MiPNet20.12 IOC103 Manaus

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.

The O2k is set up in the seminar room for the training and pilot project.
Preparation of chemicals.
Oxygen calibration of the O2k with full attention.
Ramon goes fishing.
A small specimen of Tambaqui is selected.
The fish is killed and opened.
The heart is immediately immersed in ice cold BIOPS.
A 20 mg portion of the heart is prepared.
Mechanical separation of cardiac fibers.
A balance between carefull separation and speed is important, before determination of the wet weight (c. 20 mg).
The tissue is ready for homogenization with the Shredder.
The homogenate (5 ml) is distributed at alternating 500 µl steps into the two O2k-Chambers.

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.

A small sepcimen, at a developmental stage that may have just started air breathing, is selected for the pilot study.
The entire heart is just 20 mg wet weight.
The heart becomes quickly hemoglobin depleted during dissection in BIOPS.
The Arapaima heart has a texture different from the Tambaqui.
Ramon prepares the tissue for wet weight measurement.
Aleicia operates the Shredder for tissue homogenization.
And Aleicia distributes the homogenate into the two O2k-Chambers.
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.
Aleicia and Derek are amazed by the real-time results obtained with the O2k.
Following a re-oxygenation, the delicate uncoupler titration is started.
Ramon adds 'Events' and sets 'Marks' for real-time DatLab analysis.

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 W_w/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