Microxymetry Techniques for Cardiomyocyte Respiration

描述(由申请人提供)：心肌细胞耗氧量和产生的活性氧物种(ROS)的测量在缺血性心脏病的病理生理学中具有重要意义，因为在缺血/再灌流过程中会产生大量的氧自由基，产生的自由基损害心脏功能。虽然氧消耗和自由基产生是同时发生的，但到目前为止，还没有一种方法可以在一次实验中同时测量氧消耗和自由基产生。本拨款建议的目的是探索一种名为“微氧计量学”的新EPR技术的应用，以实现这一目标，即在单个实验中实时定量测定氧气消耗和自由基生成。该方法将电子顺磁共振测氧法和自旋捕捉法结合在一起，一步得到氧浓度和自由基浓度。这项建议的具体目的是：(I)研究不同条件下心肌细胞(包括培养细胞和新鲜从灌流心脏中分离出来的细胞)的呼吸(既包括呼吸本身，也包括添加刺激剂所刺激的呼吸)。特别是，在这些系统中，将详细研究模拟缺血如何改变心肌细胞的耗氧率。将同时研究这些细胞在生理变化条件下呼吸过程中产生的ROS，并将评估在各种有害条件下(如低氧)氧消耗与ROS生成量之间的关系。此外，模拟缺血预适应和诱导热休克蛋白对氧耗率和ROS产生的影响也将使用所提出的微氧测量法来研究；(Ii)使用所提出的微氧合酶来研究离体心线粒体的呼吸。与心肌细胞类似，心脏线粒体将受到不同的条件，如低氧和常氧PC周期，这些处理对线粒体呼吸的影响将被研究。并建议利用该技术研究复合体I和复合体I11的特异性抑制剂以及它们对状态3和状态4呼吸的影响以及随后对线粒体耗氧量的影响。总体而言，通过建立这项新技术，可以同时测量细胞和亚细胞成分的微升体积的氧气消耗和ROS产生。

DESCRIPTION (provided by applicant): Measurements of oxygen consumption and generation of reactive oxygen species (ROS) in cardiomyocytes, assume importance in pathophysiology of ischemic heart disease because large quantities of oxygen free radicals are generated during ischemia/reperfusion, and the produced free radicals impair the heart function. Though the oxygen consumption and free radicals generation occur simultaneously, till date there is no method known to simultaneously measure both oxygen consumption and free radicals generation in a single experiment. The purpose of the present grant proposal is to explore the application of a new EPR technique called "Microxymetry" to achieve this goal, i.e., the real time quantitation of both the oxygen consumption and free radicals generation, in a single experiment. The present approach combines the EPR oxymetry and spin trapping into one, to get both oxygen concentration and free radicals concentration in one step. The specific aims of the proposal are: (i) to study the respiration (both as such and stimulated by added stimulants) of cardiomyocytes (both cultured cells and freshly isolated from perfused hearts) at different conditions. Particularly, in these systems, how the simulated ischemia alters oxygen consumption rate of cardiomyocytes will be studied in detail. The ROS generated during the respiration of these ceils at physiologically altered conditions, will be studied simultaneously and the relationship between oxygen consumption and the amount of ROS formed will be evaluated at various deleterious conditions like hypoxia. Further, the effects of simulated ischemic preconditioning and induction of heat shock proteins on the oxygen consumption rate and ROS production will also be studied using the proposed microxymetry; (ii) to study the respiration of isolated heart mitochondria using the proposed microxymtery. Similar to the cardiomyocytes, the heart mitochondria will be subjected to different conditions like hypoxia and normoxiaI PC cycles and the effect of these treatments on mitochondria respiration will be studied. It is also proposed to use this technique to study the specific inhibitors on complex I and complex Ill and their effect on the state 3 and state 4 respirations and the subsequent influences on the oxygen consumption of mitochondria. Overall, by establishing this new technique, it is possible to simultaneously measure the oxygen consumption and ROS production in microliter volumes of cellular and sub-cellular components.