Cardiomyocyte‐Enriched USP20 Ameliorates Pathological Cardiac Hypertrophy by Targeting STAT3 Deubiquitination

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  Figure 1. Identification of cardiomyocyte‐enriched USP20 as a downregulated factor in mouse and human cardiac hypertrophy. A) Wild type (WT) male mice were induced hypertrophic myocardium by Ang II (please refer to the Supplementary data online for detailed methods). The heart samples from vehicle and Ang II‐administered mice were harvested for RNA transcriptome sequencing and showed the expression profiles of deubiquitinating enzymes (DUBs). We used log2 fold change as the data source of x‐axis and log10 P‐value as the data source of y‐axis. Blue dots and red dots represent upregulated and downregulated DUBs compared to the control group, respectively. Gray dots indicate DUBs with no statistically significant difference compared to the control group. B,C) RT‐qPCR analysis of Usp20 mRNA level in Ang II‐ (B) and TAC‐(C) induced hypertrophic myocardium. n = 6. **p < 0.01. D,E) The protein level of USP20 in the heart tissues was determined by immunoblotting in Ang II‐ (D) and TAC‐ (E) induced cardiac hypertrophy. n = 6. ***p < 0.001. F) RT‐qPCR analysis of Usp20 mRNA level in human hearts from control or patients diagnosed with cardiac hypertrophy. n = 3. HF = heart failure. **p < 0.01. G)…

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  Figure 2. Cardiomyocyte‐specific deficiency of USP20 exacerbates cardiac hypertrophy and dysfunction induced by Ang II. A) Schematic illustration of the experimental design of USP20 in Ang II‐induced cardiac dysfunction and myocardial hypertrophy: USP20fl/fl as control and cardiomyocyte‐specific USP20 deficiency (USP20 CKO) mice were implanted with Ang II‐infused osmotic mini‐pump (1000 ng kg/min) or saline for 4 weeks. B–D) Representative echocardiographic images from each group in mice. n = 6 (B). Quantification of ejection fraction (EF) (C) and fractional shortening (FS) (D) of each group, n = 6. E) Representative images of gross‐heart of each group. n = 6 per group. Scale bar, 2.5 mm. F) The ratio of heart weight (HW) to body weight (BW). G) The ratio of heart weight (HW) to tibial length (TL). H) Representative HE stained images of heart sections. n = 6. Scale bar, 2.5 mm and 50 µm. I,J) Representative wheat germ agglutinin (WGA) stained images of heart sections, n = 6. Scale bar, 20 µm (I), and quantification of WGA of each group (J). K,L) Representative masson stained images of myocardial interstitium in heart sections. Scale bar, 50 µm (K) and quantitative analysis (L). M) RT‐qPCR…

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  Figure 3. Deficiency of cardiomyocyte‐specific USP20 aggravates cardiac hypertrophy and dysfunction induced by TAC. A) A schematic diagram illustrating the experimental for USP20‐mediated cardiac dysfunction and myocardial hypertrophy induced by TAC: TAC or sham surgery was performed in USP20fl/fl as control and cardiomyocyte‐specific USP20 deficiency (USP20 CKO) mice for 4 weeks. B–D) Representative echocardiographic images from each group in mice (B). n = 6. Quantification of ejection fraction (EF) (C) and fractional shortening (FS) (D) of each group n = 6. E) Representative images of gross‐heart of each group. n = 6. Scale bar, 2.5 mm. F) The ratio of heart weight (HW) to body weight (BW). G) The ratio of heart weight (HW) to tibial length (TL). H) Representative HE stained images of heart sections, n = 6. Scale bar, 2.5 mm and 50 µm. I,J) Representative wheat germ agglutinin (WGA) stained images of heart sections, n = 6. Scale bar, 20 µm (I), and quantification of WGA of each group (J). K,L) Representative masson stained images of myocardial interstitium in heart sections. Scale bar, 50 µm. (K) and quantitative analysis (L). M) RT‐qPCR analysis of Myh7 and Nppa in heart tissues. n = 6.…

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  Figure 4. Identification of STAT3 as a substrate of USP20 in cardiomyocytes. A) Schematic illustration of the experimental design for the two interactomes used for USP20 substrate screening. HL‐1 cells (Dataset I) were transfected with either Flag‐vector or Flag‐USP20 following Ang II stimulation at 1 µmol for 24 h. The heart tissues (Dataset II) from mice induced by Ang II were lysed and divided into two portions. The binding proteins were extracted and digested into peptides, and analyzed by liquid chromatography‐mass spectrometry/mass spectrometry (LC‐MS/MS). The list is the potential USP20 substrates identified from the interactomes. B,C) 2D plots with the log10 signal intensity of the quantified proteins on the y axis and the molecular weight (MW) of proteins on the x axis were identified from Dataset I (B) and II (C). D) Immunoprecipitation (IP) of USP20 and STAT3 in the heart tissues from Ang II‐ or vehicle‐treated mice. E) IP of USP20 and STAT3 in Ang II‐incubated neonatal rat cardiomyocytes (NRCMs). F) Co‐IP assays were conducted using NIH3T3 cells transfected with Flag‐USP20 or His‐STAT3, as well as cells co‐transfected with Flag‐USP20 and His‐STAT3 plasmids. G,H)…

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  Figure 5. USP20 attenuates the K63‐linked deubiquitination of STAT3 at residue K177 through the active site H645. A,B) Overexpression of USP20 Flag‐plasmid was transfected into NIH3T3 cells for 24 h. The levels of Flag and STAT3 were assesses by immunoblotting (A) and the corresponding quantitative analysis (B) n = 3. n.s., no significance, ***p < 0.001. C,D) USP20 CKO and USP20fl/fl mice were induced myocardial hypertrophy by Ang II. USP20 and STAT3 expression (C) in the hearts was determined as in (A) and the quantitative analysis of STAT3 (D). n = 6. ***p < 0.001, ns., no significance. E,F) NIH3T3 cells were infected with lentivirus containing empty vector (gCtrl) or USP20‐gRNA (gUSP20) at multiplicith of infection (MOI) of 50. After antibiotic selection, USP20−/−‐NIH3T3 cells were obtained. Either gCtrl or gUSP20 were incubated with cycloheximide (CHX). The USP20 and STAT3 expression (E) was examined as in (A) and densitometric quantification of STAT3 (F). n = 3. ns., no significance. G) NIH3T3 cells were co‐transfected either gCtrl or gUSP20 with overexpression plasmids of His‐STAT3, HA‐Ub, HA‐K48, and HA‐K63, and then incubated with MG132 at 10 µmol for IP of STAT3. Ubiquitinated…

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  Figure 6. USP20 protects heart from cardiac hypertrophy by inhibiting STAT3 nuclear transcription to promote CARM1 expression. A,B) NRCMs were infected with adenovirus (Ad) encoding USP20 (Ad‐USP20) of multiplicity of infection at MOI of 50 or 100 and Ad‐null as control, then incubated with Ang II at 1 µmol for 24 h. The expression of USP20, p‐STAT3 and STAT3 was examined (A) and quantitative analysis (B). n = 3. **p < 0.01, ns., no significance. C,D) NRCMs were transfected with Ad‐Null or Ad‐USP20, and si‐NC (NC, negative control) or si‐USP20 following stimulation with Ang II at 1 µmol for 24 h. Western blotting for p‐STAT3 and Lamin B1 in nuclear (C) and the quantitative analysis (D). n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001. E) HL‐1 cells were transfected with plasmids of EV (empty vector) or USP20OE and stimulated with Ang II for cleavage under targets and tagmentation (CUT&Tag) assay. F) The motif of STAT3 peak matched with CARM1. G) CUT&Tag was performed on si‐NC or si‐USP20‐transfected HL‐1 cells to verify STAT3 binding to the promoter regions of the Carm1 gene. H) CUT&Tag‐qPCR assay for the binding of STAT3 at Carm1 promoter regions. n = 6. ***p < 0.001. I) The analysis of…

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  Figure 7. Cardiomyocyte‐specific overexpression of USP20 improves cardiac hypertrophy and dysfunction induced by Ang II. A) WT mice were injected with adeno‐associated virus serotype 9 (AAV9) mediated cardiomyocyte‐specific overexpression of USP20 (USP20OE) or empty vector (EV) (2 × 1011 v.g., i.v.). Four weeks later, the mice were induced to cardiac hypertrophy by Ang II as in Figure 2A. B) Echocardiographic images from each group in mice. C,D) Echocardiographic analysis of ejection fraction (EF) and fractional shortening (FS). E) Gross‐heart from each group. F) The ratio of heart weight (HW) to body weight (BW). G) The ratio of heart weight (HW) to tibial length (TL). H) HE stained images of heart sections. Scale bar, 2.5 mm and 50 µm. I,J) Representative wheat germ agglutinin (WGA) stained images of heart sections. Scale bar, 50 µm (I) and quantitative analysis (J). K,L) Representative masson stained images of myocardial interstitium in heart sections. Scale bar, 50 µm (K) and quantitative analysis (L). M) RT‐qPCR analysis of Myh7 and Nppa in heart tissues. n = 6. ns., no significance, *p < 0.05, ** p < 0.01, and ***p < 0.001.

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  Figure 8. USP20 ameliorates cardiac hypertrophy and dysfunction by inhibiting STAT3. A) USP20 CKO mice were implanted with Ang II‐infused osmotic mini‐pump for 4 weeks. During this period, STAT3 inhibitor stattic was administered every day (10mg kg−1, qd). B) Representative M‐mode echocardiographic images from each group in mice. C,D) Echocardiographic analysis of ejection fraction (EF) and fractional shortening (FS). E) Representative images of gross‐heart from each group. F) The ratio of heart weight (HW) to body weight (BW). G) The ratio of heart weight (HW) to tibial length (TL). H) HE stained images of heart sections. Scale bar, 2.5 mm and 50 µm. I,J) Wheat germ agglutinin (WGA) stained images of heart sections. Scale bar, 50 µm (I) and quantitative analysis (J). K,L) Representative masson stained images of myocardial interstitium in heart sections. Scale bar, 50 µm (K) and quantitative analysis (L). M) RT‐qPCR analysis of Myh7 and Nppa in heart tissues. n = 6. **p < 0.01, ***p < 0.001, n.s., no significance.