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Background Fever or inflammation state may enhance the Brugada syndrome (BrS) phenotype in some but not all patients. However, the underlying mechanism in human cardiomyocytes has not yet been clarified. Methods Human induced pluripotent stem cell (hiPSC) lines generated from fibroblasts of three BrS patients harboring variants in SCN10A (abbreviated as BrS1) and CACNB2 (abbreviated as BrS2), SCN5A (abbreviated as BrS3) and one healthy donor (abbreviated as WT) and a site-corrected (using CRISPR/Cas9) hiPSC line of each BrS patient (abbreviated as isogenic1, isogenic2 and isogenic3) were used for differentiation into cardiomyocytes (hiPSC-CMs). Western blot, patch clamp and calcium transient analyses were carried out. Results All 3 BrS cell lines showed a significantly reduced peak sodium current (I Na ) compared with isogenic or WT cells at baseline. Hyperthermia challenge (40 °C) significantly decreased I Na and enhanced arrhythmogeneity in BrS1 and BrS3 but not in BrS2 cells. The hyperthermia effects involved PKA reduction. The lipopolysaccharide (LPS) challenge exacerbated the phenotype in electrophysiological characteristics in all 3 BrS cell lines. ROS-Blocker abolished the LPS effects in all BrS hiPSC-CMs, while an interleukin-6 receptor blocker abolished the proarrhythmic effect of LPS in BrS1 and BrS3 hiPSC-CMs but not in hiPSC-CMs of BrS2. Conclusions Hyperthermia exacerbated the BrS phenotype in hiPSC-CMs carrying SCN10A and SCN5A variants, whereas LPS aggravated the phenotype in all three BrS variants through distinct mechanisms; Hyperthermia and LPS effects on BrS phenotype may be genotype-dependent. Graphical abstract

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 Nav1.5 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. SCN5A variant (c.3148G>A/p.Ala1050Thr) causes loss‐of‐function of sodium channels in human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) from BrS patient. SCN5A variant (c.3148G>A/p.Ala1050Thr) increases the channel sensitivity to hyperthermia and LPS challenge in hiPSC‐CMs from BrS patient. Autophagy and PKA pathway mediate the effect of LPS and hyperthermia on hiPSC‐CMs from BrS patient with SCN5A variant (c.3148G>A/p.Ala1050Thr), respectively.

Biallelic loss-of-function (LoF) variants in the BTRR complex members BLM, TOP3A, RMI1, and RMI2 cause Bloom syndrome (BS). The BTRR complex mainly acts on DNA replication and DNA repair processes, and dysfunction of this complex underlies, e.g., increased genomic instability and cancer predisposition associated with the BS phenotype. Here, we report CRISPR/Cas9-based genome-edited isogenic induced pluripotent stem cell (iPSC) models with compound heterozygous LoF variants in BLM, TOP3A, and RMI1. The cellular phenotype of all three knockout (KO) iPSC lines included chromosome segregation defects, increased sister chromatid exchange rates, and impaired homologous recombination repair. Using single-cell whole genome sequencing, we showed that BTRR complex deficiency causes increased copy number alterations (CNAs) in the genomes and, therefore, represents a driver for genomic instability. CNA load was further induced by applying replication stress, and we observed that BTRRKO iPSCs acquired fewer de novo CNA events compared to wild-type cells, suggesting a possible limitation of genomic instability induction. Importantly, induced and non-induced CNAs in single-cell genomes were not stochastically distributed throughout the genome, but instead enriched at fragile sites. This finding might offer an opportunity for the development of novel NGS-based approaches to measure rates of genomic instability in disease conditions.

Noonan syndrome patients harboring causative variants in LZTR1 are particularly at risk to develop severe and early-onset hypertrophic cardiomyopathy. However, the underling disease mechanisms of LZTR1 missense variants driving the cardiac pathology are poorly understood. Hence, therapeutic options for Noonan syndrome patients are limited. In this study, we investigated the mechanistic consequences of a novel homozygous causative variant LZTR1L580P by using patient-specific and CRISPR/Cas9-corrected iPSC-cardiomyocytes. Molecular, cellular, and functional phenotyping in combination with in silico prediction of protein complexes uncovered a unique LZTR1L580P-specific disease mechanism provoking the cardiac hypertrophy. The homozygous variant was predicted to alter the binding affinity of the dimerization domains facilitating the formation of linear LZTR1 polymer chains. The altered polymerization resulted in dysfunction of the LZTR1-cullin 3 ubiquitin ligase complexes and subsequently, in accumulation of RAS GTPases, thereby provoking global pathological changes of the proteomic landscape ultimately leading to cellular hypertrophy. Importantly, uni- or biallelic genetic correction of the LZTR1L580P missense variant rescued the molecular and cellular disease-associated phenotype, providing proof-of-concept for CRISPR-based gene therapies.Competing Interest StatementThe authors have declared no competing interest.

Noonan syndrome (NS), the most common among the RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770C>T missense change. We characterize the molecular, structural and functional consequences of aberrant RAF1 –associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes, and myocardial tissue biopsies. The disease phenotype was partly reverted by using both MEK inhibition, and a gene-corrected isogenic RAF1L257S cell line. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease. These insights represent a basis to develop future targeted therapeutic approaches.