ИСТИНА

Cell volume is regulated by multiple interrelated processes taking place in the cell cytoplasm and in the submembrane region. Regulation of the activity of plasma membrane ATPases is one of the important mechanisms for the maintenance of cell volume. This regulation in turn depends on the ATP production of mitochondria. It is known that mitochondrial production of ATP is heavily dependent on the dynamically changing redox status of the electron transport chain (ETC) cytochromes. At present, main methods of mitochondria studies are fluorescent microscopy with small organic dyes or genetically encoded fluorescent proteins or absorption spectroscopy and measurements of O2 and ETC substrate consumption on isolated ETC complexes. These techniques require cell disruption or affect cell properties. Therefore, for the investigation of mitochondria-related processes of cell volume regulation it is important to develop novel, non-invasive experimental approaches. We present a non-invasive, label-free approach to study redox state of reduced cytochromes c, c1 and b of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach we perform studies of cardiomyocytes representing different morphological and functional states. Raman mapping reveals that these cardiomyocytes differ in the amounts of reduced cytochromes c, c1 and b. The rod-shaped cardiomyocytes possess uneven distribution of reduced cytochromes c, c1 and b in cell center and periphery. Moreover, we demonstrated the decrease in the relative amounts of reduced cytochromes c, c1 and b in the rod-shaped cardiomyocytes caused by H2O2-induced oxidative stress before any visible morphological changes. Results of Raman mapping and time-dependent study of reduced cytochromes of complexes II and III and cytochrome c in cardiomyocytes are in a good agreement with our fluorescence indicator studies and other published data.