Effect of ouabain on metabolic oxidative state in living cardiomyocytes evaluated by time-resolved spectroscopy of endogenous NAD(P)H fluorescence

Abstract

Abstract. Time-resolved spectrometry of endogenous nicotinamide dinucleotide phosphate [NAD(P)H] fluorescence is a useful method to evaluate metabolic oxidative state in living cells. Ouabain is a well-known pharmaceutical drug used in the treatment of cardiovascular disease, the effects of which on myocardial metabolism were recently demonstrated. Mechanisms implicated in these actions are still poorly understood. We investigate the effect of ouabain on the metabolic oxidative state of living cardiac cells identified by time-resolved fluorescence spectroscopy of mitochondrial NAD(P)H. Spectral unmixing is used to resolve individual NAD(P)H fluorescence components. Ouabain decreased the integral intensity of NAD(P)H fluorescence, leading to a reduced component amplitudes ratio corresponding to a change in metabolic state. We also noted that lactate/pyruvate, affecting the cytosolic NADH gradient, increased the effect of ouabain on the component amplitudes ratio. Cell oxidation levels, evaluated as the percentage of oxidized NAD(P)H, decreased exponentially with rising concentrations of the cardiac glycoside. Ouabain also stimulated the mitochondrial NADH production. Our study sheds a new light on the role that ouabain plays in the regulation of metabolic state, and presents perspective on a noninvasive, pharmaceutical approach for testing the effect of drugs on the mitochondrial metabolism by means of time-resolved fluorescence spectroscopy in living cells.

Fingerprinting of metabolic states by NAD(P)H fluorescence lifetime spectroscopy in living cells: A review

“…This reducing condition is characterized by decrease in the cell oxidation levels towards fully reduced state and, consequently, by rise in NAD(P)H fluorescence intensity. In this situation, the resolved fluorescence lifetime of the NAD(P)H in ''bound'' state was found without change or increased (Chorvatova et al, 2013b), and the redox ratio shifted towards more ''free'' NAD(P)H; this was observed both in rat (Chorvatova et al, 2013b), as well as in human (Cheng et al, 2007) cardiomyocytes. Flavin fluorescence remains mostly unchanged, leading to an increased NADH/FAD ratio.…”

“…In these conditions, the cell is put in a fully oxidized state, with NADH dehydrogenated at maximum by flavoprotein complexes, leading to lower NAD(P)H fluorescence intensity. In this situation, fluorescence lifetime of the resolved component related to NAD(P)H in the ''bound'' state has been shown to be decreased, while that in the ''free'' state increased (Chorvatova et al, 2013b), resulting in prolonged mean lifetime. In this oxidizing condition, increase in the cell oxidation results in complete or partial depletion of oxidized nucleotides: fluorescence of NADH decreases, while that of FAD increases, leading to reduced NADH/FAD ratio.…”

“…In cardiac cells, stimulation of NADH production by 3-␤-hydroxybutyrate/acetoacetate gradient leads to the rise in the NAD(P)H intensity, which was accompanied by no change of the fluorescence lifetime of the resolved NAD(P)H components (Chorvatova et al, 2013b), resulting in no significant change in the mean lifetime. In DLD-1 cells, inhibition of glycolysis with dichloroacetate caused switch to an OXPHOS signature with lower amounts of ''free'' NADH (Stringari et al, 2012).…”

“…In DLD-1 cells, inhibition of glycolysis with dichloroacetate caused switch to an OXPHOS signature with lower amounts of ''free'' NADH (Stringari et al, 2012). Shift towards more ''free'' NAD(P)H can result in an increase in the ''free/bound'' component amplitude ratio (a2/a1), or it remains without change (Chorvatova et al, 2012(Chorvatova et al, , 2013b. In addition, constant flavin fluorescence results in the rise in NADH/FAD ratio.…”

An automated multiwell plate reading flim microscope for live cell autofluorescence lifetime assays

“…Over the past two decades the literature relating to°uorescence lifetime measurements of NADH, both in vitro and in vivo, has grown extensively, with many studies across di®erent cell lines and utilizing di®erent metabolic modulators establishing a link between changes in°u orescence lifetime and changes in cell metabolism. [8][9][10][11] Since aberrant metabolism is a hallmark of cancer, there has been considerable interest in exploiting measurements of cellular auto°uorescence lifetime to read out changes in metabolic state for the study, diagnosis and monitoring of cancer; such changes have been associated with a shift from oxidative phosphorylation to glycolysis. 12 Recently, the link between metabolic state and stem cell fate has o®ered an additional motivation for exploiting NADH lifetime-based readouts.…”