Difference between revisions of "MiPNet20.12 IOC103 Manaus"
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{{OROBOROS header page name}} | |||
{{Publication | {{Publication | ||
|title=[[Image:O2k-Network.png|right|60px|link=O2k-Network|O2k-Network]] | |title=[[Image:O2k-Network.png|right|60px|link=O2k-Network|O2k-Network]] | ||
'''Manaus BR''', 2015 Jul 06- | '''Manaus BR''', 2015 Jul 06-10. To the heart of the Amazon. O2k-Workshop on HRR and O2k-Fluorometry, '''IOC103'''. | ||
|authors= | |authors=Oroboros | ||
|year=2015-07-06 | |year=2015-07-06 | ||
|journal=Mitochondr Physiol Network | |journal=Mitochondr Physiol Network | ||
|abstract=[[File:Arapaima | |abstract=[[File:Arapaima 4515.JPG |left |350px |Arapaima, Pirarucu, or Paiche, Manaus]] | ||
2015 Jul 06- | '''2015 Jul 06-10. To the heart of the Amazon: comparative cardiac mitochondrial physiology of Amazon fish. O2k-Workshop on HRR and start-up project, IOC103.''' | ||
|mipnetlab=BR Manaus Val AL, AT Innsbruck | |mipnetlab=BR Manaus Val AL, AT Innsbruck Oroboros | ||
}} | }} | ||
{{Labeling | {{Labeling | ||
|instruments=Oxygraph-2k | |instruments=Oxygraph-2k | ||
|additional=ORO, IOC, | |additional=ORO, IOC, PBI-Shredder, O2k-Network, 2015 | ||
}} | }} | ||
[[File:Amazon 4403.JPG |left |240px | [[File:Amazon 4403.JPG |left |240px |Amazon, Manaus]] | ||
<big><big>'''To the heart of the Amazon'''</big></big> | <big><big>'''To the heart of the Amazon'''</big></big> | ||
Mitochondrial function is compared in mitochondrial preparations of fish heart. According to the suggestion by Dr. Adalberto | :::: Mitochondrial function is compared in mitochondrial preparations of fish heart. According to the suggestion by Dr. Adalberto L Val, one of each of the major groups should be selected: Potamotrygonidae, Osteoglossidae, Characiformes, Siluriformes, Perciformes (Cichlidae). The results obtained with a basic [[SUIT protocol]] will be compared with mitochondrial respiratory control patterns in other species, including human and mouse heart mitochondria. This study explores evolutionary variations of mitochondrial respiratory control patterns in different species, to define mitochondrial fitness in terms of adaptations to different environments and life styles. The results may provide a key towards delineating mitochondrial respiratory control patterns in health in different species and tissues, as a basis to classify mitochondrial risk factors as causes and consequences of disease. | ||
<br /> | |||
<br /> | |||
[[Image:The world as a laboratory.jpg|right|180px|link=http://wiki.oroboros.at/index.php/The_world_as_a_laboratory|Science and adventure]] | |||
__TOC__ | __TOC__ | ||
== Venue and organization == | == Venue and organization == | ||
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<br /> | <br /> | ||
<br /> | <br /> | ||
:* [[Val | :* [[Val Adalberto L |Adalberto L Val]], Director | ||
:* [[ | :* [[Almeida-Val Vera MF |Vera MF Almeida-Val]] | ||
:* [[Baptista RB |Ramon B Baptista]] | :* [[Baptista RB |Ramon B Baptista]] | ||
<br /> | <br /> | ||
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<br /> | <br /> | ||
<br /> | <br /> | ||
== | == O2k-Workshop and pilot project == | ||
[[File:IOC103 0331.JPG|left|240px]] | [[File:IOC103 0331.JPG|left|240px]] | ||
Day 1: Assembly of the O2k | Day 1: Assembly of the O2k | ||
From left to right: Erich Gnaiger, Reginaldo da Silva Oliveira, Derek Felipe Campos, Wadir Heinrichs Cardas, Ramon B Baptista. | From left to right: Erich Gnaiger, Reginaldo da Silva Oliveira, Derek Felipe Campos, Wadir Heinrichs Cardas, Ramon B Baptista. | ||
<br /> | |||
Day 2: SUIT protocol for comparative mitochondrial physiology - a pilot experiment with 20 mg wet weight of cardiac tissue from Tambaqui (''Colossoma macropomum''). Tissue homogenate was prepared with the PBI Shredder. | Day 2: SUIT protocol for comparative mitochondrial physiology - a pilot experiment with 20 mg wet weight of cardiac tissue from Tambaqui (''Colossoma macropomum''). Tissue homogenate was prepared with the PBI Shredder. | ||
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High reproducibility of mitochondrial respiratory control in cardiac tissue from Tambaqui. | High reproducibility of mitochondrial respiratory control in cardiac tissue from Tambaqui. | ||
<br /> | |||
<br /> | |||
<br /> | |||
=== Tambaqui === | |||
[[File:Tambaqui 4447.JPG|left|300px]] Tambaqui, ''Colossoma macropomum''. | |||
<br /> | |||
<gallery mode=packed widths="240px" heights="200px"> | |||
File:IOC103 0289.JPG|The O2k is set up in the seminar room for the training and pilot project. | |||
File:IOC103 0411.JPG|Preparation of chemicals. | |||
File:IOC103 0350.JPG|Oxygen calibration of the O2k with full attention. | |||
File:IOC103 0425.JPG|Ramon goes fishing. | |||
File:IOC103 0432.JPG|A small specimen of Tambaqui is selected. | |||
File:IOC103-Tambaqui-heart 4449.JPG|The fish is killed and opened. | |||
File:IOC103-Tambaqui-heart 4451.JPG|The heart is immediately immersed in ice cold BIOPS. | |||
File:IOC103-Tambaqui-heart 4457.JPG|A 20 mg portion of the heart is prepared. | |||
File:IOC103 0467.JPG|Mechanical separation of cardiac fibers. | |||
File:IOC103 0495.JPG|A balance between carefull separation and speed is important, before determination of the wet weight (c. 20 mg). | |||
File:IOC103 0507.JPG|The tissue is ready for homogenization with the Shredder. | |||
File:IOC103 0289.JPG|The homogenate (5 ml) is distributed at alternating 500 µl steps into the two O2k-Chambers. | |||
</gallery> | |||
<br /> | |||
[[File: | |||
[[File:IOC103- | [[File:Arapaima 4620-36.jpg |right|400px|''Arapaima gigas'', Manaus]] | ||
=== Arapaima or Pirarucu === | |||
[[File:Arapaima-mouth.jpg|left|300px |''Arapaima gigas'', Manaus]] | |||
[[File:Arapaima 4510.JPG |left|300px|''Arapaima gigas'', Manaus]] | |||
The Arapaima or Pirarucu is native to the Amazon, and is among the largest freshwater fish in the world (>2.5 m). As an adaptation to the seasonal hypoxic conditions, Arapaima has developed a swim bladder with lung-like function associated with air breathing. | |||
<br /> | |||
<gallery mode=packed widths="240px" heights="200px"> | |||
File:IOC103 Arapaima-heart 4534.JPG|A small sepcimen, at a developmental stage that may have just started air breathing, is selected for the pilot study. | |||
File:IOC103 Arapaima-heart 4539.JPG|The entire heart is just 20 mg wet weight. | |||
File:IOC103 Arapaima-heart 4540.JPG|The heart becomes quickly hemoglobin depleted during dissection in BIOPS. | |||
File:IOC103 Arapaima-heart 4541.JPG|The Arapaima heart has a texture different from the Tambaqui. | |||
File:IOC103 Arapaima-heart 4527.JPG|Ramon prepares the tissue for wet weight measurement. | |||
File:IOC103 Arapaima-Shredder 4531.JPG|Aleicia operates the Shredder for tissue homogenization. | |||
File:IOC103 Arapaima-HRR 4546.JPG|And Aleicia distributes the homogenate into the two O2k-Chambers. | |||
File:IOC103 Arapaima-HRR 4550.JPG|After adding CI-linked substrates (glutamate&malate), ADP, and cytochrome ''c'', a re-oxygenation is followed by titration of pyruvate. Aleicia is just adding succinate. | |||
File:IOC103 Arapaima-HRR 4552.JPG|Aleicia and Derek are amazed by the real-time results obtained with the O2k. | |||
File:IOC103 Arapaima-HRR 4553.JPG|Following a re-oxygenation, the delicate uncoupler titration is started. | |||
File:IOC103 Arapaima-HRR 4555.JPG|Ramon adds 'Events' and sets 'Marks' for real-time DatLab analysis. | |||
</gallery> | |||
[[File:IOC103-intro 4444.JPG |right|240px]] | [[File:IOC103-intro 4444.JPG |right|240px]] | ||
== Lecturers and tutors == | == Lecturers and tutors == | ||
* [[Gnaiger Erich]], CEO, | * [[Gnaiger Erich]], CEO, Oroboros Instruments | ||
* [[Gnaiger C |Carolina Gnaiger]], Assistant, Fotography, | * [[Gnaiger C |Carolina Gnaiger]], Assistant, Fotography, Oroboros Instruments | ||
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== SUIT protocol for the cardiac mitochondrial project == | == SUIT protocol for the cardiac mitochondrial project == | ||
Heart homogante from various fish species | [[File:SUIT-diagram.jpg |left|300px |Substrate-coupling control diagram]] | ||
[[ | Heart homogante from various fish species is analyzed with a basic substrate-uncoupler-inhibitor titration ([[SUIT]]) protocol. A [[SUIT protocol library |SUIT protocol library]] CI-CI<small>&</small>II-CII SUIT protocol was selected: GM +D +c +P +S +U +Rot +Ama (further details: [[MiPNet09.12 O2k-Titrations |»O2k-Titrations]]. | ||
[[ | |||
Cardiac tissue from Arapaima and Tambaqui was prepared in [[BIOPS]] on ice, 15 to 20 mg blotted wet weight were transferred into 500 µl ice cold [[MiR05Cr]] (60 mM lactobionic acid was replaced by 60 mM KCl) in the tube of the [[PBI-Shredder_O2k-Set | PBI-Shredder O2k-Set]], where the tissue was further separated by a pre-cooled pari of scissors, and tissue homogenate was prepared according to standard procedures [[MiPNet17.15 PBI-Shredder Mouse-heart-brain-liver]]. The homogenate was distributed immediately into the two 2-ml chambers of the O2k (pipetting 500 µl aliquots alernating into the two chambers, with multiple suctions between titrations). Data recording with DatLab was started when the homogenate was placed into the O2k-Chambers regulated at 28 °C, the homogenate was stirred for about 3 min with a gas phase, and the SUIT protocol was started immediately at air saturation upon closing the chamber. Oxygen levels were maintained approximately between 100 and 350 µM. | |||
The training programme and pilot project were conducted from July 8 to 10, 2015. | |||
[[File:Arapaima 4515m.jpg |left|300px |''Arapaima gigas'', Manaus]] | |||
[[File:Arapaima-heart 2015-07-10 P1-02B.jpg |800 px]] | |||
Mouse heart homogenate (37 °C) is shown for comparison. | [[File:Tambaqui 4447m.jpg |left|300px |''Colossoma macropomum'', Manaus]] | ||
[[File:Tambaqui-heart 2015-07-09 P1-02A.jpg |800 px]] | |||
[[File:Arctic-charr Koerbersee2014-IOC80(s).jpg |left|300px |Arctic charr]] | |||
[[File:Trout-heart 2011-10-05 P1-02A.jpg |800 px]] | |||
[[File:Mouse-C57BL6.jpg |left|300px |Mouse C57 BL6]] | |||
[[File:Mouse-heart_2011-11-16_P2-02A.jpg |800 px]] | |||
Trout heart homogenate (15 °C, 4 mg ''W''<sub>w</sub>/ml; 2011-10-05 P1-02A). Very low LEAK and ROX. No ADP limitation at D1 (1 mM) with glutamate&malate (GM), but glutamate&malate&pyruvate (GMP) was not saturated at 2 mM ADP (D2), as shown by the increase of flux when doubling the ADP concentration (4 mM; D4). No oxygen limitation. Low ''P/E'' ratio, indicating limitation of OXPHOS by the phosphorylation system. The optimum uncoupler (U; FCCP) concentration is higher than in liver homogenate (2.5 versus 1.0 µM), but 3 µM exerts already a pronounced time-dependent inhibitory effect (not due to limitation by oxygen, as shown by the lower flux after reoxygenation). Mouse heart homogenate (37 °C) is shown for comparison. | |||
=== References === | === References === | ||
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* » [[O2k-Publications: Comparative MiP;environmental MiP |Comparative mitochondrial physiology]] | * » [[O2k-Publications: Comparative MiP;environmental MiP |Comparative mitochondrial physiology]] | ||
* » [[O2k-Publications: Fishes]] | * » [[O2k-Publications: Fishes]] | ||
* » [[MiPNet17.03 Shredder vs | * » [[MiPNet17.03 Shredder vs Fibers]] | ||
=== Instrumental quality control === | |||
<gallery mode=packed widths="240px" heights="200px"> | |||
File:IOC103 O2-calibration-R1-over-time.jpg |O<sub>2</sub>–calibrations of the [[OroboPOS]] at air saturation (R1, 28 °C). '''A''': R1 [V] as a function of time after first calibration. '''B''': Change of R1 [%] after first calibration. Stability was better than 3%. Zero oxygen calibrations: 1.0 and 1.1% signal of the signal at air saturation (not shown). »[[MiPNet06.03 POS-calibration-SOP]] | |||
File:205-07-10 P1-01 Instr-O2-background.jpg|Instrumental O<sub>2</sub> background test at high oxygen. »[[MiPNet19.18E O2 flux analysis - DatLab 6]] | |||
File:205-07-10 P1-01 Instr-O2-background-graphs.jpg|Instrumental O<sub>2</sub> background: real-time analysis in DatLab 6 of background oxygen flux (A) or residuals (B) as a function of oxygen concentration. »[[MiPNet14.06 Instrumental O2 background]] | |||
File:IOC103 Instr-O2-background.jpg|Instrumental O<sub>2</sub> background. The stippled lines show the reference background (factory settings for calibration of the slope, 37 °C, extrapolated from the experimental 'normoxic' raange below air saturation). '''A''' and '''B''': Background oxygen flux as a function of oxygen concentration in a single background experiment (previous figures, 28 °C). '''C''': Summary of all values obtained during IOC103. These parameters (full lines) were used for final flux analysis (calibration of slope in DatLab). »[[MiPNet14.06 Instrumental O2 background]] | |||
</gallery> | |||
[[Image:O2k-Workshops.png|left|130px|link=O2k-Workshops|O2k-Workshops]] | [[Image:O2k-Workshops.png|left|130px|link=O2k-Workshops|O2k-Workshops]] | ||
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Please click »[[IOC Recommended Reading| here]]« | Please click »[[IOC Recommended Reading| here]]« | ||
'''»''' [[MitoPedia: Respiratory states]][[Image:P.jpg|link=OXPHOS capacity|OXPHOS]] [[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:E.jpg|link= | '''»''' [[MitoPedia: Respiratory states]][[Image:P.jpg|link=OXPHOS capacity|OXPHOS]] [[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:E.jpg|link=ET capacity|ETS]] [[Image:L.jpg|link=LEAK respiration|LEAK]] [[Image:ROX.jpg|link=Residual oxygen consumption|ROX]] | ||
[[Image:MitoGlobal.jpg|right|80px|link=MitoGlobal|MitoGlobal]] O2k-Workshops are listed as [[MitoGlobal Events]].[[Category:O2k-Workshops]] | [[Image:MitoGlobal.jpg|right|80px|link=MitoGlobal|MitoGlobal]] O2k-Workshops are listed as [[MitoGlobal Events]].[[Category:O2k-Workshops]] | ||
== Some impressions == | == Some impressions == | ||
=== INPA Science forest === | [[File:To the heart of the Amazon - Fish market.jpg |570px |Fish market, Manaus]] | ||
[[File:INPA- | === INPA Science forest and Manaus Zoo === | ||
[[File:To the heart of the Amazon - Manaus Zoo.jpg |570px |Manaus Zoo]] | |||
<gallery mode=packed widths="240px" heights="200px"> | |||
File:Amazon 4666.JPG | |||
File:INPA-Manatee 0355.JPG |Manatee project | |||
File:INPA-Science forest 0403.JPG |Piranias in a traditional hut | |||
File:INPA-Turtles 0389.JPG |Turtles in the forest | |||
File:Black-jaguar 4691.JPG |Black jaguar, Amazon | |||
File:Amazon 4676.JPG |Jaguar, Amazon | |||
</gallery> | |||
=== Perspectives === | === Perspectives === | ||
<gallery mode=packed widths="240px" heights="200px"> | |||
File:RioNegro-Manaus 4619.JPG |Rio Negro, port of Manaus: maximum water level in July 2015, compared to 2012. | |||
File:Amazon-boat 4384.JPG | |||
Should we get a boat (Vendo | File:Amazon-boat 4434.JPG |Should we get a boat (Vendo) for a MitoLab on the Amazon? | ||
File:Amazon-boat 4406.JPG | |||
</gallery> |
Latest revision as of 00:16, 12 January 2022
MiPNet20.12 IOC103 Manaus
Manaus BR, 2015 Jul 06-10. To the heart of the Amazon. O2k-Workshop on HRR and O2k-Fluorometry, IOC103. |
Oroboros (2015-07-06) Mitochondr Physiol Network
Abstract:
2015 Jul 06-10. To the heart of the Amazon: comparative cardiac mitochondrial physiology of Amazon fish. O2k-Workshop on HRR and start-up project, IOC103.
• O2k-Network Lab: BR Manaus Val AL, AT Innsbruck Oroboros
Labels:
HRR: Oxygraph-2k
ORO, IOC, PBI-Shredder, O2k-Network, 2015
To the heart of the Amazon
- Mitochondrial function is compared in mitochondrial preparations of fish heart. According to the suggestion by Dr. Adalberto L Val, one of each of the major groups should be selected: Potamotrygonidae, Osteoglossidae, Characiformes, Siluriformes, Perciformes (Cichlidae). The results obtained with a basic SUIT protocol will be compared with mitochondrial respiratory control patterns in other species, including human and mouse heart mitochondria. This study explores evolutionary variations of mitochondrial respiratory control patterns in different species, to define mitochondrial fitness in terms of adaptations to different environments and life styles. The results may provide a key towards delineating mitochondrial respiratory control patterns in health in different species and tissues, as a basis to classify mitochondrial risk factors as causes and consequences of disease.
Venue and organization
Venue
- Instituto Nacional de Pesquisas de Amazonia (INPA)
- Laboratório de Ecofisiologia e Evolução Molecular (LEEM)
- Ministerio da Ciencia e Tecnologia
- Av. Andre Araujo, 2938
- 69067-375 Manaus, AM, Brazil
Local organizers and contact
O2k-Workshop and pilot project
Day 1: Assembly of the O2k
From left to right: Erich Gnaiger, Reginaldo da Silva Oliveira, Derek Felipe Campos, Wadir Heinrichs Cardas, Ramon B Baptista.
Day 2: SUIT protocol for comparative mitochondrial physiology - a pilot experiment with 20 mg wet weight of cardiac tissue from Tambaqui (Colossoma macropomum). Tissue homogenate was prepared with the PBI Shredder.
Days 3-4: Project: Mitochondrial respiratory control pattern in Amazon fish heart.
High reproducibility of mitochondrial respiratory control in cardiac tissue from Tambaqui.
Tambaqui
Tambaqui, Colossoma macropomum.
Arapaima or Pirarucu
The Arapaima or Pirarucu is native to the Amazon, and is among the largest freshwater fish in the world (>2.5 m). As an adaptation to the seasonal hypoxic conditions, Arapaima has developed a swim bladder with lung-like function associated with air breathing.
Lecturers and tutors
- Gnaiger Erich, CEO, Oroboros Instruments
- Carolina Gnaiger, Assistant, Fotography, Oroboros Instruments
Participants
- Vera M Fonseca de Almeida e Val - [email protected]
- Adalberto L Val - [email protected]
- Aleicia Holland - [email protected]
- Ramon Barros Baptista - [email protected]
- Derek Selipe Compos - [email protected]
- Waldir Heinrichs Cacdas - [email protected]
- Reginaldo da Silva Oliveira - [email protected]
- Marcio Soares Ferrreira - [email protected]
- Andrea Gnaiger - [email protected]
- Carolina Gnaiger - [email protected]
- Erich Gnaiger - [email protected]
SUIT protocol for the cardiac mitochondrial project
Heart homogante from various fish species is analyzed with a basic substrate-uncoupler-inhibitor titration (SUIT) protocol. A SUIT protocol library CI-CI&II-CII SUIT protocol was selected: GM +D +c +P +S +U +Rot +Ama (further details: »O2k-Titrations.
Cardiac tissue from Arapaima and Tambaqui was prepared in BIOPS on ice, 15 to 20 mg blotted wet weight were transferred into 500 µl ice cold MiR05Cr (60 mM lactobionic acid was replaced by 60 mM KCl) in the tube of the PBI-Shredder O2k-Set, where the tissue was further separated by a pre-cooled pari of scissors, and tissue homogenate was prepared according to standard procedures MiPNet17.15 PBI-Shredder Mouse-heart-brain-liver. The homogenate was distributed immediately into the two 2-ml chambers of the O2k (pipetting 500 µl aliquots alernating into the two chambers, with multiple suctions between titrations). Data recording with DatLab was started when the homogenate was placed into the O2k-Chambers regulated at 28 °C, the homogenate was stirred for about 3 min with a gas phase, and the SUIT protocol was started immediately at air saturation upon closing the chamber. Oxygen levels were maintained approximately between 100 and 350 µM.
The training programme and pilot project were conducted from July 8 to 10, 2015.
Trout heart homogenate (15 °C, 4 mg Ww/ml; 2011-10-05 P1-02A). Very low LEAK and ROX. No ADP limitation at D1 (1 mM) with glutamate&malate (GM), but glutamate&malate&pyruvate (GMP) was not saturated at 2 mM ADP (D2), as shown by the increase of flux when doubling the ADP concentration (4 mM; D4). No oxygen limitation. Low P/E ratio, indicating limitation of OXPHOS by the phosphorylation system. The optimum uncoupler (U; FCCP) concentration is higher than in liver homogenate (2.5 versus 1.0 µM), but 3 µM exerts already a pronounced time-dependent inhibitory effect (not due to limitation by oxygen, as shown by the lower flux after reoxygenation). Mouse heart homogenate (37 °C) is shown for comparison.
References
- On respiratory control of human heart mitochondria
- Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43:1729–38. »Bioblast link«
Instrumental quality control
O2–calibrations of the OroboPOS at air saturation (R1, 28 °C). A: R1 [V] as a function of time after first calibration. B: Change of R1 [%] after first calibration. Stability was better than 3%. Zero oxygen calibrations: 1.0 and 1.1% signal of the signal at air saturation (not shown). »MiPNet06.03 POS-calibration-SOP
Instrumental O2 background test at high oxygen. »MiPNet19.18E O2 flux analysis - DatLab 6
Instrumental O2 background: real-time analysis in DatLab 6 of background oxygen flux (A) or residuals (B) as a function of oxygen concentration. »MiPNet14.06 Instrumental O2 background
Instrumental O2 background. The stippled lines show the reference background (factory settings for calibration of the slope, 37 °C, extrapolated from the experimental 'normoxic' raange below air saturation). A and B: Background oxygen flux as a function of oxygen concentration in a single background experiment (previous figures, 28 °C). C: Summary of all values obtained during IOC103. These parameters (full lines) were used for final flux analysis (calibration of slope in DatLab). »MiPNet14.06 Instrumental O2 background
Recommended IOC reading
Please click » here«
» MitoPedia: Respiratory states
O2k-Workshops are listed as MitoGlobal Events.