Project 3: Identifying and quantifying ROS in cardiac myocytes and fibroblasts: A comprehensive study

PI Göttingen: I. Bogeski; PI London: J.R. Burgoyne; PhD student: Z. Bonilla del Rio

Scientific background and preliminary results

Cells generate a number of functionally-relevant reactive oxygen species (ROS). They all have different chemical properties which determine their biological effects. Nevertheless, the superoxide anion radical (●O2¯) and hydrogen peroxide (H2O2) are the most common ROS and are established as important signalling molecules. These two ROS are generated by the mitochondrial respiratory chain, the Nox and by other enzymatic redox reactions within organelles, cytosol but also in the extracellular space. In mitochondria, ●O2¯ is generated when a single electron is transferred from one of the electron transfer chain complexes to molecular oxygen in the mitochondrial intramembrane space or within the mitochondrial matrix. Nox, on the other hand, produce ●O2¯ by transferring electrons from NADPH to O2. Depending on the cell type, Nox can generate ●O2¯ within the ER, phagosomes, endosomes and/or on the outer side of the plasma membrane. Due to its charge and high reactivity, ●O2¯ molecules do not diffuse for a long distance and are almost fully and rapidly converted into H2O2. Because of this high reactivity, with a calculated half-life of around 2 μs, direct determination of the ●O2¯ is difficult. On the other hand, H2O2 is also highly reactive, but is significantly more stable than ●O2¯. Considering the relevance of ROS for many biological processes, their precise identification and quantification are essential to fully understand their role in physiology and pathology. ROS act as oxidants for proteins, lipids, and DNA; whereby the potentials between the redox couples determine the outcome of the chemical reaction. In this regard, ROS concentration is a very important parameter which will determine if a redox reaction takes place or not, as well as the type of oxidative modification. Hence, precise determination of cellular ROS is also indispensable to predict biological outcomes. Due to their physico-chemical properties, ROS require very sensitive, quantitative, and dynamic measuring techniques to allow detection with a good spatial and temporal resolution. Presently, the most popular ROS measuring techniques include the utilization of genetically encoded protein sensors, fluorescent dyes, electron paramagnetic resonance (EPR), and electrochemistry. More indirect techniques include mass spectrometry and redox histochemistry. These approaches all have their advantages and disadvantages; thus, drawing conclusions from datasets where only one detecting technique has been used is not advised.

The role of redox regulation, and thereby ROS, in cardiac physiology and pathology has been explored in the past. Accordingly, ROS generation, but also their elimination in cardiac cells has been inspected under various experimental conditions and in different experimental systems. Cardiac ROS have been identified and their functional role has been evaluated in whole organs, tissues, single cells, cell populations, and organelles. The major sources in cardiac cells have also been investigated and include Nox2, Nox4 and mitochondria. It is presently clear that ROS are involved in the pathogenesis of cardiovascular diseases such as heart failure, hypertrophy, arrhythmias as well as atherosclerosis. However, in most of these studies ROS have been identified using only one detection technique and by using very variable experimental settings. Other
limitations include low sensitivity or resolution of ROS detection. Moreover, the equipment, culture conditions, and other environmental factors such as O2, temperature, pH, etc. strongly vary between the different studies and thereby affect the accuracy and universality of the conclusions.

Hypotheses of the PhD project

In the project proposed, the doctoral researcher will test the following hypotheses:

1. A four techniques-based approach will provide novel insights into the spatio-temporal parameters of ROS production in cardiac myocytes and fibroblasts.

2. ROS production by intact cells and cellular organelles strongly depends on environmental factors an on pathology-induced cell alterations.

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