Pacsin 2 is required for the maintenance of a normal cardiac function in the developing mouse heart.

The Pacsin proteins (Pacsin 1, 2 and 3) play an important role in intracellular trafficking and thereby signal transduction in many cells types. This study was designed to examine the role of Pacsin 2 in cardiac development and function. We investigated the development and electrophysiological properties of Pacsin 2 knockout (P2KO) hearts and single cardiomyocytes isolated from 11.5 and 15.5days old fetal mice. Immunofluorescence experiments confirmed the lack of Pacsin 2 protein expression in P2KO cardiac myocytes in comparison to wildtype (WT). Western blotting demonstrates low expression levels of connexin 43 and T-box 3 proteins in P2KO compared to wildtype (WT). Electrophysiology measurements including online Multi-Electrode Array (MEA) based field potential (FP) recordings on isolated whole heart of P2KO mice showed a prolonged AV-conduction time. Patch clamp measurements of P2KO cardiomyocytes revealed differences in action potential (AP) parameters and decreased pacemaker funny channel (If), as well as L-type Ca2+ channel (ICaL), and sodium channel (INa). These findings demonstrate that Pacsin 2 is necessary for cardiac development and function in mouse embryos, which will enhance our knowledge to better understand the genesis of cardiovascular diseases.

Multiple nickel-sensitive targets elicit cardiac arrhythmia in isolated mouse hearts after pituitary adenylate cyclase-activating polypeptide-mediated chronotropy.

The pituitary adenylate cyclase-activating polypeptide (PACAP)-27 modulates various biological processes, from the cellular level to function specification. However, the cardiac actions of this neuropeptide are still under intense studies. Using control (+|+) and mice lacking (-|-) either R-type (Cav2.3) or T-type (Cav3.2) Ca2+ channels, we investigated the effects of PACAP-27 on cardiac activity of spontaneously beating isolated perfused hearts. Superfusion of PACAP-27 (20nM) caused a significant increase of baseline heart frequency in Cav2.3(+|+) (156.9±10.8 to 239.4±23.4 bpm; p<0.01) and Cav2.3(-|-) (190.3±26.4 to 270.5±25.8 bpm; p<0.05) hearts. For Cav3.2, the heart rate was significantly increased in Cav3.2(-|-) (133.1±8.5 bpm to 204.6±27.9 bpm; p<0.05) compared to Cav3.2(+|+) hearts (185.7±11.2 bpm to 209.3±22.7 bpm). While the P wave duration and QTc interval were significantly increased in Cav2.3(+|+) and Cav2.3(-|-) hearts following PACAP-27 superfusion, there was no effect in Cav3.2(+|+) and Cav3.2(-|-) hearts. The positive chronotropic effects observed in the four study groups, as well as the effect on P wave duration and QTc interval were abolished in the presence of Ni2+ (50µM) and PACAP-27 (20nM) in hearts from Cav2.3(+|+) and Cav2.3(-|-) mice. In addition to suppressing PACAP's response, Ni2+ also induced conduction disturbances in investigated hearts. In conclusion, the most Ni2+-sensitive Ca2+ channels (R- and T-type) may modulate the PACAP signaling cascade during cardiac excitation in isolated mouse hearts, albeit to a lesser extent than other Ni2+-sensitive targets.

Cardiac functions of voltage-gated Ca(2+) channels: role of the pharmacoresistant type (E-/R-Type) in cardiac modulation and putative implication in sudden unexpected death in epilepsy (SUDEP).

Voltage-gated Ca(2+) channels (VGCCs) are ubiquitous in excitable cells. These channels play key roles in many physiological events like cardiac regulation/pacemaker activity due to intracellular Ca(2+) transients. In the myocardium, the Cav1 subfamily (L-type: Cav1.2 and Cav1.3) is the main contributor to excitation-contraction coupling and/or pacemaking, whereas the Cav3 subfamily (T-type: Cav3.1 and Cav3.2) is important in rhythmically firing of the cardiac nodal cells. No established cardiac function has been attributed to the Cav2 family (E-/R-type: Cav2.3) despite accumulating evidence of cardiac dysregulation observed upon deletion of the Cav2.3 gene, the only member of this family so far detected in cardiomyocytes. In this review, we summarize the pathophysiological changes observed after ablation of the E-/R-type VGCC and propose a cardiac mechanism of action for this channel. Also, considering the role played by this channel in epilepsy and its reported sensitivity to antiepileptic drugs, a putative involvement of this channel in the cardiac mechanism of sudden unexpected death in epilepsy is also discussed.

Beat Rate Variability in Murine Embryonic Stem Cell-Derived Cardiomyocytes: Effect of Antiarrhythmic Drugs.

BACKGROUND/AIMS: Heart rate variability (HRV) refers to the fluctuation of the time interval between consecutive heartbeats in humans. It has recently been discovered that cardiomyocytes derived from human embryonic and induced pluripotent stem cells show beat rate variability (BRV) that is similar to the HRV in humans. In the present study, clinical aspects of HRV were transferred to an in vitro model. The aims of the study were to explore the BRV in murine embryonic stem cell (mESC)-derived cardiomyocytes and to demonstrate the influence of antiarrhythmic drugs on BRV as has been shown in clinical trials previously. METHODS: The Microelectrode Array (MEA) technique was used to perform short-term recordings of extracellular field potentials (FPs) of spontaneously beating cardiomyocytes derived from mESCs (D3 cell line, αPig-44). Offline analysis was focused on time domain and nonlinear methods. RESULTS: The Poincaré-Plot analysis of measurements without pharmacological intervention revealed that three different shapes of scatter plots occurred most frequently. Comparable shapes have been described in clinical studies before. The antiarrhythmic drugs Ivabradine, Verapamil and Sotalol augmented BRV, whereas Flecainide decreased BRV parameters at low concentrations (SDSD 79.0 ± 8.7% of control at 10(-9) M, p < 0.05) and increased variability measures at higher concentrations (SDNN 258.8 ± 42.7% of control at 10(-5) M, p < 0.05). Amiodarone and Metoprolol did not alter BRV significantly. CONCLUSIONS: Spontaneously beating cardiomyocytes derived from mESCs showed BRV that appears to be similar to the HRV known from humans. Antiarrhythmic drugs affected BRV parameters similar to clinical observations. Therefore, our study demonstrates that this in vitro model can contribute to a better understanding of electrophysiological properties of mESC-derived cardiomyocytes and might serve as a valuable tool for drug safety screening.

Identification of genomic biomarkers for anthracycline-induced cardiotoxicity in human iPSC-derived cardiomyocytes: an in vitro repeated exposure toxicity approach for safety assessment.

The currently available techniques for the safety evaluation of candidate drugs are usually cost-intensive and time-consuming and are often insufficient to predict human relevant cardiotoxicity. The purpose of this study was to develop an in vitro repeated exposure toxicity methodology allowing the identification of predictive genomics biomarkers of functional relevance for drug-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The hiPSC-CMs were incubated with 156 nM doxorubicin, which is a well-characterized cardiotoxicant, for 2 or 6 days followed by washout of the test compound and further incubation in compound-free culture medium until day 14 after the onset of exposure. An xCELLigence Real-Time Cell Analyser was used to monitor doxorubicin-induced cytotoxicity while also monitoring functional alterations of cardiomyocytes by counting of the beating frequency of cardiomyocytes. Unlike single exposure, repeated doxorubicin exposure resulted in long-term arrhythmic beating in hiPSC-CMs accompanied by significant cytotoxicity. Global gene expression changes were studied using microarrays and bioinformatics tools. Analysis of the transcriptomic data revealed early expression signatures of genes involved in formation of sarcomeric structures, regulation of ion homeostasis and induction of apoptosis. Eighty-four significantly deregulated genes related to cardiac functions, stress and apoptosis were validated using real-time PCR. The expression of the 84 genes was further studied by real-time PCR in hiPSC-CMs incubated with daunorubicin and mitoxantrone, further anthracycline family members that are also known to induce cardiotoxicity. A panel of 35 genes was deregulated by all three anthracycline family members and can therefore be expected to predict the cardiotoxicity of compounds acting by similar mechanisms as doxorubicin, daunorubicin or mitoxantrone. The identified gene panel can be applied in the safety assessment of novel drug candidates as well as available therapeutics to identify compounds that may cause cardiotoxicity.

"Watching the Detectives" report of the general assembly of the EU project DETECTIVE Brussels, 24-25 November 2015.

SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and "-omics" technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich "-omics" databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.

Effects of synthetic neural adhesion molecule mimetic peptides and related proteins on the cardiomyogenic differentiation of mouse embryonic stem cells.

BACKGROUND/AIMS: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. METHODS: In the present study, using a transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP), we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGL(L), hNgf_C2, EnkaminE, Plannexin and C3) on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. RESULTS: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF) peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. CONCLUSION: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to cardiac pathologies where BDNF levels are impaired.

Functional expression and regulation of hyperpolarization-activated cyclic nucleotide-gated channels (HCN) in mouse iPS cell-derived cardiomyocytes after UTF1 -neo selection.

BACKGROUND/AIMS: In vitro reprogramming of somatic cells holds great potential to serve as an autologous source of cells for tissue repair. However, major difficulties in achieving this potential include obtaining homogeneous and stable cells for transplantation. High electrical activity of cells such as cardiomyocytes (CMs) is crucial for both, safety and efficiency of cell replacement therapy. Moreover, the function of the cardiac pacemaker is controlled by the activities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we have examined changes in HCN gene expression and function during cardiomyogenesis. METHODS: We differentiated murine iPS cells selected by an undifferentiated transcription factor 1 (UTF1) -promoter-driven G418 resistance to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. RESULTS: As key cardiac markers alpha-actinin and cardiac troponin T could be identified in derived CMs. Immunocytochemical staining of CMs showed the presence of all HCN subunits (HCN1-4). Electrophysiology experiments revealed developmental changes of action potentials and If currents as well as functional hormonal regulation and sensitivity to If channel blockers. CONCLUSION: We conclude that iPS cells derived from UTF-selection give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed If current in a development-dependent manner; and have all phenotypes with the pacemaker as predominant subtype. This might be of great importance for transplantation purposes.

Effects of plant extracts and derivatives on cardiac K+, Nav, and Cav channels: a review.

Natural products (NPs) are endless sources of compounds for fighting against several pathologies. Many dysfunctions, including cardiovascular disorders, such as cardiac arrhythmias have their modes of action regulation of the concentration of electrolytes inside and outside the cell targeting ion channels. Here, we highlight plant extracts and secondary metabolites' effects on the treatment of related cardiac pathologies on hERG, Nav, and Cav of cardiomyocytes. The natural product's pharmacology of expressed receptors like alpha-adrenergic receptors causes an influx of Ca2+ ions through receptor-operated Ca2+ ion channels. We also examine the NPs associated with cardiac contractions such as myocardial contractility by reducing the L-type calcium current and decreasing the intracellular calcium transient, inhibiting the K+ induced contractions, decreasing amplitude of myocyte shortening and showed negative ionotropic and chronotropic effects due to decreasing cytosolic Ca2+. We examine whether the NPs block potassium channels, particular the hERG channel and regulatory effects on Nav1.7.

Pharmacoresistant Cav 2·3 (E-type/R-type) voltage-gated calcium channels influence heart rate dynamics and may contribute to cardiac impulse conduction.

Voltage-gated Ca(2+) channels regulate cardiac automaticity, rhythmicity and excitation-contraction coupling. Whereas L-type (Cav 1·2, Cav 1·3) and T-type (Cav 3·1, Cav 3·2) channels are widely accepted for their functional relevance in the heart, the role of Cav 2·3 Ca(2+) channels expressing R-type currents remains to be elucidated. We have investigated heart rate dynamics in control and Cav 2·3-deficient mice using implantable electrocardiogram radiotelemetry and pharmacological injection experiments. Autonomic block revealed that the intrinsic heart rate does not differ between both genotypes. Systemic administration of isoproterenol resulted in a significant reduction in interbeat interval in both genotypes. It remained unaffected after administering propranolol in Cav 2·3(-|-) mice. Heart rate from isolated hearts as well as atrioventricular conduction for both genotypes differed significantly. Additionally, we identified and analysed the developmental expression of two splice variants, i.e. Cav 2·3c and Cav 2·3e. Using patch clamp technology, R-type currents could be detected in isolated prenatal cardiomyocytes and be related to R-type Ca(2+) channels. Our results indicate that on the systemic level, the pharmacologically inducible heart rate range and heart rate reserve are impaired in Cav 2·3 (-|-) mice. In addition, experiments on Langendorff perfused hearts elucidate differences in basic properties between both genotypes. Thus, Cav 2·3 does not only contribute to the cardiac autonomous nervous system but also to intrinsic rhythm propagation.

In vitro effect of hydroxychloroquine on pluripotent stem cells and their cardiomyocytes derivatives.

Introduction: Hydroxychloroquine (HDQ) is an antimalarial drug that has also shown its effectiveness in autoimmune diseases. Despite having side effects such as retinopathy, neuromyopathy and controversial cardiac toxicity, HDQ has been presented and now intensively studied for the treatment and prevention of coronavirus disease 2019 (COVID-19). Recent works revealed both beneficial and toxic effects during HDQ treatment. The cardiotoxic profile of HDQ remains unclear and identifying risk factors is challenging. Methods: Here, we used well-established cell-cultured to study the cytotoxic effect of HDQ, mouse induced pluripotent stem cells (miPSC) and their cardiomyocytes (CMs) derivatives were exposed to different concentrations of HDQ. Cell colony morphology was assessed by microscopy whereas cell viability was measured by flow cytometry and impedance-based methods. The effect of HDQ on beating activity of mouse and human induced pluripotent stem cell-derived CMs (miPSC-CMs and hiPSC-CMs, respectively) and mouse embryonic stem cell-derived CMs (mESC-CMs) were captured by the xCELLigence RTCA and microelectrode array (MEA) systems. Results and discussion: Our results revealed that 20 µM of HDQ promotes proliferation of stem cells used suggesting that if appropriately monitored, HDQ may have a cardioprotective effect and may also represent a possible candidate for tissue repair. In addition, the field potential signals revealed that higher doses of this medication caused bradycardia that could be reversed with a higher concentration of ß-adrenergic agonist, Isoproterenol (Iso). On the contrary, HDQ caused an increase in the beating rate of hiPSC-CMs, which was further helped upon application of Isoproterenol (Iso) suggesting that HDQ and Iso may also work synergistically. These results indicate that HDQ is potentially toxic at high concentrations and can modulate the beating activity of cardiomyocytes. Moreover, HDQ could have a synergistic inotropic effect with isoproterenol on cardiac cells.

Effect of Ethanolic Extract of Vernonia amygdalina on the Proliferation, Viability and Function of Mouse Induced Pluripotent Stem Cells and Cardiomyocytes.

Vernonia amygdalina (V. amygdalina) leaves are commonly used in traditional medicine around the world for the treatment of a plethora disorders, including heart disease. The aim of this study was to examine and evaluate the cardiac effect of V. amygdalina leaf extracts using mouse induced pluripotent stem cells (miPSCs) and their cardiomyocytes' (CMs) derivatives. We used a well-established stem cell culture to assess the effect of V. amygdalina extract on miPSC proliferation, EB formation and the beating activity of miPS cell-derived CMs. To study the cytotoxic effect of our extract, undifferentiating miPSCs were exposed to different concentrations of V. amygdalina. Cell colony formation and EB morphology were assessed using microscopy, whereas the cell viability was accessed with an impedance-based method and immunocytochemistry following treatment with different concentrations of V. amygdalina. Ethanolic extract of V. amygdalina induced toxicity in miPSCs, as revealed by a decrease in cell proliferation and colony formation, and an increase in cell death at a concentration of ≥20 mg/mL. At a concentration of 10 mg/mL, the rate of beating EBs was observed with no significant difference regarding the yield of cardiac cells. In addition, V. amygdalina did not affect the sarcomeric organization, but induced positive or negative effects on miPS cell-derived CMs' differentiation in a concentration-dependent manner. Taken together, our findings demonstrate that the ethanolic extract of V. amygdalina affected cell proliferation, colony forming and cardiac beating capacities in a concentration-dependent manner.

Circulating microRNAs and cardiomyocyte proliferation in heart failure patients related to 10 years survival.

AIMS: Mechanochemical signalling drives organogenesis and is highly conserved in mammal evolution. Regaining recovery in myocardial jeopardy by inducing principles linking cardiovascular therapy and clinical outcome has been the dream of scientists for decades. Concepts involving embryonic pathways to regenerate adult failing hearts became popular in the early millennium. Since then, abundant data on stem cell research have been published, never reaching widespread application in heart failure therapy. Another conceptual access, using mechanotransduction in cardiac veins to limit myocardial decay, is pressure-controlled intermittent coronary sinus occlusion (PICSO). Recently, we reported acute molecular signs and signals of PICSO activating regulatory miRNA and inducing cell proliferation mimicking cardiac development in adult failing hearts. According to a previously formulated hypothesis, 'embryonic recall', this study aimed to define molecular signals involved in endogenous heart repair during PICSO and study their relation to patient survival. METHODS AND RESULTS: We previously reported a study on the acute molecular effects of PICSO in an observational non-randomized study. Eight out of the thirty-two patients with advanced heart failure undergoing cardiac resynchronization therapy (CRT) were treated with PICSO. Survival was monitored over 10 years, and coronary sinus blood samples were collected during intervention before and after 20 min and tested for miRNA signalling and proliferation when co-cultured with cardiomyocytes. A numerically lower death rate post-CRT and PICSO as compared with control CRT only, and a non-significant reduction in all-cause mortality risk of 42% was observed (37.5% vs. 54.0%, relative risk = 0.58, 95% confidence interval: 0.17-2.05; P = 0.402). Four miRNAs involved in cell cycle, proliferation, morphogenesis, embryonic development, and apoptosis significantly increased concomitantly in survivors and PICSO compared with a decrease in non-survivors (hsa-miR Let7b, P < 0.01; hsa-miR- 421, P < 0.006; hsa-miR 363-3p, P < 0.03 and hsa-miR 19b-3p P < 0.01). In contrast, three miRNAs involved in proliferation and survival, determining cell fate, and recycling endosomes decreased in survivors and PICSO (hsa miR 101-3p, P < 0.03; hsa-miR 25-3p, P < 002; hsa-miR 30d-5p P < 0.04). In vitro cellular proliferation increased in survivors and lowered in non-survivors showing a pattern distinction, discriminating longevity according to up to 10-year survival in heart failure patients. CONCLUSIONS: This study proposes that generating regenerative signals observed during PICSO intervention relate to patient outcomes. Morphogenetic pathways induced by periods of flow reversal in cardiac veins in a domino-like pattern transform embryonic into regenerative signals. Studies supporting the conversion of mechanochemical signals into regenerative molecules during PICSO are warranted to substantiate predictive power on patient longevity, opening new therapeutic avenues in otherwise untreatable heart failure.

In vitro model for assessing arrhythmogenic properties of drugs based on high-resolution impedance measurements.

BACKGROUND/AIMS: Cardiac dysfunction is one of the main cause of drug candidate failures in the preclinical and/or clinical studies and responsible for the retraction of large number of drugs from the market. The prediction of arrhythmic risk based on preclinical trials during drug development remains limited despite intensive and costly investigation. Moreover, methods for analyzing beating behavior of cardiomyocytes (CMs) in culture to diagnose arrhythmias are not well developed. METHODS: In this study, we combined two emerging technologies, induced pluripotent stem (iPS) cell-derived CMs and impedance-based real-time (xCELLigence RTCA Cardio Instrument) monitoring of CM electrical activity, to assess the effect of drugs known affect cardiac activity such as isoproterenol, carbachol, terfenadine, sotalol and doxorubicin. Cells were exposed to a drug in a single dose or repeated dose scenarios and data were analyzed using RTCA Cardio software, Poincaré plot and detrended fluctuation analysis. RESULTS: The results revealed significant changes in beating parameters of iPS-CMs induced by reference compounds. Heptanol, gap junction blocker, completely disrupted the synchronous beating pattern of iPS-CMs. Decrease of beating rate, amplitude and beat-to-beat signal variations of iPS-CMs monolayer observed in the presence of doxorubicin revealed severe abnormality detected by the system. Additionally, the irregular beating rhythms recorded in the presence of Terfenadine and Sotalol at high concentration, reflect abnormalities in cell contraction and/or relaxation which may lead to arrhythmia. CONCLUSIONS: All these results indicated that xCELLigence RTCA Cardio system combined with iPS cells, has the potential to be an attractive high-throughput tool for studying CMs during prolonged culture times and to screen potential drugs for cardiotoxic side effects.

The influence of light on the beat rate variability of murine embryonic stem cell derived cardiomyocytes.

BACKGROUND: The human heart rhythm can be quantified by analyzing the heart rate variability (HRV). A major influencing factor of the HRV is the circadian rhythm. The ocular light and the hormone melatonin play decisive roles in the circadian rhythm. The beat rate variability (BRV) is considered to be the in vitro equivalent of the HRV. Previous studies have demonstrated the influence of melatonin on cardiomyocytes. Also, the influence of light on cardiomyocytes has been described before. Nevertheless, the effect of light on the BRV of cardiomyocytes has not yet been examined. MATERIAL AND METHODS: The BRV of spontaneously beating cardiomyocytes was measured with microelectrode arrays over a time period of 30 min. The experiments were either performed with light exposure (with and without an infrared filter) or in complete darkness. RESULTS: The BRV was higher and the beating frequency was lower when the cardiomyocytes were exposed to the full spectrum of light, compared to the measurements in darkness as well as to the measurements with an infrared filter. In contrast, the differences of BRV between the measurements in darkness and the measurements with an infrared filter were not as distinct. CONCLUSIONS: This is the first study demonstrating the influence of light on the beating rhythm of heart tissue in vitro. The results indicate that especially the infrared spectrum of light alters the BRV. These findings could be of interest for clinical applications such as the field of optical pacing as well as in neonatal patient care.

Syndapin-2 mediated transcytosis of amyloid-ß across the blood-brain barrier.

A deficient transport of amyloid-ß across the blood-brain barrier, and its diminished clearance from the brain, contribute to neurodegenerative and vascular pathologies, such as Alzheimer's disease and cerebral amyloid angiopathy, respectively. At the blood-brain barrier, amyloid-ß efflux transport is associated with the low-density lipoprotein receptor-related protein 1. However, the precise mechanisms governing amyloid-ß transport across the blood-brain barrier, in health and disease, remain to be fully understood. Recent evidence indicates that the low-density lipoprotein receptor-related protein 1 transcytosis occurs through a tubulation-mediated mechanism stabilized by syndapin-2. Here, we show that syndapin-2 is associated with amyloid-ß clearance via low-density lipoprotein receptor-related protein 1 across the blood-brain barrier. We further demonstrate that risk factors for Alzheimer's disease, amyloid-ß expression and ageing, are associated with a decline in the native expression of syndapin-2 within the brain endothelium. Our data reveals that syndapin-2-mediated pathway, and its balance with the endosomal sorting, are important for amyloid-ß clearance proposing a measure to evaluate Alzheimer's disease and ageing, as well as a target for counteracting amyloid-ß build-up. Moreover, we provide evidence for the impact of the avidity of amyloid-ß assemblies in their trafficking across the brain endothelium and in low-density lipoprotein receptor-related protein 1 expression levels, which may affect the overall clearance of amyloid-ß across the blood-brain barrier.

In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells.

BACKGROUND/AIMS: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. METHODS: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. RESULTS: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca(2+) release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca(2+)-induced Ca(2+)-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. CONCLUSION: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

Triazole, imidazole, and thiazole-based compounds as potential agents against coronavirus.

The expansion of the novel coronavirus known as SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), COVID-19 (coronavirus disease 2019), or 2019-nCoV (2019 novel coronavirus) is a global concern over its pandemic potential. The need for therapeutic alternatives to stop this new pandemic is urgent. Nowadays, no efficacious therapy is available, and vaccines and drugs are underdeveloped to cure or prevent SARS-CoV-2 infections in many countries. Some vaccines candidates have been approved; however, a number of people are still skeptical of this coronavirus vaccines. Probably because of issues related to the quantity of the vaccine and a possible long-term side effects which are still being studied. The previous pandemics of infections caused by coronavirus, such as SARS-CoV in 2003, the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, HCoV-229E, and HCoV-OC43 were described in the 1960 s, -HCoV-NL63 isolated in 2004, and HCoV-HKU1identified in 2005 prompted researchers to characterize many compounds against these viruses. Most of them could be potentially active against the currently emerging novel coronavirus. Five membered nitrogen heterocycles with a triazole, imidazole, and thiazole moiety are often found in many bioactive molecules such as coronavirus inhibitors. This present work summarizes to review the biological and structural studies of these compound types as coronavirus inhibitors.

Modeling genetic cardiac channelopathies using induced pluripotent stem cells - Status quo from an electrophysiological perspective.

Long QT syndrome (LQTS), Brugada syndrome (BrS), and catecholaminergic polymorphic ventricular tachycardia (CPVT) are genetic diseases of the heart caused by mutations in specific cardiac ion channels and are characterized by paroxysmal arrhythmias, which can deteriorate into ventricular fibrillation. In LQTS3 and BrS different mutations in the SCN5A gene lead to a gain-or a loss-of-function of the voltage-gated sodium channel Nav1.5, respectively. Although sharing the same gene mutation, these syndromes are characterized by different clinical manifestations and functional perturbations and in some cases even present an overlapping clinical phenotype. Several studies have shown that Na+ current abnormalities in LQTS3 and BrS can also cause Ca2+-signaling aberrancies in cardiomyocytes (CMs). Abnormal Ca2+ homeostasis is also the main feature of CPVT which is mostly caused by heterozygous mutations in the RyR2 gene. Large numbers of disease-causing mutations were identified in RyR2 and SCN5A but it is not clear how different variants in the SCN5A gene produce different clinical syndromes and if in CPVT Ca2+ abnormalities and drug sensitivities vary depending on the mutation site in the RyR2. These questions can now be addressed by using patient-specific in vitro models of these diseases based on induced pluripotent stem cells (iPSCs). In this review, we summarize different insights gained from these models with a focus on electrophysiological perturbations caused by different ion channel mutations and discuss how will this knowledge help develop better stratification and more efficient personalized therapies for these patients.

The influence of melatonin on the heart rhythm - An in vitro simulation with murine embryonic stem cell derived cardiomyocytes.

BACKGROUND: In healthy individuals, a major factor influencing the heart rate variability (HRV) is the circadian rhythm. The role of melatonin as an essential component of the circadian rhythm in the adult human organism and the beneficial effects of a treatment with melatonin during the fetal period is well described. Toxic effects of melatonin are discussed less frequently. Since pharmacological studies cannot be carried out on pregnant women, the establishment of an equivalent in vitro model is important. We therefore tested whether melatonin can influence the beat rate variability (BRV) of spontaneously beating cardiomyocytes derived from murine embryonic stem cells (mESCs) and whether melatonin exhibits toxic effects in this in vitro model. METHODS: Microelectrode Arrays recorded extracellular field potentials of spontaneously beating cardiomyocytes. Melatonin was applied in a concentration range from 10-11 M to 10-5 M. The analysis of the BRV focused on time domain methods. RESULTS: In line with clinical observations, melatonin decreased the beating frequency and increased the BRV. The effect of melatonin up to a concentration of 10-6 M was reversible, whereas the application of higher concentrations induced an irreversible effect. CONCLUSION: The study underlines the potential of this in vitro model to help explore the development of circadian rhythms and their modulation by melatonin in the embryonic phase. The results imply that melatonin influences the heart rhythm as early as during the embryonic heart development. Furthermore, the results indicate a potentially toxic effect of melatonin that has not been described in detail before.