Background: Pathological cardiac hypertrophy can lead to heart failure and is one of the leading causes of death globally. Understanding the molecular mechanism of pathological cardiac hypertrophy will contribute to the treatment of heart failure. DUBs (deubiquitinating enzymes) are essential to cardiac pathophysiology by precisely controlling protein function, localization, and degradation. This study set out to investigate the role and molecular mechanism of a DUB, USP25 (ubiquitin-specific peptidase 25), in pathological cardiac hypertrophy. Methods: The role of USP25 in myocardial hypertrophy was evaluated in murine cardiomyocytes in response to Ang II (angiotensin II) and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of heart failure patients. Liquid chromotography with mass spectrometry/mass spectrometry analysis combined with Co-IP was used to identify SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2A), an antihypertrophy protein, as an interacting protein of USP25. To clarify the molecular mechanism of USP25 in the regulation of SERCA2a, we constructed a series of mutant plasmids of USP25. In addition, we overexpressed USP25 and SERCA2a in the heart with adenoassociated virus serotype 9 vectors to validate the biological function of USP25 and SERCA2a interaction. Results: We revealed increased protein level of USP25 in murine cardiomyocytes subject to Ang II and transverse aortic constriction stimulation and in hypertrophic myocardium tissues of patients with heart failure. USP25 deficiency aggravated cardiac hypertrophy and cardiac dysfunction under Ang II and transverse aortic constriction treatment. Mechanistically, USP25 bound to SERCA2a directly via its USP (ubiquitin-specific protease) domain and cysteine at position 178 of USP25 exerts deubiquitination to maintain the stability of the SERCA2a protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby maintaining calcium handling in cardiomyocytes. Moreover, restoration of USP25 expression via adenoassociated virus serotype 9 vectors in USP25 ^−/− mice attenuated Ang II-induced cardiac hypertrophy and cardiac dysfunction, whereas myocardial overexpression of SERCA2a could mimic the effect of USP25. Conclusions: We confirmed that USP25 inhibited cardiac hypertrophy by deubiquitinating and stabilizing SERCA2a.

BACKGROUND: Macrophages play a crucial role in atherosclerotic plaque formation, and the death of macrophages is a vital factor in determining the fate of atherosclerosis. GSDMD (gasdermin D)-mediated pyroptosis is a programmed cell death, characterized by membrane pore formation and inflammatory factor release. METHODS: ApoE ^−/− and Gsdmd ^−/− ApoE ^−/− mice, bone marrow transplantation, and AAV (adeno-associated virus serotype 9)-F4/80-shGSDMD (shRNA-GSDMD) were used to examine the effect of macrophage-derived GSDMD on atherosclerosis. Single-cell RNA sequencing was used to investigate the changing profile of different cellular components and the cellular localization of GSDMD during atherosclerosis. RESULTS: First, we found that GSDMD is activated in human and mouse atherosclerotic plaques and Gsdmd ^−/− attenuates the atherosclerotic lesion area in high-fat diet–fed ApoE ^−/− mice. We performed single-cell RNA sequencing of ApoE ^−/− and Gsdmd ^−/− ApoE ^−/− mouse aortas and showed that GSDMD is principally expressed in atherosclerotic macrophages. Using bone marrow transplantation and AAV-F4/80-shGSDMD, we identified the potential role of macrophage-derived GSDMD in aortic pyroptosis and atherosclerotic injuries in vivo. Mechanistically, GSDMD contributes to mitochondrial perforation and mitochondrial DNA leakage and subsequently activates the STING (stimulator of interferon gene)-IRF3 (interferon regulatory factor 3)/NF-κB (nuclear factor kappa B) axis. Meanwhile, GSDMD regulates the STING pathway activation and macrophage migration via cytokine secretion. Inhibition of GSDMD with GSDMD-specific inhibitor GI-Y1 (GSDMD inhibitor Y1) can effectively alleviate the progression of atherosclerosis. CONCLUSIONS: Our study has provided a novel macrophage-derived GSDMD mechanism in the promotion of atherosclerosis and demonstrated that GSDMD can be a potential therapeutic target for atherosclerosis.

In coronary arteries, plaque disruption, the major acute clinical manifestations of atherosclerosis, leads to a subsequent cardiac event, such as acute myocardial infarction (AMI) and unstable angina pectoris (UA). Numerous reports have shown that high expression of MMP-9 (matrix metalloproteinase-9), MMP-13 (matrix metalloproteinase-13) and EMMPRIN (extracellular matrix metalloproteinase induce) in monocyte/macrophage results in the plaque progression and destabilization. Curcumin exerts well-known anti-inflammatory and antioxidant effects and probably has a protective role in the atherosclerosis. The purpose of our study was to investigate the molecular mechanisms by which curcumin affects MMP-9, MMP13 and EMMPRIN in PMA (phorbol 12-myristate 13-acetate) induced macrophages. Human monocytic cells (THP-1 cells) were pretreated with curcumin or compound C for 1 h, and then induced by PMA for 48 h. Total RNA and proteins were collected for real-time PCR and Western blot analysis, respectively. In the present study, the exposure to curcumin resulted in attenuated JNK, p38, and ERK activation and decreased expression of MMP-9, MMP-13 and EMMPRIN in PMA induced macrophages. Moreover, we demonstrated that AMPK (AMP-activated protein kinase) and PKC (Protein Kinase C) was activated by PMA during monocyte/macrophage differentiation. Furthermore, curcumin reversed PMA stimulated PKC activation and suppressed the chronic activation of AMPK, which in turn reduced the expression of MMP-9, MMP-13 and EMMPRIN. Therefore, it is suggested that curcumin by inhibiting AMPK-MAPK (mitogen activated protein kinase) and PKC pathway may led to down-regulated EMMPRIN, MMP-9 and MMP-13 expression in PMA-induced THP-1 cells.