AGING AND OXIDATION IN SKELETAL AND CARDIAC MUSCLE

我们的长期目标是了解蛋白质和脂质的作用 氧化在与年龄有关的修改钙调节在心脏。 我们的重点将是肌浆网（SR）Ca-ATPase，一个活跃的 对维持低静息水平起关键作用的转运蛋白 细胞内钙和正常的心肌松弛。 的影响 氧化、老化和对氧化应激的敏感性将被检查 SR膜的蛋白质和脂质成分。 这项工作大部分 将依赖于与骨骼类似研究的结果进行比较， SR，其中骨骼Ca-ATP酶同种型更丰富，膜 组成更简单。 我们的三个具体目标是：第一，确定 Ca-ATP酶的氧化修饰，其调节蛋白， 受磷蛋白和SR脂质，通过暴露分离的SR启动 活性氧物质。 我们将强调活性氧 其化学性质简单，可以定量。 这些免费 自由基将靶向脂质双层或水溶性的 介质，以便特异性地氧化跨膜或可溶性 缩氨酸 通过这种方式，我们寻求深入了解氧化损伤的机制 到细胞蛋白质。 活性氧与 和功能是不可用的生物老化的样品，其中大的 反应的数量，加上细胞修复的存在， 机制使我们对蛋白质形成过程的理解变得复杂 损害 第二，我们将定义细胞环境的作用， 改变SR膜对氧化条件的敏感性。 这种方法，利用原代培养的心肌细胞，将提供一个 有机会检查氧化条件对蛋白质的影响， 脂质周转 利用从这些模型系统中获得的知识，我们 将（第三），确定年龄相关的氧化修饰， 相关的结构和功能后果。 使用两种模型 生物老化的系统将强调定量识别 自由基介导的蛋白质修饰的产物， 脂质。 荧光和自旋标记电子的联合使用 顺磁共振光谱将提供有关 蛋白质构象和双层动力学。 更加了解 与年龄相关的心肌钙调节分子缺陷， 与人类健康有关，因为生物分子的氧化被认为 在衰老和心肌梗死中起着重要作用。 这种理解 对于设计有效的治疗方法来延迟发病是必要的 以及心肌功能下降和对 氧化应激

Our long-term goal is to understand the role of protein and lipid oxidation in age-related modifications of calcium regulation in the heart. Our focus will be on the sarcoplasmic reticulum (SR) Ca-ATPase, an active transport protein that is central to the maintenance of low resting levels of intracellular calcium and normal myocardial relaxation. Effects of oxidation, aging, and susceptibility to oxidative stress will be examined for the protein and lipid components of SR membranes. Much of this work will rely on comparison with results from similar studies with skeletal SR, where the skeletal Ca-ATPase isoform is more abundant and the membrane composition is simpler. Our three specific aims are: FIRST, to identify oxidative modifications of the Ca-ATPase, its regulatory protein, phospholamban, and SR lipids, initiated by exposure of isolated SR vesicles to reactive oxygen species. We will emphasize reactive oxygen species whose chemistry is simple and allows quantification. These free radicals will be targeted to either the lipid bilayer or to the aqueous medium, in order to specifically oxidize membrane-spanning or soluble peptides. In this way we seek insight into mechanisms of oxidative damage to cellular proteins. The relationship between reactive oxygen species and function is not available from biologically aged samples where a large number of reactions, coupled with the presence of cellular repair mechanisms complicate our understanding of processes leading to protein damage. SECOND, we will define the role of the cellular environment in modifying the susceptibility of SR membranes to oxidative conditions. This approach, utilizing primary cultures of myocytes, will provide an opportunity to examine the effect of oxidative conditions on protein and lipid turnover. Using the knowledge gained from these model systems, we will (THIRDLY), identify age-related oxidative modifications, and their associated structural and functional consequences. Work with both model and biologically aged systems will emphasize quantitative identification of the products of free radical-mediated modifications of proteins and lipids. The combined use of fluorescence and spin-label electron paramagnetic resonance spectroscopies will provide information regarding protein conformation and bilayer dynamics. An increased understanding of age-related molecular defects of calcium regulation in cardiac muscle is relevant to human health, since oxidation of biomolecules is thought to play a major role in aging and myocardial infarction. This understanding is necessary for the design of effective therapies for delaying the onset and progress of decreased myocardial function and susceptibility to oxidative stress.