Objective To insure early detection and hence efficient prevention of allograft rejection in transplanted heart, investigate possible applications of NAD(P)H fluorescence components analysis at the level of living cardiac cells to propose new approaches for diagnosis of rejection. Methods NAD(P)H was studied for noninvasive fluorescent probing of the mitochondrial function. Human cardiomyocyte were isolated from one additional endomyocardial biopsy (EMB) of 14 pediatric patients with heart ransplantation. Rat cardiomyocyte (n=5, 13-14 week old) were also isolated by the same approach for human myocytes. Autofluorescence(AF) was recorded in living cardiomyocytes following excitation with 375 nm UVlight and detection by spectrallyresolved time correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and lifetimes. Rat cardiac cells were divided into four groups: normoxic condition, normoxia with Rotenone, ischemic condition and ischemia with Rotenone. Comparison of cardiomyocyte AF between human and rat; compared kinetics of rat cardiomyocytes AF in normoxic conditions to ischemiamimicking ones, induced at physiological temperatures by reducing cell pH and oxygen content; comparison of cardiomyocyte AF dynamic changes in transplanted pediatric patients presenting either no rejection (R0) or mild rejection (R1). Results We have achieved appropriate isolation of living cardiomyocytes from human biopsies, as well as from rat cardiac tissues and determined their AF. At least a 3-exponential decay with 0.5-0.7ns, 1.9-2.4 ns and 9.0-15.0 ns lifetime pools is necessary to describe human cardiomyocyte AF within 420560 nm spectral range. Rat cardiomyocyte steadystate AF in ischemiamimicking condition was significantly increased when compared normoxic ones (Plt;0.05); application of Rotenone induced a significant increase in AF intensity in ischemic and normoxic condition, however no significant difference between the two groups (Plt;0.05).Human cardiomyocyte AF was found significantly lower in comparison to experimental rat model in the same condition(Plt;0.05). A correlation between changes in steadystate NAD(P)H fluorescence and rejection grades was found when comparison of R1 to R0. R1 showed significantly increased fluorescence intensity (Plt;0.05), without change in the spectra shape, results can be comparable to the effect of ischemiamimic conditions. Conclusion Our studies clearly demonstrated that spectrallyresolved fluorescence spectral analysis coupled to fluorescence lifetime are high sensitive approaches to examine mitochondrial metabolic oxidative state directly in living human cardiomyocytes with good reproducibility. Human cardiomyocytes are more metabolically active than the rat ones, while this activity (and thus ATP production) seems lowered during rejection process. In perspective, the advantage of this method is the possibility of its combination to multiphoton confocal microscopy, which can result in the adaptation of this approach directly to tissue biopsy, as well as in vivo directly via cardiac catheterization without the necessity of cell isolation. This approach provides promising new tool for clinical diagnosis and treatment of allograft rejection, and will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level.
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［NAD(P)H］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(in vitro)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。