TITLE: High resolution respirometry of isolated mitochondria from adult Octopus maya (Class: Cephalopoda) systemic heart
AUTHORS: Ana Karen Meza-Buendía, Omar Emiliano Aparicio-Trejo, Fernando Díaz-Herrera, Claudia Caamal-Monsreal, José Pedraza-Chaverri, Carolina Álvarez-Delgado, Kurt Paschke, Carlos Rosas

[DESCRIPTION]
Mitochondrial respirometry is key to understand how environmental factors model energetic cellular process. In the case of ectotherms, thermal tolerance has been hypothesized to be intimately linked with mitochondria capability to produce enough adenosine triphosphate (ATP) to respond to the energetic demands of animals in high temperatures. Recent studies made in Octopus maya proposed the hypothesis postulating that high temperatures could restrain female reproduction due to the limited capacity of the animals’ heart to sustain oxygen flow to the body, affecting in this manner energy production in the rest of the organs, including the ovarium. Until now, no reports have shown temperature effects and other environmental variables on cephalopod mitochondria activity because of the lack of a method to evaluate mitochondrial respiratory parameters on those groups of species. In this sense and for the first time, this study developed a method to obtain mitochondrial respirometry data of adult Octopus maya’s heart. This protocol illustrates a step-by-step procedure to get high yield and functional mitochondria of cephalopod heart and procedure for determining the corresponding respiratory parameters. The isolation procedures described here require two hours, demonstrating that confident and replicable results can be obtained with this method.

[STEPS]
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
1. 
 
Starve octopus overnight before the isolation experiment.
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
2. Sacrifice an adult Octopus maya specimen (about 1 kg) previously anesthetized with 3% alcohol and quickly remove the systemic heart from the mantle cavity. 

 

SECTION: Isolation of mitochondria from the systemic heart of adult octopus
3. Place the systemic heart immediately on a Petri dish on ice and add 1 mL of mitochondrial Isolation Buffer A to rinse the organ.
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
4. Cut the systemic heart into pieces with scissors and mince into smaller pieces with a scalpel, which should be done while the Petri dish is on ice.
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
5. Transfer the cut pieces of the organ to a homogenization tube with 2 ml of cold mitochondrial isolation buffer A.

 

SECTION: Isolation of mitochondria from the systemic heart of adult octopus
6. Homogenize the systemic heart using Potter-Elvehjem PTFE pestle and glass tube (Sigma-Aldrich P7859-1EA) homogenizer operated by a drill at 500 rpm. Three to four stocks are made to homogenize the previously minced tissue. Homogenization is done in a container with ice and the ice homogenization tube must not be removed. 

 

SECTION: Isolation of mitochondria from the systemic heart of adult octopus
7. Transfer the homogenate by decantation to a pre-cooled 2-ml Eppendorf tube® and centrifuge at 392 rfc at 4 °C and kept on ice for 5 min. If the centrifuge is not nearby, keep the tube with the homogenate cold.
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
8. Transfer the supernatant obtained from the previous step to another pre-cooled 2-ml Eppendorf tube® with a micropipette and keep on ice. 

 

SECTION: Isolation of mitochondria from the systemic heart of adult octopus
9. Centrifuge the transferred supernatant at 7,939 rfc for 15 min at 4 °C ('mitochondrial pellet formation').
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
10. Discard the supernantant by decantation and wash off the pellet. First add 1 ml of coldmitochondrial isolation buffer B and re-suspend the pellet gently with a soft bristle brush (natural bristles).
 

SECTION: Isolation of mitochondria from the systemic heart of adult octopus
11. Re-suspend the pellet, add 1 ml of cold mitochondrial isolation buffer B. Subsequently shake gently and quickly to homogenize and keep on ice.
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
12. Centrifuge at 7,938 rfc at 4 °C for 15 minutes.
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
13. Discard the supernatant by decantation and conserve the pellet. 

 

SECTION: Isolation of mitochondria from the systemic heart of adult octopus
14. Add 160 µL of cold mitochondrial isolation buffer to concentrate the sample and resuspend the pellet in the same way as in steps 10 and 11. Keep cold.
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
15. Measure mitochondrial concentration using the Bradford method (Bradford 1976). According to our own experimental results, mitochondrial suspensions from the systemic heart of Octopus maya adults contain aproximatly 14 mg protein/ml per 1 gram of minced tissue. Mitochondria are now ready to be used in experiments of respirometry; use the preparation within 1–4 h for better functional responses.

 

SECTION: Measuring mitochondrial respiration: High-resolution respirometry (HRR)
17. 
 

26. Add 5 µl ADP (500 mM ADP stock solution) to obtain a final concentration of 1.25 mM. A faster oxygen consumption is observed and represents Respiratory State 3’ (S3’), where ATP production is the principal contribution of oxygen consumption. 
 

SECTION: Substrate/inhibitor titration (SUIT) analysis
21. 

 




 



SECTION: Substrate/inhibitor titration (SUIT) analysis
23. Use a Hamilton microsyringe (Oroboros Instruments), add Proline (Pro) to a final concentration of 5 mM. 
 

SECTION: Isolation of mitochondria from the systemic heart of adult octopus
16. NOTE: A diagram of the summary steps of the isolation of mitochondria from Octopus maya is shown in Fig 1.
  

SECTION: Equipment setup: Calibration of polarographic oxygen sensors
18. Add 2 mL of mitochondrial respiration buffer (MiR05) to the chamber (this protocol was developed using a 2 ml volume), and the O₂ sensors are calibrated.

 

SECTION: Measuring mitochondrial respiration: High-resolution respirometry (HRR)
18. 
SECTION: Equipment setup: Calibration of polarographic oxygen sensors
19. Wait for an equilibrium with atmospheric oxygen and the required experimental temperature; the system reaches the steady basal consumption state of the system in operation, a point where the O₂ consumption rate is constant.
SECTION: Equipment setup: Calibration of polarographic oxygen sensors
20. Start recording of oxygen consumption. Verify that the recording is stable and that no drifts are apparent.
SECTION: Substrate/inhibitor titration (SUIT) analysis
22. Use an appropriate Hamilton microsyringe (Oroboros Instrument), add mitochondria (Mtc) to obtain a final concentration between 300-500 μg ml -1. This step is followed by a rapid and transient decrease in oxygen content of the chamber followed by a slower decrease caused by respiration of the mitochondria, commonly referred to as Respiratory State 1. 
 

SECTION: Substrate/inhibitor titration (SUIT) analysis
25. Record for ~ 2 min.
SECTION: Substrate/inhibitor titration (SUIT) analysis
24. Observe a faster rate of oxygen consumption because of basal activity of the respiratory chain to counteract proton leakage from the inner mitochondrial membrane, which represents Respiratory State 2’ (S2’).
31. Record for ~5 min and then stop recording.
27. Record until the rate of oxygen consumption begins to drop.
28. Add 1 µl of oligomycinA(5 mM oligomycin stock solution) to obtain a final concentration of 2.5 µM and induce Respiratory State 4’ (S4’o). With this procedure OXPHOS is inhibited by oligomycin and the rate of oxygen consumption begins to rapidly plateau (steady state).
29. Record for ~ 2 min.
30. Add 2.5 µM rotenone (Rot) plus 12.5 µM antimycin A (Ant) to obtain residual or non-mitochondrial respiration (ROX). Both compounds inhibit the electron transport system flux and induce a rapid decrease in oxygen consumption rate until it remains constant.
32. The Respiratory States S3’ and S4’o, were corrected for the respiratory state ROX (residual non-mitochondrial respiration): S3= S3’-ROX and S4= S4’o-ROX. The respiratory control parameter (RC) was defined as S3/S4o,while respiration directly attributable to OXPHOS was defined as S3-S4o, which is the phosphorylation state parameter (P). See Fig 2.