Procedure:
Before transfer to the experimental chamber
1. Set up the chiller 1 at 10oC and chiller 2 at 12oC.
2. Maintain the temperature of perfusate reservoirs, perfusate lines, and saline bath are at 10oC.
3. Turn on the circulation valves for chiller 1 and turn on the bypass valves for chiller 2
4. Set up the inline perfusate delivery at the constant pressure of 0 cmH2O and outflow line at 30 cmH2O.
5. Ensure that perfusate is flowing through the lines adequately
6. Clamp off the outflow line
Transfer
7. Transfer and fully immersed the fish in the saline bath of constant temperature 10oC
8. Connect the input cannula immediately to the line delivering perfusate while completely immersed in water
9. Connect the output cannula to the outflow line while completely immersed in water
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Turn off the circulation for chiller 1 and turn on the circulation for chiller 2, and start sampling
11. Increase the temperature of chiller 2 to 12.5oC and keep it at the same temperature for 6 minutes OR alternatively, raise the initial chiller 2 temperature at a faster rate.
12. Keep raising the temperature at the rate of 0.5oC every 6 minutes until arrhythmia is reached and determine TArr.
13. As TArr is approached, stop and bring the temperature of the heart down to 5oC lower than TArr. (e.g. if TArr 25 oC then temperature of the heart should be 20 oC) (switching chillers here )
14. Following step 12 determine if the same TArr is reached. Repeat this step acceptable number of times.
• If TArr is not the same after repetitions, then follow the single intervention procedure.
• However, If TArr is same after repetitions, then follow the multiple intervention procedures.
Single intervention procedure:
15. Use cholinergic agonists to slow down the heart rate and determine TArr for each case:
a. Inject Carbachol after step