| BackgroundSince ubiquitin (Ub) was discovered in the early 1970’s, the ubiquitination proteasome system (UPS), which consists of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), proteasomes, and deubiquitinating enzymes (DUBs), has emerged as a critical regulator in virtually all aspects of cell biology. The process of ubiquitination is catalyzed by the sequential action of E1s, E2s, and E3s. DUBs mediate the removal and processing of ubiquitin from ubiquitinated proteins. There are eight E1s, a dozen different types of E2s, and hundreds of E3s in the human. Approximately 100 functional DUBs are coded in human genome.It has been well demonstrated that ubiquitin, E1s, several E2s, E3s, and proteasomes play an important role in the regulation of cardiac homeostasis and dysfunction; however, less is known about the role of DUBs in the heart. Despite the finding of upregulated levels of a few DUBs including A20/tumor necrosis factor alpha induced protein 3 (TNFAIP3), ubiquitin-specific protease 5 (USP5), USP20, and ubiquitin carboxyl terminal hydrolyase L1 (UCH-L1) in the failing heart, A20 is the only DUB that has been extensively studied in the heart. While A20 appears to be a negative regulator of cardiac pathological remodeling and dysfunction most likely via its ability to suppress the activation of NF-κB, mitogen-activated protein kinases (MAPKs) and transforming growth factor-β (TGF-β) signaling, the pathophysiological implications of other DUBs in the heart remain unknown.CYLD, a DUB, was originally identified as a gene mutated in familial cylindromatosis, a genetic condition that predisposes individuals for the development of tumors of skin appendages, termed cylindroma. Subsequent studies have revealed that CYLD regulates diverse physiological processes, ranging from cell cycle progression and immune response to spermatogenesis and osteoclastogensis, and also plays a key role in the pathogenesis of cancer, lung fibrosis, and inflammatory bowel disease. At the molecular level, the signaling functions of CYLD are cell type-and stimuli-dependent. Nevertheless, it is generally accepted that CYLD primarily acts as a negative regulator of IκB kinase (IKK) complex thereby suppressing NF-KB-driven signaling while it also is capable of inhibiting other signal cascades including MAPK and TGF-β signaling. Given the similarity between aforementioned CYLD-and A20-operated signaling regulations, one could hypothesize that CYLD functions like A20 as a suppressor of cardiac pathological remodeling and dysfunction.Therefore, the purpose of this study was to explore a potential role of CYLD in cardiac protection. However, unlike A20 which is critical for cardiac protection, our results indicate that CYLD instead acts as a mediator of cardiac dysfunction. At a molecular level, CYLD minimally regulates NF-κB cascades in the heart, whereas it could inhibit ERK-and p38-mediated expression of c-jun, c-fos, and c-myc that control the expression of Nrf2, a key transcription factor of antioxidant defense system in the heart, thereby enhancing oxidative stress. Thus, CYLD is a novel mediator of oxidative stress in the heart by suppressing Nrf2-operated anti-oxidative capacity independent of NF-κB signaling, which contributes to cardiac maladaptive remodeling and dysfunction.Objectives1. Our findings demonstrate for the first time that CYLD mediates cardiac maladaptive remodeling and dysfunction.2. It most likely via enhancing myocardial oxidative stress in response to pressure overload.3. At the molecular level, CYLD interrupts the ERK-and p38-/AP-l and c-Myc pathways to suppress Nrf2-operated anti-oxidative capacity, thereby enhancing oxidative stress in the heart. Thereby identifying a potential, novel target for the treatment of TAC-induced cardiac maladaptive remodeling and dysfunction.Materials and methods1. Human Heart Specimens2.Generate CYLD knockout (CYLD-/-) mice.3. Set up transverse aortic constriction (TAC) model.4. Echocardiography for transverse aortic constriction (TAC) model.5. TAC model pathological feature.6. Histology and Immunochemistry for myocardial hypertrophy, fibrosis, apoptosis, oxidative stress and CYLD expression.7. Gene expression detected by Q-PCR.8.Deep Sequencing (Seq) Analysis.9. Protein expression detected by Western blot.10. Neonatal rats myocardial cells culture.11. Virus and siRNA Preparation.Adenovirus Infection and Plasmid Transfection.12. Intracellular Reactive Oxygen Species (ROS) Measurement.13. [3H]-Leucine uptake assay.14. Lipid Overloading.15. Cell viability measured by LDH assay.16.Statistical differences were analyzed by one-way ANOVA followed by Bonferroni test for multiple comparisons using GraphPad Prism software.Results1. Upregulation of CYLD expression in failing murine and human hearts.2. Ameliorated myocardial oxidative stress, cardiac maladaptive remodeling, and heart failure in CYLD-/-mice after pathological pressure overload3. Upregulations of c-fos, c-jun, c-myc, and Nrf2 in the heart of CYLD-/-mice4. Silencing CYLD upregulates Nrf2 expression via activation of ERK and p38/AP1 and c-Myc pathways in cardiomyocytes5. Silencing CYLD suppresses oxidative stress via upregulation of Nrf2 in cardiomyocytesConclusion1. Our findings demonstrate for the first time that CYLD mediates cardiac maladaptive remodeling and dysfunction, most likely via enhancing myocardial oxidative stress in response to pressure overload.2. At the molecular level, CYLD interrupts the ERK-and p38-/AP-1 and c-Myc pathways to suppress Nrf2-operated anti-oxidative capacity, thereby enhancing oxidative stress in the heart. |
