Direct Measurements of Heme Concentration in Heart Cells by Raman Spectroscopy

Resonance Raman spectroscopy is a powerful technique that yields specific molecular information on samples under study. It has been used as a powerful tool to understand the structure and states of heme proteins. The vibrational bands of heme are well known. However, excitation wavelengths of 488 and 405 nm have not been used before to provide physiologically relevant information in terms of the concentration and state of heme proteins in cardiomyocytes. 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, and the mechanism of cardioprotective effect of nitric oxide donor in isolated ventricular cardiomyocytes exposed to metabolic inhibition and re-energisation. In order to understand the effect of the different environments on cells and hemoproteins, pre-resonance and resonance Raman tweezing microspectroscopy were applied to monitor the intracellular redox state of hemoglobin from single red blood cells. Further, hypoxia and reoxygenation in an isolated red blood cell were investigated using pre-resonance Raman tweezing and a microfluidic flow cell. Moreover, for the first time the effect of nitric oxide donor on single red blood cells under hyperoxic and hypoxic condations was investigated using Raman tweezing. In terms of cardiomyocytes, for the first time the results show that Raman microspectroscopy using an excitation wavelength of 488 nm can be used to: (1) characterize different cellular responses to reoxygenation of hypoxic cardiomyocytes, (2) investigate the difference between cardiomyocytes that did not recover contractility, and the other cell did recover contractility exposed to metabolic inhibtion and re-energisation, and (3) investigate the mechanism of cardioprotective effect of nitric oxide donor in isolated ventricular cardiomyocytes exposed to metabolic inhibition and re-energisation. A short wavelength of 405 nm was found to result in rapid damage to the cardiomyocytes