Wüst Rob C I, Helmes Michiel, Stienen Ger J M
Department of Physiology, Institute for Cardiovascular Research, VU University Medical Centre, Amsterdam, the Netherlands.
J Physiol. 2015 Apr 15;593(8):1829-40. doi: 10.1113/jphysiol.2014.286153. Epub 2015 Mar 13.
A photometry-based technique was developed to measure nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence and contractile properties simultaneously in intact rat trabeculae at a high time resolution. This provides insight into the function of mitochondrial complex I and II. Maximal complex I and complex II activities were determined in saponin-permeabilized right ventricular tissue by respirometry. In trabeculae, complex II function was considerably smaller than the maximal complex II activity, suggesting large complex II reserve capacity. Up-down asymmetry in NADH and FAD kinetics suggests a complex interaction between mitochondrial and contractile function. These data show that simultaneous measurement of contractile properties and NADH and FAD kinetics in cardiac trabeculae provides a mean to study the differences in complex I and II function in intact preparations in health and disease.
The functional properties of cardiac mitochondria in intact preparations have been mainly studied by measurements of nicotinamide adenine dinucleotide (NADH) autofluorescence, which reflects mitochondrial complex I function. To assess complex II function, we extended this method by measuring flavin adenine dinucleotide (FAD)-related autofluorescence in electrically stimulated cardiac trabeculae isolated from the right ventricle from the rat at 27°C. NADH and FAD autofluorescence and tension responses were measured when stimulation frequency was increased from 0.5 Hz to 1, 2 or 3 Hz for 3 min, and thereafter decreased to 0.5 Hz. Maximal complex I and complex II activity in vitro were determined in saponin-permeabilized right ventricular tissue by respirometry. NADH responses upon an increase in stimulation frequency showed a rapid decline, followed by a slow recovery towards the initial level. FAD responses followed a similar time course, but in the opposite direction. The amplitudes of early rapid changes in the NADH and FAD concentration correlated well with the change in tension time integral per second (R(2) = 0.833 and 0.660 for NADH and FAD, respectively), but with different slopes for the up and down transient. Maximal velocity of the increase in FAD concentration (16 ± 4 μm s(-1) ), measured upon an increase in stimulation frequency from 0.5 to 3 Hz was considerably smaller than that of the decrease in NADH (78 ± 13 μm s(-1) ). The respiration measurements indicated that the maximal velocity of NADH utilization (143 ± 14 μm s(-1) ) was 2 times smaller than that of FADH2 (291 ± 19 μm s(-1) ). This indicates that in cardiac mitochondria considerable complex II activity reserve is present.
开发了一种基于光度法的技术，可在高时间分辨率下同时测量完整大鼠小梁中烟酰胺腺嘌呤二核苷酸（NADH）和黄素腺嘌呤二核苷酸（FAD）的自发荧光以及收缩特性。这有助于深入了解线粒体复合体I和II的功能。通过呼吸测定法测定皂素通透的右心室组织中的最大复合体I和复合体II活性。在小梁中，复合体II的功能远小于最大复合体II活性，表明复合体II具有很大的储备能力。NADH和FAD动力学的上下不对称表明线粒体功能与收缩功能之间存在复杂的相互作用。这些数据表明，同时测量心脏小梁的收缩特性以及NADH和FAD动力学为研究健康和疾病状态下完整制剂中复合体I和II功能的差异提供了一种手段。
完整制剂中心脏线粒体的功能特性主要通过测量烟酰胺腺嘌呤二核苷酸（NADH）自发荧光来研究，该荧光反映线粒体复合体I的功能。为了评估复合体II的功能，我们通过在27°C下测量从大鼠右心室分离的电刺激心脏小梁中与黄素腺嘌呤二核苷酸（FAD）相关的自发荧光来扩展此方法。当刺激频率从0.5 Hz增加到1、2或3 Hz持续3分钟，然后降至0.5 Hz时，测量NADH和FAD自发荧光以及张力反应。通过呼吸测定法测定皂素通透的右心室组织中的体外最大复合体I和复合体II活性。刺激频率增加时NADH反应迅速下降，随后缓慢恢复到初始水平。FAD反应遵循相似的时间进程，但方向相反。NADH和FAD浓度早期快速变化的幅度与每秒张力时间积分的变化密切相关（NADH和FAD的R²分别为0.833和0.660），但上升和下降瞬变的斜率不同。刺激频率从0.5增加到3 Hz时测得的FAD浓度增加的最大速度（16±4μm s⁻¹）远小于NADH降低的速度（78±13μm s⁻¹）。呼吸测量表明，NADH利用的最大速度（143±14μm s⁻¹）比FADH₂的最大速度（291±19μm s⁻¹）小2倍。这表明心脏线粒体中存在相当大的复合体II活性储备。
