HiPSC-derived cardiomyocyte to model Brugada syndrome: both asymptomatic and symptomatic mutation carriers reveal increased arrhythmogenicity.

Brugada syndrome is an inherited cardiac arrhythmia disorder that is mainly associated with mutations of the cardiac voltage-gated sodium channel alpha subunit 5 (SCN5A) gene. The clinical symptoms include ventricular fibrillation and an increased risk of sudden cardiac death. Human-induced pluripotent stem cell (hiPSC) lines were derived from symptomatic and asymptomatic individuals carrying the R1913C mutation in the SCN5A gene. The present work aimed to observe the phenotype-specific differences in hiPSC-derived cardiomyocytes (CMs) obtained from symptomatic and asymptomatic mutation carriers. In this study, CM electrophysiological properties, beating abilities and calcium parameters were measured. Mutant CMs exhibited higher average sodium current densities than healthy CMs, but the differences were not statistically significant. Action potential durations were significantly shorter in CMs from the symptomatic individual, and a spike-and-dome morphology of action potential was exclusively observed in CMs from the symptomatic individual. More arrhythmias occurred in mutant CMs at single cell and cell aggregate levels compared with those observed in wild-type CMs. Moreover, there were no major differences in ionic currents or intracellular calcium dynamics between the CMs of asymptomatic and symptomatic individuals after the administration of adrenaline and flecainide.In conclusion, mutant CMs were more prone to arrhythmia than healthy CMs but did not explain why only one of the mutation carriers was symptomatic.

Identification by mass spectrometry and immunoblotting of xenogeneic antigens in the N- and O-glycomes of porcine, bovine and equine heart tissues.

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by ß-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors.

The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Galα1,3Galß1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize O-glycans released by reductive ß-elimination, and new diagnostic ions to distinguish Galα1,3Gal- from Galα1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Galα1,3Galß1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases.

The Junctophilin-2 Mutation p.(Thr161Lys) Is Associated with Hypertrophic Cardiomyopathy Using Patient-Specific iPS Cardiomyocytes and Demonstrates Prolonged Action Potential and Increased Arrhythmogenicity.

Hypertrophic cardiomyopathy (HCM) is one of the most common genetic cardiac diseases; it is primarily caused by mutations in sarcomeric genes. However, HCM is also associated with mutations in non-sarcomeric proteins and a Finnish founder mutation for HCM in non-sarcomeric protein junctophilin-2 (JPH2) has been identified. This study aimed at assessing the issue of modelling the rare Finnish founder mutation in cardiomyocytes (CMs) differentiated from iPSCs; therefore, presenting the same cardiac abnormalities observed in the patients. To explore the abnormal functions in JPH2-HCM, skin fibroblasts from a Finnish patient with JPH2 p.(Thr161Lys) were reprogrammed into iPSCs and further differentiated into CMs. As a control line, an isogenic counterpart was generated using the CRISPR/Cas9 genome editing method. Finally, iPSC-CMs were evaluated for the morphological and functional characteristics associated with JPH2 mutation. JPH2-hiPSC-CMs displayed key HCM hallmarks (cellular hypertrophy, multi-nucleation, sarcomeric disarray). Moreover, JPH2-hiPSC-CMs exhibit a higher degree of arrhythmia and longer action potential duration associated with slower inactivation of calcium channels. Functional evaluation supported clinical observations, with differences in beating characteristics when compared with isogenic-hiPSC-CMs. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as a translationally relevant platform to study genetic cardiac diseases.

Electrophysiological Changes of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes during Acute Hypoxia and Reoxygenation.

Ischemic heart disease is the most common cardiovascular disease and a major burden for healthcare worldwide. However, its pathophysiology is still not fully understood, and human-based models for disease mechanisms and treatments are needed. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to model acute ischemia-reperfusion in our novel cell culture assembly. The assembly enables exchange of oxygen partial pressure for the cells within minutes, mimicking acute ischemic event. In this study, hypoxia was induced using 0% O2 gas for three hours and reoxygenation with 19% O2 gas for 24 hours in serum- and glucose-free medium. According to electrophysiological recordings, hypoxia decreased the hiPSC-CM-beating frequency and field potential (FP) amplitude. Furthermore, FP depolarization time and propagation slowed down. Most of the electrophysiological changes reverted during reoxygenation. However, immunocytochemical staining of the hypoxic and reoxygenation samples showed that morphological changes and changes in the sarcomere structure did not revert during reoxygenation but further deteriorated. qPCR results showed no significant differences in apoptosis or stress-related genes or in the expression of glycolytic genes. In conclusion, the hiPSC-CMs reproduced many characteristic changes of adult CMs during ischemia and reperfusion, indicating their usefulness as a human-based model of acute cardiac ischemia-reperfusion.

hiPSC-Derived Cardiomyocyte Model of LQT2 Syndrome Derived from Asymptomatic and Symptomatic Mutation Carriers Reproduces Clinical Differences in Aggregates but Not in Single Cells.

Mutations in the HERG gene encoding the potassium ion channel HERG, represent one of the most frequent causes of long QT syndrome type-2 (LQT2). The same genetic mutation frequently presents different clinical phenotypes in the family. Our study aimed to model LQT2 and study functional differences between the mutation carriers of variable clinical phenotypes. We derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from asymptomatic and symptomatic HERG mutation carriers from the same family. When comparing asymptomatic and symptomatic single LQT2 hiPSC-CMs, results from allelic imbalance, potassium current density, and arrhythmicity on adrenaline exposure were similar, but a difference in Ca2+ transients was observed. The major differences were, however, observed at aggregate level with increased susceptibility to arrhythmias on exposure to adrenaline or potassium channel blockers on CM aggregates derived from the symptomatic individual. The effect of this mutation was modeled in-silico which indicated the reactivation of an inward calcium current as one of the main causes of arrhythmia. Our in-vitro hiPSC-CM model recapitulated major phenotype characteristics observed in LQT2 mutation carriers and strong phenotype differences between LQT2 asymptomatic vs. symptomatic were revealed at CM-aggregate level.

A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins.

Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO) cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b), CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1), core 3 (B3GNT6), core 4 (GCNT1 and B3GNT6), or extended core 1 (B3GNT3) chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc) or type 2 (Galb4GlcNAc) outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.

O-glycan repertoires on a mucin-type reporter protein expressed in CHO cell pools transiently transfected with O-glycan core enzyme cDNAs.

Glyco-engineering of host cells is used to increase efficacy, decrease immunogenicity and increase circulatory half-lives of protein biopharmaceuticals. The effect of transiently expressed O-glycan core chain glycosyltransferases on O-glycan biosynthesis pathways in CHO cells is reported. Liquid chromatography-mass spectrometry and Western blotting were used to map the O-glycome of a mucin-type fusion protein transiently co-transfected with ß1,3-N-acetylglucosaminyltransferase 3 (extended C1 ß3GnT3), core 2 ß1,6-N-acetylglucosaminyltransferase I (C2 ß3GnT1) or core 3 ß1,3-N-acetylglucosaminyltransferase 6 (C3 ß3GnT6) in CHO cells. Extended core 1 (GlcNAcß1,3Galß1,3GalNAc) and core 3 (GlcNAcß1,3GalNAc), and increased expression of core 2 [Galß1,3(GlcNAcß1,6)GalNAc], O-glycans were generated on P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL1/mIgG2b). Endogenous poly-N-acetyllactosamine (poly-LacNAc) synthase elongated extended core 1 and core 3 generating O-glycans with up to five LacNAc repeats. Low amounts of core 3 O-glycans appeared upon extended C1 ß3GnT3 expression. The α2,6-sialylated type 2 chain was detected upon co-transfection with the ß-galactoside α2,6-sialyltransferase I. N-acetylglucosamine-6-O-sulfotransferase 2 transferred sulfate to carbon 6 of GlcNAc in poly-LacNAc sequences. CHO cells with its known O-glycan repertoire can be used to express recombinant mucin-type proteins together with selected glycosyltransferases in order to recreate carbohydrate determinants on defined O-glycan chains. They will become important tools for assessing the core chain-dependent binding activity of carbohydrate-binding proteins.