【摘要】 目的研究实验动物心肌线粒体氧化还原状态,监测缺血时线粒体功能改变的早期信号。方法线粒体呼吸链主要电子供应者烟酰胺腺嘌呤(磷酸)二核苷酸,或NAD(P)H荧光为无创荧光标记,用光谱分辨的时间相关单光子计数(TCSPC)检测紫外光(UV)激发的心肌自发荧光(AF)光谱和荧光寿命。结果需用至少3个荧光寿命池0.4~0.7 ns,1.2~1.9 ns和8.0~13.0 ns描述心肌AF。相关衰减光谱(DAS)显示4个NAD(P)H荧光固有光谱,分别为峰值470 nm的短荧光寿命池,及450 nm,470 nm和490 nm的中间和长荧光寿命池。酮体增加线粒体NADH产量,提高AF强度,但不改变荧光寿命。线粒体呼吸阻断剂Rotenone,显著增加AF强度和缩短平均荧光寿命。氧化磷酸化解偶联剂Dinitrophenol(DNP),显著降低AF强度,在520 nm处增宽荧光光谱并显著延长平均荧光寿命。这些结果和NADH荧光动力学离体实验(in vitro)及模拟缺血状态实验有可比性。结论研究可解释NADH构象改变,以及从NADH到异戎酸脱氢酶(LipDH)结合的黄素蛋白间能量转移的荧光动力学变化。光谱分辨的荧光寿命显微技术提供了在细胞水平上研究心肌能量代谢或线粒体功能障碍的新工具。更多还原

【Abstract】 Objective To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiological conditions,such as ischemia.%Methods Fluorescence of nicotinamide adenine dinucleotide(phosphate),or NAD(P)H,the principal electron donor in mitochondrial respiration responsible for vital ATP supply of cardiomyocytes,is studied for non-invasive fluorescent probing of the mitochondrial function.Examination of NAD(P)H fluorescence in living cardiomyocytes following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting(TCSPC),is based on the simultaneous measurement of the fluorescence spectra and lifetime.%Results The dynamic characteristics of NAD(P)H fluorescence decay in living rat cardiomyocytes show that at least a 3-exponential decay model,with 0.4-0.7 ns,1.2-1.9 ns and 8.0-13.0 ns lifetimes,is necessary to describe cardiomyocyteautofluorescence(AF).Decay-associated spectra(DSA) revealed the presence of 4 spectrally-distinct populations of NADH molecules in cardiomyocytes with spectral maximum at 470 nm for short-lifetime pool for the first time,and emission peaks at 450 nm,470 nm and 490 nm for intermediate and long-lifetime pools.Increased mitochondrial NADH content ratio by ketone bodies enhanced the AF intensity,without the significant change in fluorescent lifetimes.Rotenone,the inhibitor of Complex I of the mitochondrial respiratory chain,increased AF and shortened the average fluorescence lifetime.Dinitrophenol(DNP),an uncoupling agent of the mitochondrial oxidative phosphorylation,lowered AF,broadened the spectral shoulder at 520 nm and increased the average lifetime.These effects,comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro,were also examined under ischemia-mimetic conditions.% Conclusion Our findings anticipate a contribution of both conformational NADH changes and energy transfer from NADH to lipoamide dehydrogenase(LipDH)-bound flavins,to explain observed fluorescence kinetics.Presented spectrally resolved fluo-rescence lifetime approach provides promising new tool for analysis of mitochondrial NAD(P)Hin living cardiomyocytes,and hence for investigation of energy metabolismand mito-chondrial dysfunction at a cellular level.更多还原