Human leukocyte antigen F-associated transcript 10 regulates the I Ks potassium channel by competing for Kv7.1 ubiquitination

Figures

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  Figure 1.. Effects of FAT10 overexpression on IKs current densities. (a) Schematic diagram of the generation of hiPSC-CMs generated. (b) Representative immunoblots of TNNI3, α-actin and MLC2v expression in hiPSC-CMs as detected by WB. (c) Representative immunofluorescence assays of hiPSC-CMs expressing α-actin (left, red), TNNI3 (middle, red) and MLC2v (right, green). (d) Mean current density versus voltage relationships for hiPSC-CMs transfected with ad-Fat10 (n = 15) or ad-con (n = 12). (e) Representative current traces were recorded in hiPSC-CMs transfected with ad-Fat10 or ad-con. (f) Representative APs were recorded in hiPSC-CMs when transfected with ad-Fat10 (right) (n = 9) or ad-con (left) (n = 9). (g) The duration of APD50 and APD90 in hiPSC-CMs when transfected with ad-Fat10 or ad-con. (h) Mean current density versus voltage relationships for HEK293T-Fat10−/− cells. (i) Representative current traces recorded in HEK293T-Fat10−/− cells expressing KCNQ1 with KCNE1. *p < 0.05, **p < 0.01, ***p < 0.001 and n.s. = not significant.

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  Figure 2.. FAT10 regulates Kv7.1 protein expression in cardiomyocytes. (a) Representative immunoblots (left) and quantitative assessment of the protein (right) levels of Kv7.1 in NRCMs treated with ad-Fat10. (b) Kv7.1 mRNA level as measured by qRT-PCR in cultured NRCMs after treatment with ad-Fat10. (c) Representative immunoblots (left) and quantitative assessment of the protein (right) levels of Kv7.1 in NRCMs treated with si-Fat10. (d) Kv7.1 mRNA levels were measured by qRT-PCR in cultured NRCMs after treatment with si-Fat10. (e) Representative immunoblots (left) and quantitative assessment of the protein (right) levels of Kv7.1 in NRCMs treated with TNF-α/IFN-γ. (f) Kv7.1 mRNA levels were measured by qRT-PCR in cultured NRCMs after treatment with TNF-α/IFN-γ. *p < 0.05, **p < 0.01, ***p < 0.001 and n.s. = not significant. The data shown are the mean of three independent experiments.

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  Figure 3.. FAT10 inhibits Kv7.1 ubiquitination. (a,b) Representative immunoblots (left) and quantitative assessment of the protein (right) levels of Kv7.1 in NRCMs treated with CHX (20 µM) or MG132 (15 µM) for the indicated times. (c) Representative immunoblots (left) and quantitative assessment of the protein (right) levels of Kv7.1 in NRCMs treated with MG132 (15 µM) for the indicated times. (d) Co-IP between endogenous ubiquitin and FAT10. (e–h) Representative immunoblots or quantitative data of Kv7.1 expression in NRCMs after adenoviral overexpression (e,f) or knockdown (g,h) of Fat10. Cells were treated with or without MG132. (i) Cardiomyocytes were transfected with the indicated adenovirus constructs. The cells in each group were treated with MG132. Kv7.1 ubiquitination was detected. (j) Representative immunoblots of the Kv7.1 ubiquitination levels in Fat10+/+ and Fat10−/− HEK293 cells treated with or without TNF-α/IFN-γ. (k) Representative immunoblots of the Kv7.1 ubiquitination levels in the indicated groups of Fat10−/− HEK293 cells. *p < 0.05, **p < 0.01, ***p < 0.001 and n.s. = not significant. The data shown are the mean of three independent experiments.

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  Figure 4.. FAT10 stabilizes Kv7.1 by competing with ubiquitin for binding. (a) Co-IP for Kv7.1 and FAT10 in cultured cardiomyocytes. (b) Colocalization of FAT10 and Kv7.1 in cardiomyocyte cultures; FAT10(1:50) is shown in red, Kv7.1(1:200) is shown in green, and DAPI nuclear counterstaining is shown in blue. (c) The association between FAT10 and Kv7.1 in HEK293 cells expressing Flag-FAT10 and His-KCNQ1 was detected by Co-IP. (d) NRCMs were transfected with ad-ΔFat10. Co-IP assays were performed to analyse the binding of FAT10 with Kv7.1. (e) The association between Flag and His in HEK293 cells expressing Flag-ΔFAT10 and His-KCNQ1 was detected by Co-IP. (f) H9C2 cells were transfected with increasing amounts of Flag-Fat10 plasmid. The cells were lysed for immunoprecipitation using anti-Ub and anti-FAT10 beads to detect Kv7.1 binding. (g,h) Fat10+/+HEK293 cells were transfected with Si-Ub or Si-scramble and treated with IFN-γ/TNF-α for the indicated times. Co-IP was performed to detect Ub–Kv7.1 and the FAT10–Kv7.1 complex. (i,j) Fat10−/−HEK293 cells were transfected with Si-Ub/Si-con and the Flag-Fat10 plasmid. Co-IP was performed to detect Ub–Kv7.1 and FAT10–Kv7.1 complex formation. HEK293…

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  Figure 5.. Effects of FAT10 overexpression on IKs current densities under hypoxic stress. (a) Mean current density versus voltage relationships for hiPSC-CMs in the ad-con group (n = 11) and ad-con + hypoxia group (n = 12). (b) Representative current traces recorded in hiPSC-CMs in the ad-con group and ad-con + hypoxia group. (c) Mean current density versus voltage relationships for hiPSC-CMs in the ad-con group (n = 12) and ad-Fat10 + hypoxia group (n = 15). (d) Representative current traces recorded in hiPSC-CMs in the ad-con + hypoxia group and ad-Fat10 + hypoxia group. *p < 0.05, **p < 0.01, ***p < 0.001 and n.s. = not significant.

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  Figure 6.. Graphical abstract by which FAT10 stabilizes Kv7.1. The graphical diagram illustrates that the overexpression of FAT10 results in a reduction in Ub–Kv7.1 complexes and an increase in FAT10–Kv7.1 complexes. Thus, FAT10 inhibits Kv7.1 ubiquitination, enhancing the expression of Kv7.1 and ultimately strengthening the IKs currents.