Measuring recovery in mitochondrial respiration after varying lengths of reperfusion time
Location
Philadelphia
Start Date
13-5-2015 1:00 PM
Description
Recent research on ischemia-reperfusion injury has been successful in unraveling the mechanisms of action on a cellular level, but the picture is not yet complete. The damage to cells is not fully reversed when blood flow is restored. Instead, reperfusion of the tissue paradoxically leads to apoptosis. This apoptosis has been tied to damage to mitochondria due to ROS generation during anaerobic respiration. Studies have shown that reperfusion-induced apoptosis can constitute up to 50% of the final size of the infarct. It is critical to investigate the effects of ischemia-reperfusion on mitochondrial respiration in order to better understand how to prevent irreversible reperfusion injury. We propose using an Oroboros Oxygraph-2K (O-2K) respirometer to measure the effects of anoxia with varied reperfusion time on mitochondrial respiration. Our experiments will be carried out on in vitro murine atrial cardiomyocytes, which will allow us to accurately control and isolate our experimental parameters. Each experiment will follow a pre-determined “SUIT” protocol. SUIT refers to substrate, uncoupler, inhibitor titrations. These protocols are designed to measure various functional states of respiration. Increasing reperfusion times will increase damage to the tissue. However, we postulate that reperfusion after an anoxic event has a paradoxical effect on the cells, and that respiratory impairment following anoxia is reversed during increased renormoxia. Our immediate goals for this project are: 1) establish high-resolution respirometry protocols and optimize for our model system, 2) generate respirometry data using various cell types (i.e. HL-1, PC-3) to demonstrate proficiency, and 3) observe respiration in cells following anoxia with varying length of reperfusion times. The cells are stressed by introduction into an anoxic environment. The cap of the T75 flask is replaced with a wax plug with two holes and sealed with parafilm. A gas tank containing a mixture of 95% CO2/5% N2 is outfitted with a regulator and flow meter. This is connected to the flask via one of the holes in the plug. The other hole is used to outgas the flask using a long tube to prevent backflow. Along the tube, we splice in a vacuum flask containing a Mitsubishi Anaero-indicator. This indicator turns blue when the atmosphere has been removed of oxygen. The flask is placed on an agitator, allowing for better gas equilibration between cell media and the atmosphere. We use an Oroboros Oxygraph-2K to measure mitochondrial respiration in two samples simultaneously. Using the concentration information, we measure two 1 x 106 cell samples. These samples are added to the reaction chamber of the O2K. Cell growth media is added to bring the volume up to 2 mL. Each chamber is open to the atmosphere and the starting oxygen concentration is allowed to equilibrate. The chambers are then closed so that a ROUTINE respiration rate may be measured. This state is measured for 10 minutes. Oligomycin is titrated (final conc. = 2.5 µM), which inhibits ATP synthase and induces a LEAK state (i.e. State 4 respiration). The LEAK respiration is measured for 5 minutes. We measure the maximum electron transport chain oxidation rate (ETS) by titration of CCCP. This uncoupler is added in 1 µl increments. Each addition causes a temporary increase in respiration. CCCP is titrated in this manner until a maximal respiration rate is observed. Finally, we use rotenone to measure the background level of oxidation. Rotenone is added for a final concentration of 0.5 µM. Rotenone prevents electrons from transferring from Complex I to the Q Junction, effectively stopping the oxidation of NADH. This residual oxygen consumption, or ROX, is due to oxidative side reactions unrelated to the electron transport chain.
Measuring recovery in mitochondrial respiration after varying lengths of reperfusion time
Philadelphia
Recent research on ischemia-reperfusion injury has been successful in unraveling the mechanisms of action on a cellular level, but the picture is not yet complete. The damage to cells is not fully reversed when blood flow is restored. Instead, reperfusion of the tissue paradoxically leads to apoptosis. This apoptosis has been tied to damage to mitochondria due to ROS generation during anaerobic respiration. Studies have shown that reperfusion-induced apoptosis can constitute up to 50% of the final size of the infarct. It is critical to investigate the effects of ischemia-reperfusion on mitochondrial respiration in order to better understand how to prevent irreversible reperfusion injury. We propose using an Oroboros Oxygraph-2K (O-2K) respirometer to measure the effects of anoxia with varied reperfusion time on mitochondrial respiration. Our experiments will be carried out on in vitro murine atrial cardiomyocytes, which will allow us to accurately control and isolate our experimental parameters. Each experiment will follow a pre-determined “SUIT” protocol. SUIT refers to substrate, uncoupler, inhibitor titrations. These protocols are designed to measure various functional states of respiration. Increasing reperfusion times will increase damage to the tissue. However, we postulate that reperfusion after an anoxic event has a paradoxical effect on the cells, and that respiratory impairment following anoxia is reversed during increased renormoxia. Our immediate goals for this project are: 1) establish high-resolution respirometry protocols and optimize for our model system, 2) generate respirometry data using various cell types (i.e. HL-1, PC-3) to demonstrate proficiency, and 3) observe respiration in cells following anoxia with varying length of reperfusion times. The cells are stressed by introduction into an anoxic environment. The cap of the T75 flask is replaced with a wax plug with two holes and sealed with parafilm. A gas tank containing a mixture of 95% CO2/5% N2 is outfitted with a regulator and flow meter. This is connected to the flask via one of the holes in the plug. The other hole is used to outgas the flask using a long tube to prevent backflow. Along the tube, we splice in a vacuum flask containing a Mitsubishi Anaero-indicator. This indicator turns blue when the atmosphere has been removed of oxygen. The flask is placed on an agitator, allowing for better gas equilibration between cell media and the atmosphere. We use an Oroboros Oxygraph-2K to measure mitochondrial respiration in two samples simultaneously. Using the concentration information, we measure two 1 x 106 cell samples. These samples are added to the reaction chamber of the O2K. Cell growth media is added to bring the volume up to 2 mL. Each chamber is open to the atmosphere and the starting oxygen concentration is allowed to equilibrate. The chambers are then closed so that a ROUTINE respiration rate may be measured. This state is measured for 10 minutes. Oligomycin is titrated (final conc. = 2.5 µM), which inhibits ATP synthase and induces a LEAK state (i.e. State 4 respiration). The LEAK respiration is measured for 5 minutes. We measure the maximum electron transport chain oxidation rate (ETS) by titration of CCCP. This uncoupler is added in 1 µl increments. Each addition causes a temporary increase in respiration. CCCP is titrated in this manner until a maximal respiration rate is observed. Finally, we use rotenone to measure the background level of oxidation. Rotenone is added for a final concentration of 0.5 µM. Rotenone prevents electrons from transferring from Complex I to the Q Junction, effectively stopping the oxidation of NADH. This residual oxygen consumption, or ROX, is due to oxidative side reactions unrelated to the electron transport chain.