MiPNet20.12 IOC103 Manaus

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MiPNet20.12 IOC103 Manaus

Publications in the MiPMap
O2k-Network

Manaus BR, 2015 Jul 06-10. To the heart of the Amazon. O2k-Workshop on HRR and O2k-Fluorometry, IOC103.

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OROBOROS (2015-07-06) Mitochondr Physiol Network

Abstract:
Arapaima, Pirarucu, or Paiche, Manaus

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 

Additional: ORO, IOC, PBI-Shredder, O2k-Network, 2015 

Amazon, Manaus

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.

Science and adventure

Venue and organization

BR Manaus Val AL
Entrance to LEEM

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



BR Manaus Val AL

Local organizers and contact






O2k-Workshop and pilot project

IOC103 0331.JPG

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 4447.JPG
Tambaqui, Colossoma macropomum.




Arapaima gigas, Manaus

Arapaima or Pirarucu

Arapaima gigas, Manaus
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.


IOC103-intro 4444.JPG

Lecturers and tutors


Participants

IOC103 4543.JPG
IOC103 4558.JPG
  • Vera M Fonseca de Almeida e Val - veraval@inpa.gov.br
  • Adalberto L Val - dalval.inpa@gmail.com
  • Aleicia Holland - a.holland@cqu.edu.au
  • Ramon Barros Baptista - ramon.baptista@gmail.com
  • Derek Selipe Compos - compos.derek@gmail.com
  • Waldir Heinrichs Cacdas - dinho_c@hotmail.com
  • Reginaldo da Silva Oliveira - reginaldo.oliveiria@inpa.gov.br
  • Marcio Soares Ferrreira - marcio@inpa.gov.br
  • Andrea Gnaiger - andrea.gnaiger@oroboros.at
  • Carolina Gnaiger - carolina.gnaiger@oroboros.at
  • Erich Gnaiger - erich.gnaiger@oroboros.at


SUIT protocol for the cardiac mitochondrial project

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 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 10, 2015.

Arapaima gigas, Manaus

Arapaima-heart 2015-07-10 P1-02B.jpg

Colossoma macropomum, Manaus

Tambaqui-heart 2015-07-09 P1-02A.jpg

Arctic charr

Trout-heart 2011-10-05 P1-02A.jpg

Mouse C57 BL6

Mouse-heart 2011-11-16 P2-02A.jpg

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

O2k-Workshops

Recommended IOC reading

Please click » here«

» MitoPedia: Respiratory statesOXPHOS ROUTINE ETS LEAK ROX


MitoGlobal
O2k-Workshops are listed as MitoGlobal Events.

Some impressions

Fish market, Manaus

INPA Science forest and Manaus Zoo

Manaus Zoo

Perspectives