应用心肌自发荧光评估存活心肌线粒体氧化代谢状态

Assessment of Mitochondrial Metabolic Oxidative State in Living Cardiomyocytes with Cardiomyocyte Autofluorescence

目的：应用心肌自发荧光（AF）研究心肌线粒体氧化代谢状态，监测线粒体功能改变的早期信号。方法：烟酰胺腺嘌呤（磷酸）二核苷酸ENAD（P）HI作为荧光探针，用光谱分辨的时间相关单光子计数（TCSPC）记录375nm紫外激光激发的心肌AF光谱和荧光寿命，测试影响线粒体呼吸时AF动态衰减。结果：在420～560nm光谱区域，至少需用3个荧光寿命池0．4～0．7ns，1．2～1．9ns和8．0～13．0ns描述细胞AF。线粒体呼吸阻断剂鱼藤酮可显著增加AF强度，缩短平均荧光寿命。氧化磷酸化解偶联剂二硝基酚可显著降低AF强度，在520nm处增宽荧光光谱，延长平均荧光寿命。这些结果和NADH荧光动力学离体实验（invitro）有可比性。结论：光谱分辨的荧光寿命技术测定心肌NAD（P）H荧光有很好的重复性，在细胞水平上增加了心肌氧化代谢或线粒体功能障碍的知识，为临床诊断和治疗线粒体功能障碍开拓了新视野。

Objective： To study fingerprinting of mitochondrial metabolic oxidative state in living cardiomyocytes with cadiomyocyte autofluorescence （AF） in order to monitor cellular fluorescence of nicotinamide adenine dinucleotide （phosphate） [NAD（P）H], the principal electron donor in mitochondrial respiration which is responsible for vital ATP supply to sensitively and reflects early signs of mitochondrial function in pathophysiological conditions, such as ischemia, diabetes and heart failure. Methods： NAD（P）H was studied as a marker for non-invasive fluorescent probing of the mitochondrial function. NAD（P）H fluorescence was recorded in living cardiomyocytes following excitation with 375 nm UV-light and detection by spectrally-resolved time-correlated single photon counting （TCSPC）, based on the simultaneous measurement of the fluorescence spectra and fluorescence lifetimes. Modulation of mitochondrial respiration was tested by studying dynamic characteristics of NAD（P）H fluorescence decay in living cardiomyocytes. Results： At least a 3-exponential decay model, with 0.4 - 0.7 ns, 1.2 - 1.9 ns and 8.0 - 13.0 ns lifetime pools was necessary to describe cardiomyocyte AF within 420 - 560 nm spectral range. Rotenone, the inhibitor of Complex Ⅰ of the mitochondrial respiratory chain, increased AF intensity and shortened the average fluorescence lifetime. Dinitrophenol （DNP）, an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF intensity, broadened the spectral shoulder at 520 nm and increased the average fluorescence lifetime. These effects were comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro. Conclusion： Spectrally-resolved fluorescence lifetime technique provides promising new tool for analysis of mitochondrial NAD（P）H fluorescence with good reproducibility in living cardiomyocytes. This approach will enhance our knowledge about cardiomyocyte oxidative me- tabolism and/or its dysfunction at a cellular level. In the future, this approach can prove helpful in the clinical diagnosis and treatment of mitochondrial disorder.