Hilfiker-Kleiner Denise, Hilfiker Andres, Schieffer Bernhard, Engel David, Mann Douglas L, Wollert Kai C, Drexler Helmut
Department of Cardiology and Angiology, Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, 30625 Hannover, Germany.
Cardiovasc Res. 2002 Feb 1;53(2):460-9. doi: 10.1016/s0008-6363(01)00463-1.
Tumor necrosis factor alpha(TNFalpha) is thought to play a key role in the pathogenesis of cardiac failure. In the myocardium, TNFalpha enhances the expression of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) has been shown to affect beta-agonist-dependent cardiac contractility and relaxation. It is not clear, however, whether TNFalpha mediated NO release has sustained cardiac effects, by altering expression of cardiomyocyte specific genes such as alpha-myosin heavy chain (alphaMHC).
Neonatal rat ventricular cardiomyocytes (CM) were stimulated with TNFalpha and/or the NOS inhibitor nitro-L-arginine (L-NNA). Protein binding to the E-box enhancer element in the alphaMHC promoter was evaluated by electrophoretic mobility shift assay (EMSA) and transcriptional activity of the E-box consensus motif was determined by luciferase assay. mRNA levels of the endogenous alphaMHC gene were assessed by RT-PCR. In vivo studies were performed in transgenic mice with cardiac specific over-expression of TNFalpha.
CM treated with TNFalpha exhibited decreased levels of alphaMHC transcripts (69 +/- 8% of control), the effect of TNFalpha was reversed by L-NNA (94 +/- 14% of control). As shown by EMSA, TNFalpha reduced protein binding to the alphaMHC E-box enhancer motif via NO dependent pathways. Addition of the NO-donor sodium nitroprusside (SNP) to CM nuclear extracts dose dependently disrupted protein binding to the alphaMHC E-box. Furthermore, exposure of CM to TNFalpha or SNP decreased transcription from an E-box luciferase-reporter construct (TNFalpha: 74 +/- 12%; SNP 250 microM: 72 +/- 10%; SNP 500 microM: 66 +/- 11% of control). In myocardial tissue of TNFalpha transgenic mice, increased nitrotyrosine staining, decreased protein binding to the alphaMHC E-box motif and reduced expression of alphaMHC (62 +/- 26%) were observed.
The present study shows that TNFalpha reduces alphaMHC transcript levels in cardiomyocytes. Our data obtained in cultured CM and in TNFalpha transgenic mice support the notion that TNFalpha exerts these effects by NO and E-box dependent mechanisms in vitro and possibly in vivo.
肿瘤坏死因子α（TNFα）被认为在心力衰竭的发病机制中起关键作用。在心肌中，TNFα可增强诱导型一氧化氮合酶（iNOS）的表达。一氧化氮（NO）已被证明会影响β-激动剂依赖的心脏收缩和舒张。然而，尚不清楚TNFα介导的NO释放是否通过改变心肌细胞特异性基因如α-肌球蛋白重链（αMHC）的表达而对心脏产生持续影响。
用TNFα和/或一氧化氮合酶抑制剂硝基-L-精氨酸（L-NNA）刺激新生大鼠心室心肌细胞（CM）。通过电泳迁移率变动分析（EMSA）评估与αMHC启动子中E盒增强子元件的蛋白结合情况，并通过荧光素酶测定法确定E盒共有基序的转录活性。通过逆转录聚合酶链反应（RT-PCR）评估内源性αMHC基因的mRNA水平。在心脏特异性过度表达TNFα的转基因小鼠中进行体内研究。
用TNFα处理的CM显示αMHC转录本水平降低（为对照的69±8%），L-NNA可逆转TNFα的这种作用（为对照的94±14%）。如EMSA所示，TNFα通过NO依赖途径减少与αMHC E盒增强子基序的蛋白结合。向CM核提取物中添加NO供体硝普钠（SNP）可剂量依赖性地破坏与αMHC E盒的蛋白结合。此外，将CM暴露于TNFα或SNP会降低E盒荧光素酶报告构建体的转录（TNFα：为对照的74±12%；250μM SNP：为对照的72±10%；500μM SNP：为对照的66±11%）。在TNFα转基因小鼠的心肌组织中，观察到硝基酪氨酸染色增加、与αMHC E盒基序的蛋白结合减少以及αMHC表达降低（为对照的62±26%）。
本研究表明TNFα可降低心肌细胞中αMHC转录本水平。我们在培养的CM和TNFα转基因小鼠中获得的数据支持以下观点，即TNFα在体外和可能在体内通过NO和E盒依赖机制发挥这些作用。
