Nitric oxide and superoxide effects on the failing heart

描述(申请人提供)：能量代谢异常被怀疑是导致衰竭心脏的心肌功能障碍进展的原因。我们观察到，心力衰竭与心肌ATP含量降低、游离ADP增加以及心肌耗氧量(MVO2)减少有关。最近的研究表明，一氧化氮(NO)可以与氧在细胞色素C氧化酶上竞争，从而限制线粒体的呼吸。尽管内皮型一氧化氮合酶(ENOS)活性在心力衰竭时降低，但诱导型一氧化氮合酶(INOS)在心力衰竭中的表达已有报道。在初步研究中，我们发现选择性的iNOS抑制会导致衰竭心脏的MVO2增加，但在正常心脏中不会。因此，有人建议进行研究，以检验这一假设，即在起搏诱导的犬心力衰竭中，NO可以限制MVO2和ATP的产生。使用选择性诱导型一氧化氮合酶抑制剂1400W、选择性一氧化氮合酶抑制剂VINYI-L-NIO，以及非选择性一氧化氮合酶硝基-L-精氨酸的抑制，将证明在衰竭心脏中，诱导型一氧化氮合酶或神经型一氧化氮合酶是一氧化氮的主要来源。由于NO与超氧化物(02-)反应生成过氧亚硝酸盐，这也可能损害线粒体的呼吸，研究将确定清除O2-或提供过氧亚硝酸盐分解催化剂是否会增加衰竭心脏的MVO2。由于线粒体呼吸抑制会损害ATP的合成，31P核磁共振波谱将用于评估选择性iNOS和非选择性NOS阻断以及降解过氧亚硝酸盐对MVO2和心肌[ADP]的影响。如果NO、O2-或过氧亚硝酸盐对线粒体呼吸的抑制作用削弱了ATP的产生，导致胞质内游离ADP的增加，那么阻断NO的合成、清除O2-或用02-清除剂和/或iNOS抑制剂减少过氧亚硝酸盐的产生将导致[ADP]降低，同时MVO2增加。因此，31P核磁共振波谱将被用来检验这一假设，即NOS抑制导致胞浆[ADP]下降，同时MVO2增加。在体内和体外使用选择性的iNOS、nNOS和非选择性的NOS抑制剂(带皮的纤维和分离的线粒体)将证明这种NO效应是由iNOS、nNOS还是线粒体NOS介导的。进一步的研究将确定CHF是否会改变冠脉和心肌中eNOS、nNOS、iNOS、CuZn-SOD、Mn-SOD和EC-SOD的mRNA、蛋白表达和活性。

DESCRIPTION (provided by applicant): Abnormalities of energy metabolism are suspected to contribute to progression of myocardial dysfunction in the failing heart. We have observed that heart failure is associated with decreased myocardial ATP content and increased free ADP, as well as a reduction of myocardial oxygen consumption (MVO2). Recent studies have demonstrated that nitric oxide (NO) can compete with oxygen at cytochrome C oxidase to limit mitochondrial respiration. Although endothelial NO synthase (eNOS) activity is decreased in the failing heart, inducible NO synthase (iNOS) expression has been reported in the failing heart. In preliminary studies we found that selective iNOS inhibition resulted in an increase of MVO2 in failing hearts but not in normal hearts. Consequently, studies are proposed to test the hypothesis that NO acts to limit MVO2 and ATP production in pacing-induced heart Failure in dogs. Use of both the selective iNOS inhibitor 1400W, the selective nNOS inhibitor vinyI-L-NIO, as well as nonselective NOS inhibition with nitro-L-arginine, will demonstrate whether iNOS or nNOS is the principle source of NO in the failing heart. Because NO reacts with superoxide (02-) to form peroxynitrite, which might also impair mitochondrial respiration, studies will determine whether scavenging 02- or supplying a peroxynitrite decomposition catalyst can increase MVO2 in the failing heart. Since inhibition of mitochondrial respiration would impair ATP synthesis, 31P nuclear magnetic resonance (NMR) spectroscopy will be used to assess the effect of selective iNOS and nonselective NOS blockade, as well as degrading peroxynitrite, on MVO2 and myocardial [ADP]. If inhibition of mitochondrial respiration by NO, O2- or peroxynitrite impairs ATP production, resulting in an increase of free cytosolic ADP, then blocking NO synthesis, scavenging 02- or decreasing peroxynitrite production with an 02- scavenger and/or iNOS inhibitor would cause a decrease of [ADP] with a simultaneous increase of MVO2. Consequently, 31P NMR spectroscopy will be used to test the hypothesis that NOS inhibition results in a decrease of cytosolic [ADP] at the same time that MVO2 is increased. The use of selective iNOS, nNOS and nonselective NOS inhibitors in vivo and in vitro (skinned fibers and isolated mitochondria) will demonstrate whether this NO effect is mediated by iNOS, nNOS or mitochondrial NOS. Additional studies will determine whether mRNA and protein expression and activity for eNOS, nNOS, iNOS, CuZn-SOD, Mn-SOD and Ec-SOD are altered in coronary vessels and myocardium by the presence of CHF.