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

Abstract

Raman microspectroscopy has been used to monitor changes in the redox and ligandcoordination 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 III . An observation of Mb III bands in the Raman spectrum was made for all the cardiomyocytes that recovered contractile function, whilst the absence of Mb 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.3

Effect of Substrate Stiffness on Redox State of Single Cardiomyocyte: A Scanning Electrochemical Microscopy Study

“…8 The interaction of reactive oxygen species (ROS) and antioxidants (e.g., glutathione) in cells play a vital role in regulating intracellular redox microenvironment, 9 which may induce various cell repercussions such as oxidative stress and apoptosis. 10,11 Thus, it is of great importance to understand the relationship between the redox state of cardiomyocytes and their mechanical microenvironment.…”

“…The mechanical microenvironment of cardiomyocytes also changes with the pathological conditions (e.g., overaccumulation of ECM caused by myocardial fibrosis). , In addition, the redox reactions involved in biosynthetic and cellular metabolic processes are critical for both acute cardiac adaptations and chronic cardiac remodeling of cardiomyocytes . The interaction of reactive oxygen species (ROS) and antioxidants (e.g., glutathione) in cells play a vital role in regulating intracellular redox microenvironment, which may induce various cell repercussions such as oxidative stress and apoptosis. , Thus, it is of great importance to understand the relationship between the redox state of cardiomyocytes and their mechanical microenvironment.…”

Raman Spectroscopy and Microscopy Applications in Cardiovascular Diseases: From Molecules to Organs

“…Several studies were performed to understand the intracellular redox state of myoglobin and cytochrome c (or the heme complex). Researchers preconditioned ex vivo cardiomyocytes with drugs to induce NO release at the single-cell level and monitored it using Raman microspectroscopy [63]. They also monitored the reduced levels of cytochrome c from hypoxia-induced rat cardiomyocytes and reoxygenated those cells to understand the cellular response thereafter [35].…”