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.

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In the aim to study calcium distribution and dynamics in single living left-ventricular rat cardiomyocytes, we have applied a new method for discrimination of the calcium probe fluorescence signal from the intrinsically fluorescing cell constituents. In a case study investigating calcium-sensitive dye Fluo-3, we have characterized the spectral and the lifetime fingerprints of the Fluo-3 fluorescence and of the cell flavin autofluorescence by spectrally-resolved confocal microscopy and/or by multiwavelength time-correlated single photon counting. We demonstrate that employing such spectral database, a low-intensity Fluo-3 signals comparable to the cell autofluorescence can be successfully resolved in cardiomyocytes using spectral linear unmixing algorithm.

Ventricular fibrillation (VF) is the main cause of sudden cardiac death. We hypothesized that VF induced by large scars in an isolated porcine heart model could aid the understanding of VF in human hearts associated with structural disease. The explanted hearts were perfused with blood and Tyrode solution at 37C, and optically imaged with a voltage-sensitive fluorescence dye (di4-ANEPPS excited at 530nm with 150W halogen lamp). The emitted signal was filtered (610nm) and recorded with high speed cameras (MiCAM02, Brain-Vison, Jp) at 0.7mm spatial resolution. No optical signals could be recorded from the core of chronic infarcts or RF lesions. A total of 10 hearts were used: 4 controls, 3 with lesions generated via RF ablation and 3 with chronic infarcts. We observed the propagation of the depolarization waves and analyzed the VF waveforms at the border zone (BZ) and normal myocardium. We analyzed the VF waves in the frequency domain by calculating the dominant frequency (DF) on select regions of interest using Matlab (Mathworks, Ca). Our results showed that DF is smaller at the BZ compared to healthy tissue. Referenced to the average DF in the control hearts (10.07þ/-0.54 Hz), the DF was slightly smaller in healthy myocardium of infarct hearts (i.e., 8.9þ/ -0.71Hz) and significantly smaller at the border zone (i.e., 6.03 þ/-0.86Hz). In ablated hearts, mean DF in normal myocardium was 9.16þ/-0.7Hz and 7.24þ/-0.66Hz at BZ, respectively. We suggest that these differences are related to the heterogeneous restitution properties as well as the changes in tissue structure at the BZ. The BZ of chronic scars is comprised of a mixture of viable and necrotic fibers; whereas in the acute settings of RF lesions, inflammation and edema are present at the BZ without alteration of fiber directions.