https://doi.org/10.1007/s00392-024-02526-y
1Universitätsklinikum Mannheim I. Medizinische Klinik Mannheim, Deutschland; 2Universitätsklinikum Mannheim GmbH I. Medizinische Klinik Mannheim, Deutschland; 3Universitätsmedizin Göttingen Herzzentrum Göttingen - Stem Cell Unit Göttingen, Deutschland; 4Klinikum der Ruhr-Universität Bochum Medizinische Klinik II, Kardiologie Bochum, Deutschland
Background
Brugada Syndrome is an inherited channelopathy. The incidence of the disease is significantly higher in males, but its underlying mechanisms remain to be elucidated. This study aims to explore the influence of testosterone on phenotypic features of Brugada Syndrome with SCN5A gene variants and elucidate potential mechanisms.
Brugada Syndrome is an inherited channelopathy. The incidence of the disease is significantly higher in males, but its underlying mechanisms remain to be elucidated. This study aims to explore the influence of testosterone on phenotypic features of Brugada Syndrome with SCN5A gene variants 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 variant (c.3148G>A/p.Ala1050Thr), a healthy donor (WT), and a site- corrected hiPSC line. These hiPSC lines were differentiated into cardiomyocytes (hiPSC-CMs), followed by a 24-hour treatment with testosterone (Te, 10/100 nM). Subsequent Western blot and patch clamp analyses were conducted for characterization.
Human-induced pluripotent stem cell (hiPSC) lines were generated from fibroblasts obtained from a Brugada Syndrome (BrS) patient with SCN5A variant (c.3148G>A/p.Ala1050Thr), a healthy donor (WT), and a site- corrected hiPSC line. These hiPSC lines were differentiated into cardiomyocytes (hiPSC-CMs), followed by a 24-hour treatment with testosterone (Te, 10/100 nM). Subsequent Western blot and patch clamp analyses were conducted for characterization.
Results
Treatment with 100 nM Te on BrS hiPSC-CMs led decrease in Nav1.5 expression, I Na and V max of APs. The administration of the testosterone antagonist, flutamide, effectively nullified the effects induced by Te. Remarkably, Te failed to exert any significant impact on Nav1.5 expression and I Na 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, compared with the effect observed with BafA1 treatment alone. Additionally, activation of autophagy through rapamycin increased the expression of Nav1.5 and I Na in BrS hiPSC-CMs, whereas inhibition of autophagy reduced the BrS phenotype, underscoring the pivotal role of autophagy modulation. The 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. Moreover, the inhibition of AMPK effectively mitigated the exacerbating effects of Te on the BrS phenotype.
Treatment with 100 nM Te on BrS hiPSC-CMs led decrease in Nav1.5 expression, I Na and V max of APs. The administration of the testosterone antagonist, flutamide, effectively nullified the effects induced by Te. Remarkably, Te failed to exert any significant impact on Nav1.5 expression and I Na 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, compared with the effect observed with BafA1 treatment alone. Additionally, activation of autophagy through rapamycin increased the expression of Nav1.5 and I Na in BrS hiPSC-CMs, whereas inhibition of autophagy reduced the BrS phenotype, underscoring the pivotal role of autophagy modulation. The 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. Moreover, the inhibition of AMPK effectively mitigated the exacerbating effects of Te on the BrS phenotype.
Conclusion
Testosterone may exacerbate the BrS phenotype in hiPSC-CMs with SCN5A variants through the regulation of autophagy. The mTOR/AMPK signaling pathway plays a crucial role in mediating these effects.
Testosterone may exacerbate the BrS phenotype in hiPSC-CMs with SCN5A variants through the regulation of autophagy. The mTOR/AMPK signaling pathway plays a crucial role in mediating these effects.