Difference between revisions of "MiPNet20.12 IOC103 Manaus"
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|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 06-07. '''To the heart of the Amazon: comparative cardiac mitochondrial physiology of Amazon fish.''' O2k-Workshop on HRR and start-up project, IOC103. | 2015 Jul 06-07. '''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 OROBOROS | |mipnetlab=BR Manaus Val AL, AT Innsbruck OROBOROS | ||
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=== Tambaqui === | === Tambaqui === | ||
[[File:Tambaqui 4447.JPG|left|300px]] Tambaqui, ''Colossoma macropomum''. | [[File:Tambaqui 4447.JPG|left|300px]] Tambaqui, ''Colossoma macropomum''. | ||
<br /> | |||
<gallery> | <gallery> | ||
File:IOC103 0289.JPG|The O2k is set up in the seminar room for the training and pilot project. | File:IOC103 0289.JPG|The O2k is set up in the seminar room for the training and pilot project. | ||
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=== Arapaima or Pirarucu === | === Arapaima or Pirarucu === | ||
[[File:Arapaima-mouth.jpg|left|300px |''Arapaima gigas'', Manaus]] | [[File:Arapaima-mouth.jpg|left|300px |''Arapaima gigas'', Manaus]] | ||
[[File:Arapaima | [[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. | 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> | <gallery> | ||
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 4534.JPG|A small sepcimen, at a developmental stage that may have just started air breathing, is selected for the pilot study. | ||
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== SUIT protocol for the cardiac mitochondrial project == | == SUIT protocol for the cardiac mitochondrial project == | ||
Heart homogante from various fish species is analyzed with a basic SUIT protocol. The training programme and pilot project were conducted | [[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 [[diametral |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 HRR-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 11 2015. | |||
[[File:Arapaima 4515m.jpg |left|300px |''Arapaima gigas'', Manaus]] | [[File:Arapaima 4515m.jpg |left|300px |''Arapaima gigas'', Manaus]] | ||
[[File:Arapaima-heart 2015-07-10 P1-02B.jpg |800 px]] | [[File:Arapaima-heart 2015-07-10 P1-02B.jpg |800 px]] | ||
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'''»''' [[MitoPedia: Respiratory states]][[Image:P.jpg|link=OXPHOS capacity|OXPHOS]] [[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:E.jpg|link=ETS capacity|ETS]] [[Image:L.jpg|link=LEAK respiration|LEAK]] [[Image:ROX.jpg|link=Residual oxygen consumption|ROX]] | '''»''' [[MitoPedia: Respiratory states]][[Image:P.jpg|link=OXPHOS capacity|OXPHOS]] [[Image:R.jpg|link=ROUTINE respiration|ROUTINE]] [[Image:E.jpg|link=ETS capacity|ETS]] [[Image:L.jpg|link=LEAK respiration|LEAK]] [[Image:ROX.jpg|link=Residual oxygen consumption|ROX]] | ||
=== Instrumental quality control === | |||
[[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]] |
Revision as of 10:46, 31 July 2015
MiPNet20.12 IOC103 Manaus
Manaus BR, 2015 Jul 06-07. To the heart of the Amazon. O2k-Workshop on HRR and O2k-Fluorometry, IOC103. |
OROBOROS (2015-07-06) Mitochondr Physiol Network
Abstract:
2015 Jul 06-07. 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, MiPNet, 2015, Next
To the heart of the Amazon
Mitochondrial function is compared in mitochondrial preparations of fish heart. According to the suggestion by Dr. Adalberto Luis 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 HRR-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 11 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«
Recommended IOC reading
Please click » here«
» MitoPedia: Respiratory states
Instrumental quality control
O2k-Workshops are listed as MitoGlobal Events.