Beyond Intensity Imaging: Dissipative Equilibrium of NADH/NAD⁺ as a Metabolic Sensor for Ischemic Response in Cardiac Tissue

AbstractNADH autofluorescence imaging is a promising tool for detecting metabolic dysfunction but is sensitive to optical artifacts. We demonstrate that glutamate dehydrogenase (GDH) drives the NADH/NAD⁺ balance toward a dissipative equilibrium under external catalytic NADH→NAD⁺ conversion, enabling GDH activity assessment in cardiac tissue. This approach has potential applications in organ preservation and understanding metabolic stress responses.Optical mappingNADH fluorescence was recorded for hearts in Tyrode’s salt solution (Sigma-Aldrich Co., USA) using an Olympus MVX-10 fluorescence microscope (Olympus Co., Tokyo, Japan) equipped with a high-speed EM-CCD camera, Andor iXon-3 (Andor Technology Ltd., Belfast, UK), at a sampling rate of 67 frames per second. The final imaging resolution was 256×256 pixels (2×2-pixel binning, 5× electron multiplication), with an exposure time of ~14.9 ms. The excitation light was generated by a LIGHTNING-CURE LC-L1V5 UV diode (Hamamatsu, Japan), with an excitation wavelength of 365±5 nm. To minimize the absorption of excitation radiation by the tissue, an optically transparent mixture of Tyrode’s and Normacor solutions (1:4 ratio) was used for perfusion—this minimized the absorption of both excitation and emission radiation by red blood cells. To extract NADH fluorescence, a dichroic mirror (Chroma T455lp, Olympus Co., Tokyo, Japan) and an emission filter (Chroma ET480/40m, Olympus Co., Tokyo, Japan) with a passband of 40 nm centered at 480 nm were used. The estimated emission efficiency was 27.8%, representing a necessary compromise to suppress flavin fluorescence. Signal amplification was fully ensured by the applied binning (2×2 pixels).Heart isolation and perfusion The cardiac isolation and aortic cannulation protocol began with general anesthesia (isoflurane) of a laboratory rat (Wistar, *n* = 6). An important aspect of the study was the rational use of laboratory animals; therefore, the experimental testing of hypotheses was carried out according to pre-selected protocols (given in the following sections) on a minimum sufficient number of animals. The motivation for the protocols drawn up and their application is given in the following sections. The subsequent steps of cardiac retrieval included: (1) making an incision from the xiphoid process to the lateral edges of the ribs, (2) cutting the ribs along the left and right anterior axillary lines to facilitate thoracotomy, and (3) lifting the chest. These steps ensured complete access to the heart. The cardiac retrieval procedure was completed by separating portions of the vena cava and aorta; after this, the heart was washed with an oxygenated solution of Tyrode's salts (Sigma-Aldrich Co., USA) containing heparin (0.4–0.5 IU/mL) and transferred to a 65-mm Petri dish with the same solution. The heart was then cannulated (using a catheter needle with a soft polymer sheath, needle size 18G), and for additional fixation of the heart, a knot was tied on the cannula using surgical thread. The heart was then connected to a perfusion circuit with oxygenation. No more than 10 minutes elapsed from the moment of heart removal until the start of perfusion through the cannula. 1.2. Perfusion of Isolated HeartsAfter cannulation, the heart was perfused according to the Langendorff method. For this purpose, a perfusion setup was used, combining a perfusion circuit and an optical cardiac mapping system. The perfusion circuit included a Masterflex L/S dual-circuit peristaltic pump with a Masterflex L/S Easy-Load II pump head (Cole-Parmer Instrument Company, Vernon Hills, IL, USA). The total perfusate volume was 50 mL, and the perfusion rate was 1 mL/min. A Cole-Parmer Polystat Standard thermostat (Cole-Parmer Instrument Company, Vernon Hills, IL, USA) and a Tokai Hit thermoplate (Tokai Hit, Japan) were used to maintain the fluid temperature at 37°C. A Cole-Parmer Bubble Trap and an Oxygenating Bubbler (Cole-Parmer Instrument Company, Vernon Hills, IL, USA) were used to oxygenate the solution and eliminate gas bubbles in the perfusate. Long-term preservation of the heart was achieved by adding the cardioplegic solution Normacor (OAO CardioSystemPharma, Khimki, Russia) to the perfusate in a 1:4 ratio. To maintain a constant temperature of 37°C throughout the perfusion circuit, a second water-jacketed perfusion circuit based on the Cole-Parmer Polystat Standard thermostat (Cole-Parmer Instrument Company, Vernon Hills, IL, USA) was used. The gas mixture (95% O₂ and 5% CO₂) supply to the perfusate was controlled using a dissolved oxygen meter (Cole-Parmer Instrument Company, Vernon Hills, IL, USA). HEPES (Sigma-Aldrich Co., USA) was used to prepare Tyrode’s salt solution, and the pH was maintained at 7.4 throughout the experiment. A water column was used to maintain pressure in the rat heart. The optical mapping system included an Olympus MVX-10 MacroView fluorescence microscope (Olympus Co., Tokyo, Japan) and a high-speed EM-CCD camera, Andor iXon-3 (Andor Technology Ltd., Belfast, UK).烟酰胺腺嘌呤二核苷酸（NADH, Nicotinamide Adenine Dinucleotide）自体荧光成像是检测代谢功能异常的极具前景的工具，但易受光学伪影干扰。本研究证实，在体外催化性NADH→NAD⁺转化过程中，谷氨酸脱氢酶（GDH, Glutamate Dehydrogenase）可将NADH/NAD⁺平衡推向耗散平衡态，从而实现心肌组织中GDH活性的评估。该方法在器官保存以及理解代谢应激反应方面具有潜在应用价值。 光学标测 本研究使用奥林巴斯MVX-10荧光显微镜（Olympus MVX-10, Olympus Co., Tokyo, Japan）搭配高速EM-CCD相机Andor iXon-3（Andor Technology Ltd., Belfast, UK），对置于台氏盐溶液（Tyrode’s salt solution, Sigma-Aldrich Co., USA）中的心脏进行NADH荧光记录，采样速率为67帧/秒。最终成像分辨率为256×256像素（采用2×2像素合并，电子倍增倍数为5），曝光时长约14.9 ms。激发光源采用LIGHTNING-CURE LC-L1V5紫外二极管（Hamamatsu, Japan），激发波长为365±5 nm。为减少组织对激发辐射的吸收，我们使用台氏溶液与Normacor溶液按1:4比例混合的光学透明灌注液，该混合液可同时降低红细胞对激发光与发射光的吸收。 为提取NADH荧光信号，本研究使用二向色镜Chroma T455lp（Chroma T455lp, Olympus Co., Tokyo, Japan）以及中心波长为480 nm、通带宽度为40 nm的发射滤光片Chroma ET480/40m（Chroma ET480/40m, Olympus Co., Tokyo, Japan）。经估算，该配置的发射效率为27.8%，这是抑制黄素荧光所需的必要折中方案。信号放大可通过采用的2×2像素合并方案得到充分保障。 心脏分离与灌注 本研究的心脏分离及主动脉插管流程始于对实验大鼠（Wistar品系，*n*=6）实施异氟烷（isoflurane）全身麻醉。本研究注重实验动物的合理使用，因此假说验证实验均按照预先选定的方案（详见后续章节），使用最小足够数量的实验动物开展。实验方案的制定依据与应用细节将在后续章节详述。 心脏获取的后续步骤包括：(1) 从剑突至肋骨侧缘做切口；(2) 沿左右腋前线切断肋骨以开胸；(3) 提拉胸腔，以此完全暴露心脏。心脏获取流程的最后步骤为分离腔静脉与主动脉部分段，随后用含肝素（0.4–0.5 IU/mL）的充氧台氏盐溶液（Sigma-Aldrich Co., USA）冲洗心脏，并将其转移至盛有相同溶液的65 mm培养皿中。随后使用带有柔软聚合物鞘的18G穿刺针导管对心脏进行插管，并用手术线在插管上打结以进一步固定心脏。之后将心脏连接至充氧灌注环路。从心脏取出至开始通过插管灌注的时间间隔不超过10分钟。 1.2. 离体心脏灌注 插管完成后，按照朗德多夫（Langendorff）法对心脏进行灌注。本研究使用的灌注装置集成了灌注环路与光学心脏标测系统。灌注环路包含Masterflex L/S双回路蠕动泵及其配套的Masterflex L/S Easy-Load II泵头（Cole-Parmer Instrument Company, Vernon Hills, IL, USA）。灌注液总容积为50 mL，灌注速率为1 mL/min。使用Cole-Parmer Polystat Standard恒温水浴（Cole-Parmer Instrument Company, Vernon Hills, IL, USA）与Tokai Hit热板（Tokai Hit, Japan）将溶液温度维持在37°C。使用Cole-Parmer气泡捕集器与充氧气泡器（Cole-Parmer Instrument Company, Vernon Hills, IL, USA）对灌注液进行充氧并去除气体气泡。 通过向灌注液中按1:4比例加入心肌停搏液Normacor（OAO CardioSystemPharma, Khimki, Russia）可实现心脏的长期保存。为维持整个灌注环路的温度恒定在37°C，我们额外采用了基于Cole-Parmer Polystat Standard恒温水浴（Cole-Parmer Instrument Company, Vernon Hills, IL, USA）的第二套水套式灌注环路。 灌注液的气体混合物（95% O₂与5% CO₂）供应通过溶解氧检测仪（Cole-Parmer Instrument Company, Vernon Hills, IL, USA）进行控制。使用4-羟乙基哌嗪乙磺酸（HEPES, Sigma-Aldrich Co., USA）配制台氏盐溶液，实验全程将pH值维持在7.4。使用水柱维持大鼠心脏的灌注压力。光学标测系统包含奥林巴斯MVX-10宏观视野荧光显微镜（Olympus MVX-10 MacroView, Olympus Co., Tokyo, Japan）以及高速EM-CCD相机Andor iXon-3（Andor Technology Ltd., Belfast, UK）。