Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.

De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.

Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm.

Tumor heterogeneity presents a challenge for inferring clonal evolution and driver gene identification. Here, we describe a method for analyzing the cancer genome at a single-cell nucleotide level. To perform our analyses, we first devised and validated a high-throughput whole-genome single-cell sequencing method using two lymphoblastoid cell line single cells. We then carried out whole-exome single-cell sequencing of 90 cells from a JAK2-negative myeloproliferative neoplasm patient. The sequencing data from 58 cells passed our quality control criteria, and these data indicated that this neoplasm represented a monoclonal evolution. We further identified essential thrombocythemia (ET)-related candidate mutations such as SESN2 and NTRK1, which may be involved in neoplasm progression. This pilot study allowed the initial characterization of the disease-related genetic architecture at the single-cell nucleotide level. Further, we established a single-cell sequencing method that opens the way for detailed analyses of a variety of tumor types, including those with high genetic complex between patients.

Single-cell exome sequencing reveals single-nucleotide mutation characteristics of a kidney tumor.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and has very few mutations that are shared between different patients. To better understand the intratumoral genetics underlying mutations of ccRCC, we carried out single-cell exome sequencing on a ccRCC tumor and its adjacent kidney tissue. Our data indicate that this tumor was unlikely to have resulted from mutations in VHL and PBRM1. Quantitative population genetic analysis indicates that the tumor did not contain any significant clonal subpopulations and also showed that mutations that had different allele frequencies within the population also had different mutation spectrums. Analyses of these data allowed us to delineate a detailed intratumoral genetic landscape at a single-cell level. Our pilot study demonstrates that ccRCC may be more genetically complex than previously thought and provides information that can lead to new ways to investigate individual tumors, with the aim of developing more effective cellular targeted therapies.

Enhanced brain-targeting and efficacy of cannabidiol via RVG-Exo/CBD nanodelivery system.

This study introduces an innovative brain-targeted drug delivery system, RVG-Exo/CBD, utilizing rabies virus glycoprotein (RVG)-engineered exosomes for encapsulating cannabidiol (CBD). The novel delivery system was meticulously characterized, confirming the maintenance of exosomal integrity, size, and successful drug encapsulation with a high drug loading rate of 83.0 %. Evaluation of the RVG-Exo/CBD's brain-targeting capability demonstrated superior distribution and retention in brain tissue compared to unmodified exosomes, primarily validated through in vivo fluorescence imaging. The efficacy of this delivery system was assessed using a behavioral sensitization model in mice, where RVG-Exo/CBD notably suppressed methamphetamine-induced hyperactivity more effectively than CBD alone, indicating a reduction in effective dose and enhanced bioavailability. Overall, the RVG-Exo/CBD system emerges as a promising strategy for enhancing the therapeutic efficacy and safety of CBD, particularly for neurological applications, highlighting its potential for addressing the limitations associated with traditional CBD administration in clinical settings.

Association of domain-specific physical activity with albuminuria among prediabetes and diabetes: a large cross-sectional study.

BACKGROUND: Albuminuria, the presence of excess of protein in urine, is a well-known risk factor for early kidney damage among diabetic/prediabetic patients. There is a complex interaction between physical activity (PA) and albuminuria. However, the relationship of specific-domain PA and albuminuria remained obscure. METHODS: Albuminuria was defined as urinary albumin/creatinine ratio (ACR) > 30 mg/g. PA was self-reported by participants and classified into transportation-related PA (TPA), occupation-related PA (OPA), and leisure-time PA (LTPA). Weighted logistic regression was conducted to compute the odds ratios (ORs) and 95% confidence intervals (CIs). Restricted cubic spline (RCS) was used to evaluate the dose-response of PA domains with the risk of albuminuria. RESULTS: A total of 6739 diabetic/prediabetic patients (mean age: 56.52 ± 0.29 years) were enrolled in our study, including 3181 (47.20%) females and 3558 (52.80%) males. Of them, 1578 (23.42%) were identified with albuminuria, and 5161(76.58%) were without albuminuria. Diabetic/prediabetic patients who adhered the PA guidelines for total PA had a 22% decreased risk of albuminuria (OR = 0.78, 95%CI 0.64-0.95), and those met the PA guidelines for LTPA had a 28% decreased of albuminuria (OR = 0.72, 95%CI 0.57-0.92). However, OPA and TPA were both not associated with decreased risk of albuminuria. RCS showed linear relationship between the risk of albuminuria with LTPA. CONCLUSIONS: Meeting the PA guideline for LTPA, but not OPA and TPA, was inversely related to the risk of albuminuria among diabetic/prediabetic patients. Additionally, achieving more than 300 min/week of LTPA conferred the positive effects in reducing albuminuria among diabetic/prediabetic patients.

Small conductance calcium-activated potassium channel contributes to stress induced endothelial dysfunctions.

Patients with Takotsubo syndrome displayed endothelial dysfunction, but underlying mechanisms have not been fully clarified. This study aimed to explore molecular signalling responsible for catecholamine excess induced endothelial dysfunction. Human cardiac microvascular endothelial cells were challenged by epinephrine to mimic catecholamine excess. Patch clamp, FACS, ELISA, PCR, and immunostaining were employed for the study. Epinephrine (Epi) enhanced small conductance calcium-activated potassium channel current (ISK1-3) through activating α1 adrenoceptor. Phenylephrine enhanced edothelin-1 (ET-1) and reactive oxygen species (ROS) production, and the effects involved contribution of ISK1-3. H2O2 enhanced ISK1-3 and ET-1 production. Enhancing ISK1-3 caused a hyperpolarization, which increases ROS and ET-1 production. BAPTA partially reduced phenylephrine-induced enhancement of ET-1 and ROS, suggesting that α1 receptor activation can enhance ROS/ET-1 generation in both calcium-dependent and calcium-independent ways. The study demonstrates that high concentration catecholamine can activate SK1-3 channels through α1 receptor-ROS signalling and increase ET-1 production, facilitating vasoconstriction.

Roles and Mechanisms of Dopamine Receptor Signaling in Catecholamine Excess Induced Endothelial Dysfunctions.

Endothelial dysfunction may contribute to pathogenesis of Takotsubo cardiomyopathy, but mechanism underlying endothelial dysfunction in the setting of catecholamine excess has not been clarified. The study reports that D1/D5 dopamine receptor signaling and small conductance calcium-activated potassium channels contribute to high concentration catecholamine induced endothelial cell dysfunction. For mimicking catecholamine excess, 100 µM epinephrine (Epi) was used to treat human cardiac microvascular endothelial cells. Patch clamp, FACS, ELISA, PCR, western blot and immunostaining analyses were performed in the study. Epi enhanced small conductance calcium-activated potassium channel current (ISK1-3) without influencing the channel expression and the effect was attenuated by D1/D5 receptor blocker. D1/D5 agonists mimicked the Epi effect, suggesting involvement of D1/D5 receptors in Epi effects. The enhancement of ISK1-3 caused by D1/D5 activation involved roles of PKA, ROS and NADPH oxidases. Activation of D1/D5 and SK1-3 channels caused a hyperpolarization, reduced NO production and increased ROS production. The NO reduction was membrane potential independent, while ROS production was increased by the hyperpolarization. ROS (H2O2) suppressed NO production. The study demonstrates that high concentration catecholamine can activate D1/D5 and SK1-3 channels through NADPH-ROS and PKA signaling and reduce NO production, which may facilitate vasoconstriction in the setting of catecholamine excess.

miR-194-3p regulates epithelial-mesenchymal transition in embryonic epicardial cells via p120/ß-catenin signaling.

The epicardium is integral to cardiac development and facilitates endogenous heart regeneration and repair. While miR-194-3p is associated with cellular migration and invasion, its impact on epicardial cells remains uncharted. In this work we use gain-of-function and loss-of-function methodologies to investigate the function of miR-194-3p in cardiac development. We culture embryonic epicardial cells in vitro and subject them to transforming growth factor ß (TGF-ß) treatment to induce epithelial-mesenchymal transition (EMT) and monitor miR-194-3p expression. In addition, the effects of miR-194-3p mimics and inhibitors on epicardial cell development and changes in EMT are investigated. To validate the binding targets of miR-194-3p and its ability to recover the target gene-phenotype, we produce a mutant vector p120-catenin-3'UTR-MUT. In epicardial cells, TGF-ß-induced EMT results in a notable overexpression of miR-194-3p. The administration of miR-194-3p mimics promotes EMT, which is correlated with elevated levels of mesenchymal markers. Conversely, miR-194-3p inhibitor attenuates EMT. Further investigations reveal a negative correlation between miR-194-3p and p120-catenin, which influences ß-catenin level in the cell adhesion pathway. The suppression of EMT caused by the miR-194-3p inhibitor is balanced by silencing of p120-catenin. In conclusion, miR-194-3p directly targets p120-catenin and modulates its expression, which in turn alters ß-catenin expression, critically influencing the EMT process in the embryonic epicardial cells via the cell adhesion mechanism.

A novel multi-dimensional coal and gangue X-ray sorting algorithm based on CdZnTe photon counting detectors.

BACKGROUND: The gangue content in coal seriously affects the calorific value produced by its combustion. In practical applications, gangue in coal needs to be completely separated. The pseudo-dual-energy X-ray method does not have high sorting accuracy. OBJECTIVE: This study aims to propose a novel multi-dimensional coal and gangue X-ray sorting algorithm based on CdZnTe photon counting detectors to solve the problem of coal and gangue sorting by X-ray. METHODS: This complete algorithm includes five steps: (1) Preferred energy bins, (2) transmittance sorting, (3) one-dimensional R-value sorting, (4) two-dimensional R-value sorting, and (5) three-dimensional R-value sorting. The output range of each step is determined by prior information from 65 groups of coal and gangue. An additional 110 groups of coal and gangue are employed experimentally to validate the algorithm's accuracy. RESULTS: Compared with the 60% sorting accuracy of the Pseudo-dual-energy method, the new algorithm reached a sorting accuracy of 99%. CONCLUSIONS: Study results demonstrate the superiority of this novel algorithm and its feasibility in practical applications. This novel algorithm can guide other two-substance X-ray sorting applications based on photon counting detectors.

PLANT NATRIURETIC PEPTIDE A and Its Putative Receptor PNP-R2 Antagonize Salicylic Acid-Mediated Signaling and Cell Death.

The plant stress hormone salicylic acid (SA) participates in local and systemic acquired resistance, which eventually leads to whole-plant resistance to bacterial pathogens. However, if SA-mediated signaling is not appropriately controlled, plants incur defense-associated fitness costs such as growth inhibition and cell death. Despite its importance, to date only a few components counteracting the SA-primed stress responses have been identified in Arabidopsis (Arabidopsis thaliana). These include other plant hormones such as jasmonic acid and abscisic acid, and proteins such as LESION SIMULATING DISEASE1, a transcription coregulator. Here, we describe PLANT NATRIURETIC PEPTIDE A (PNP-A), a functional analog to vertebrate atrial natriuretic peptides, that appears to antagonize the SA-mediated plant stress responses. While loss of PNP-A potentiates SA-mediated signaling, exogenous application of synthetic PNP-A or overexpression of PNP-A significantly compromises the SA-primed immune responses. Moreover, we identify a plasma membrane-localized receptor-like protein, PNP-R2, that interacts with PNP-A and is required to initiate the PNP-A-mediated intracellular signaling. In summary, our work identifies a peptide and its putative cognate receptor as counteracting both SA-mediated signaling and SA-primed cell death in Arabidopsis.

Analysis and correction of misalignment-induced aberrations in two-mirror telescopes with stop on the secondary mirror.

We study the problem of misalignment aberration analysis and correction of the two-mirror telescopes with stop on the secondary mirror. The variation law of the system's aberration field is analyzed with nodal aberration theory when the primary mirror with an astigmatic figure error is misaligned. The analytic expression among the system wave aberration, misalignments, and astigmatism figure error is given, and the correction model of system misalignment aberration is established. The simulation experiment shows that the relative error of the prediction of system misalignment coma and astigmatism based on this model is less than 4.1%.

Epicardial adipose tissue in patients with chronic obstructive pulmonary disease: systematic review with meta­analysis and trial sequential analysis.

BACKGROUND: Limited data suggest that chronic obstructive pulmonary disease (COPD) patients have pathologic elevated epicardial adipose tissue (EAT), which is splanchnic fat tissue with anti-inflammatory properties and regulating free fatty acids functions. Therefore, there is a need for meta-analysis to explore the relationship between EAT and COPD. METHODS: Online databases were systematically searched for studies about EAT in COPD patients published up to October 5th, 2022. The EAT data of the COPD patient group and the control group were included. Trial sequential analysis (TSA) and meta-analysis were applied to assess the difference in EAT between patients with and without COPD. TSA software and Stata 12.0 were used in all statistical analyses. RESULTS: The final analysis included 5 studies (n = 596 patients). COPD patients had significantly more EAT than control subjects (SMD: 0.0.802; 95% CI: 0.231, 1.372; P = 0.006; TSA-adjusted 95% CI 1.20, 1.80; P < 0.0001). And higher CRP levels in COPD patients than non-COPD patients, whereas triglycerides and LDL were not significantly different between patients with and without COPD. CONCLUSION: EAT is abnormally elevated in COPD patients, which may be related to systemic inflammatory responses in COPD. PROSPERO NUMBER: CRD42021228273.

A novel multi-view X-ray digital imaging stitching algorithm.

BACKGROUND: In fan beam X-ray imaging applications, several X-ray images sometimes need to be stitched together into a panoramic image because of the size limitations of the detector. OBJECTIVE: This study aims to propose a novel multi-view X-ray digital imaging stitching algorithm (MVS) based on the CdZnTe photon counting linear array detectors to solve the problem of fan beam X-ray stitching deformation. METHODS: The panoramic image is generated in four steps including (1) multi-view projection data acquisition, (2) overlapping positioning, (3) weighted fusion and (4) projected pixel value calculation. Images of a globe and foot are scanned by fan beam X-rays and a CdZnTe detector. The proposed method is applied to stitch together the scanned images of the globe. Three other methods are also used for comparison. Finally, this MVS algorithm is also used in the stitching of scanned images of the foot. RESULTS: Compared with the 50% stitching accuracy of other methods, the new MVS algorithm reached a stitching accuracy of 94.4%. Image distortion on the globe and feet is also eliminated and thus image quality is significantly improved. CONCLUSIONS: This study proposes a new multi-view X-ray digital imaging stitching algorithm. Study results demonstrate the superiority of this new algorithm and its feasibility in practical applications.

Epigenetic mechanism of L-type calcium channel ß-subunit downregulation in short QT human induced pluripotent stem cell-derived cardiomyocytes with CACNB2 mutation.

AIMS: A loss-of-function mutation in L-type calcium (Ca2+) channel subunit gene CACNB2 has been reported to cause short QT syndrome subtype 5 (SQT5). However, the mechanism underlying the loss-of-function of the Ca2+ channel has not been clarified. In the present study, we aim to explore the DNA methylation mechanism of L-type Ca2+ channel downregulation in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of SQT5. METHODS AND RESULTS: The hiPSC-CMs were generated from a healthy donor and a SQT5 patient carrying the CACNB2 variant c.1439C > T/p.S480L. The variant was genetically corrected using ribonucleoprotein-based CRISPR/Cas9 technique to obtain an isogenic control cell line. The action potential (AP) and Ca2+ current were measured by patch clamp. Protein expression levels were determined by western blotting. Dot blotting and bisulfite sequence were performed for epigenetic study. Our results showed that AP durations at 10% repolarization (APD10) and 50% repolarization (APD50) were significantly shortened in SQT5 cells and both the expression level of the ß-subunit and channel current of L-type Ca2+ channel were reduced. Besides, an increased level of whole-genome DNA methylation and DNA methylation of CpG island in the promoter region of CACNB2 gene was detected. Overexpression of demethylation enzyme could rescue the decreased expression of CACNB2 and the L-type Ca2+ current. CONCLUSION: In SQT5 hiPSC-CMs carrying the CACNB2-S480L variant, the decreased L-type Ca2+ current resulting from decreased CACNB2 protein expression was caused by enhanced methylation in the promoter region of the CACNB2 gene and upregulation of DNA methyltransferases might be one of the mechanisms.

Optical design method of a three-mirror anastigmatic telescope with low misalignment sensitivity based on the nodal aberration theory.

We propose a design method for a three-mirror anastigmatic telescope with low misalignment sensitivity and deduce the analytic expression between the misalignment aberration and its optical parameters based on the nodal aberration theory. We establish an optical system as-built performance evaluation model. Using this model as the system's as-built performance evaluation indicator, we can get an optical system that could have both low misalignment sensitivity and good image quality after optimization. The design results of a field bias three-mirror anastigmatic telescope show that the misalignment aberration of the system can be reduced by changing the spacing of the mirrors. When the spacing between the primary mirror and the secondary mirror increases and the spacing from the secondary mirror to the third mirror and the third mirror to the image plane decreases, the misalignment sensitivity will drop significantly. If the mirror spacing is changed by 10%, the misalignment sensitivity of the telescope optimized by our method is only about 85% of that of the traditional method.

Toxicity of avermectin to Eriocheir sinensis and the isolation of a avermectin-degrading bacterium, Ochrobactrum sp. AVM-2.

Avermectin is widely used in the prevention and treatment of parasites diseases in aquaculture. However, the residual avermectin has a serious impact on the growth and quality of aquatic animals including Eriocheir sinensis. This study shows that the LC50 of avermectin to E. sinensis for 24, 48, 72 and 96 h was 21.88, 13.40, 9.11 and 7.10 mg/L, respectively. After avermectin stress, the activities of superoxide dismutase (SOD), catalase (CAT) and phenol oxidase (PO) in the hepatopancreas of E. sinensis increased and reached the peak on the 6th day. The content of malondialdehyde (MDA) accumulated with the increase of exposure time and concentration of avermectin. After 15 days of avermectin exposure, hepatopancreas was damaged seriously. These results indicated that avermectin had toxicity to E. sinensis. In order to solve the pollution problem caused by residual avermectin, a degrading bacterium AVM-2 was separated from the sediment of E. sinensis breeding pond. The strain was confirmed to be Ochrobactrum sp by morphology observation, physiological and biochemical identification and 16 S rDNA sequences analysis. When the pH value was 7, the temperature was 30 â„ƒ, the concentration of substrate was low, the quantity of inoculation was high, Ochrobactrum sp. AVM-2 had better degradation effect on avermectin. When the addition of Ochrobactrum sp. AVM-2 was 2.34 × 108 CFU/L, the residual avermectin in muscle and hepatopancreatine significantly decreased, and the degradation rate was about 66%. In summary, Ochrobactrum sp. AVM-2 could be used to solve the residual problem of avermectin and ensure the food safety of E. sinensis.

A Preclinical Study on Brugada Syndrome with a CACNB2 Variant Using Human Cardiomyocytes from Induced Pluripotent Stem Cells.

Aims: Some gene variants in the sodium channels, as well as calcium channels, have been associated with Brugada syndrome (BrS). However, the investigation of the human cellular phenotype and the use of drugs for BrS in presence of variant in the calcium channel subunit is still lacking. Objectives: The objective of this study was to establish a cellular model of BrS in the presence of a CACNB2 variant of uncertain significance (c.425C > T/p.S142F) using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and test drug effects using this model. Methods and results: This study recruited cells from a patient with Brugada syndrome (BrS) and recurrent ventricular fibrillation carrying a missense variant in CACNB2 as well as from three healthy independent persons. These cells (hiPSC-CMs) generated from skin biopsies of healthy persons and the BrS patient (BrS-hiPSC-CMs) as well as CRISPR/Cas9 corrected cells (isogenic control, site-variant corrected) were used for this study. The hiPSC-CMs from the BrS patient showed a significantly reduced L-type calcium channel current (ICa-L) compared with the healthy control hiPSC-CMs. The inactivation curve was shifted to a more positive potential and the recovery from inactivation was accelerated. The protein expression of CACNB2 of the hiPSC-CMs from the BrS-patient was significantly decreased compared with healthy hiPSC-CMs. Moreover, the correction of the CACNB2 site-variant rescued the changes seen in the hiPSC-CMs of the BrS patient to the normal state. These data indicate that the CACNB2 gene variant led to loss-of-function of L-type calcium channels in hiPSC-CMs from the BrS patient. Strikingly, arrhythmia events were more frequently detected in BrS-hiPSC-CMs. Bisoprolol (beta-blockers) at low concentration and quinidine decreased arrhythmic events. Conclusions: The CACNB2 variant (c.425C > T/p.S142F) causes a loss-of-function of L-type calcium channels and is pathogenic for this type of BrS. Bisoprolol and quinidine may be effective for treating BrS with this variant.

Nitrate mediated resistance against Fusarium infection in cucumber plants acts via photorespiration.

Fusarium wilt is one of the major biotic factors limiting cucumber (Cucumis sativus L.) growth and yield. The outcomes of cucumber-Fusarium interactions can be influenced by the form of nitrogen nutrition (nitrate [NO3- ] or ammonium [NH4+ ]); however, the physiological mechanisms of N-regulated cucumber disease resistance are still largely unclear. Here, we investigated the relationship between nitrogen forms and cucumber resistance to Fusarium infection. Our results showed that on Fusarium infection, NO3- feeding decreased the levels of the fungal toxin, fusaric acid, leaf membrane oxidative, organelle damage and disease-associated loss in photosynthesis. Metabolomic analysis and gas-exchange measurements linked NO3- mediated plant defence with enhanced leaf photorespiration rates. Cucumber plants sprayed with the photorespiration inhibitor isoniazid were more susceptible to Fusarium and there was a negative correlation between photorespiration rate and leaf membrane injury. However, there were positive correlations between photorespiration rate, NO3- assimilation and the tricarboxylic acid (TCA) cycle. This provides a potential electron sink or the peroxisomal H2 O2 catalysed by glycolate oxidase. We suggest that the NO3- nutrition enhanced cucumber resistance against Fusarium infection was associated with photorespiration. Our findings provide a novel insight into a mechanism involving the interaction of photorespiration with nitrogen forms to drive wider defence.

Molecular characterization, expression analysis and immune effect of Galectin-8 from Japanese flounder (Paralichthys olivaceus).

Galectin-8 gene belongs to the agglutinin family, which can specifically recognize ß-galactoside bonds and play essential roles in many biological processes. In this study, we researched the sequence characteristics and immune-related function of Galectin-8 gene in Japanese flounder Paralichthys olivaceus, named PoGalectin-8. The results showed that the open reading frame of PoGalectin-8 was 891 bp, which encoding a protein with 296 amino acid residues and containing typical HXNPR and WGXEE motifs in the N-terminal and C-terminal CRD domains. Sequence alignment showed that PoGalectin-8 was conserved in different aquatic animals and exhibited the highest similarity (95.27%) with Seriola dumerili. PoGalectin-8 expressed in all detected tissues and exhibited the highest expression level in spleen, followed by skin and kidney. After infected by Edwardsiella tarda, the expression of PoGalectin-8 was down-regulated in the spleen and skin tissues of P. olivaceus. Further to study its immune-related functions, the recombinant PoGalectin-8 (rPoGalectin-8) was expressed and purified. The rPoGalectin-8 can specifically bind to lipopolysaccharide and peptidoglycan, the main components of cell walls from Gram-negative and Gram-positive bacteria. Bacteria binding and the microbial agglutinating experiments showed that the rPoGalectin-8 could bind and agglutinate all examined Gram-positive and Gram-negative bacteria. This study implied that PoGalectin-8, as a pattern recognition receptor, may play important roles during immune responses against bacterial infection, which laid a foundation for further functional identification of Galectin-8 in aquatic animal immunity.

Alpha 1-adrenoceptor signalling contributes to toxic effects of catecholamine on electrical properties in cardiomyocytes.

AIMS: This study aimed to investigate possible roles and underlying mechanisms of alpha-adrenoceptor coupled signalling for the pathogenesis of Takotsubo syndrome (TTS). METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were treated with a toxic concentration of epinephrine (Epi, 0.5 mM for 1 h) to mimic the setting of TTS. Patch-clamp technique, polymerase chain reaction (PCR) and Fluorescence-activated cell sorting (FACS) were employed for the study. High concentration Epi suppressed the depolarization velocity, prolonged duration of action potentials and induced arrhythmic events in hiPSC-CMs. The Epi effects were attenuated by an alpha-adrenoceptor blocker (phentolamine), suggesting involvement of alpha-adrenoceptor signalling in arrhythmogenesis related to QT interval prolongation in the setting of TTS. An alpha 1-adrenoceptor agonist (phenylephrine) but not an alpha 2-adrenoceptor agonist (clonidine) mimicked Epi effects. Epi enhanced ROS production, which could be attenuated by the alpha- adrenoceptor blocker. Treatment of cells with H2O2 (100 µM) mimicked the effects of Epi on action potentials and a reactive oxygen species (ROS)-blocker (N-acetyl-I-cysteine, 1 mM) prevented the Epi effects, indicating that the ROS signalling is involved in the alpha-adrenoceptor actions. Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidases were involved in alpha 1-adrenoceptor signalling. A protein kinase C (PKC) blocker suppressed the effects of Epi, phenylephrine and ROS as well, implying that PKC participated in alpha 1-adrenoceptor signalling and acted as a downstream factor of ROS. The abnormal action potentials resulted from alpha 1-adrenoceptor activation-induced dysfunctions of ion channels including the voltage-dependent Na+ and L-type Ca2+ channels. CONCLUSIONS: Alpha 1-adrenoceptor signalling plays important roles for arrhythmogenesis of TTS. Alpha-adrenoceptor blockers might be clinically helpful for treating arrhythmias in patients with TTS.