Mapping of Redox State of Mitochondrial Cytochromes in Live Cardiomyocytes Using Raman Microspectroscopy

Nadezda A Brazhe,Alexey R Brazhe,Ninett L Find,Marek Treiman,Olga V Sosnovtseva,Georgy V Maksimov,Valdur Saks

doi:10.1371/journal.pone.0041990

Nadezda A Brazhe, Alexey R Brazhe + Show 5 more

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https://doi.org/10.1371/journal.pone.0041990

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Abstract

This paper presents a nonivasive approach to study redox state of reduced cytochromes , and 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 rod- and round-shaped cardiomyocytes, representing different morphological and functional states. Raman mapping and cluster analysis reveal that these cardiomyocytes differ in the amounts of reduced cytochromes , and . The rod-shaped cardiomyocytes possess uneven distribution of reduced cytochromes , and in cell center and periphery. Moreover, by means of Raman spectroscopy we demonstrated the decrease in the relative amounts of reduced cytochromes , and in the rod-shaped cardiomyocytes caused by H2O2-induced oxidative stress before any visible changes. Results of Raman mapping and time-dependent study of reduced cytochromes of complexes II and III and cytochrome in cardiomyocytes are in a good agreement with our fluorescence indicator studies and other published data.

Highlights

In respiring mitochondria, the continuous electron flow through the electron transport chain (ECT) down the redox potential span of approximately 1100 mV [1] allows the energy to be captured by a proton gradient, making a protonmotive force (Dp) available for ATP production [2]
In the second set of experiments we studied redox state changes of mitochondrial cytochromes during early stage of oxidative stress caused by application of hydrogen peroxide (H2O2), when cell morphology was still not visibly affected
Peak assignment in Raman spectrum of cardiomyocytes We first defined those bands of CM Raman spectra that were most sensitive to the redox state of cytochromes c, c1 and bhigh, blow of complex III and cytochrome b of complex II

Summary

Introduction

The continuous electron flow through the electron transport chain (ECT) down the redox potential span of approximately 1100 mV [1] allows the energy to be captured by a proton gradient, making a protonmotive force (Dp) available for ATP production [2]. Ogawa et al [18] have shown that Raman spectra of cardiomyocytes (CM) from healthy and infarct regions of myocardium differ, and have attributed this to the different functional states of mitochondria. These authors have shown that peaks at 750 and 1125 cm originate from cytochromes c, c1 and b [18]. No further analysis of these peaks has been presented

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