Aug 17, 2022

Public workspaceHigh resolution respirometry of isolated mitochondria from adult Octopus maya (Class: Cephalopoda) systemic heart

DOI
dx.doi.org/10.17504/protocols.io.kxygxzb2zv8j/v1
  • Ana Karen Meza-Buendia1,
  • Omar Emiliano Aparicio-Trejo2,
  • Fernando Díaz1,
  • Claudia Caamal-Monsreal3,4,
  • José Pedraza-Chaverri5,
  • Carolina Álvarez-Delgado6,
  • Kurt Paschke7,8,
  • Carlos Rosas3,4
  • 1Laboratorio de Ecofisiología de Organismos Acuáticos, Departamento de Biotecnología Marina, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Baja California, México;
  • 2Departamento de Fisiopatología Cardio-Renal, Instituto Nacional de Cardiología “Ignacio Chávez”, Mexico City 14080, Mexico;
  • 3Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, Universidad Nacional Autónoma de México, Sisal, Mexico;
  • 4Laboratorio de Resilencia Costera (LANRESC, CONACYT, Sisal, Mexico;
  • 5Laboratorio F-315, Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, 04510, Ciudad de México, Mexico;
  • 6Departamento de Innovación Biomédica, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Carretera Tijuana-Ensenada 3918, Zona Playitas, Baja California, Mexico;
  • 7Instituto de Acuicultura, Universidad Austral de Chile, Puerto Montt, Chile;
  • 8Centro FONDAP de Investigación de AltasLatitudes (IDEAL), Punta Arenas, Chile
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DOI: dx.doi.org/10.17504/protocols.io.kxygxzb2zv8j/v1
Protocol Citation: Ana Karen Meza-Buendia, Omar Emiliano Aparicio-Trejo, Fernando Díaz, Claudia Caamal-Monsreal, José Pedraza-Chaverri, Carolina Álvarez-Delgado, Kurt Paschke, Carlos Rosas 2022. High resolution respirometry of isolated mitochondria from adult Octopus maya (Class: Cephalopoda) systemic heart. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzb2zv8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 13, 2022
Last Modified: August 17, 2022
Protocol Integer ID: 66580
Keywords: Isolation of mitochondria, Respirometry, Adult octopus, Isolation of mitochondria, Octopus maya, Octopus, High resolution respirometry , heart, respiratory states
Abstract
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.
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

Materials
Chemicals for Mitochondrial Isolation Buffer and Mitochondrial Respiratory Buffer:

  1. ReagentSucroseSigma AldrichCatalog #S9378
  2. ReagentPotassium chloride(KCl Sigma cat. no. P5405)SigmaCatalog #P5405
  3. ReagentEthylene-bis(oxyethylenenitrilo)tetraacetic acidSigma AldrichCatalog #E3889
  4. Reagent4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acidSigmaCatalog #H4034
  5. ReagentBovine serum albumin (BSA) Sigma – AldrichCatalog #A7030
  6. ReagentMagnesium chlorideCatalog #208337
  7. Lactobionic acid (Sigma, cat. no. 15316)
  8. ReagentTaurineSigma – AldrichCatalog #T0625
  9. ReagentPotassium phosphate monobasicSigma – AldrichCatalog #P5379

Chemicals for respiratory substrates and inhibitors:

  • ReagentL-prolineSigma – AldrichCatalog #P0380
  • ReagentAdenosine 5 ’diphosphate potassium salt(ADP)Sigma – AldrichCatalog #A5285
  • ReagentOligomycin from Streptomyces diastatochromogenesSigma – AldrichCatalog #O4876
  • ReagentAntimycin A from Streptomyces sp.Sigma – AldrichCatalog #A8674
  • Rotenone (C23H22O6, Sigma, cat. no. 45656)

Other chemicals:

  1. Ethanol (70 %)
  2. Bi-distilled water.
  3. ReagentPotassium hydroxide (KOH)Sigma – AldrichCatalog #484016

Preparation of mitochondrial isolation buffers:

Mitochondrial Isolation Buffer A:

Mix all the reagents in Table 1 except BSA- Fatty Acid-Free. Once dissolved, the pH of the buffer is adjusted to 7.4. The pH is adjusted with 5 M KOH. If not used immediately, make aliquots in 50 mL falcon® tubes and stored at Temperature-80 °C for up to six months.

If used on the same day of preparation (after pH adjustment), take an aliquot (50 mL), and add the corresponding amount of BSA (1 g/L). Store the other aliquots at Temperature-80 °C .

Mitochondrial isolation buffer B:

Mix all the reagents from Table 1 without adding the BSA- Fatty Acid-Free. Adjust pH to 7.4 with 5M KOH. If not used immediately prepare aliquots in 50 mL falcon® tubes and store at Temperature-80 °C for up to six months. If used on the same day as preparation, it can be used after pH adjustment.

Table 1. Modified isolation buffer from Mommsen and Hochachka (1981):

ABCDEF
ComponentMWg/molMolarity (mM)OsmolesOsmolarityAmount for 200 mL final volume [g]
Sucrose342.3500150034.23
KCl74.5515023002.23
EGTA380.352120.15
HEPES238.3251251.19
826 mOsmoles
BSA 1g/L 0.2
Experiment day:

Both mitochondrial isolation buffers are thawed at room temperature or in a 36°C water bath. Once the mitochondrial isolation buffer A is thawed, add the BSA- Fatty Acid-Free (Table 1). Dissolve 0.05 g for a 50 mL aliquot. Mitochondrial isolation buffer B once completely thawed can be used.

When both isolation buffers are used on the day of the experiment, they are kept cold. Once opened, they can be refrozen at -20°C and used within six days.

MiR05 mitochondrial respiratory buffer preparation based on Gnaigner et al. (2018):

  1. Transfer all the reagents from Table 2 except the BSA-free fatty acids into a beaker.
  2. Add Amount230 mL of double distilled water.
  3. Dissolve with magnetic stirring at Temperature30 °C .
  4. Add Amount3.75 mL of 5 M KOH at Temperature30 °C and stir for 90 min.
  5. Adjust pH 7.1 with Concentration5 Molarity (M) KOH at Temperature30 °C using a pH electrode. The pH adjustment can be slow (90 min). NOTE: pH must be stable for at least 5 min. Do not leave the pH electrode in the solution for the 90-min waiting time.
  6. Separate 50 mL aliquots in Falcon (Falcon Tubes Pvt Ltd) tubes and store at Temperature-80 °C (the MiR05 will have a 6- month shelf life).

.
6.

Table 2. Mitochondrial respiration buffer MiR05 (Gnaiger et al. 2018):
ABCD
ComponentMW g/mol Final concentration [mM]Amount for 250 mL final volume [g]
EGTA380.40.50.047
MgCl295.230.071
Lactobionic acid358.3605.375
Taurine125.1200.625
KH2PO4136.1100.34
HEPES238.3201.191
Sucrose342.31109.413
BSA 1g/L0.25
Use on the day of experiment:

  1. Thaw at room temperature or in a water bath at Temperature36 °C .
  2. Once thawed add Amount0.05 g of BSA in Amount50 mL of MiR05.
  3. Keep cold until use.

Preparation of substrates and inhibitors for high-resolution respirometry:


The substrates and inhibitors used in this methodology are prepared according to the information available online from Oroboros Instrument. However, its preparations are described below, for more information consult https://wiki.oroboros.at/index.php/OROBOROS_INSTRUMENTS.

  • 2 M Proline stock solution:
Dissolve Amount1151.30 mg of Proline in 5 ml of bio-distilled water. Prepare Amount500 µL aliquots and store at Temperature-20 °C .

  • 500 mM ADP stock solution:
Dissolve Amount501.3 mg of ADP in Amount1.2 mL of bio-distilled water (ADP does not dissolve at this stage), neutralize with Concentration5 Molarity (M) KOH (~ 450 µl) and check pH 7. Adjust the final volume to 2 ml. Prepare Amount200 µL aliquots and store at Temperature-80 °C .

  • 5 mM Oligomycin stock solution:
Dissolve Amount4 mg of oligomycin in Amount1 mL of ethanol 70%. Prepare Amount200 µL aliquots and store at Temperature-20 °C .

  • 1 mM Rotenone stock solution:
Dissolve 0.39 mg of rotenone in 1 ml of ethanol 70%. Prepare 200µl aliquots and store at -20 ° C.

  • 5 mM Antimycin A stock solution:
Dissolve Amount5.4 mg of antimycin A in Amount2 mL ml of 70% ethanol solution. Prepare Amount200 µL aliquots and store at Temperature-20 °C .








Before start
Pre-chill glassware before starting the procedure.
Isolation of mitochondria from the systemic heart of adult octopus
Isolation of mitochondria from the systemic heart of adult octopus
Starve octopus DurationOvernight before the isolation experiment.
Overnight
Sacrifice an adult Octopus maya specimen (about Amount1 kg ) previously anesthetized with 3% alcohol and quickly remove the systemic heart from the mantle cavity.
Note
CRITICAL STEP: To obtain mitochondria from the systemic heart of O. maya, a minimum of Amount0.5 g of tissue is used.

Critical
Place the systemic heart immediately on a Petri dish TemperatureOn ice and add Amount1 mL of mitochondrial Isolation Buffer A to rinse the organ.

Pipetting
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 TemperatureOn ice .

Transfer the cut pieces of the organ to a homogenization tube with Amount2 mL of cold mitochondrial isolation buffer A.
Note
NOTE: Homogenization, as well as the following steps, must be carried out at Temperature4 °C .

Pipetting
Homogenize the systemic heart using Potter-Elvehjem PTFE pestle and glass tube (Sigma-Aldrich P7859-1EA) homogenizer operated by a drill at Centrifigation500 rpm .
Centrifigation
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.
Note
CRITICAL STEP: The drill pistil must enter rotating to avoid forming bubbles and generating surface tension causing the isolated mitochondria to burst.

Critical
Transfer the homogenate by decantation to a pre-cooled 2 ml Eppendorf tube® and centrifuge at Centrifigation392 rcf at Temperature4 °C for Duration00:05:00 .
Note
NOTE If the centrifuge is not nearby, keep the tube with the homogenate cold.

5m
Centrifigation
Pipetting
Transfer the supernatant obtained from the previous step to another pre-cooled 2 ml Eppendorf tube® with a micropipette and keep TemperatureOn ice .
Note
CRITICAL STEP: Hold the Eppendorf tube® by the top of the cap to avoid heating it and keep it on ice.


Pipetting
Critical
Centrifuge the transferred supernatant at Centrifigation7938 rcf for Duration00:15:00 at Temperature4 °C ('mitochondrial pellet formation').
15m
Centrifigation
Discard the supernatant by decantation and wash off the pellet.
Wash
First add Amount1 mL of cold mitochondrial isolation buffer B and re-suspend the pellet gently with a soft bristle brush (natural bristles).
Note
CRITICAL STEP: Decantation should be quick and avoid leaving the tube without ice as much as possible.
CRITICAL STEP: Resuspension of the pellet is performed on ice without lifting the Eppendorf tube®.

Pipetting
Critical
Re-suspend the pellet, add Amount1 mL of cold mitochondrial isolation buffer B. Subsequently shake gently and quickly to homogenize and keep TemperatureOn ice .

Pipetting
Centrifuge at Centrifigation7938 rcf at Temperature4 °C for Duration00:15:00 .
15m
Centrifigation
Discard the supernatant by decantation and conserve the pellet.
Note
CRITICAL STEP: Decantation should be quick and avoid leaving the tube out of the ice as much as possible.

Add Amount160 µL of cold mitochondrial isolation buffer to concentrate the sample and resuspend the pellet in the same way as in steps 10 and 11.
Note
NOTE: Keep cold.

Pipetting
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 ~Amount14 mg protein/ml per Amount1 g of minced tissue. Mitochondria are now ready to be used in experiments of respirometry. Use the preparation within Duration01:00:00 –Duration04:00:00 for better functional responses.
Note
CRITICAL STEP: Keep the mitochondrial fraction TemperatureOn ice .
NOTE: A diagram of the summary steps of the isolation of mitochondria from Octopus maya is shown in Fig 1.

Fig 1. Mitochondrial isolation of a systemic heart from an adult Octopus maya.

5h
Critical
Measuring mitochondrial respiration: High-resolution respirometry (HRR)
Measuring mitochondrial respiration: High-resolution respirometry (HRR)

Note
The following protocol is designed to be used in a commercially available HRR device, the Oxygraph™ O2k (Oroboros Instruments, Innsbruck, AT), which uses a polarographic oxygen sensor to detect oxygen (O₂) flux of ± 1 pmol O₂·s⁻¹·mL⁻¹.
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 Temperature24 °C ).
Equipment setup: Calibration of polarographic oxygen sensors
Equipment setup: Calibration of polarographic oxygen sensors
Add Amount2 mL of mitochondrial respiration buffer (MiR05) to the chamber (this protocol was developed using a Amount2 mL volume), and the O₂ sensors are calibrated.
Note
NOTE: Mitochondrial Respiration Buffer MiR05 should be used instead of distilled water for calibration.

Pipetting
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.
Start recording of oxygen consumption.
Verify that the recording is stable and that no drifts are apparent.
Substrate/inhibitor titration (SUIT) analysis
Substrate/inhibitor titration (SUIT) analysis
9m
9m

Note
This section provides a SUIT protocol for the analysis of oxidative phosphorylation (OXPHOS) in Octopus maya systemic heart mitochondria, being a tool for understanding the mitochondrial respiratory control of this species. See Table 1, to consult the concentrations of the substrates and inhibitors used in this protocol.

Table 1. Action and concentration of agents used for measuring mitochondrial respiration of isolated mitochondria from the systemic heart of Octopus maya.
ABC
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

Use an appropriate Hamilton microsyringe (Oroboros Instrument), add mitochondria (Mtc) to obtain a final concentration between Amount300 μg/ml -Amount500 μg/ml .
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.
Note
NOTE: Amount600 µg to Amount1000 µg of total protein are recommended.

Use a Hamilton microsyringe (Oroboros Instruments), add Proline (Pro) to a final concentration of Concentration5 millimolar (mM) .
Note
CRITICAL STEP: The corresponding respiratory substrates must be immediately added to avoid mitochondrial membrane potential depolarization.

NOTE: The addition of proline starts proline pathway (entry in electron transport system direct into Q-junction) and the glutamate-anaplerotic pathway (stimulates CI-linked respiration). Proline is oxidized to 1-delta pyrroline 5 carboxylate by proline dehydrogenase of the inner mitochondrial membrane reducing FAD to FADH2, where 1-delta pyrroline 5 carboxylate is converted to glutamate by 1 pyrroline 5 carboxylate dehydrogenase. Additionally, FADH2 is oxidized to stimulate quinone reduction, activating Q-junction.

Critical
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’).
Record for ~ Duration00:02:00 .
2m
Add Amount5 µL ADP (Concentration500 millimolar (mM) ADP stock solution) to obtain a final concentration of Concentration1.25 millimolar (mM) .
Pipetting
A faster oxygen consumption is observed and represents Respiratory State 3’ (S3’), where ATP production is the principal contribution of oxygen consumption.
Note
CRITICAL STEP: The rate of oxygen consumption should be faster than the rate of consumption observed when adding the substrate alone, indicating that well-coupled mitochondria have been obtained.

Critical
Record until the rate of oxygen consumption begins to drop.
Add Amount1 µL of oligomycin A (Concentration5 millimolar (mM) oligomycin stock solution) to obtain a final concentration of Concentration2.5 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).
Record for ~ Duration00:02:00 .
2m
Add Concentration2.5 micromolar (µM) rotenone (Rot) plus Concentration12.5 micromolar (µM) antimycin A (Ant) to obtain residual or non-mitochondrial respiration (ROX).
Pipetting
Both compounds inhibit the electron transport system flux and induce a rapid decrease in oxygen consumption rate until it remains constant.
Record for ~ Duration00:05:00 and then stop recording.
5m
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.
Note
NOTE: To correctly determine O₂ consumption rate in each Respiratory State, it is necessary to ensure that the steady-state is reached.

NOTE: To avoid hypoxia in the chambers, they must be reoxygenated (by chamber opening) if O2 concentration falls below Concentration20 micromolar (µM) .


Fig 2. Schematic representations of the method used to determine the rate of oxygen consumption in each respiratory state (S2’, S3’, S4’o and ROX); the blue line corresponds to O2 concentration (µM), while the red line corresponds to oxygen consumption rate (pmol O₂ s⁻¹ mg⁻¹). Mtc: mitochondria, Pro: proline, ADP: adenosine diphosphate, Oligo: oligomycin, Rot: rotenone and Ant: antimycin A. S2’= State 2’; S3’ = State 3’; S4’o = state 4 oligomycin-induced; ROX= residual non- mitochondrial respiration; Rot + Anyt = rotenone plus antimycin.