DOI: 10.5281/zenodo.15802785
title: Beyond Intensity Imaging: Dissipative Equilibrium of NADH/NAD⁺ as a Metabolic Sensor for Ischemic Response in Cardiac Tissue

description: 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).