OXYGEN CONCENTRATION & REDOX METABOLISM WITH TRITYL PROBES IN CARDIAC MYOCYTES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 这个合作项目的目标是成像活的心肌细胞或来自正常心脏和心肌病心脏的肌细胞簇中的O2和氧化还原代谢。正常的心肌细胞(来自大鼠)通常约为20x25x150微米。对于肥厚型心肌病(CM)，这些细胞的大小可以增加50%以上。我们将在ACERT使用最近开发的ESRM技术，以及最近开发的细胞内三叔丁基自由基探针和三甲基氮氧双自由基来提供氧和氧化还原代谢的亚细胞和细胞外成像，这将补充我们目前在离体和活体心脏上进行的“组织水平”成像，采用低场/低分辨率体内ESR成像。ESR显微镜将用于我们最近开发的新型三苯基酯和三苯基-氮氧化物双自由基，从而能够测量细胞和组织中的O2浓度和氧化还原代谢。这是我们上面提到的赠款的延期。 具体来说，我们会： 1.用我们新近开发的用作细胞内自旋标记和血氧测定仪的三苯基酯探针孵育和负载心肌细胞，然后使用ESRM测量它们的位置和细胞内局部的O2浓度。细胞外三叔丁基CT-03也将进行类似的测量。 2.使用我们新的细胞内氧化还原代谢的三苯基氮氧化物双自由基进行类似的测量。 3.将细胞置于不同的Ca(II)水平和0.5~5 Hz的搏动刺激下，测定其对正常心肌细胞和心肌细胞内O2和氧化还原代谢的影响。 几个重要的问题将被解决，包括：细胞内的O2浓度和氧化还原代谢是否与细胞外的值不同？细胞内部是否存在空间差异？这些值会随着心肌细胞收缩活动而改变吗？这些值是否随着Ca(II)浓度的变化而变化？

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this collaborative project is to image O2 and redox metabolism in live cardiac myocytes or myocyte clusters from normal hearts and hearts with cardiomyopathy. Normal cardiac myocytes (from rats) are typically about 20 x 25 x 150 microns. With hypertrophic cardiomyopathy (CM) these cells can increase in size by over 50%. We will make use of recently developed ESRM technology at ACERT and recently developed intra-cellular trityl radical probes and trityl-nitroxide biradicals to provide subcellular and extra cellular imaging of oxygen and redox metabolism that will complement our present "tissue level" imaging on isolated and in vivo hearts, conducted with low field/lower resolution in-vivo ESR imaging. ESR microscopy will be employed with our recently developed novel trityl esters and trityl-nitroxide biradicals that enable measurements of O2 concentrations and redox metabolism in cells and tissues. This is an extension of our grants noted above. Specifically, we will: 1. Incubate and load cardiac myocytes with our recently developed trityl ester probes that we have shown to serve as intracellular spin labels and oximetry probes and then employ ESRM to measure their location and the localized intracellular O2 concentration. Similar measurements will be performed with the extracellular trityl CT-03. 2. Perform similar measurements with our new trityl nitroxide biradicals of intracellular redox metabolism. 3. Subject the cells to different Ca(II) levels and stimulation with beating at rates of 0.5 Hz to 5 Hz and measure the effects on O2 and redox metabolism within the normal and CM myocytes. Several important questions will be addressed including: Is intracellular O2 concentration and redox metabolism different than extracellular values? Are there spatial differences within the cell? Do these values change with myocyte contractile activity? Do these values change with Ca(II) concentration?