Experiment design for quantifying ischemic and reperfusion injury in cardiomyocytes

Ischemic damage to the heart occurs when blood flow is obstructed. As a result, oxygen and nutrient delivery to cardiomyocytes is diminished, and metabolic waste accumulates, effectively reducing the pH. When flow returns, and reperfusion occurs, cardiomyocytes can become severely damaged. To better understand the cellular mechanisms involved during this type of injury, an ischemia and reperfusion (I/R) system was developed for use on isolated cardiomyocytes. The experimental system was designed to predictably control experimental factors including temperature, pH, dissolved gas and nutrient content. By mounting the system on an inverted microscope, it is able to monitor physiological signals including any cytosolic event with an available fluorescent dye as well as any contractile and morphological changes visible through light microscopy. The ability of the system to measure fluorescent indicators was demonstrated with BCECF and Fura-2 to quantify changes in intracellular pH and calcium, respectively. Images were taken in brightfield to monitor any potential morphological changes. Furthermore, the ability of the apparatus to sustain a hypoxic (low oxygen) environment as well as switch to a physiological one was confirmed using an oxygen probe. The results from this research have established an experimental system as well as protocols for supporting experimental designs to quantify the contributions from various isolated and synchronous assaults associated with ischemic and reperfusion injury. Specifically, a detailed experimental design is presented as future work that will clarify the individual and interactive effects of accumulating lactate and potassium ions in the extracellular fluid during ischemic and reperfusion injury