The Deubiquitinase UCHL1 Drives Susceptibility to Atrial Fibrillation by Stabilizing CaMKII‐δ

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  FIGURE 1. Upregulation of UCHL1 expression in atrial myocytes from mice and patients with atrial fibrillation (AF). (A) Microarray expression‐profile analysis of deubiquitinating enzyme (DUB) genes in atrial tissues of Ang II‐ and saline‐infused mice (n = 4 mice per group). p values were determined by unpaired two‐tailed Student's t‐test. (B) Heatmap showing the protein levels of all DUBs in the two atrial‐tissue groups identified by proteomic analysis (n = 3 mice per group). (C) Venn diagram showing the overlap of the differentially expressed DUB genes and proteins in the two atrial‐tissue groups. p <0.05 as the cutoff for both RNA and protein. (D) qPCR analysis of the mRNA expression of UCHL1 in the atria of the two groups (n = 6 mice per group). (E) Representative immunoblot of UCHL1 in atria of the two groups (n = 6 mice per group). (F) Representative immunoblot of UCHL1 in primary atrial cardiomyocytes (ACMs) stimulated with saline or Ang II (100 nm) for 12 or 24 h (n = 6 independent experiments). (G) Representative images and quantification of immunofluorescence staining of UCHL1 (red) and α‐actinin (green) in ACMs 24 h after saline or Ang II treatment (n = 60 ACMs per group). (H)…

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  FIGURE 2. Atrial cardiomyocyte‐specific deficiency of UCHL1 alleviates Ang II‐induced AF susceptibility. (A) Schematic diagram of the induction procedure for the AF mouse model. Atrial myocyte‐specific UCHL1‐knockout mice (UCHL1fl/fl /NPPA‐Cre+, UCHL1‐CKO; 8‐week‐old male) and age‐matched littermates (UCHL1fl/fl , CTR, male) were infused with saline or Ang II (2000 ng/kg/min) for 3 weeks and then subjected to electrical stimulation. (B) Representative immunoblot (left) and quantification (right) of UCHL1 protein levels in atria (n = 6 mice per group). (C) Representative intracardiac electrogram recordings in CTR and UCHL1‐CKO mice after infusion and burst pacing. The red dotted lines indicate AF, and the green solid lines indicate SR. (D) Percentage of successful AF induction and duration of AF (n = 20 mice per group). (E) Representative images of M‐mode echocardiography (Echo) of left atrial diameter (LAD, left) and quantification of LAD (right, n = 6 mice per group). (F–H) Representative images (left) and quantification (right) of histological staining, including Masson's staining (blue), immunofluorescence staining for CD68‐positive cells (red for CD68, blue for DAPI), and DHE staining…

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  FIGURE 3. Cardiac‐specific UCHL1 overexpression aggravates Ang II‐induced AF susceptibility. (A) Schematic diagram of rAAV9 injection and AF mouse model establishment. Wild‐type (WT) (C57BL/6J, 8‐week‐old male) mice were injected with rAAV9‐EV (empty vector) or rAAV9‐UCHL1 for 2 weeks and then infused with saline and Ang II for another 3 weeks. (B) Representative immunoblots (left) and quantification (right) of UCHL1 protein levels using anti‐Flag antibody in atria (n = 6 mice per group). (C) Representative intracardiac atrial electrogram recordings in rAAV9‐EV or rAAV9‐UCHL1‐injected mice after burst pacing. Red dotted line represents AF, and green solid line represents SR. (D) Percentage of successful AF induction (left) and duration of AF (right; n = 20 mice per group). (E) Representative images of M‐mode echocardiography of LAD (left) and quantification (right) of LAD (n = 6 mice per group). (F) Representative images of Masson's staining (left) and quantification (right) of atrial fibrosis (n = 6 mice per group). Scale bar: 20 µm. (G) Representative images of IHC staining for CD68‐positive cells (n = 6 mice per group). Scale bar: 20 µm. (H) Representative images of DHE staining (left)…

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  FIGURE 4. UCHL1 directly interacts with CaMKII‐δ. (A) Schematic illustration of interactome analysis for UCHL1 and identification of a peptide sequence (DLKPENLLLASK) corresponding to CaMK2d (CaMKII‐δ). (B) Relative abundance of 11 proteins immunoprecipitated by anti‐UCHL1 compared with IgG control, as indicated by UCHL1‐immunoprecipitation (IP) score/IgG‐IP score ratios. (C) Prediction of the interaction between UCHL1 proteins (2–221 aa) and CaMKII‐δ proteins (8–301 aa) using the ZDOCK server. The predicted structure was visualized using PyMOL software. (D) IP analysis for the interaction between UCHL1 and CaMKII‐δ in atria with anti‐UCHL1 antibody or IgG control (n = 3 independent experiments). (E) IP analysis for the interaction between CaMKII‐δ and UCHL1 in ACMs using anti‐CaMKII‐δ antibody or IgG (n = 3 independent experiments). (F) IP assay for the interaction between UCHL1 and CaMKII‐δ in Ang II‐stimulated ACMs (n = 3 independent experiments). (G) IP assay of the interaction between UCHL1 and CaMKII‐δ. HEK293T cells were co‐transfected with Flag‐tagged full‐length (FL) or GFP‐tagged truncated UCHL1 and Myc‐tagged FL CaMKII‐δ. Lysates were immunoprecipitated with anti‐Flag or…

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  FIGURE 5. UCHL1 promotes CaMKII‐δ deubiquitination and stabilization. (A) Schematic illustration of the target protein profile of UCHL1 in cultured ACMs using ubiquitin (Ub) antibody‐conjugated beads to enrich modified peptides, followed by high‐resolution liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis. (B) Venn diagram depicting the proteins with reduced ubiquitination in UCHL1‐overexpressing ACMs and UCHL1‐interacting proteins in ACMs. (C) Mass spectrometry identified ubiquitination of CaMKII‐δ at lysine 251 (K251) in Ad‐EV‐ or Ad‐UCHL1‐infected ACMs, and a reduction of the modified peptide was observed in UCHL1‐overexpressing ACMs using HRM XICs. (D) Immunoprecipitation of lysates from HEK293T cells transfected with Myc‐CaMKII‐δ (WT), Myc‐CaMKII‐δ (K251R), Flag‐UCHL1, and HA‐Ub; immunoprecipitation was performed with an anti‐Myc antibody, followed by immunoblot analysis with anti‐HA, anti‐Myc, and anti‐Flag antibodies. (E, F) Ubiquitination assay (left) and quantification (right) of CaMKII‐δ in cultured ACMs infected with an adenovirus containing an empty vector (Ad‐EV), Ad‐UCHL1, Ad‐siRNA‐control, or Ad‐siRNA‐UCHL1, and treated with MG132 (10 µm) 6 h before harvest…

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  FIGURE 6. UCHL1 regulates atrial calcium handling through activation of CaMKII‐RyR2 signaling in mice. (A) Representative immunoblots (left) and quantitative analysis (right, n = 6 mice per group) of CaMKII‐δ, total and phosphorylated (p)‐RyR2 (S2814) protein levels in the atria of CTR and UCHL1‐CKO mice after saline or Ang II infusion. (B) Representative immunoblotting (left) and quantification (right, n = 6 mice per group) of CaMKII‐δ, total and p‐RyR2 (S2814) protein levels in the atria of rAAV9‐EV‐ and rAAV9‐UCHL1‐injected mice after saline or Ang II infusion. (C) Representative immunoblots (top) and quantification (bottom, n = 6 mice per group) of Flag (UCHL1), CaMKII‐δ, total and p‐RyR2 (S2814) protein levels in the atria of four groups after Ang II infusion. (D) Representative heatmaps of conduction velocity (CV) and calcium (Ca2+) transient duration at 90% repolarization (CaD90) at 8 Hz pacing cycle length (PCL) of 125 ms in the atria from CTR and UCHL1‐CKO mice after saline or Ang II infusion (top). Quantification of CV and CaD90 during the CaT phase (bottom, n = 6 mice per group). (E) Representative heatmaps of CV and CaD90 at 8 Hz PCL of 125 ms in the atria of rAAV9‐EV‐ and…

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  FIGURE 7. UCHL1 aggravates Ang II‐induced AF by stabilizing CaMKII‐δ. (A) Schematic illustration for rAAV9 injection and AF mouse model generation. WT mice (C57BL/6J, 8‐week‐old male) were injected with rAAV9‐EV or rAAV9‐UCHL1 (under the cardiac‐specific cTNT promoter) together with rAAV9‐siControl or rAAV9‐siCaMKII‐δ (under the cardiac‐specific cTNT promoter) for 2 weeks and then treated with Ang II for another 3 weeks. (B) Representative images of intracardiac electrogram recordings after burst pacing. Red dotted line: AF; green solid line: SR. (C) Percentage of successful AF induction (top, n = 20 mice per group) and quantitative analysis of total AF duration (bottom, n = 20 mice per group). (D) Representative images of M‐mode echocardiography (left) and quantification (right) of left atrial diameter (LAD, mm; n = 6 mice per group). (E) Representative images and quantification of Masson's staining in left atria (n = 6 mice per group). (F) Immunofluorescent staining (left) and quantification (right) of CD68‐positive macrophages in left atria (n = 6 mice per group). (G) DHE staining (left) and quantification of superoxide levels (right) in atrial tissues (n = 6 mice per group). (H) qPCR…

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  FIGURE 8. Administration of 6RK73 suppresses angiotensin II‐induced AF. (A) Experimental scheme of drug treatment in WT (C57BL/6J, 8‐week‐old male) mice treated with 6RK73, a covalent irreversible and specific inhibitor of UCHL1 (0.5 mg/kg, three times per week) for 2 days, and then infused with Ang II (2000 ng/kg/min) and 6RK73 for 3 weeks. (B) Representative immunoblots and quantification of UCHL1 protein levels using anti‐UCHL1 and anti‐GAPDH antibodies (n = 6 mice per group). (C) Representative intracardiac atrial electrogram recordings of mice treated as in A and subjected to burst pacing. Red dotted lines represent AF, and green solid lines represent sinus rhythm (SR). (D) Percentage of successful AF induction (top, n = 20 mice per group) and the duration of AF (bottom). (E) Representative M‐mode echocardiography (ECHO) images of LAD (mm) in the left atrium (n = 6 mice per group). (F–H) Representative histological images (left) and quantification (right, n = 6 mice per group), including Masson's staining for fibrosis (blue), IF staining of CD68‐positive macrophages (red for CD68 staining, blue for DAPI), and DHE staining (red for ROS) in 6RK73‐ or DMSO‐treated mice infused with…