What is coronary heart disease?
CHD is the most common of cardiovascular diseases, causing over seven million deaths a year worldwide1. It involves the narrowing of the coronary arteries due to a gradual build-up of fatty material on the interior surface of the vessels (atherosclerosis, Fig. 1). As these arteries are responsible for supplying the heart with oxygen-rich blood, this narrowing of the arteries makes it harder for the blood to flow through, reducing the oxygen supply to heart muscle cells (cardiac ischemia). The resulting mitochondrial dysfunction and oxidative stress cause inflammation and, if a piece of this fatty material breaks away from the plaque, it can cause a complete blockage of the vessel. This leads to the death of surrounding muscle cells and, potentially, a heart attack (myocardial infarction). As such, there is significant interest in the various pathways involved in the progression of CHD, with many researchers investigating possible clinically-relevant biomarkers and therapeutic targets, such as the cardiac-specific regulatory protein troponin2 or non-coding RNA and mRNA from monocytes3.
Following a period of acute ischemia, restoration of the blood flow (reperfusion) can also damage the cells, as the rapid increase of oxygen creates reactive oxygen species (ROS) and inflammation due to oxidative stress. Understanding the physiology of ischemia and reperfusion injury (IRI) is, therefore, another key area of clinical interest but, until recently, it has been difficult to initiate, maintain and reverse acute hypoxic environments in in vitro studies, leading to an over-reliance on animal models4.
Developing a cell-based model
When performing cell-based assays, it is important that the conditions are as close to the in vivo environment as possible to ensure that the results are relevant. Investigating the effects of IRI, therefore, requires an experimental setup that can imitate rapid changes in oxygen, while also recording its biological effects.
BMG LABTECH’s powerful CLARIOstar® Plus multi-mode microplate reader – equipped with advanced atmospheric control unit (ACU) – provides a powerful and convenient solution to this issue. The ACU can regulate and independently control oxygen and carbon dioxide gas levels within the microplate reading chamber, allowing oxygen levels to be rapidly and precisely controlled to mimic normal in vivo conditions, ischemia or reperfusion events5. This makes it possible to investigate the effects that oxygen deprivation and reperfusion injury can have on cells, elucidating the role of various cellular processes and identifying potential drug targets to minimize the effects of patients suffering from CHD.
Ischemia-reperfusion research on cell-based models requires a multiplexed approach. The first requirement is the ability to perform real-time measurements of cellular oxygenation under biologically relevant or hypoxic conditions. A popular approach is to use MitoXpress® Intra Intracellular Oxygen Assay (Agilent Technologies), a kit comprising of a chemically stable, oxygen-sensitive fluorescent dye capable of permeating into the cells. The fluorescence of this probe (~645 nm) is quenched by oxygen, meaning that the measured signal is inversely proportional to the intracellular oxygen concentration6.
Mitochondrial dysfunction and oxidative stress are key areas of interest for research into IRI. The membrane-permeable JC-1 dye (Invitrogen) is widely used in apoptosis studies to monitor mitochondrial health, providing an indicator of mitochondrial membrane potential in a variety of cell types, including cardiac myocytes7. Potential-dependent accumulation of the positively charged dye in the electronegative interiors of healthy mitochondria leads to a shift in the dye’s fluorescent emission from green (~529 nm) to red (~590 nm). As this membrane potential is lost during oxidative stress8, the red: green fluorescence intensity ratio can be used to investigate the role of mitochondrial dysfunction in IRI.
Reactive Oxygen Species Detection is another key marker of IRI, and the fluorescent probe dihydroethidium (dHEt) (Sigma-Aldrich) is commonly used to detect superoxides (produced by partial reduction of oxygen by mitochondria during oxidative phosphorylation) and hydrogen peroxide (caused by dismutation of superoxides)9. The presence of ROS is represented as total DHE fluorescence (~606 nm), allowing detailed metabolic characterization of IRI.
The power of multiplexing
The discrete excitation/emission profiles of these fluorescent dyes, in combination with the precise control of oxygenation offered by the CLARIOstar reader and ACU, allows multiparametric studies of IRI in a microplate format. Learn more in this scientific talk:
This approach has already been used to investigate the role of inflammatory markers and various enzymes associated with oxidative stress, as well as the potential of using activators of the RISK (reperfusion injury salvage kinase) signaling pathway, such as by insulin, to protect against IRI. The user-defined oxygen profile can be executed automatically by the ACU, consistently and effectively mimicking deprivation and re-oxygenation to gain reliable and biologically-applicable data. The high throughput, real-time nature of this method is helping researchers to accelerate ischemia-reperfusion studies, allowing larger-scale screening studies and opening up the possibility of analyzing multiple parameters in parallel, to ensure the maximum output of each experiment.
To find out more about how BMG Labtech readers could advance your research into CHD, read our collection of peer-reviewed articles.
- Krumm et. al. (2017): The CLARIOstar with ACU exposes cells to ischemia-reperfusion conditions and monitors their oxygenation, BMG LABTECH App Note 309.
- Maurer et. al.(2016): Real-time measurement of intracellular O2 in mammalian cells, BMG LABTECH App Note 290.