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

Raman microspectroscopy has been used to monitor changes in the redox and ligand-coordination states of the heme complex in myoglobin during the pre-conditioning of ex vivo cardiomyocytes with pharmacological drugs that release nitric oxide (NO). These chemical agents are known to confer protection on heart tissue against ischemia-reperfusion injury. Subsequent changes in the redox and ligand-coordination states during experimental simulations of ischemia and reperfusion have also been monitored. We found that these measurements, in real time, could be used to evaluate the pre-conditioning treatment of cardiomyocytes, and predict the likelihood of cell survival following a potentially-lethal period of ischemia. Evaluation of the pre-conditioning treatment was done at the single-cell level. The binding of NO to myoglobin, giving a 6-coordinate ferrous-heme complex, was inferred from the measured Raman bands of a cardiomyocyte by comparison to pure solution of the protein in the presence of NO. A key change in the Raman spectrum was observed after perfusion of the NO-donor was completed, where if the pre-conditioning treatment was successful then the bands corresponding to the nitrosyl complex were replaced by bands corresponding to metmyoglobin, Mb[SUPERSCRIPT: III]. An observation of Mb[SUPERSCRIPT: III] bands in the Raman spectrum was made for all the cardiomyocytes that recovered contractile function, whilst the absence of Mb[SUPERSCRIPT: III] bands always indicated that the cardiomyocyte would be unable to recover contractile function, following the simulated conditions of ischemia and reperfusion in these experiments.