Ubiquitin E3 Ligase Chip Inhibition Mechanism Of Action Of The Angiotensin Ii-induced Cardiac Remodeling

Background:The carboxyl terminus of heat shock protein70-interacting protein (CHIP), an E3ligase/chaperone, can degradate multiple protein substrates through proteasome, playing an important role in pathological progress and ailments conditions like cardiomyocyte injury, apoptosis, neoplasm and thermo-stress. However, the functional role of CHIP in cardiac fibrosis, apoptosis and inflammation induced by angiotensin Ⅱ (Ang Ⅱ) remains unclear. Therefore, we focus on the molecular mechanism about CHIP in cardiac remodeling induced by Ang Ⅱ.Methods:We use10weeks old wild type (WT) mice and CHIP-overexpressed transgenic (CHIP-TG) mice, randomly divided into4groups, and each group has eight mice:(1) normal saline (NS)+WT;(2) NS+CHIP-TG;(3) Ang II+WT;(4) Ang Ⅱ+CHIP-TG. These mice were infused with Ang Ⅱ (1500ng/kg per minute) or saline. After one week, B ultrasound is used to test the heart function of animals; Heart sections were stained with hematoxylin and eosin (H&E) and Masson trichrome in order to observe the invasion of the inflammatory cells and collagen deposition. The number of macrophages (Mac-2) and the levels of monocyte chemoattractant protein1(MCP-1), intercellular adhesion molecule1(ICAM-1), a-smooth muscle actin (a-SMA), Collagen I and TGF-(3were measured by immunohistochemistry. The levels of Collage I, TGF-β, MCP-1, ICAM-1, IL-1(3and IL-6were measured by PT-PCR. The TUNEL staining was used for apoptosis. Neonatal rat cardiomyocytes were isolated by enzymatic disassociation from1to3-day-old Sprague-Dawley rats. Recombinant small interfering RNA (siRNA) adenovirus (Ad), including Ad-siRNA-GFP control and Ad-siRNA-CHIP. Cells were transfected with Ad-siRNA-GFP control or Ad-siRNA-CHIP as described. After24h, cells were treated with Ang Ⅱ (100nM) or saline for6to24h. The levels of Collage I, TGF-p, MCP-1, ICAM-1, IL-1β and IL-6were measured by PT-PCR. The TUNEL staining was used for apoptosis. In addition, neonatal cardiomyocytes were treated with p38MAPK inhibitor (SB203580,10μ M) or JNK inhibitor (SP600125,20μ M) for30min prior to Ang Ⅱ (100nM) stimulation, and then incubated under the stimulation of Ang Ⅱ for6to24h.The levels of IL-1β and IL-6were measured by PT-PCR. The TUNEL staining was used for apoptosis. At the last, the mechanisms underlying these beneficial actions were associated with CHIP-mediated inhibition of NF-κB and mitogen-activated protein kinase (p38MAPK and JNK) signaling.Results:1) In treatment with NS, there were no differences in cardiac function, pathology and the invasion of the inflammatory cells between WT and CHIP-TG groups.2) Seven days after Ang Ⅱ infusion, the cardiac function, fibrosis area, apoptosis and the invasion of the inflammatory cells of Ang Ⅱ+WT group are increased obviously compared with NS+WT group.3) Compared with Ang Ⅱ+WT group, the fibrosis area and the level of a-SMA, collagen I and TGF-β of Ang Ⅱ+TG group is obviously decreased. The number of macrophages (Mac-2) and the levels of proinflammatory cytokines (MCP-1, ICAM-1, IL-1β and IL-6) are obviously decreased. The apoptosis of myocardial cell is obviously decreased.4) CHIP siRNA knockdown markedly increased Ang Ⅱ-induced apoptosis and the expression of proinflammatory cytokines (MCP-1, ICAM-1, IL-1β and IL-6), as compared with siRNA control.5) The mechanisms underlying these beneficial actions were associated with CHIP-mediated inhibition of NF-κB and mitogen-activated protein kinase (p38MAPK and JNK) signaling.Conclusion:CHIP plays an important role for in regulating Ang II-triggered hypertensive cardiac fibrosis, inflammation and apoptosis.