Nitric oxide and superoxide effects on the failing heart

描述（由申请方提供）：怀疑能量代谢异常有助于衰竭心脏中心肌功能障碍的进展。我们已经观察到心力衰竭与心肌ATP含量降低和游离ADP增加以及心肌耗氧量（MVO 2）降低相关。最近的研究表明，一氧化氮（NO）可以在细胞色素C氧化酶上与氧竞争，限制线粒体呼吸。虽然内皮型一氧化氮合酶（eNOS）的活性下降，在衰竭的心脏，诱导型一氧化氮合酶（iNOS）的表达已被报道。在初步研究中，我们发现选择性iNOS抑制导致衰竭心脏的MVO 2增加，但在正常心脏中不增加。因此，提出了研究来检验这一假设，即在犬起搏诱导的心力衰竭中，NO起到限制MVO 2和ATP产生的作用。选择性iNOS抑制剂1400 W、选择性nNOS抑制剂vinyI-L-NIO以及用硝基-L-精氨酸的非选择性NOS抑制的使用将证明iNOS或nNOS是否是衰竭心脏中NO的主要来源。由于NO与超氧化物（O2-）反应形成过氧亚硝酸盐，这也可能损害线粒体呼吸，研究将确定清除O2-或提供过氧亚硝酸盐分解催化剂是否可以增加衰竭心脏中的MVO 2。由于抑制线粒体呼吸会损害ATP合成，因此将使用31 P核磁共振（NMR）光谱评估选择性iNOS和非选择性NOS阻断以及降解过氧亚硝酸盐对MVO 2和心肌[ADP]的影响。如果通过NO、O2-或过氧亚硝酸盐抑制线粒体呼吸损害ATP产生，导致游离胞质ADP的增加，则用O2-清除剂和/或iNOS抑制剂阻断NO合成、清除O2-或减少过氧亚硝酸盐产生将导致[ADP]减少，同时MVO 2增加。因此，将使用31 P NMR光谱来检验NOS抑制导致细胞溶质[ADP]减少同时MVO 2增加的假设。在体内和体外（皮肤纤维和分离的线粒体）使用选择性iNOS、nNOS和非选择性NOS抑制剂将证明这种NO作用是否由iNOS、nNOS或线粒体NOS介导。进一步的研究将确定CHF的存在是否改变了冠状血管和心肌中eNOS、nNOS、iNOS、CuZn-SOD、Mn-SOD和Ec-SOD的mRNA和蛋白质表达和活性。

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.