Auxiliary HRR-Tools Mitochondrial Physiology Network 17.15: 1-6 (2012-2013) 2013 OROBOROS Version 3: 2013-01-15 Tissue Homogenates for Diagnosis of Mitochondrial Respiratory Function: Mouse Heart, Brain and Liver Andrea Eigentler,1 Mona Fontana-Ayoub,2 Erich Gnaiger1,2 1 Medical University of Innsbruck Department of Visceral, Transplant and Thoracic Surgery D. Swarovski Research Laboratory, 6020 Innsbruck, Austria 2 OROBOROS INSTRUMENTS Corp high-resolution respirometry Schöpfstr 18, A-6020 Innsbruck, Austria Email: erich.gnaiger@oroboros.at www.oroboros.at http://wiki.oroboros.at/index.php/K-Regio_MitoCom_Tyrol 1. Introduction A high-quality preparation of tissue homogenate may represent an optimum compromise for a variety of respirometric and fluorometric studies. These considerations provided the rationale for initiating a study with the PBI-Shredder, an auxiliary HRR-Tool providing a standardized approach to prepare homogenates of various tissues (e.g. heart, liver, brain) with high reproducibility of mitochondrial yield and mitochondrial function as evaluated with HRR. 2. Sample preparation 2.1. Organ harvest Heart, liver and brain are excised from the sacrificed mouse and added into Falcon tubes containing sufficient ice-cold BIOPS (mouse heart) or respiration medium (mouse liver and brain) to cover the entire tissue sample. Keep on ice and minimize transportation and storage time as far as possible. instruments@oroboros.at www.oroboros.at MiPNet17.15 2.2. PBI-Shredder: an auxiliary tool for HRR 2 Tissue preparation and homogenization (shredding) Prepare tissue samples of about 4 mg wet weight (Ww) for mouse heart muscle and 8 mg Ww for mouse brain and liver for two O2k-Chambers (half the Ww if one Shredder Tube should be used for one O2k-Chamber). Preparation of tissue homogenate and determination of the wet weight (Ww) has to be done according to MiPNet17.02. 3. Experimental setup with the Oxygraph-2k Respiration of all tissue homogenates was measured in respiration medium at 37 °C and in a normoxygen range. For detailed describtion see MiPNet17.02. 4. Experimental protocol Substrate-uncoupler-inhibitor-titration (SUIT) protocols were used to evaluate the mitochondrial function of mouse heart, mouse brain and mouse liver homogenate. All substrates, inhibitors and the uncoupler used in the protocols with mouse heart, brain and liver homogenate can be found in MiPNet09.12. 5. Results In the present methodological evaluation of tissue homogenates prepared with the PBI-Shredder SG3, results are expressed per mg of tissue applied for preparation per O2k-chamber (2 ml). Representative results of two chambers are presented for each tissue homogenate. 6. Mouse heart homogenate High-resolution respirometry 300 450 240 360 180 270 120 180 60 90 0 0:00 0:10 PMG 0:21 D2 D3 D4 0:32 Range [h:min]: 1:05 c S 0:43 D5 F0.25 F0.5 Rot 0:54 Ama 0 1:05 O2 Flux per mass (C) [pmol/(s*mg)] 6.1. Mna Fig. 1: SUIT protocol with homogenate of mouse myocardium. Overlay of O2ktraces of two chambers. Oxygen concentration [µM] (black and blue line) and oxygen flux per mass [pmols-1mg-1] (red and green line). MiR05Cr, 37 °C. OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k MiPNet17.15 PBI-Shredder: an auxiliary tool for HRR 3 400 1.2 300 200 chamber C chamber D 100 Flux control ratio O2 flux per mass [pmol/(s*mg)] -1 6.2. O2 Mouse flux per tissue mass [pmols-1mgMouse ] and FCRhomogenate heart homogenate heart 1.0 0.8 0.6 chamber C 0.4 chamber D 0.2 0.0 0 Fig. 2: O2 flux per mass [pmols1mg-1] and flux control ratio normalized to PMGSE and corrected for ROX, of chamber C and chamber D, in mouse heart homogenate. Coupling control ratios and substrate control ratios FCR with ROX correction CI ETS OXPHOS 0.59 LEAK 0.07 PMG CI+II CII 1.00 0.52 1.00 E 1.00 Flux control ratio Coupling control 6.3. P L PMGS S 0.80 L 0.60 P 0.40 E 0.20 E P 0.00 Substrate control PMG L PMGS S(Rot)_E Fig. 3: Coupling control and substrate control ratios for mouse homogenate, calculated as mean of chamber C and chamber D. heart 7. Mouse brain (cortex) homogenate High-resolution respirometry 250 70 200 56 150 42 100 28 50 14 0 0:00 0:11 PM 0:23 D1 D2 G D3 D3 cc 0:35 Range [h:min]: 1:10 S D4 F0.5 F1 Rot 0:46 Ama 0:58 0 1:10 O2 Flux per mass (A) [pmol/(s*mg)] 7.1. Mna Fig. 4: Overlay of O2k-trace of chamber A and chamber B. Oxygen concentration [µM] (black and blue line) and oxygen flux per mass [pmols-1mg-1] (red and green line) of mouse brain (cortex) homogenate in MiR05. OROBOROS INSTRUMENTS www.oroboros.at MiPNet17.15 4 O2 flux per tissue mass [pmols-1Mouse mg-1]brain andhomogenate FCR Mouse brain homogenate 7.2. 1.2 60 40 chamber A 20 chamber B Flux control ratio O2 flux per mass [pmol/(s*mg)] PBI-Shredder: an auxiliary tool for HRR 1.0 0.8 0.6 chamber A 0.4 chamber B 0.2 0 0.0 Fig. 5: O2 flux per mass [pmols-1mg-1] and flux control ratio normalized to PMGSE and corrected for ROX, of chamber A and chamber B, in mouse brain (cortex) homogenate. 7.3. Coupling control ratios and substrate control ratios FCR with ROX correction CI ETS OXPHOS 0.28 LEAK 0.16 PM 0.38 CI+II CII 1.00 0.60 1.00 Flux control ratio Coupling control CI E 0.99 P L PMG PMGS 0.80 0.60 L 0.40 P E 0.20 E P 0.00 S PM PMG Substrate control L PMGS S(Rot) Fig. 6: Coupling control and substrate control ratios for mouse brain (cortex) homogenate, calculated as mean of chamber A and chamber B. 8. Mouse liver homogenate High-resolution respirometry 250 160 200 128 150 96 100 64 50 32 0 0:00 0:13 Amr HRPGM 0:27 D1 D2 D3 P D4 S 0:41 Range [h:min]: 1:23 reoxy D5 F0.5 F1 reoxy 0:55 F1.5 Rot Mna 0 1:23 1:09 Ama H2O2 O2 Flux per mass (A) [pmol/(s*mg)] 8.1. H2O2 Fig. 7: Overlay of O2k-trace of chamber A and chamber B. Oxygen concentration [µM] (black and blue line) and oxygen flux per mass [pmols-1mg-1] (red and green line) of mouse liver homogenate in MiR05. OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k MiPNet17.15 5 O flux per tissue mass [pmols-1mg-1] and FCR 2 Mouse liver homogenate Mouse liver homogenate 140 1.2 120 1.0 100 80 60 chamber A 40 chamber B Flux control ratio O2 flux per mass [pmol/(s*mg)] 8.2. PBI-Shredder: an auxiliary tool for HRR 0.8 0.6 chamber A 0.4 20 0.2 0 0.0 chamber B Fig. 8: O2 flux per mass [pmols-1mg-1] and flux control ratio normalized to PMGSE and corrected for ROX, of chamber A and chamber B, in mouse liver homogenate. 8.3. Coupling control ratios and substrate control ratios CI CI ETS OXPHOS 034 LEAK 0.07 GM 0.47 CI+II CII 1.00 0.67 0.86 1.00 E P L GMP GMPS S Flux control ratio Coupling control FCR with ROX correction 0.80 0.60 L 0.40 P E 0.20 E P 0.00 Substrate control GM GMP L GMPS S(Rot) Fig. 9: Coupling control and substrate control ratios for mouse liver homogenate, calculated as mean of chamber A and chamber B. 9. Conclusions The application of an additional tool to remove the serrated Shredder Ram as well as the Shredder Cap after homogenization increased the mitochondrial yield by washing out the homogenate completely from both sides of the Lysis Disk. The cytochrome c effect could be diminished in mouse heart homogenate and no cytochrome c effect occurred in mouse liver and mouse brain homogenate. 10. Acknowledgements Contribution to K-Regio project MitoCom Tyrol, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF). www.oroboros.at/?MitoCom-Tyrol OROBOROS INSTRUMENTS www.oroboros.at MiPNet17.15 PBI-Shredder: an auxiliary tool for HRR 6 11. References Mitochondr Physiol Network – MiPNet Manuals and Protocols MiPNet09.12: Oxygraph-2k titrations. Mitochondria, permeabilized cells and biopsies. Mitochondr Physiol Network 09.12. MiPNet17.02: PBI-Shredder HRR-Set: Preparation of tissue homogenates for diagnosis of mitochondrial respiratory function. 12. Author contributions and publication versions Eigentler A performed experiments and wrote the manuscripts. Fontana-Ayoub M performed experiments. Gnaiger E designed experiments and edited the manuscript. Results for fish (trout) homogenate of liver and heart are published in MiPNet17.03: Doerrier C, Draxl A, Wiethuechter A, Eigentler A, Gnaiger E (2012) Mitochondrial respiration in permeabilized fibres versus homogenate from fish myocardium and liver. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03. Preliminary results for mouse heart homogenate versus permeabilized fibres can be found in the Appendix of MiPNet17.03. OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k