Negatively Curved Octagon-Incorporated Aza-nanographene and its Assembly with Fullerenes.

A negatively curved aza-nanographene (NG) containing two octagons was synthesized by a regioselective and stepwise cyclodehydrogenation procedure, in which a double aza[7]helicene was simultaneously formed as an intermediate. Their saddle-shaped structures with negative curvature were unambiguously confirmed by X-ray crystallography, thereby enabling the exploration of the structure-property relationship by photophysical, electrochemical and conformational studies. Moreover, the assembly of the octagon-embedded aza-NG with fullerenes was probed by fluorescence spectral titration, with record-high binding constants (Ka=9.5×103 M-1 with C60, Ka=3.7×104 M-1 with C70) found among reported negatively curved polycyclic aromatic compounds. The tight association of aza-NG with C60 was further elucidated by X-ray diffraction analysis of their co-crystal, which showed the formation of a 1 : 1 complex with substantial concave-convex interactions.

Photoresponse of Solution-Synthesized Graphene Nanoribbon Heterojunctions on Diamond Indicating Phototunable Photodiode Polarity.

Graphene nanoribbon heterostructures and heterojunctions have attracted interest as next-generation molecular diodes with atomic precision. Their mass production via solution methods and prototypical device integration remains to be explored. Here, the bottom-up solution synthesis and characterization of liquid-phase-processable graphene nanoribbon heterostructures (GNRHs) are demonstrated. Joint photoresponsivity measurements and simulations provide evidence of the structurally defined heterostructure motif acting as a type-I heterojunction. Real-time, time-dependent density functional tight-binding simulations further reveal that the photocurrent polarity can be tuned at different excitation wavelengths. Our results introduce liquid-phase-processable, self-assembled heterojunctions for the development of nanoscale diode circuitry and adaptive hardware.

Novel insights in the pathomechanism of Brugada syndrome and fever-related type 1 ECG changes in a preclinical study using human-induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Brugada syndrome (BrS) is causing sudden cardiac death (SCD) mainly at young age. Studying the underlying mechanisms associated with BrS type I electrocardiogram (ECG) changes in the presence of fever and roles of autophagy for BrS remains lacking. OBJECTIVES: We sought to study the pathogenic role of an SCN5A gene variant for BrS with fever-induced type 1 ECG phenotype. In addition, we studied the role of inflammation and autophagy in the pathomechanism of BrS. METHODS: Human-induced pluripotent stem cell (hiPSC) lines from a BrS patient harboring a pathogenic variant (c.3148G>A/p. Ala1050Thr) in SCN5A and two healthy donors (non-BrS) and a CRISPR/Cas9 site-corrected cell line (BrS-corr) were differentiated into cardiomyocytes (hiPSC-CMs) for the study. RESULTS: Reductions of Nav 1.5 expression, peak sodium channel current (INa ) and upstroke velocity (Vmax ) of action potentials with an increase in arrhythmic events were detected in BrS compared to non-BrS and BrS-corr cells. Increasing the cell culture temperature from 37 to 40°C (fever-like state) exacerbated the phenotypic changes in BrS cells. The fever-effects were enhanced by protein kinase A (PKA) inhibitor but reversed by PKA activator. Lipopolysaccharides (LPS) but not increased temperature up to 40°C enhanced the autophagy level in BrS-hiPSC-CMs by increasing reactive oxidative species and inhibiting PI3K/AKT signalling, and hence exacerbated the phenotypic changes. LPS enhanced high temperature-related effect on peak INa shown in BrS hiPSC-CMs. Effects of LPS and high temperature were not detected in non-BrS cells. CONCLUSIONS: The study demonstrated that the SCN5A variant (c.3148G>A/p.Ala1050Thr) caused loss-of-function of sodium channels and increased the channel sensitivity to high temperature and LPS challenge in hiPSC-CMs from a BrS cell line with this variant but not in two non-BrS hiPSC-CM lines. The results suggest that LPS may exacerbate BrS phenotype via enhancing autophagy, whereas fever may exacerbate BrS phenotype via inhibiting PKA-signalling in BrS cardiomyocytes with but probably not limited to this variant.

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.

Lipopolysaccharide Modifies Sodium Current Kinetics through ROS and PKC Signalling in Induced Pluripotent Stem-Derived Cardiomyocytes from Brugada Syndrome Patient.

Studies have suggested a connection between inflammation and arrhythmogenesis of Brugada syndrome (BrS). However, experimental studies regarding the roles of inflammation in the arrhythmogenesis of BrS and its underlying mechanism are still lacking. This study aimed to investigate the influence of inflammation on BrS-phenotype features using human-induced stem cell-derived cardiomyocytes (hiPSC-CMs) from a BrS-patient carrying an SCN10A variant (c.3749G > A). After LPS treatment, the peak sodium current decreased significantly in SCN10A-hiPSC-CMs, but not in healthy donor-hiPSC-CMs. LPS also changed sodium channel gating kinetics, including activation, inactivation, and recovery from inactivation. NAC (N-acetyl-l-cysteine), a blocker of ROS (reactive oxygen species), failed to affect the sodium current, but prevented the LPS-induced reduction of sodium channel currents and changes in gating kinetics, suggesting a contribution of ROS to the LPS effects. Hydrogen peroxide (H2O2), a main form of ROS in cells, mimicked the LPS effects on sodium channel currents and gating kinetics, implying that ROS might mediate LPS-effects on sodium channels. The effects of H2O2 could be attenuated by a PKC blocker chelerythrine, indicating that PKC is a downstream factor of ROS. This study demonstrated that LPS can exacerbate the loss-of-function of sodium channels in BrS cells. Inflammation may play an important role in the pathogenesis of BrS.

Identification of SCN5a p.C335R Variant in a Large Family with Dilated Cardiomyopathy and Conduction Disease.

INTRODUCTION: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. METHODS: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. RESULTS: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na+ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. CONCLUSION: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.

Effects of Antiarrhythmic Drugs on hERG Gating in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes From a Patient With Short QT Syndrome Type 1.

Aims: The short QT syndrome type 1 (SQT1) is linked to hERG channel mutations (e.g., N588K). Drug effects on hERG channel gating kinetics in SQT1-cells have not been investigated. Methods: This study used hiPSC-CMs of a healthy donor and a SQT1-patient carrying the N588K mutation and patch clamp to examine the drug effects on hERG channel gating kinetics. Results: Ajmaline, amiodarone, ivabradine, flecainide, quinidine, mexiletine and ranolazine inhibited the hERG channel current (IKr) less strongly in hiPSC-CMs from the SQTS1-patient (SQT1-hiPSC-CMs) comparing with cells from the healthy donor (donor-hiPSC-CMs). Quinidine and mexiletine reduced, but ajmaline, amiodarone, ivabradine and ranolazine increased the time to peak of IKr similarly in SQT1-hiPSC-CMs and donor-hiPSC-CMs. Although regarding the shift of activation and inactivation curves, tested drugs showed differential effects in donor- and SQT1-hiPSC-CMs, quinidine, ajmaline, ivabradine and mexiletine but not amiodarone, flecainide and ranolazine reduced the window current in SQT1-hiPSC-CMs. Quinidine, ajmaline, ivabradine and mexiletine differentially changed the time constant of recovery from inactivation, but all of them increased the time constant of deactivation in SQT1-hiPSC-CMs. Conclusion: The window current-reducing and deactivation-slowing effects may be important for the antiarrhythmic effect of ajmaline, ivabradine, quinidine and mexiletine in SQT1-cells. This information may be helpful for selecting drugs for treating SQT1-patients with hERG channel mutation.

Nucleoside Diphosphate Kinase B Contributes to Arrhythmogenesis in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes from a Patient with Arrhythmogenic Right Ventricular Cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare, inheritable cardiac disorder characterized by ventricular tachyarrhythmias, progressive loss of cardiomyocytes with fibrofatty replacement and sudden cardiac death. The exact underlying mechanisms are unclear. METHODS: This study investigated the possible roles of nucleoside diphosphate kinase B (NDPK-B) and SK4 channels in the arrhythmogenesis of ARVC by using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). RESULTS: In hiPSC-CMs from a patient with ARVC, the expression levels of NDPK-B and SK4 channels were upregulated, the cell automaticity was increased and the occurrence rate of arrhythmic events was enhanced. Recombinant NDPK-B applied into hiPSC-CMs from either healthy donors or the patient enhanced SK4 channel current (ISK4), cell automaticity and the occurrence of arrhythmic events, whereas protein histidine phosphatase 1 (PHP-1), a counter actor of NDPK-B, prevented the NDPK-B effect. Application of PHP-1 alone or a SK4 channel blocker also reduced cell automaticity and arrhythmic events. CONCLUSION: This study demonstrated that the elevated NDPK-B expression, via activating SK4 channels, contributes to arrhythmogenesis in ARVC, and hence, NDPK-B may be a potential therapeutic target for treating arrhythmias in patients with ARVC.

Studying Brugada Syndrome With an SCN1B Variants in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

BACKGROUND: Among rare channelopathies BrS patients are at high risk of sudden cardiac death (SCD). SCN5A mutations are found in a quarter of patients. Other rare gene mutations including SCN1B have been implicated to BrS. Studying the human cellular phenotype of BrS associated with rare gene mutation remains lacking. OBJECTIVES: We sought to study the cellular phenotype of BrS with the SCN1B gene variants using human-induced pluripotent stem cell (hiPSCs)-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: A BrS patient suffering from recurrent syncope harboring a two variants (c.629T > C and c.637C > A) in SCN1B, which encodes the function-modifying sodium channel beta1 subunit, and three independent healthy subjects were recruited and their skin biopsies were used to generate hiPSCs, which were differentiated into cardiomyocytes (hiPSC-CMs) for studying the cellular electrophysiology. A significantly reduced peak and late sodium channel current (INa) and a shift of activation curve to more positive potential as well as a shift of inactivation curve to more negative potential were detected in hiPSC-CMs of the BrS patient, indicating that the SCN1B variants impact the function of sodium channels in cardiomyocytes. The reduced INa led to a reduction of amplitude (APA) and upstroke velocity (V max ) of action potentials. Ajmaline, a sodium channel blocker, showed a stronger effect on APA and Vmax in BrS cells as compared to cells from healthy donors. Furthermore, carbachol was able to increase arrhythmia events and the beating frequency in BrS. CONCLUSION: Our hiPSC-CMs from a BrS-patient with two variants in SCN1B recapitulated some key phenotypic features of BrS and can provide a platform for studies on BrS with SCN1B variants.

A cellular model of Brugada syndrome with SCN10A variants using human-induced pluripotent stem cell-derived cardiomyocytes.

AIMS: Brugada syndrome (BrS) is associated with a pronounced risk to develop sudden cardiac death (SCD). Up to 21% of patients are related to mutations in SCN5A. Studies identified SCN10A as a contributor of BrS. However, the investigation of the human cellular phenotype of BrS in the presence of SCN10A mutations remains lacking. The objective of this study was to establish a cellular model of BrS in presence of SCN10A mutations using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS AND RESULTS: Dermal fibroblasts obtained from a BrS patient suffering from SCD harbouring the SCN10A double variants (c.3803G>A and c.3749G>A) and three independent healthy control subjects were reprogrammed to hiPSCs. Human-induced pluripotent stem cells were differentiated into cardiomyocytes (hiPSC-CMs).The hiPSC-CMs from the BrS patient showed a significantly reduced peak sodium channel current (INa) and a significantly reduced ATX II (sea anemone toxin, an enhancer of late INa) sensitive as well as A-887826 (a blocker of SCN10A channel) sensitive late sodium channel current (INa) when compared with the healthy control hiPSC-CMs, indicating loss-of-function of sodium channels. Consistent with reduced INa the action potential amplitude and upstroke velocity (Vmax) were significantly reduced, which may contribute to arrhythmogenesis of BrS. Moreover, Ajmaline effects on action potentials were stronger in BrS-hiPSC-CMs than in healthy control cells. This is in agreement with the higher susceptibility of patients to sodium channel blocking drugs in unmasking BrS. CONCLUSION: Patient-specific hiPSC-CMs are able to recapitulate single-cell phenotype features of BrS with SCN10A mutations and may provide novel opportunities to further elucidate the cellular disease mechanism.

Drug Testing in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes From a Patient With Short QT Syndrome Type 1.

Short QT syndrome (SQTS) predisposes afflicted patients to sudden cardiac death. Until now, only one drug-quinidine-has been shown to be effective in patients with SQTS type 1(SQTS1). The objective of this study was to use human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with SQTS1 to search for potentially effective drugs for the treatment of SQTS1 patients. Patch clamp and single-cell contraction measurements were employed to assess drug effects. Ivabradine, mexiletine, and ajmaline but not flecainide, ranolazine, or amiodarone prolonged the action potential duration (APD) in hiPSC-CMs from an SQTS1 patient. Ivabradine, ajmaline, and mexiletine inhibited KCNH2 channel currents significantly, which may underlie their APD-prolonging effects. Under proarrhythmic epinephrine stimulation in spontaneously beating SQTS1 hiPSC-CMs, ivabradine, mexiletine, and ajmaline but not flecainide reduced the epinephrine-induced arrhythmic events. The results demonstrate that ivabradine, ajmaline, and mexiletine may be candidate drugs for preventing tachyarrhythmias in SQTS1 patients.