The overall goal of this procedure is to obtain simultaneous real time measures of myocardial rates of glucose oxidation and fatty acid oxidation, as well as cardiac contractual function in an isolated working rat heart. This method can help answer key questions in the cardiovascular field. Such as how the heart metabolically adapts to acute changes in energy demands and how these responses affected in pathological conditions.
The main advantage of this technique is that it provides the investigator total control over the environmental variables such as the nature and the concentrations of substrates, drugs, or hormones delivered to the heart. The most would be Bhavisha Bakrania a post doctoral fellow from the cardiovascular Reno resource center at the University of Mississippi Medical Center. To begin, start the pump to prime the perfusion apparatus and to oxygenate the buffer.
After preparing the materials, anesthetize a rat and prepare it for surgery. Clip the abdomen free of hair and perform a mid line incision with lateral extension. Move the stomach and intestine aside to access the inferior vena cava and inject 200 USP units of Heparin.
Wait five to 10 seconds, then using scissors cut the diaphragm and the sides of the rib cage to expose the contents of the chest cavity. Next, delicately grab the heart using the hand and excise both the heart and lungs together. Cut at the level of the descending aorta, being careful not to damage the aortic arch and ascending aorta in the process.
Immediately transfer the heart and lungs to a dissection dish filled with ice cold KH buffer. After the heart stops beating, learn to work quickly so it can be restarted within a minute or two. Now submerge the heart and lungs in a new dish of ice cold KH buffer.
Quickly trim off any large pieces of lung tissue, then cut the descending aorta right above the aortic arch. For the following steps, take the necessary precautions for use of radioactives. Next, using forceps carefully open the aorta and slide the heart up onto the cannula.
Be very careful not pass the aortic route with a cannula, as this may cause hypoperfusion of the heart and damage to the aortic valves. Now, secure the junction with a micro clip and initiate the Lagendorff perfusion of the heart. Without rushing, secure the aorta to the aortic cannula using a 3 0 silk suture and remove the micro clip.
Then locate the pulmonary vein and trim away non cardiac tissues as needed. Leave enough tissue to anchor the left atrium to the cannula. The cannulation of the left atrium is the second most critical step of the protocol as improper insertion of the cannula or the presence of a leak will weaken contractions and compromise the quantification of metabolic rates.
Next, prepare the left atrial line cannula, just as done for the right cannula. Then with forceps, delicately grab the opening of the pulmonary vein and slide the heart up on the atrial cannula. Move the tissues as needed without bending the aorta or overly straining the heart.
Then secure the cannula with a 3 0 silk suture. Finally, open the atrial line while simultaneously switching the aortic line to working mode. If buffer leaks out from the left atrium, close the atrial line and switch back to Lagendorff mode.
Then use more sutures to better secure the cannula and try switching to working mode again. If the experiment includes direct measurement of LV function with a catheter, then begin by puncturing the LV apex using a 26 gauge needle, introduce a pressure volume catheter and align the shaft with the LV longitudinal axis. Then position the distal electrode E1 just below the aortic valve, adjacent to the endocardial border and position the proximal electrode E4 just inside the ventricular wall.
Now seal the heart in a water jacketed chamber. Then start monitoring functional parameters in the software. Once baseline parameters have been stable for at least five minutes, start recording data.
To measure the coronary flow, record the time it takes to collect 20 milliliters of effluent. Then take two millimeters of the effluent into a syringe via the three way stopcock and discard the remainder. Immediately transfer the two milliliter sample into a micro centrifuge tube and take a half milliliter aliqout to determine the rate of glucose oxidation.
Store the rest on ice. Continue taking coronary effluence samples periodically and before finishing, inject 10 micro liters of hypertonic saline into the atrial line. Just before it attaches to the cannula.
Use the response to this bolus to calculate the parallel conductance for accurate determination of cardiac volume. Next, unseal the heart chamber and remove the catheter in the LV.Then cut the heart off the cannulas and drop it into ice cold KH buffer. Now, quickly dry the heart on a paper towel and measure its wet weight.
Once weighed, proceed to dissect the required tissue samples and then freeze the remaining tissue using tongs cooled on dry ice. In preparation, load glass scintillation vials with one milliliter of 10x hyamine hydroxide. Prepare one for each coronary effluence sample and two for controls.
As soon as a sample vial is loaded, place it directly into a loaded scintillation vial. For the analysis, delicately cap the vial with a rubber sleeve stopper and use parafilm to seal the vial. Now, using a one milliliter syringe with a long 23 gauge needle, inject 200 micro liters of 60%perchloric acid through the rubber sleeve.
Let all the sample vials sit overnight. The next day, carefully retrieve each sample tube with tweezers and wash their bottoms off over the open vials with one milliliter of scintillation cocktail to retrieve all of the hydroxide of hyamine. Then add nine more milliliters of scintillation cocktail to each vial.
To get the specific activity, add 0.5 milliliters of profusion buffer into two of the vials. Now shake the vials vigorously and let the vial settle for at least six hours before taking measurements. To determine the myocardial oleate oxidation rates, first prepare an anionic exchange resin column for each sample.
Just load the tips of three milliliters syringe tubes with glass wool. Then add the resin water slurry up to the two milliliter mark in each column using a transfer pipette. Next, wash the resin by passing two milliliters of ultra pure water through each column three times.
Then place scintillation vials under the columns and load the columns with a half milliliter of coronary effluent. For the background measurement, use perfusion buffer instead. Now, elute the columns successively with one half milliliter, one milliliter, and finally two milliliters of ultra pure water.
Discard the columns. Then add 13 milliliters of scintillation cocktail to the vials. Add 0.5 milliliters of perfusion buffer directly to two vials filled with 13 milliliters of liquid scintillation cocktail for determination of the specific activity.
Vigorously shake the vials, let them settle for six plus hours and take measurements. Hearts were isolated from two 16 week rats and subjected to different stress conditions. For one heart, an acute increase in work load was simulated by increasing the after load by 40%and by adding epinephrine to the perfusion buffer.
The heart rate immediately increased by more than 50%with the work jump induction. The cardiac power increased by more than 40%at the work jump, which was accompanied by an increase in both rates of glucose and oleate oxidation. The relative increase from base line for glucose oxidation was striking.
The other heart was subjected to 15 minutes of global ischemia and 30 minutes of reperfusion. Under these experimental conditions, a near normal pulse pressure was restored a few minutes into the reperfusion. Within 10 minutes, cardiac power rapidly increased to above pre-ischemia values before returning to near baseline values.
This resulted from increased oleate oxidation. Where as glucose oxidation was quick to resume to pre-ischemic activity. Once mastered, isolation of the heart to the initial reperfusion in the working mode can be done in less than five minutes if performed properly.
Following this procedure, other methods such as RNA and protein expression analyses can be done in order to answer additional questions, such as the effect of a drug or stress condition on molecular signaling pathways. Don't forget that working with radio tracers can be extremely hazardous and precautions such as wearing full personal protective equipments should be always taken while performing this procedure.
