Pathogenic roles and mechanisms of fever and inflammation in Brugada syndrome with different gene variants

Y. Li (Mannheim)¹, L. Rose (Mannheim)¹, T. Prädel (Mannheim)¹, M. Kleinsorge (Göttingen)², X. Fan (Mannheim)³, Z. Meng (Mannheim)³, C. Yan (Mannheim)³, R. Liu (Mannheim)³, X. Lei (Mannheim)³, B. Zhao (Mannheim)³, G. Yang (Mannheim)³, Z. Liao (Mannheim)¹, H. Dinkel (Mannheim)¹, A. Busley (Göttingen)⁴, R. Zhong (Mannheim)¹, F. Zhang (Mannheim)¹, Q. Xu (Mannheim)¹, L. Maywald (Mannheim)¹, A. Aweimer (Bochum)⁵, M. Huang (Mannheim)¹, A. Moscu-Gregor (Planegg-Martinsried)⁶, N. Hamdani (Bochum)⁷, L. Schneider (Bochum)⁸, Y. Zemedi (Bochum)⁷, S. Zhazykbayeva (Bochum)⁹, A. Hohn (Mannheim)¹, Z. Yang (Mannheim)¹, L. Qiao (Mannheim)¹, A. Mügge (Bochum)¹⁰, L. Cyganek (Göttingen)¹¹, I. Akin (Mannheim)³, X. Zhou (Mannheim)³, I. El-Battrawy (Bochum)^12
1Universitätsklinikum Mannheim I. Medizinische Klinik Mannheim, Deutschland; ²Universitätsmedizin Göttingen Herzzentrum, Klinik für Kardiologie und Pneumologie Göttingen, Deutschland; ³Universitätsklinikum Mannheim GmbH I. Medizinische Klinik Mannheim, Deutschland; ⁴Universitätsmedizin Göttingen Klinik für Kardiologie und Pneumologie Göttingen, Deutschland; ⁵Berufsgenossenschaftlliches Universitätsklinikum Bergmannsheil gGmbH Medizinische Klinik II, Kardiologie und Angiologie Bochum, Deutschland; ⁶Ludwig-Maximilians-Universität München Kardiovaskuläre Physiologie Planegg-Martinsried, Deutschland; ⁷Kath. Klinikum Bochum Cellular Physiology Bochum, Deutschland; ⁸Berufsgenossenschaftliches Universitätsklinikum Bergmannsheil Medizinische Klinik II, Kardiologie und Angiologie Bochum, Deutschland; ⁹Ruhr University Bochum Department of Cellular and Translational Physiology Bochum, Deutschland; ¹⁰Institut für Physiologie, Institut für Forschung und Lehre (IFL), Ruhr-Universität Bochum Abteilung für Zelluläre und Translationale Physiologie, Molekulare und Experimentelle Kardiologie Bochum, Deutschland; ¹¹Universitätsmedizin Göttingen Herzzentrum Göttingen - Stem Cell Unit Göttingen, Deutschland; ¹²Klinikum der Ruhr-Universität Bochum Medizinische Klinik II, Kardiologie Bochum, Deutschland

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. This study was designed to investigate whether the effects of fever or inflammation on BrS features vary with different gene variants and to explore potential mechanisms underlying fever or inflammation effects.

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.

Findings The hiPSC-CMs from all BrS hiPSCs showed a significantly reduced peak sodium current (I_Na) compared with isogenic1 or WT at baseline. hiPSC-CMs of BrS2 showed also a significant reduction in L-type calcium channel currents (I_Ca-L). Arrhythmia-like events were detected more frequently in hiPSC-CMs from all three BrS patients. These data confirmed the BrS phenotype. When the temperature of hiPSC-CMs culture was increased from 37°C to 40°C for 24 hours, a significant decrease of I_Na was detected in hiPSC-CMs of BrS1 and BrS3, but not in BrS2. Increased arrhythmia-like events and interval variability as a sign of high arrhythmogenicity were recorded in hiPSC-CMs of BrS1 and BrS3 but not in BrS2 after increasing the temperature from 37°C to 40°C. At 40°C, the protein kinase A (PKA) level was reduced in hiPSC-CMs of BrS1. A PKA activator abolished the changes and a PKA inhibitor enhanced the BrS phenotype as well as the 40°C phenotype. Treating the BrS1 cells with lipopolysaccharide (LPS) showed a further reduction of peak I_Na, and increased arrhythmic events. Treating hiPSC-CMs of BrS2 with LPS reduced I_Ca-L and APD50, increased arrhythmic events and interval variability. ROS-Blocker abolished the LPS effects in all BrS hiPSC-CMs, while an interleukin-6 receptor blocker abolished the proarrhythmic effect of LPS only in BrS1 and BrS3 hiPSC-CMs but not in hiPSC-CMs of BrS2.

Interpretation Hyperthermia exacerbated BrS phenotype in hiPSC-CMs carrying the SCN5A or SCN10A but not CACNB2 variant. The enhanced BrS phenotype at high temperature (40°C) may be related to reduced PKA activity in BrS patients. LPS enhanced the BrS phenotype in hiPSC-CMs through increasing ROS level. Interleukin 6 contributed to ROS effects in cells with the SCN5A and SCN10A but not CACNB2 variants.