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