TITLE: High resolution respirometry of isolated mitochondria from adult Octopus maya (Class: Cephalopoda) systemic heart
AUTHORS: Ana Karen Meza-Buendia, Omar Emiliano Aparicio-Trejo, Fernando Díaz, 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. In a recent study made in Octopus maya was 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 (Meza-Buendia et al. 2021). Unfortunately, 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 in those species’ groups. 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 procedure described in this paper takes approximately 3 to 4 hours from isolation of intact mitochondria to measurement of mitochondrial oxygen consumption.

[BEFORE_START]
Pre-chill glassware before starting the procedure.

[GUIDELINES]
Citations:
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7; 72:248-54. doi: 10.1006/abio.1976.9999. PMID: 942051
Gnaiger E, Plangger I, Hunger M. Mitochondrial respiration medium: MiR05-Kit. 2018. Mitochondrial Physiol Network 22.10(02):1-2.
Meza-Buendia AK, Trejo-Escamilla I, Piu M, Caamal-Monsreal C, Rodríguez-Fuentes G, et al. Why high temperatures limit reproduction in cephalopods? The case of Octopus maya. Aquac Res. 2021; 52(11):5111-5123. https://doi.org/10.1111/are.15387
Mommsen TP, Hochachka PW. Respiratory and enzymatic properties of squid heart mitochondria. Eur J Biochem. 1981 Nov;120(2):345-50. doi: 10.1111/j.14321033.1981.tb05710.x. PMID: 7318831


[STEPS]
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
1. Starve octopus   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
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.
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
7. Transfer the homogenate by decantation to a pre-cooled 2 ml Eppendorf tube® and centrifuge at 392 rcf at 4 °C for 5 min.
 

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 7938 rcf for 15 min at 4 °C ('mitochondrial pellet formation').
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
10. Discard the supernatant by decantation and wash off the pellet.
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 7938 rcf at 4 °C for 15 min.
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.
 

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 ~14 mg protein/ml per 1 g of minced tissue. Mitochondria are now ready to be used in experiments of respirometry. Use the preparation within 60 min–240 min for better functional responses.
 

  

SECTION: Measuring mitochondrial respiration: High-resolution respirometry (HRR)
16. 
 
To adapt the protocol to other commercial equipment, please see the manufacturer’s specifications. The equipment should be turned on before the mitochondrial isolation starts, so it reaches the selected experimental working temperature (the data shown in this document were determined at temperature of 24 °C).
SECTION: Equipment setup: Calibration of polarographic oxygen sensors
17. 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: Equipment setup: Calibration of polarographic oxygen sensors
18. Wait for an equilibrium with atmospheric oxygen and the required experimental temperature.
SECTION: Equipment setup: Calibration of polarographic oxygen sensors
19. Start recording of oxygen consumption.
SECTION: Substrate/inhibitor titration (SUIT) analysis
20. 
 

Table 1. Action and concentration of agents used for measuring mitochondrial respiration of isolated mitochondria from the systemic heart of Octopus maya.
     Reagent      Action      Final concentration        Proline      Amino acid substrate      5 mM        ADP      Substrate for the generation of   ATP      1.25 mM        Antimycin      Complex III inhibitor      12.5 µM        Rotenone      Complex I inhibitor      2.5 µM        Oligomycin      ATP synthase inhibitor      2.5 µM   

SECTION: Substrate/inhibitor titration (SUIT) analysis
21. Use an appropriate Hamilton microsyringe (Oroboros Instrument), add mitochondria (Mtc) to obtain a final concentration between 300 μg/ml-500 μg/ml .
SECTION: Substrate/inhibitor titration (SUIT) analysis
22. Use a Hamilton microsyringe (Oroboros Instruments), add Proline (Pro) to a final concentration of 5 millimolar (mM). 
 

SECTION: Substrate/inhibitor titration (SUIT) analysis
23. 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’).
SECTION: Substrate/inhibitor titration (SUIT) analysis
24. Record for ~ 2 min.
SECTION: Substrate/inhibitor titration (SUIT) analysis
25. Add 5 µL ADP (500 millimolar (mM) ADP stock solution) to obtain a final concentration of 1.25 millimolar (mM).
SECTION: Substrate/inhibitor titration (SUIT) analysis
26. Record until the rate of oxygen consumption begins to drop.
SECTION: Substrate/inhibitor titration (SUIT) analysis
27. Add 1 µL of oligomycin A (5 millimolar (mM) oligomycin stock solution) to obtain a final concentration of 2.5 micromolar (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).
SECTION: Substrate/inhibitor titration (SUIT) analysis
28. Record for ~ 2 min.
SECTION: Substrate/inhibitor titration (SUIT) analysis
29. Add 2.5 micromolar (µM) rotenone (Rot) plus 12.5 micromolar (µM) antimycin A (Ant) to obtain residual or non-mitochondrial respiration (ROX).
SECTION: Substrate/inhibitor titration (SUIT) analysis
30. Record for ~ 5 min and then stop recording.
SECTION: Substrate/inhibitor titration (SUIT) analysis
31. 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.
 


  

SECTION: Isolation of mitochondria from the systemic heart of adult octopus
6.1. Three to four stocks are made to homogenize the previously minced tissue.
SECTION: Isolation of mitochondria from the systemic heart of adult octopus
6.2. 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
10.1. First add 1 mL of cold mitochondrial isolation buffer B and re-suspend the pellet gently with a soft bristle brush (natural bristles).
 

SECTION: Substrate/inhibitor titration (SUIT) analysis
25.1. A faster oxygen consumption is observed and represents Respiratory State 3’ (S3’), where ATP production is the principal contribution of oxygen consumption.
 

SECTION: Equipment setup: Calibration of polarographic oxygen sensors
18.1. 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
19.1. Verify that the recording is stable and that no drifts are apparent.
SECTION: Substrate/inhibitor titration (SUIT) analysis
21.1. 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
29.1. Both compounds inhibit the electron transport system flux and induce a rapid decrease in oxygen consumption rate until it remains constant.