A reference-free optical method for assessing cardiac ischemia via NADH autofluorescence dynamics

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 (GDH) drives the NAD+/NADH balance toward a competing semi-equilibrium (CSE) state when an external catalytic process promotes the NADH → NAD+ conversion. This CSE 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 CSE. These results present a new approach to the optical assessment of tissue metabolic activity based on autofluorescence imaging of NADH. Our method allows the assessment of cardiac tissue ischemia without knowledge of the photolysis rate, thereby overcoming the inherent limitations of optical detection in living tissues.