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

Abstract The conversion of nicotinamide adenine dinucleotide (NAD⁺) to its reduced form (NADH) by dehydrogenases is a key step in numerous redox reactions and, consequently, in cellular energy conversion. NADH autofluorescence imaging represents a promising method for the optical detection of metabolic dysfunction in living tissues. However, it is sensitive to the total NAD(H) content as well as to variations in absorption and light scattering, which may fluctuate independently. A major objective is therefore to identify invariant quantities that are responsive to reversible ischemic tissue injury while circumventing the limitations of intensity-based imaging. We show experimentally and in silico that glutamate dehydrogenase drives the NAD⁺/NADH balance toward a dissipative equilibrium when an external catalytic process promotes the NADH → NAD⁺ conversion. This dissipative equilibrium state is uniquely determined by the total NAD(H) pool, the GDH concentration, and the external catalytic activity. Experimental validation using UV-induced NADH photolysis (300–500 mW/cm²) implementing the NADH → NAD⁺ reaction showed that GDH activity can be estimated in the epicardium of ex vivo Langendorff-perfused hearts by analyzing the dissipative equilibrium. These results present a new approach to optical assessment of tissue metabolic activity based on autofluorescence imaging of NADH. Our method allows assessment of cardiac tissue ischemia without knowledge of the photolysis rate, thereby overcoming the inherent limitations of optical detection in living tissues.