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

A Almohammedi, A Hudson, N Storey. University of Leicester, Leicester, UK

Following ischaemia / reperfusion injury, as in a myocardial infraction, cardiac myocytes undergo oxidative stress which leads to several potential outcomes including; necrotic or apoptotic cell death or dysregulated calcium homeostasis or disruption of the electron transport chain. All the pathophysiological changes caused by oxidative stress are not fully understood. The aim of this study was to develop a novel technique, resonance Raman microspectroscopy, to investigate the cellular responses of cardiac myocytes to hypoxia and reoxygenation.

Adult male Wistar rats (250-300 g) were sacrificed by cervical dislocation and single ventricular myocytes isolated by enzymatic dissociation. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Resonance Raman spectroscopy was sensitive enough to measure the redox state of both myoglobin and cytochrome c of isolated adult rat cardiac myocytes during hypoxia and reoxygenation. We were able to distinguish the vibrational bands by comparison to the spectra of pure proteins; for deoxygenated myoglobin, MbII (1356 cm-1), oxygenated myoglobin, MbII-O2 (1372 cm-1), metmyoglobin, MbIII (1372 cm-1), reduced cytochrome c, CytII (1363 cm-1), and oxidised cytochrome c, CytIII (1374 cm-1).

The presence of a ν4 band centred at 1356 cm-1 in the spectrum of both the cardiac myocyte and deoxymyoglobin, MbII, suggests that the dominant protein observed is deoxygenated myoglobin. The ν4 band in the solution spectra of ferrocytochrome c, CytII, and ferricytochrome c, CytIII, are found at the higher wavenumber values of 1363 cm-1 and 1374 cm-1, respectively, compared with the centre wavenumber of 1356 cm-1 measured in the cardiac myocyte spectrum. A comparison of the position and relative intensities of the bands between 1540 and 1650 cm-1 was evidence that cytochrome c is not making a substantial contribution to the measured Raman spectrum for the untreated cardiac myocyte.

After hypoxia and reoxygenation there was the appearance of a strong shoulder on the ν4 band at 1372 cm-1. There was also a change in the vibrational bands in the region 1540 - 1650 cm-1 in particular the intensity increased for bands at 1587 cm-1 and 1622 cm-1. This indicated that there was an increased contribution in the cardiac myocyte from oxygenated myoglobin. In addition, the appearance of cytochrome c was observed after period of chemical hypoxia with sodium dithionite (8 mM), but not with Tyrode solution bubbled N2 gas. Separate ν4 bands were resolved at 1356 (MbII), 1363 (CytII) and 1372 cm-1 (MbII-O2) accompanied by an increase in the ν11 band at 1548 cm-1 (CytII), ν2/ν37/ν19 bands at 1587 cm-1 (MbII-O2 and CytII), and ν10 bands at 1622 (CytII) and 1642 cm-1 (MbII-O2). The difference spectrum showed that there was increased intensity at 1360 relative to 1370 cm-1 suggesting the release of cytochrome c is greater during the reoxygenation of the cell following chemical hypoxia with sodium dithionite compared to bubbling nitrogen gas.

In conclusion, the resonance Ramen spectroscopic technique was adapted for assaying redox changes to cytochrtome C and myoglobin after hypoxia / reoxygenation. The redox potential of cytochrome C and myoglobin both changed upon hypoxia / reoxygenation. There is potential for future use of the resonance Ramen spectroscopic technique as an analytical method to monitor the effects of varying levels of oxygen and nutrients supplied to cardiac myocytes during either the preconditioning or the reperfusion of ischaemic tissue. These results also have an impact on the assessment of experimental simulations of hypoxia and ischaemia in cells, practically chemical versus N2 bubbled solution to simulate hypoxia.