Author + information
- Mengqiu Dang1,
- Xiangjun Zeng3,
- Hongxia Wang3,
- Huihua Li4,5,
- Fenghe Du1,2,
- Buxing Chen1 and
- Caixia Guo1
- 1Department of Cardiology, Beijing Tian Tan Hospital, Capital Medical University, 6 Tiantan Xili, Dongcheng District, Beijing, China
- 2Department of Geriatrics, Beijing Tian Tan Hospital, Capital Medical University, 6 Tiantan Xili, Dongcheng District, Beijing, China
- 3Department of Physiology and Pathophysiology, Capital Medical University, Beijing, China
- 4Department of Cardiology, Institute of cardiovascular Disease, First Affiliated Hospital of Dalian Medical University, Dalian, China
- 5Department of Nutrition and Food Hygiene, School of Public Health, Advanced Institute of Medical Sciences, Dalian Medical University, Dalian, China
Apoptosis was one of the most important injuries induced by ischemia/reperfusion. Previous studies have demonstrated that sRAGE inhibited cardiomyocytes apoptosis through up-regulating ubiquitin proteasome system (UPS). However, the mechanisms remained unknown. The aim of this study was to clarify the mechanisms of sRAGE in inhibiting myocardial ischemia/reperfusion induced apoptosis through mediating UPS activities.
Ischemia/reperfusion model was performed by ligation of left anterior descending coronary artery (LAD) in adult male C57BL/6 mice (8-10 weeks). sRAGE and neutralizing antibody (25μg/mouse, i.p.) for IFN-γ or saline were administrated before ligation. In cultured neonatal cardiomyocytes, ischemia was simulated with “ischemia buffer” for 2 hours followed by reperfusion for 24 hours with or without overexpression of sRAGE by adenovirus. In addition, adenovirus siRNA-β1i and adenovirus siRNA-β5i were used to knockdown these proteasome subunits. The expression of IFN-γ was detected both in vivo and in vitro by elisa and RT-PCR, respectively. Cardiac function, infarct size and apoptosis after sRAGE and IFN-γ antibody treatment were assessed by echocardiography, Evens-blue/TTC staining and TUNEL staining, respectively. The expression of caspase-3 was also tested by western-blot to evaluate the apoptosis in vivo and in vitro. Proteasome activities including caspase-like, trypsin-like and chymotrypsin-like activities were also assessed.
sRAGE significantly up-regulated the expression of IFN-γ mRNA in cultured cardiomyocytes and protein level in mice serum. Neutralization of IFN-γ with anti-IFN-γ antibody abolished the rescue effects of sRAGE for cardiac dysfunction, infarct size and apoptosis provoked by myocardial ischemia/reperfusion. Meanwhile, neutralization of IFN-γ markedly reversed the upregulation of the β1i and β5i expression induced by sRAGE in heart, which accompanied with decreasing chymotrypsin-like proteasome activity. In addition, the absence of β5i remarkably abolished the protective effects of sRAGE on ischemia/reperfusion induced apoptosis. But absence of β1i did not show such effects.
Our data suggested that sRAGE mediated UPS activation through up-regulating the expression of IFN-γ. The increased expression of β5i induced by IFN-γ was the major proteasome subunits involved in the inhibition of apoptosis by sRAGE.