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The expression of USP28 is increased in cardiac hypertrophy and is principally distributed in cardiomyocytes. (A) Heatmap showing the mRNA profile of DUB genes in Ang II-induced mouse hypertrophic hearts from our published transcriptome (n = 3; GSE221396). (B) The mRNA expression of Usp28 in Ang II-or TACinduced mouse cardiac hypertrophy (n = 6; ** P < 0.01, *** P < 0.001). (C) Western blot and quantitation of USP28 protein expression in mouse hypertrophic myocardium (n = 6; *** P < 0.001). (D) The Usp28 mRNA level in human hypertrophic myocardium (NCH = non-cardiac hypertrophy, CH = cardiac hypertrophy; n = 4; *** P < 0.001). (E) scRNA-seq was performed in TAC mice hearts (For each group, single-cell suspensions from 3-4 hearts were pooled as 1 sample). Left, the UMAP dimensional reduction showing 5 main cell types of heart, including cardiomyocytes (CM), fibroblasts (FB), macrophages (MP), endothelial cells (EC) and pericytes (PC), and their specific marker genes. Approximate 17000 single heart cells (CM: 4484, EC: 3666, FB: 4320, MP: 2691, PC: 2154) were analyzed. Right, Biaxial scatter plot showing the expression pattern of Usp28. (F) Immunofluorescence staining of USP28 (red) and α-actin (green) in cardiac sections treated with Ang II (upper) or TAC (below). (G) NRPCs were transfected with siRNAs of NC (negative control) or USP28 followed by Ang II (1μM, 24h). TRITC-labeled rhodamine phalloidin staining (left) and the quantitative analysis (right) showed the surface area of NRPCs (n = 6; *** P < 0.001).
Source publication
Rationale: Cardiac hypertrophy is an important pathological basis for heart failure. Most physiological activities of cardiomyocytes are regulated by proteins and their post-translational modification. Deubiquitinating enzymes (DUBs) are involved in protein stability maintenance and closely related to myocardial hypertrophy. In this study, we aimed...
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... first analyzed the mRNA profile of DUB genes in hearts of mice with or without Ang II infusion from our published transcriptome dataset (GSE221396) [21], in which we found that the up-regulation of Usp28 was most prominent (Fig. 1A). Next, we validated that Usp28 mRNA levels were increased in Ang II-or TAC-induced mouse hearts, when compared to the controls (Fig. 1B). The up-regulated protein level of USP28 was also confirmed in Ang II-or TAC-induced mouse hearts (Fig. 1C). Likewise, USP28 mRNA expression was increased in human hypertrophic myocardium (Fig. 1D). ...
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... the mRNA profile of DUB genes in hearts of mice with or without Ang II infusion from our published transcriptome dataset (GSE221396) [21], in which we found that the up-regulation of Usp28 was most prominent (Fig. 1A). Next, we validated that Usp28 mRNA levels were increased in Ang II-or TAC-induced mouse hearts, when compared to the controls (Fig. 1B). The up-regulated protein level of USP28 was also confirmed in Ang II-or TAC-induced mouse hearts (Fig. 1C). Likewise, USP28 mRNA expression was increased in human hypertrophic myocardium (Fig. 1D). To examine the cellular localization of up-regulated USP28 in heart, we performed a scRNA-seq of approximate 17000 single heart cells from ...
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... dataset (GSE221396) [21], in which we found that the up-regulation of Usp28 was most prominent (Fig. 1A). Next, we validated that Usp28 mRNA levels were increased in Ang II-or TAC-induced mouse hearts, when compared to the controls (Fig. 1B). The up-regulated protein level of USP28 was also confirmed in Ang II-or TAC-induced mouse hearts (Fig. 1C). Likewise, USP28 mRNA expression was increased in human hypertrophic myocardium (Fig. 1D). To examine the cellular localization of up-regulated USP28 in heart, we performed a scRNA-seq of approximate 17000 single heart cells from TAC-treated mice. Notably, the Usp28 gene was principally distributed in cardiomyocytes (CM) (Fig. 1E). In ...
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... prominent (Fig. 1A). Next, we validated that Usp28 mRNA levels were increased in Ang II-or TAC-induced mouse hearts, when compared to the controls (Fig. 1B). The up-regulated protein level of USP28 was also confirmed in Ang II-or TAC-induced mouse hearts (Fig. 1C). Likewise, USP28 mRNA expression was increased in human hypertrophic myocardium (Fig. 1D). To examine the cellular localization of up-regulated USP28 in heart, we performed a scRNA-seq of approximate 17000 single heart cells from TAC-treated mice. Notably, the Usp28 gene was principally distributed in cardiomyocytes (CM) (Fig. 1E). In line with scRNA-seq, immunofluorescence staining showed that the increased USP28 ...
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... mouse hearts (Fig. 1C). Likewise, USP28 mRNA expression was increased in human hypertrophic myocardium (Fig. 1D). To examine the cellular localization of up-regulated USP28 in heart, we performed a scRNA-seq of approximate 17000 single heart cells from TAC-treated mice. Notably, the Usp28 gene was principally distributed in cardiomyocytes (CM) (Fig. 1E). In line with scRNA-seq, immunofluorescence staining showed that the increased USP28 immunoreactivity was predominantly noted in α-actin + cardiomyocytes (Fig. 1F). In vitro, USP28 protein expression was increased in Ang II-challenged cardiomyocyte ( Fig. S1A). In addition, Ang II-induced expression of USP28 was mainly increased in ...
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... USP28 in heart, we performed a scRNA-seq of approximate 17000 single heart cells from TAC-treated mice. Notably, the Usp28 gene was principally distributed in cardiomyocytes (CM) (Fig. 1E). In line with scRNA-seq, immunofluorescence staining showed that the increased USP28 immunoreactivity was predominantly noted in α-actin + cardiomyocytes (Fig. 1F). In vitro, USP28 protein expression was increased in Ang II-challenged cardiomyocyte ( Fig. S1A). In addition, Ang II-induced expression of USP28 was mainly increased in cardiomyocytes, rather than non-cardiomyocytes (Fig. S1B). Functionally, we found that silencing USP28 suppressed Ang II or ISO-induced increase in surface area of ...
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... mice. Notably, the Usp28 gene was principally distributed in cardiomyocytes (CM) (Fig. 1E). In line with scRNA-seq, immunofluorescence staining showed that the increased USP28 immunoreactivity was predominantly noted in α-actin + cardiomyocytes (Fig. 1F). In vitro, USP28 protein expression was increased in Ang II-challenged cardiomyocyte ( Fig. S1A). In addition, Ang II-induced expression of USP28 was mainly increased in cardiomyocytes, rather than non-cardiomyocytes (Fig. S1B). Functionally, we found that silencing USP28 suppressed Ang II or ISO-induced increase in surface area of NRPCs ( Fig. 1G and Fig. S1C-D). Likewise, USP28 knockdown reduced the mRNA levels of hypertrophic ...
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... staining showed that the increased USP28 immunoreactivity was predominantly noted in α-actin + cardiomyocytes (Fig. 1F). In vitro, USP28 protein expression was increased in Ang II-challenged cardiomyocyte ( Fig. S1A). In addition, Ang II-induced expression of USP28 was mainly increased in cardiomyocytes, rather than non-cardiomyocytes (Fig. S1B). Functionally, we found that silencing USP28 suppressed Ang II or ISO-induced increase in surface area of NRPCs ( Fig. 1G and Fig. S1C-D). Likewise, USP28 knockdown reduced the mRNA levels of hypertrophic genes (Myh7/Nppa) induced by Ang II in vitro ( Fig. S1E-F). Together, these data indicated that cardiomyocyte USP28 expression is ...
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... 1F). In vitro, USP28 protein expression was increased in Ang II-challenged cardiomyocyte ( Fig. S1A). In addition, Ang II-induced expression of USP28 was mainly increased in cardiomyocytes, rather than non-cardiomyocytes (Fig. S1B). Functionally, we found that silencing USP28 suppressed Ang II or ISO-induced increase in surface area of NRPCs ( Fig. 1G and Fig. S1C-D). Likewise, USP28 knockdown reduced the mRNA levels of hypertrophic genes (Myh7/Nppa) induced by Ang II in vitro ( Fig. S1E-F). Together, these data indicated that cardiomyocyte USP28 expression is increased in cardiac hypertrophy and may mediate the hypertrophy of ...
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... of USP28 was mainly increased in cardiomyocytes, rather than non-cardiomyocytes (Fig. S1B). Functionally, we found that silencing USP28 suppressed Ang II or ISO-induced increase in surface area of NRPCs ( Fig. 1G and Fig. S1C-D). Likewise, USP28 knockdown reduced the mRNA levels of hypertrophic genes (Myh7/Nppa) induced by Ang II in vitro ( Fig. S1E-F). Together, these data indicated that cardiomyocyte USP28 expression is increased in cardiac hypertrophy and may mediate the hypertrophy of ...
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... Scheme for the mechanism of USP28 deubiquitinates TRIM21. II (1μM, 24h)-induced HL-1 cells expressing Flag-USP28 (USP28 OE ) or EV (NES: normalized enrichment score; FDR: false discovery rate). B-F: NRPCs were transfected with siRNAs of NC (negative control) or USP28 followed by Ang II (1μM, 24h) (n = 5 for C-D, n = 6 for B and F; ** P < 0.01, *** P < 0.001, ns = no significant). ...
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... The mRNA levels of Nrf2 and Nqo1. (G) NRPCs were transfected with EV or USP28 OE for 24, and then treated with ROS scavenger NAC (1h; HY-B0215, MedChemExpress) following by the Ang II stimulation (1μM, 24h). TRITC-labeled rhodamine phalloidin staining (left) and the quantitative analysis (right) showed the surface area of NRPCs (n = 6; *** P < 0.001). ...
Citations
... USP25 is elevated in hypertrophic myocardium, where it stabilizes SERCA2a by removing K48-linked ubiquitin chains, thereby inhibiting cardiac hypertrophy [44]. Additionally, USP28 and UCHL1 expression increases in hypertrophic and failing hearts [47,48]. OTUD7B has been reported to regulate antiviral immunity [16], inflammation [49], and cancer cell proliferation [14]. ...
Background
Cardiac hypertrophy, a leading cause of heart failure, threatens global public health. Deubiquitinating enzymes (DUBs) are critical in cardiac pathophysiology by regulating protein stability, function, and degradation. Here, we investigated the role and regulating mechanism of ovarian tumor domain-containing 7B (OTUD7B) in cardiac hypertrophy by modulating fatty acid metabolism.
Methods
Mice subjected to transverse aortic constriction (TAC) and cardiomyocytes treated with phenylephrine (PE) were used to explore the role of OTUD7B in myocardial hypertrophy. The potential molecular mechanisms underlying OTUD7B's regulation of cardiac hypertrophy were explored through transcriptome analysis and further validated in cardiomyocytes.
Results
Reduced OTUD7B expression was observed in hypertrophic hearts following TAC surgery. Cardiac-specific OTUD7B deficiency exacerbated, while OTUD7B overexpression mitigated, pressure overload-induced hypertrophy and cardiac dysfunction both in vivo and in vitro. OTUD7B knockdown resulted in ferroptosis, as evidenced by decreased mitochondrial cristae, increased Fe²⁺ ion content, lipid peroxide accumulation, while OTUD7B overexpression inhibited ferroptosis. Mechanistically, transcriptomic analysis identified OTUD7B plays a role in the regulation of fatty acid metabolism and pathological cardiac hypertrophy. OTUD7B was found to directly bind to HNF4α, a transcription factor regulating fatty acid oxidation-related genes. Further, OTUD7B exerted deubiquitination activity to stabilize the HNF4α protein by removing K48-linked ubiquitin chains, thereby preventing its degradation via the proteasomal pathway and linking the HNF4α degradation and ferroptosis. Finally, ferroptosis inhibitors, ferrostatin-1, alleviated OTUD7B inhibition-induced ferroptosis, fatty acid metabolism suppression, and myocardial hypertrophy.
Conclusions
We confirmed that OTUD7B is involved in the regulation of ferroptosis in pressure overload-induced cardiac hypertrophy and highlighted that OTUD7B alleviates cardiac hypertrophy by regulating ferroptosis and fatty acid oxidation through deubiquitination and stabilization of HNF4α.
... Despite the growing number of reports on DUBs in cardiovascular diseases in recent years 19,20 [13]. Our research revealed that endogenous USP20 in the heart was located and more abundant in cardiomyocytes and served as a protective regulator involved in DIC. ...
Background The severe cardiotoxicity of doxorubicin (Dox) significantly restricts its clinical application. Deubiquitinating enzymes (DUBs) play pivotal roles in cardiac pathophysiology because of their precise regulation of protein function, localization and degradation. Objectives The objective of this study was to investigate the role and molecular mechanism of ubiquitin-specific peptidase 20 (USP20), a DUB, in doxorubicin-induced cardiotoxicity. Methods Cardiomyocyte-specific USP20-knockout (USP20-CKO) mice were utilized to assess the role of USP20 in doxorubicin-induced cardiomyopathy (DIC). Coimmunoprecipitation (co-IP) combined with liquid chromatography‒mass spectrometry/mass spectrometry (LC‒MS/MS) analysis was employed to screen the substrate protein of USP20. Furthermore, mutant plasmids of USP20 were constructed to elucidate the molecular mechanism underlying the regulation of human antigen R (HuR) by USP20. Finally, an AAV9 vector was used to overexpress USP20 in the hearts of cardiac-specific HuR-knockout mice to assess the interaction between USP20 and HuR. Results The results revealed a decrease in USP20 expression in Dox-stimulated mouse cardiomyocytes. Cardiomyocyte-specific USP20 knockout resulted in increased cardiomyocyte ferroptosis and led to DIC. Mechanistically, USP20 directly interacted with HuR through its ubiquitin-specific protease structural domain. Deubiquitination at position 154 was crucial for maintaining HuR protein stability by cleaving K48 ubiquitin chains and inhibiting proteasomal degradation. Additionally, HuR bound to GPX4 mRNA to suppress its degradation, thereby mitigating ferroptosis and contributing to alleviating DIC. Furthermore, targeted USP20 overexpression via AAV9 in cardiomyocytes significantly alleviated DIC. However, in mice with cardiomyocyte-specific HuR knockout, USP20 no longer had an anti-DIC effect, indicating that HuR, as a downstream target protein of USP20, plays an irreplaceable role in DIC. Conclusions Our findings indicate that USP20 enhances the stability of the HuR protein through deubiquitination, thereby inhibiting ferroptosis and mitigating DIC.
Although pathological cardiac hypertrophy is a key driver of heart failure, the underlying mechanisms remain incompletely elucidated. This study investigates the role and mechanism of deubiquitinating enzyme (DUB) ubiquitin‐specific protease 20 (USP20) in cardiac hypertrophy. Transcriptomic profiling of hypertrophic hearts shows significant alterations in the expression of DUBs, including a remarkable downregulation of USP20. USP20 is predominantly expressed in cardiomyocytes. Co‐immunoprecipitation (Co‐IP) followed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) is used to identify USP20 substrates. Cleavage Under Targets and Tagmentation assay (CUT&Tag) sequencing is employed to identify downstream targets of signal transducer and activator of transcription 3 (STAT3). Functionally, USP20 deficiency exacerbates cardiac hypertrophy induced by either angiotensin II (Ang II) or transverse aortic constriction (TAC), whereas USP20 overexpression alleviates hypertrophic responses. Mechanistically, USP20 deubiquitinates STAT3 by removing K63‐linked ubiquitin chains at K177 via its H645 active site, reducing STAT3 phosphorylation and nuclear translocation. This inhibites STAT3's transcriptional activity at coactivator‐associated arginine methyltransfer (Carm1) promoter, leading to upregulated CARM1 expression and mitigated hypertrophy. Importantly, the STAT3 inhibitor Stattic confirms STAT3 serves as a key substrate mediating the cardiac protective effects of USP20. These findings unveil a novel USP20/STAT3/CARM1 axis in cardiomyocytes and reveal its therapeutic potential for cardiac hypertrophy.
Protein quality control (PQC) plays a vital role in maintaining normal heart function, as cardiomyocytes are relatively sensitive to misfolded or damaged proteins, which tend to accumulate under pathological conditions. Ubiquitin-specific protease (USP) is the largest deubiquitinating enzyme family and a key component of the ubiquitin proteasome system (UPS), which is a non-lysosomal protein degradation machinery to mediate PQC in cells. USPs regulate the stability or activity of the target proteins that involve intracellular signaling, transcriptional control of inflammation, antioxidation, and cell growth. Recent studies demonstrate that the USPs can regulate fibrosis, lipid metabolism, glucose homeostasis, hypertrophic response, post-ischemic recovery and cell death such as apoptosis and ferroptosis in cardiomyocytes. Since myocardial cell loss is an important component of cardiomyopathy, therefore, these findings suggest that the UPSs play emerging roles in cardiomyopathy. This review briefly summarizes recent literature on the regulatory roles of USPs in the occurrence and development of cardiomyopathy, giving us new insights into the molecular mechanisms of USPs in different cardiomyopathy and potential preventive strategies for cardiomyopathy.
BACKGROUND
Cardiac hypertrophy constitutes the primary pathological basis for heart failure and exerts a considerable influence on morbidity and mortality. Deubiquitinating enzymes are crucial regulators of protein degradation and play a pivotal role in cardiac pathophysiology. This study aimed to clarify the involvement of a deubiquitinating enzyme, MYSM1 (Myb-like, SWIRM, and MPN domains 1), in cardiac hypertrophy and to explore the underlying mechanism.
METHODS
Cardiac hypertrophy was developed by angiotensin II or transverse aortic constriction surgery. Cardiomyocyte-specific knockdown of MYSM1 was accomplished using the adeno-associated virus serotype 9- cTNT - Mysm1 -shRNA. Co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry analysis was utilized to identify potential substrates of MYSM1.
RESULTS
First, we discovered that the expression of MYSM1 increases during cardiac hypertrophy. MYSM1 knockdown mitigated cardiac dysfunction and hypertrophy after angiotensin II administration. Cardiomyocyte-specific knockdown of MYSM1 with adeno-associated virus serotype 9 alleviated myocardial dysfunction and hypertrophy caused by transverse aortic constriction surgery. Through co-immunoprecipitation and LC-MS, poly (ADP-ribose) polymerase 1 (PARP1) was identified as a potential substrate protein of MYSM1. PARP1 initiates a novel form of programmed cell death termed parthanatos, which is characterized by excessive PARylation, nuclear translocation of AIF, and depletion of nicotinamide adenine dinucleotide. MYSM1 deubiquitinates and stabilizes PARP1 in an MPN domain-dependent manner. In addition, MYSM1 mediates cardiac hypertrophy through PARP1-dependent cardiomyocyte parthanatos.
CONCLUSIONS
This study identified the role of the MYSM1-PARP1 axis in mediating cardiac hypertrophy and suggested that MYSM1 is a promising pharmacological target for the treatment of cardiac hypertrophy.
