Fig 1: FAT10 regulates Nav1.5 protein expression in cardiomyocytes.A Coimmunoprecipitation (Co-IP) was performed to elucidate the association between FAT10 and Nav1.5 in neonatal rat cardiomyocytes (NRCMs) (left) and mouse hearts (right). B The association between FAT10 and Nav1.5 in HEK293 cells expressing Flag-Nav1.5 and HA-FAT10 was detected by Co-IP. C Photomicrographs showing the colocalization of FAT10 and Nav1.5 in cardiomyocytes (upper) and NRCMs (lower). D, E Effect of FAT10 overexpression on Nav1.5 expression levels in NRCMs under normoxic and hypoxic conditions as evaluated by WB (*p < 0.05, **p < 0.01). F, G Effect of Fat10 knockdown on the Nav1.5 expression levels in NRCMs under normoxic and hypoxic conditions as evaluated by WB (*p < 0.05, **p < 0.01). All results are expressed as the mean ± SEM of independent experiments.
Fig 2: FAT10 antagonizes Nav1.5 ubiquitination by competing with Nedd4-2.A Schematic diagram of Nav1.5 deletions and mutants. B HEK293 cells were transfected with the indicated constructs and then lysed for the Co-IP assay using anti-Flag beads to detect HA-FAT10 binding. C Representative immunoblots of FAT10 binding with Nav1.5 in HEK293 cells co-transfected with HA-Fat10 and Flag-Scn5a wild-type or the Flag-Scn5a mutation plasmid as detected by Co-IP. D GST pull-down experiments were performed to assess the association between FAT10 and Nav1.5. E Competitive binding of Nav1.5 was analyzed in a GST pull-down experiment. F, G Fat10+/+ HEK293 cells were transfected with Si-Nedd4-2 or Si-scramble and treated with IFN-?/TNF-a for the indicated times. Co-IP was performed to detect Nedd4-2–Nav1.5 and FAT10–Nav1.5 complex formation. H, I Fat10-/- HEK293 cells were transfected with Si-Nedd4-2/Si-scramble and the Flag-Fat10 plasmid. Co-IP was performed to detect Nedd4-2–Nav1.5 and FAT10–Nav1.5 complex formation. HEK293 cells were treated with IFN-?/TNF-a to induce FAT10 expression. All results are expressed as the mean ± SEM of independent experiments.
Fig 3: Fat10-deficient mice exhibit increased ventricular arrhythmia incidence and mortality rates after myocardial infarction (MI).A Schematic diagram of conditional cardiac Fat10 knockout (cFat10-/-) generated by the homologous recombination technique. B Western blotting was performed to detect the expression of FAT10 in different tissues of cFat10-/- mice and control littermates (Fat10fl/fl). C Hematoxylin–eosin staining of mouse heart sections from cFat10-/- mice and Fat10fl/fl mice. D–F Analysis of electrocardiograph (ECG) parameters from cFat10-/- mice (n = 6) and Fat10fl/fl mice (n = 10). PR interval (D); QTc interval (E); RR interval (F); (*p < 0.05). G Telemetric recordings of ECG tracings of ventricular arrhythmia in freely moving Fat10fl/fl-Sham, cFat10-/--Sham, Fat10fl/fl-MI, and cFat10-/--MI group mice (red arrows indicate PVCs, and black arrows indicate nonsustained ventricular tachycardia (VT), # indicates ventricular bigeminy). H–J Average paroxysmal PVCs (H), cumulative incidence of ventricular arrhythmias (I), and arrhythmia scores (J) of Fat10fl/fl -MI group mice (n = 23) and cFat10-/--MI group mice (n = 27) (*p < 0.05). K Kaplan–Meier curves were used to evaluate differences among the indicated groups of mice in terms of overall survival (**p < 0.01).
Fig 4: Working diagram.The graphical diagram illustrated that FAT10 protected against ischemia-induced ventricular arrhythmia by decreasing Nedd4-2–Nav1.5 complex formation.
Fig 5: Nav1.5 protein expression is reduced in Fat10-deficient ventricular myocytes.A Mass spectroscopic analysis was performed to detect protein expression in heart tissue samples from Fat10fl/fl mice (n = 3) and cFat10-/- mice (n = 3). Heatmap showing the differential expression of membrane proteins. B, C Representative immunoblots (B) and quantitative data (C) of membrane Nav1.5 protein expression in heart tissues from Fat10fl/fl and cFat10-/- mice (*p < 0.05); NCX: Na+/Ca2+ exchanger. D, E Representative immunoblots (D) and quantitative data (E) of total Nav1.5 protein expression in heart extracts from cFat10-/- and Fat10fl/fl mice as detected by WB (*p < 0.05). F, G Representative immunoblots (F) and quantitative data (G) of cytoplasmic Nav1.5 expression in heart tissues from cFat10-/- and Fat10fl/fl mice (*p < 0.05) as detected by WB. H, I Representative immunostaining showing the degree of colocalization between Nav1.5 and Na+/K+ ATPase (H) and Pearson’s correlation coefficient for colocalization (I) in isolated cardiomyocytes (scale bar = 20 µm). All results are expressed as the mean ± SEM of independent experiments.
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