USP10 mitigates Ang II–induced atrial remodeling and atrial fibrillation susceptibility by deubiquitinating NDUFS1

Figures

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  Figure 1. USP10 is decreased in patients with AF and Ang II–treated atrial cardiomyocytes. (A)The expression levels of USP10 in atrial tissues from patients with AF (n = 10) and SR controls (n = 20). (B) qPCR analysis of USP10 mRNA levels in atrial tissues from patients with AF and SR controls (n = 5/group). (C) Western blot and quantification of USP10 protein levels in atrial tissues from patients with AF and SR controls (n = 5/group). (D) qPCR analysis of Usp10 mRNA levels in atrial tissues from mice treated with Ang II or Veh (n = 4/group). (E) Representative Western blots and quantification of USP10 in atrial tissues from Ang II– or Veh-treated mice (n = 4/group). (F) Representative immunostaining images of atrial tissues from Ang II– or Veh-treated mice. Scale bar: 50 μm. (G) qPCR analysis of Usp10 mRNA levels in HL-1 atrial myocytes treated with Ang II or Veh (n = 3/group). (H) Representative Western blots and quantification of USP10 protein levels in Ang II– or Veh-treated HL-1 atrial myocytes (n = 3/group). Data represent mean ± SEM. All statistical comparisons were performed using a 2-tailed Student’s t test. **P < 0.01. USP10, ubiquitin-specific peptidase 10; FPKM, fragments per…

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  Figure 2. USP10 overexpression ameliorates Ang II–induced atrial remodeling and AF susceptibility. (A and B) Representative echocardiography images and quantification of left atrial size in WT and Usp10-CTg mice subjected to Veh or Ang II treatment (n = 7–9/group). (C–E) Representative images and quantification of Masson’s trichrome staining in left atrial sections from Usp10-CTg and WT control mice treated with Veh or Ang II (n = 4/group). Scale bars: 1 mm (C) and 100 μm (D). (F) Representative limb lead ECG traces from Usp10-CTg and WT control mice in response to Veh or Ang II treatment. (G) Fraction of mice with successful AF induction relative to the total number. (H) Quantification of AF duration in Usp10-CTg and WT control mice treated with Ang II (n = 23/group). Data are presented as mean ± SEM and were analyzed using 2-way ANOVA followed by Tukey’s multiple-comparison test (B and E), χ2 test (G), and 2-tailed Student’s t test (H). *P < 0.05, **P < 0.01. USP10, ubiquitin-specific peptidase 10; AF, atrial fibrillation; Ang II, angiotensin II; ECG, electrocardiogram; CTg, cardiomyocyte-specific transgenic.

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  Figure 3. USP10 overexpression mitigates abnormal calcium handling and DADs. (A) Representative DAD images recorded from atrial cardiomyocytes obtained from WT and Usp10-CTg mice in response to vehicle or Ang II. (B) Statistical results of DADs in the indicated groups (n = 19–23/group). (C) Representative Ca2+ transients (CaTs) evoked by 10 mM caffeine in Ca2+-free conditions in atrial cells isolated from WT and Usp10-CTg mice treated with vehicle or Ang II. (D) Statistical results of CaT amplitudes in the indicated groups (n = 15–22/group). (E) Representative Ca²+ wave images in atrial cardiomyocytes from WT and Usp10-CTg mice, recorded following 1-Hz pacing after treatment with either vehicle or Ang II. (F) Statistical results of amplitude and 50% decay time of Ca2+ waves in the indicated groups (n = 22–37/group). (G) Representative Ca2+ spark images in atrial cardiomyocytes obtained from WT and Usp10-CTg mice in response to vehicle or Ang II. Scale bar: 0.1 seconds. (H) Statistical results of Ca2+ spark–induced Ca2+ leak, Ca2+ spark frequency, Ca2+ spark amplitude, and Ca2+ spark mass in the indicated groups (n = 26–31/group). Data are presented as mean ± SEM and were analyzed using the…

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  Figure 4. USP10 overexpression contributes to the restoration of mitochondrial function. (A) Sample-to-sample distance plot of HL-1 atrial myocytes expressing Ad-USP10 or Ad-Vector analyzed by RNA-seq (n = 4/group). (B) GO enrichment analysis showing enriched pathways related to mitochondrial function, calcium ion transport, and cardiac rhythm based on RNA-seq data (n = 4/group). (C) GO enrichment analysis showing enriched pathways related to fatty acid β-oxidation, oxidative phosphorylation, and the TCA cycle (n = 4/group). (D) ATP content in atrial cardiomyocytes obtained from WT and Usp10-CTg mice in response to vehicle or Ang II (n = 6/group). (E) OCR profiles indicating mitochondrial respiratory function in atrial cells isolated from WT and Usp10-CTg mice treated with vehicle or Ang II. (F) Quantification of basal respiration and maximal respiration in atrial cells isolated from WT and Usp10-CTg mice treated with vehicle or Ang II (n = 6/group). (G) MitoSOX-based flow cytometry of ROS in atrial cells isolated from WT and Usp10-CTg mice treated with vehicle or Ang II, combining a schematic illustration of the method with quantitative data (bottom; n = 6). (H) WikiPathways enrichment…

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  Figure 5. USP10 interacts with and deubiquitinates NDUFS1. (A) Diagram illustrating the experimental design used to explore USP10-interacting proteins by IP-MS in HL-1 cells. (B) GO analysis showing the top 10 enriched terms of USP10-binding proteins. (C) UpSet plot illustrating the intersections across different biological events of USP10-binding proteins. (D and E) Co-IP and Western blot analyses showing the interaction between USP10 and NDUFS1 in HL-1 cells. (F) Western blot analysis of USP10 and NDUFS1 protein levels in HL-1 atrial myocytes expressing Ad-shUsp10 or Ad-shRNA (n = 4/group). (G) Western blot analysis of USP10 and NDUFS1 protein levels in HL-1 atrial myocytes expressing different amounts of Ad-Flag-USP10 adenovirus (n = 4/group). (H) Western blot analysis of USP10 and NDUFS1 protein levels in mouse atrial tissues in the indicated groups (n = 4/group). (I) Representative Western blot images of K48-type ubiquitination of NDUFS1 in HL-1 cells in the indicated groups. (J) Western blot analysis of NDUFS1 protein levels in HEK293T cells expressing different plasmids (n = 3/group). Data are presented as mean ± SEM and were analyzed using an unpaired, 2-tailed Student’s t test (F)…

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  Figure 6. USP10 deubiquitinates NDUFS1 at K621. (A) Predicted ubiquitination sites of NDUFS1 using GPS-Uber. (B) Molecular docking simulation and calculation of the interaction sites between MDM2 (blue) and NDUFS1 (green). (C) Western blot analysis of K48-type ubiquitination of NDUFS1 in the indicated groups. (D) Representative Western blots showing Flag-NDUFS1 protein levels in the indicated groups. (E) Molecular docking simulation and calculation of the interaction sites between USP10 (purple) and NDUFS1 (green). (F) Western blot analysis of K48-type ubiquitination of NDUFS1 in the indicated groups. (G) Western blot analysis of the ubiquitination of WT and K621R mutant immunoprecipitated with NDUFS1. (H) Alignment of K621 and adjacent amino acids of NDUFS1 across different species. USP10, ubiquitin-specific peptidase 10; NDUFS1, NADH:ubiquinone oxidoreductase subunit S1.

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  Figure 7. USP10 knockdown exacerbates abnormal calcium handling, DADs, and AF susceptibility. (A) Representative DAD images recorded from HL-1 cells expressing Ad-shRNA or Ad-shUsp10 under Ang II treatment. (B) Statistical results of DAD incidence. (C) Representative Ca2+ transient (CaT) images and statistical results of CaT amplitudes in the indicated groups (n = 15–22/group). (D) Representative Ca2+ spark images obtained from HL-1 cells expressing Ad-shRNA or Ad-shUsp10 in response to Ang II stimulation. Scale bar: 0.1 seconds. (E) Statistical results of Ca2+ spark–induced Ca2+ leak, Ca2+ spark frequency, and Ca2+ spark area in the indicated groups (n = 20–31/group). (F) Representative echocardiography images and quantification of left atrial size from WT and Usp10-CKO mice under Ang II treatment (n = 7–8/group). (G) Representative images and quantitative results of Masson’s trichrome staining on left atrial sections of Usp10-CKO and WT control mice in response to Ang II treatment (n = 4/group). (H) Representative limb lead ECG traces recorded from Usp10-CKO and WT control mice in response to Ang II treatment. (I) Fraction of mice with successful AF induction relative to the total number…