Zhu Xinsheng, Zhao Huailong, Graveline Amanda R, Buys Emmanuel S, Schmidt Ulrich, Bloch Kenneth D, Rosenzweig Anthony, Chao Wei
Dept. of Anesthesia & Critical Care, MGH, GRJ-4-462, 55 Fruit St., Boston, MA 02114, USA.
Am J Physiol Heart Circ Physiol. 2006 Oct;291(4):H1900-9. doi: 10.1152/ajpheart.00112.2006. Epub 2006 Apr 28.
Innate immune system such as Toll-like receptor 4 (TLR4) represents the first line of defense against infection. In addition to its pivotal role in host immunity, recent studies have suggested that TLR4 may play a broader role in mediating tissue inflammation and cell survival in response to noninfectious injury. We and other investigators have reported that cardiac TLR4 signaling is dynamically modulated in ischemic myocardium and that activation of TLR4 confers a survival benefit in the heart and in isolated cardiomyocytes. However, the signaling pathways leading to these effects are not completely understood. Here, we investigate the role of MyD88, an adaptor protein of TLR4 signaling, and inducible nitric oxide synthase (NOS2) in mediating TLR4-induced cardiomyocyte survival in an in vitro model of apoptosis. Serum deprivation induced a significant increase in the number of apoptotic cardiomyocytes as demonstrated by transferase-mediated dUTP nick-end labeling (TUNEL) assay, nuclear morphology, DNA laddering, and DNA-histone ELISA. Lipopolysaccharide (LPS), a TLR4 agonist, activated TLR4 signaling and led to significant reduction in apoptotic cardiomyocytes and improved cellular function of surviving cardiomyocytes with enhanced Ca(2+) transients and cell shortening. We found that both TLR4 and MyD88 are required for the LPS-induced beneficial effects as demonstrated by improved survival and function in wild-type but not in TLR4(-/-) or MyD88(-/-) cardiomyocytes. Moreover, genetic deletion or pharmacological inhibition of NOS2 abolished survival and functional rescue of cardiomyocytes treated with LPS. Taken together, these data suggest that TLR4 protects cardiomyocytes from stress-induced injury through MyD88- and NOS2-dependent mechanisms.
诸如Toll样受体4（TLR4）之类的先天免疫系统是抵御感染的第一道防线。除了在宿主免疫中发挥关键作用外，最近的研究表明，TLR4在介导对非感染性损伤的组织炎症和细胞存活方面可能发挥更广泛的作用。我们和其他研究人员报告说，心脏TLR4信号在缺血心肌中受到动态调节，并且TLR4的激活在心脏和分离的心肌细胞中具有生存益处。然而，导致这些效应的信号通路尚未完全了解。在这里，我们研究了MyD88（一种TLR4信号的衔接蛋白）和诱导型一氧化氮合酶（NOS2）在体外凋亡模型中介导TLR4诱导的心肌细胞存活中的作用。血清剥夺导致凋亡心肌细胞数量显著增加，这通过转移酶介导的dUTP缺口末端标记（TUNEL）测定、核形态、DNA梯状条带和DNA-组蛋白ELISA得以证明。脂多糖（LPS）是一种TLR4激动剂，激活TLR4信号并导致凋亡心肌细胞显著减少，并改善了存活心肌细胞的细胞功能，增强了Ca（2+）瞬变和细胞缩短。我们发现，TLR4和MyD88都是LPS诱导的有益效应所必需的，如野生型心肌细胞中存活率和功能的改善所示，但在TLR4（-/-）或MyD88（-/-）心肌细胞中则不然。此外，NOS2的基因缺失或药物抑制消除了用LPS处理的心肌细胞的存活和功能挽救。综上所述，这些数据表明TLR4通过MyD88和NOS2依赖性机制保护心肌细胞免受应激诱导的损伤。
