Spectroscopic analysis of myoglobin and cytochrome c dynamics in isolated cardiomyocytes during hypoxia and reoxygenation

Abstract

Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cytochrome c from isolated adult rat cardiomyocytes during hypoxia and reoxygenation. The nitrite reductase activity of myoglobin leads to the production of nitric oxide in cells under hypoxic conditions, which is linked to the inhibition of mitochondrial respiration. In this work, the subsequent reoxygenation of cells after hypoxia is shown to lead to increased levels of oxygen-bound myoglobin relative to the initial levels observed under normoxic conditions. Increased levels of reduced cytochrome c in ex vivo cells are also observed during hypoxia and reoxygenation by Raman microspectroscopy. The cellular response to reoxygenation differed dramatically depending on the method used in the preceding step to create hypoxic conditions in the cell suspension, where a chemical agent, sodium dithionite, leads to reduction of cytochromes in addition to removal of dissolved oxygen, and bubbling-N 2 gas leads to displacement of dissolved oxygen only. These results have an impact on the assessment of experimental simulations of hypoxia in cells. The spectroscopic technique employed in this work will be used in the future as an analytical method to monitor the effects of varying levels of oxygen and nutrients supplied to cardiomyocytes during either the preconditioning of cells or the reperfusion of ischaemic tissue.

Monitoring Changes in the Redox State of Myoglobin in Cardiomyocytes by Raman Spectroscopy Enables the Protective Effect of NO Donors to Be Evaluated

“…15 The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health. 21 The procedure yielded ~80% quiescent rod-shaped ventricular myocytes with approximate dimensions of 100 µm-length and 20 µm-width.…”

“…The flow rate was maintained at approximately 4.5 ml/minute using a peristaltic pump. Full details of the perfusion apparatus were given in the earlier publication, 15 with the exception that, in the present experiments, (i) the chamber was regulated at a temperature of ~34 °C and (ii) a pair of platinum electrodes was positioned in the chamber aligned parallel to the direction of flow. The cardiomyocytes were stimulated by an electric field at 1 Hz with a magnitude of 25 V and duration of 5 ms using a physiological stimulator (Digitimer).…”

“…Two different apparatus were employed in the experiments: either (i) a population of cardiomyocytes was monitored by brightfield imaging of a large area of the perfusion chamber using a commercial-inverted microscope, or (ii) a single cardiomyocyte was monitored by the combination of confocal Raman spectroscopy and brightfield imaging using a homebuiltinverted microscope (described previously 15 ).…”

“…20 The measured scattering cross-section of the ν 4 band obtained using a nonresonant wavelength of 488 nm was comparable to that obtained using a resonant wavelength of 405 nm, but the cardiomyocytes could be exposed to higher total energies of irradiation without causing photoinduced damage of cells. 15 The centre wavenumber of the ν 4 band is distinct for deoxygenated myoglobin (Mb II ; 1356 cm -1 ) oxygenated myoglobin or metmyoglobin (Mb II -O 2 , Mb III ; 1372 cm -1 ), ferrous cytochrome c (Cyt II ; 1363 cm -1 ) and ferric cytochrome c (Cyt III ; 1374 cm -1 ). Identification of these species is also assisted by the centre wavenumber of the bands measured in the region 1500-1650 cm -1 ; where the high-spin heme proteins, Mb II and Mb III , exhibit a similar pattern of bands, which differs from that exhibited by low-spin heme proteins, such as Mb II -O 2 .…”

“…11 The oxygenated state of myoglobin also acts as a scavenger of cellular NO and, hence, the protein plays a vital role in NO homeostasis, which is particularly important in cardiomyocytes as elevated levels inhibit mitochondrial respiration. 12,13 Non-resonance 14,15 and resonance [16][17][18][19] Raman spectroscopy have been used before in structural imaging of heart tissue 16 and to monitor dynamic changes in both isolated hearts 18 , cardiomyocytes [14][15][16][17] and exposed mitochondria. 19 In our recent work, the cellular mechanisms in ex vivo cardiomyocytes during hypoxia and hyperoxic reoxygenation were studied by Raman spectroscopy using a (non-resonant) excitation wavelength of 488 nm.…”