low, high, or very high hazard). The scoring system results also showed minimal variability across three different lots of hiPSC-CMs, indicating good reproducibility of the cell line. The predictivity values (sensitivity and specificity) for drug-induced acute cardiac risk for QT-interval prolongation and Torsade de pointes (TdPs) were >95% and statistical modeling confirmed the prediction of proarrhythmic risk. The outcomes of the NCEs also showed consistency with findings in other well-established in vitro and in vivo cardiac risk assays. Conclusion: Evaluation of a large list of reference compounds and internal NCEs has confirmed the applicability of the adaptations made to the previously published novel scoring system for the hiPSC-CMs. The validation also established the predictivity for drug-induced cardiac risks with good translation to other established preclinical in vitro and in vivo assays, confirming the application of this novel scoring system in different stem cell-CM lines for early cardiac hazard identification.

Leaf plasticity across wet and dry seasons in Croton blanchetianus (Euphorbiaceae) at a tropical dry forest.

Plant species of the Brazilian Caatinga experience seasonal wet and dry extremes, requiring seasonally different leaf characteristics for optimizing water availability. We investigated if Croton blanchetianus Baill exhibits leaf morphoanatomical traits across seasons and positioning in sunlight/natural shade. Leaves of ten 1-3 m tall plants in full sunlight and ten in natural shade were assessed in May, July (wet season), October and December (dry season) 2015 for gas exchange, leaf size, lamina and midrib cross sections (14 parameters), and chloroplast structure (5 parameters). Net photosynthesis was greater during the wet season (21.6 µm-2 s-1) compared to the dry season (5.8 µm-2 s-1) and was strongly correlated with almost all measured parameters (p < 0.01). Shaded leaves in the wet season had higher specific leaf area (19.9 m2 kg-1 in full-sun and 23.1 m2 kg-1 in shade), but in the dry season they did not differ from those in full sun (7.5 m2 kg-1 and 7.2 m2 kg-1). In the wet season, the expansion of the adaxial epidermis and mesophyll lead to larger and thicker photosynthetic area of leaves. Furthermore, chloroplast thickness, length and area were also significantly larger in full sunlight (2.1 µm, 5.1 µm, 15.2 µm2; respectively) and shaded plants (2.0 µm, 5.2 µm, 14.8 µm2; respectively) during wetter months. Croton blanchetianus exhibits seasonal plasticity in leaf structure, presumably to optimize water use efficiency during seasons of water abundance and deficit. These results suggest that the species is adaptable to the increased drought stress projected by climate change scenarios.

Seizure-induced Torsades de pointes:In a canine drug-induced long-QT1 model.

INTRODUCTION: People with epilepsy are at heightened risk of sudden death compared to the general population. The leading cause of epilepsy-related premature mortality is a sudden unexpected death in epilepsy (SUDEP). The mechanism of SUDEP remains largely unresolved and the lack of preclinical models to study the potential mechanism underlying SUDEP is a problem. METHOD: By combining electroencephalographic (EEG) and electrocardiogram (ECG) measurements within a well described LQT1 dog model, we investigated the effect of the proconvulsive compound pentylenetetrazol (PTZ), and its link to the induction of Torsades de Pointes (TdP). RESULTS: Pre-treatment with the potent and selective IKs blocker JNJ 282 induced a pronounced QT (QTc) prolongation in anaesthetized dogs (Long QT syndrome type 1 or LQT1 group) compared to dogs that were not treated (control group). Subsequent PTZ administration induced spiking on the EEG signal and seizures in both groups, but only R-on-T, salvo and TdP were observed in dogs of the LQT1 group. CONCLUSION: Our results show that a proconvulsive drug can trigger TdP-like cardiac arrhythmias, in conditions of compromised repolarization in the heart (Iks blockade). In man, TdP arrythmia's can often lead to ventricular fibrillation (VF) and sudden death. This observation suggests that long QT-intervals (genetic or drug induced) could potentially be one of the risk factors for SUDEP in epileptic patients.

Comparison of the Simulated Response of Three in Silico Human Stem Cell-Derived Cardiomyocytes Models and in Vitro Data Under 15 Drug Actions.

Objectives: Improvements in human stem cell-derived cardiomyocyte (hSC-CM) technology have promoted their use for drug testing and disease investigations. Several in silico hSC-CM models have been proposed to augment interpretation of experimental findings through simulations. This work aims to assess the response of three hSC-CM in silico models (Koivumäki2018, Kernik2019, and Paci2020) to simulated drug action, and compare simulation results against in vitro data for 15 drugs. Methods: First, simulations were conducted considering 15 drugs, using a simple pore-block model and experimental data for seven ion channels. Similarities and differences were analyzed in the in silico responses of the three models to drugs, in terms of Ca2+ transient duration (CTD90) and occurrence of arrhythmic events. Then, the sensitivity of each model to different degrees of blockage of Na+ (INa), L-type Ca2+ (ICaL), and rapid delayed rectifying K+ (IKr) currents was quantified. Finally, we compared the drug-induced effects on CTD90 against the corresponding in vitro experiments. Results: The observed CTD90 changes were overall consistent among the in silico models, all three showing changes of smaller magnitudes compared to the ones measured in vitro. For example, sparfloxacin 10 µM induced +42% CTD90 prolongation in vitro, and +17% (Koivumäki2018), +6% (Kernik2019), and +9% (Paci2020) in silico. Different arrhythmic events were observed following drug application, mainly for drugs affecting IKr. Paci2020 and Kernik2019 showed only repolarization failure, while Koivumäki2018 also displayed early and delayed afterdepolarizations. The spontaneous activity was suppressed by Na+ blockers and by drugs with similar effects on ICaL and IKr in Koivumäki2018 and Paci2020, while only by strong ICaL blockers, e.g. nisoldipine, in Kernik2019. These results were confirmed by the sensitivity analysis. Conclusion: To conclude, The CTD90 changes observed in silico are qualitatively consistent with our in vitro data, although our simulations show differences in drug responses across the hSC-CM models, which could stem from variability in the experimental data used in their construction.

Utility of Normalized TdP Score System in Drug Proarrhythmic Potential Assessment: A Blinded in vitro Study of CiPA Drugs.

Drugs that prolong QT may cause torsade de pointes (TdP). However, translation of nonclinical assessment of QT prolongation or hERG channel, targeted by QT-prolonging drugs, into clinical TdP risk has been insufficient to date. In this blinded study, we confirmed the utility of a Normalized TdP Score System in predicting drug-induced TdP risks among 34 drugs, including 28 with low, intermediate, and high TdP risks under the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative plus six compounds with names blinded to the investigators, using the rabbit ventricular wedge assay. Concentration-dependent TdP scores were determined by drug-induced changes in QT, Tp-e , and proarrhythmias. Disclosure of the names and testing concentrations was made after completion of the experiments and report to the sponsors. Drugs' normalized TdP scores were calculated thereafter based on their respective free clinical maximum concentration (Cmax ). Drugs' normalized TdP scores were calculated and ranked for 33 drugs, excluding 1 investigational drug, and the TdP risks of the 28 CiPA drugs were correctly distinguished according to their respective categories of low, intermediate, and high TdP risks under the CiPA initiative. Accordingly, we are able to propose the cutoff values of the normalized TdP scores at 1 × Cmax : ≤ 0, > 0 to < 0.65 and ≥ 0.65, respectively, for low, intermediate, and high risk. This blinded study supports utility of our Normalized TdP Score System in predicting drug-induced TdP risks in 33 drugs, including 28 used for characterization of other assays under the CiPA initiative. However, these results need to be replicated in other laboratories.

The resting membrane potential of hSC-CM in a syncytium is more hyperpolarised than that of isolated cells.

Human-induced pluripotent stem cell (hiPSC) and stem cell (hSC) derived cardiomyocytes (CM) are gaining popularity as in vitro model for cardiology and pharmacology studies. A remaining flaw of these cells, as shown by single-cell electrophysiological characterization, is a more depolarized resting membrane potential (RMP) compared to native CM. Most reports attribute this to a lower expression of the Kir2.1 potassium channel that generates the IK1 current. However, most RMP recordings are obtained from isolated hSC/hiPSC-CMs whereas in a more native setting these cells are interconnected with neighboring cells by connexin-based gap junctions, forming a syncytium. Hereby, these cells are electrically connected and the total pool of IK1 increases. Therefore, the input resistance (Ri) of interconnected cells is lower than that of isolated cells. During patch clamp experiments pipettes need to be well attached or sealed to the cell, which is reflected in the seal resistance (Rs), because a nonspecific ionic current can leak through this pipette-cell contact or seal and balance out small currents within the cell such as IK1. By recording the action potential of isolated hSC-CMs and that of hSC-CMs cultured in small monolayers, we show that the RMP of hSC-CMs in monolayer is approximately -20 mV more hyperpolarized compared to isolated cells. Accordingly, adding carbenoxolone, a connexin channel blocker, isolates the cell that is patch clamped from its neighboring cells of the monolayer and depolarizes the RMP. The presented data show that the recorded RMP of hSC-CMs in a syncytium is more negative than that determined from isolated hSC/hiPSC-CMs, most likely because the active pool of Kir2.1 channels increased.