Influence of Sex Hormones on Brugada Syndrome Phenotype

Xinhao Lei (Mannheim)1, X. Zhou (Mannheim)1

1Universitätsklinikum Mannheim I. Medizinische Klinik Mannheim, Deutschland


Background Brugada Syndrome is an autosomal dominant inherited cardiac arrhythmia disorder. The incidence of the syndrome is significantly higher in males. The potential influence of sex hormones on the phenotype of Brugada Syndrome and its underlying mechanisms remains to be elucidated. This study aims to explore the influence of sex hormones on SCN5A gene variants in Brugada Syndrome and elucidate potential mechanisms.

Methods Human-induced pluripotent stem cell (hiPSC) lines were generated from fibroblasts obtained from a Brugada Syndrome (BrS) patient with SCN5A variants (c.3148G>A/p.Ala1050Thr), a healthy donor (WT), and a site-corrected hiPSC line using CRISPR/Cas9 technology (Isogenic). These hiPSC lines were differentiated into cardiomyocytes (hiPSC-CMs), followed by a 24-hour treatment with 17β-Estradiol (E2, 1/100nM) and testosterone (Te, 10/100nM). Subsequent Western blot and patch clamp analyses were conducted for characterization.

Results Treatment with 100nM of E2 on BrS hiPSC-CMs resulted in a significant upregulation in the expression of Nav1.5, peak sodium channel currents (INa), and the maximal depolarization velocity (Vmax) of action potentials (APs). The effects of E2 were mimicked by the Estrogen Receptor (ER)β agonist diarylpropionitrile (DPN), as opposed to the ERα agonist propyl pyrazole triol (PPT). Conversely, treatment with 100 nM Te on BrS hiPSC-CMs led decrease in Nav1.5 expression, INa and Vmax of APs. The administration of the testosterone antagonist, flutamide, effectively nullified the effects induced by Te. Remarkably, neither E2 nor Te elicited any significant impact on Nav1.5 expression and INa in both the WT and Isogenic hiPSC-CMs. Further, the concurrent application of the autophagy inhibitor Bafilomycin A1 (BafA1) and Te resulted in an elevated expression of the autophagy marker LC3BII in BrS hiPSC-CMs, in contrast to the effect observed with BafA1 treatment alone. In contrast, the combined administration of BafA1 and E2 led to a reduction of autophagy levels. Additionally, the activation of autophagy through rapamycin led to an increase in the expression of Nav1.5 and INa within BrS hiPSC-CMs. Conversely, the inhibition of autophagy resulted in a decrease in the BrS phenotype, highlighting the pivotal role of autophagy modulation. Te treatment was found to increase phosphorylation levels of AMP-activated protein kinase (AMPK) but decrease the phosphorylation levels of mammalian target of rapamycin (mTOR) in BrS hiPSC-CMs. In contrast, E2 treatment led to a decrease in AMPK phosphorylation but an increase in mTOR phosphorylation in BrS hiPSC-CMs. Furthermore, the inhibition of AMPK effectively mitigated the exacerbating effects of Te on the BrS phenotype.

Conclusion Sex hormones modulate the BrS phenotype in hiPSC-CMs with SCN5A variants through autophagy regulation. The m-TOR/AMPK signaling pathway plays a pivotal role in mediating these effects.
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