Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P)H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states.

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NAD(P)H, crucial in effective management of cellular oxidative metabolism and the principal electron donors for enzymatic reactions, is a major source of autofluorescence induced in cardiac cells following excitation by UV light. Spectrally-resolved timecorrelated single photon counting was used to simultaneously measure the fluorescence spectra and fluorescence lifetimes of NAD(P)H, following excitation by a pulsed picosecond 375 nm laser diode. Spectra, as well as fluorescence lifetimes of NADH and NADPH molecules were investigated in solution at different concentrationsEffects of their respective dehydrogenation by lipoamide dehydrogenase (LipDH) or glutathione reductase (GR) were also questioned. NAD(P)H autofluorescence recorded in vitro was compared to the one measured in freshly isolated cardiac cells. We observed a good comparability between NAD(P)H parameters recorded in solution and in cells.