High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle

English version

Autoři: Adéla Krajčová ^aff001; Tomáš Urban ^aff001; David Megvinet ^aff001; Petr Waldauf ^aff001; Martin Balík ^aff002; Jan Hlavička ^aff003; Petr Budera ^aff003; Libor Janoušek ^aff004; Eva Pokorná ^aff005; František Duška ^aff001
Působiště autorů: OXYLAB – Laboratory of Mitochondrial Physiology, Department of Anaesthesia and Intensive Care, Third Faculty of Medicine, Charles University and FNKV University Hospital, Prague, Czech Republic ^aff001; Department of Anaesthesia and Intensive Care, 1 Medical Faculty, Charles University and General University Hospital, Prague, Czech Republic ^aff002; Department of Cardiac Surgery, Third Faculty of Medicine, Charles University and FNKV University Hospital, Prague, Czech Republic ^aff003; Transplantation Surgery Department, Institute for Clinical and Experimental Medicine, Prague, Czech Republic ^aff004; Department of Organ Recovery and Transplantation Databases, Institute for Clinical and Experimental Medicine, Prague, Czech Republic ^aff005
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0226142

Souhrn

Impaired myocardial bioenergetics is a hallmark of many cardiac diseases. There is a need of a simple and reproducible method of assessment of mitochondrial function from small human myocardial tissue samples. In this study we adopted high-resolution respirometry to homogenates of fresh human cardiac muscle and compare it with isolated mitochondria. We used atria resected during cardiac surgery (n = 18) and atria and left ventricles from brain-dead organ donors (n = 12). The protocol we developed consisting of two-step homogenization and exposure of 2.5% homogenate in a respirometer to sequential addition of 2.5 mM malate, 15 mM glutamate, 2.5 mM ADP, 10 μM cytochrome c, 10 mM succinate, 2.5 μM oligomycin, 1.5 μM FCCP, 3.5 μM rotenone, 4 μM antimycin and 1 mM KCN or 100 mM Sodium Azide. We found a linear dependency of oxygen consumption on oxygen concentration. This technique requires < 20 mg of myocardium and the preparation of the sample takes <20 min. Mitochondria in the homogenate, as compared to subsarcolemmal and interfibrillar isolated mitochondria, have comparable or better preserved integrity of outer mitochondrial membrane (increase of respiration after addition of cytochrome c is up to 11.7±1.8% vs. 15.7±3.1%, p˂0.05 and 11.7±3.5%, p = 0.99, resp.) and better efficiency of oxidative phosphorylation (Respiratory Control Ratio = 3.65±0.5 vs. 3.04±0.27, p˂0.01 and 2.65±0.17, p˂0.0001, resp.). Results are reproducible with coefficient of variation between two duplicate measurements ≤8% for all indices. We found that whereas atrial myocardium contains less mitochondria than the ventricle, atrial bioenergetic profiles are comparable to left ventricle. In conclusion, high resolution respirometry has been adapted to homogenates of human cardiac muscle and shown to be reliable and reproducible.

Klíčová slova:

Cardiac muscles – Cardiac surgery – Coronary artery bypass grafting – Mitochondria – Myocardium – Oxygen consumption – Pulmonary hypertension – Respirometry

Zdroje

1. Gibbs CL. Cardiac energetics. Physiol Rev. 1978;58: 174–254. doi: 10.1152/physrev.1978.58.1.174 146205

2. Suga H. Ventricular energetics. Physiol Rev. 1990;70: 247–77. doi: 10.1152/physrev.1990.70.2.247 2181496

3. Lopaschuk G, Naranjan S. Cardiac Energy Metabolism in Health and Disease. Springer. doi: 10.1007/978-1-4939-1227-8 2014.

4. Doenst T, Nguyen T, Abel E. Cardiac Metabolism in Heart Failure—Implications beyond ATP production. Circ Res. 2013;113: 709–724. doi: 10.1161/CIRCRESAHA.113.300376 23989714

5. Neubauer S. The Failing Heart—An Engine Out of Fuel. N Engl J Med. 2007;356: 1140–51. doi: 10.1056/NEJMra063052 17360992

6. Varikmaa M, Guzun R, Grichine A, Gonzalez-Granillo M, Usson Y, Boucher F, et al. Matters of the heart in bioenergetics: Mitochondrial fusion into continuous reticulum is not needed for maximal respiratory activity. J Bioenerg Biomembr. 2013;45: 319–331. doi: 10.1007/s10863-012-9494-4 23271420

7. Lionetti V, Stanley WC, Recchia FA. Modulating fatty acid oxidation in heart failure. Cardiovasc Res. 2011;90: 202–209. doi: 10.1093/cvr/cvr038 21289012

8. Quigley A, Kapsa R, Esmore D, Hale G, Byrne E. Mitochondrial respiratory chain activity in idiopathic dilated cardiomyopathy. J Card Fail. 2000;6: 47–55. doi: 10.1016/s1071-9164(00)00011-7 10746819

9. Jarreta D, Orús J, Barrientos A, Miró O, Roig E, Heras M, et al. Mitochondrial function in heart muscle from patients with idiopathic dilated cardiomyopathy. Cardiovasc Res. 2000;45: 860–865. doi: 10.1016/s0008-6363(99)00388-0 10728411

10. Tocchi A, Quarles EK, Basisty N, Gitari L, Rabinovitch PS. Mitochondrial dysfunction in cardiac aging. Biochim Biophys Acta. Elsevier B.V.; 2015;1847: 1424–1433. doi: 10.1016/j.bbabio.2015.07.009 26191650

11. Varga ZV, Ferdinandy P, Liaudet L, Pacher P. Drug-induced mitochondrial dysfunction and cardiotoxicity. Am J Physiol Hear Circ Physiol. 2015;309: 1453–1467. doi: 10.1152/ajpheart.00554.2015 26386112

12. Lemieux H, Hoppel C. Mitochondria in the human heart. J Bioenerg Biomembr. 2009;41: 99–106. doi: 10.1007/s10863-009-9211-0 19353253

13. Lanza IR, Nair KS. Functional Assessment of Isolated Mitochondria In Vitro. Methods Enzym. 2009;457: 349–372. doi: 10.1016/s0076-6879(09)05020-4 19426878

14. Figueiredo P, Ferreira R, Appell H, Duarte J. Age-induced morphological, biochemical, and functional alterations in isolated mitochondria from murine skeletal muscle. J Gerontol A Biol Sci Med Sci. 2008;63: 350–359. doi: 10.1093/gerona/63.4.350 18426958

15. Rasmussen U, Krustrup P, Kjaer M, Rasmussen H. Experimental evidence against the mitochondrial theory of aging. A study of isolated human skeletal muscle mitochondria. Exp Gerontol. 2003;38: 877–886. doi: 10.1016/s0531-5565(03)00092-5 12915209

16. Saks V, Veksler V, Kuznetsov A, Kay L, Sikk P, Tiivel T, et al. Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem. 1998;184: 81–100. doi: 10.1007/978-1-4615-5653-4_7 9746314

17. Kuznetsov AV., Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS. Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc. 2008;3: 965–976. doi: 10.1038/nprot.2008.61 18536644

18. Cardinale DA, Gejl KD, Ørtenblad N, Ekblom B, Blomstrand E, Larsen FJ. Reliability of maximal mitochondrial oxidative phosphorylation in permeabilized fibers from the vastus lateralis employing high-resolution respirometry. Physiol Rep. 2018;6: e13611. doi: 10.14814/phy2.13611 29464938

19. Pecinová A, Drahota Z, Nůsková H, Pecina P, Houštěk J. Evaluation of basic mitochondrial functions using rat tissue homogenates. Mitochondrion. 2011;11: 722–8. doi: 10.1016/j.mito.2011.05.006 21664301

20. Ziak J, Krajcova A, Jiroutkova K, Nemcova V, Dzupa V, Duska F. Assessing the function of mitochondria in cytosolic context in human skeletal muscle: Adopting high-resolution respirometry to homogenate of needle biopsy tissue samples. Mitochondrion. Elsevier; 2015;21: 106–112. doi: 10.1016/j.mito.2015.02.002 25701243

21. Pesta D, Gnaiger E. High-resolution respirometry: OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol. 2012;810: 25–58. doi: 10.1007/978-1-61779-382-0_3 22057559

22. Gnaiger E, Kuznetsov A V, Schneeberger S, Seiler Rü, Brandacher G, Steurer W, et al. Mitochondria in the Cold. In: Heldmaier G, Klingenspor M (eds) Life in the Cold, 1st edn. Springer, Berlin, Heidelberg. 2000. s. 431–442. doi: 10.1007/978-3-662-04162-8_45

23. Gnaiger E. Polarographic Oxygen Sensors, the Oxygraph, and High-Resolution Respirometry to Assess Mitochondrial Function. In: Dykens JA, Will Y (eds) Drug-Induced Mitochondrial Dysfunction, 1st edn. John Wiley & Sons, Inc. 2008. s. 325–352. doi: 10.1002/9780470372531.ch12

24. Ruas JS, Siqueira-Santos ES, Amigo I, Rodrigues-Silva E, Kowaltowski AJ, Castilho RF. Underestimation of the maximal capacity of the mitochondrial electron transport system in oligomycin-treated cells. PLoS One. 2016;11: 1–20. doi: 10.1371/journal.pone.0150967 26950698

25. Wikström M, Casey R. The oxidation of exogenous cytochrome c by mitochondria. Resolution of a long-standing controversy. FEBS Lett. 1985;183: 293–298. doi: 10.1016/0014-5793(85)80796-1 2985431

26. Perry CGR, Kane DA, Lanza IR, Neufer PD. Methods for assessing mitochondrial function in diabetes. Diabetes. 2013;62: 1041–1053. doi: 10.2337/db12-1219 23520284

27. Larsen S, Kraunsøe R, Gram M, Gnaiger E, Helge J, Dela F. The best approach: homogenization or manual permeabilization of human skeletal muscle fibers for respirometry? Anal Biochem. 2014;1: 64–8. doi: 10.1016/j.ab.2013.10.023 24161612

28. Palmer J, Tandler B, Hoppel C. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem. 1977;252: 8731–9. 925018

29. Picard M, Taivassalo T, Gouspillou G, Hepple RT. Mitochondria: isolation, structure and function. J Physiol. 2011;589: 4413–4421. doi: 10.1113/jphysiol.2011.212712 21708903

30. Srere PA. Citrate Synthase. Methods Enzym. 1969;13: 3–11. doi: 10.1016/0076-6879(69)13005-0

31. Kuznetsov AV, Kunz WS, Saks V, Usson Y. Cryopreservation of mitochondria and mitochondrial function in cardiac and skeletal muscle fibers. Anal Biochem. 2003;2697. doi: 10.1016/S0003-2697(03)00326-9

32. Toleikis A, Dzeja P, Praskevicius A, Jasaitis A. Mitochondrial functions in ischemic myocardium. I. Proton electrochemical gradient, inner membrane permeability, calcium transport and oxidative phosphorylation in isolated mitochondria. J Mol Cell Cardiol. 1979;11: 57–76. doi: 10.1016/0022-2828(79)90452-8 423257

33. Chance B, Hagihara B. Activation and inhibition of succinate oxidation following adenosine diphosphate supplements to pigeon heart mitochondria. J Biol Chem. 1962;237: 3540–3545. doi: 10.1016/0006-291x(60)90091-7 14019996

34. Chidsey CA, Weinbach EC, Pool PE, Morrow AG. Biochemical studies of energy production in the failing human heart. J Clin Invest. 1966;45: 40–50. doi: 10.1172/JCI105322 5901178

35. Mela L, Seitz S. Isolation of Mitochondria with Emphasis on Heart Mitochondria from Small Amounts of Tissue. Methods Enzym. 1979;55: 39–46. doi: 10.1016/0076-6879(79)55006-x

36. Chappell JB. The Oxidation of Citrate, Isocitrate and cis-Aconitate by Isolated Mitochondria. Biochem J. 1964;90: 225–237. doi: 10.1042/bj0900225 4378636

37. Scheubel RJ, Tostlebe M, Simm A, Rohrbach S, Prondzinsky R, Gellerich FN, et al. Dysfunction of mitochondrial respiratory chain complex I in human failing myocardium is not due to disturbed mitochondrial gene expression. J Am Coll Cardiol. Elsevier Masson SAS; 2002;40: 2174–2181. doi: 10.1016/S0735-1097(02)02600-1

38. Wilson M, Cascarano J, Wooten W, Pickett C. Quantitative isolation of liver mitochondria by zonal centrifugation. Anal Biochem. 1978;85: 255–64. doi: 10.1016/0003-2697(78)90297-x 629384

39. Tonkonogi M, Fernström M, Walsh B, Ji L, Rooyackers O Hammarqvist F Wernerman J, Sahlin K. Reduced oxidative power but unchanged antioxidative capacity in skeletal muscle from aged humans. Pflugers Arch. 2003;446: 261–9. doi: 10.1007/s00424-003-1044-9 12684796

40. Krieger D, Tate C, McMillin-Wood J, Booth F. Populations of rat skeletal muscle mitochondria after exercise and immobilization. J Appl Physiol. 1980;48: 23–8. doi: 10.1152/jappl.1980.48.1.23 6444398

41. Picard M, Taivassalo T, Ritchie D, Wright KJ, Thomas MM, Romestaing C, et al. Mitochondrial structure and function are disrupted by standard Isolation methods. PLoS One. 2011;6: 1–12. doi: 10.1371/journal.pone.0018317 21512578

42. Saks V, Guzun R, Timohhina N, Tepp K, Varikmaa M, Monge C, et al. Structure-function relationships in feedback regulation of energy fluxes in vivo in health and disease: Mitochondrial Interactosome. Biochim Biophys Acta. Elsevier B.V.; 2010;1797: 678–697. doi: 10.1016/j.bbabio.2010.01.011 20096261

43. Veksler V, Kuznetsov A, Sharov V, Kapelko V, Saks V. Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers. Biochim Biophys Acta. 1987;892: 191–6. doi: 10.1016/0005-2728(87)90174-5 3593705

44. Mathers KE, Staples JF. Saponin-permeabilization is not a viable alternative to isolated mitochondria for assessing oxidative metabolism in hibernation. Biol Open. 2015;4: 858–864. doi: 10.1242/bio.011544 25979709

45. From AM, Maleszewski JJ, Rihal CS. Current status of endomyocardial biopsy. Mayo Clin Proc. 2011;86: 1095–1102. doi: 10.4065/mcp.2011.0296 22033254

46. Murphy J, Frantz R, Cooper L. Endomyocardial biopsy. In: Murphy J, Lloyd M (eds). Mayo Clinic Cardiology: Concise Textbook, 3rd edn. Mayo Clinic Scientific Press, pp 1481–1487.

47. Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E. Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol. 2011;43: 1729–38. doi: 10.1016/j.biocel.2011.08.008 21871578

48. Boushel R, Gnaiger E, Schjerling P, Skovbro M, Kraunsøe R, Dela F. Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle. Diabetologia. 2007;50: 790–796. doi: 10.1007/s00125-007-0594-3 17334651

49. Perry CGR, Kane DA, Lin C, Kozy R, Brook L, Lark DS, et al. Inhibiting Myosin-ATPase Reveals Dynamic Range of Mitochondrial Respiratory Control in Skeletal Muscle. Biochem J. 2013;437. doi: 10.1042/BJ20110366 21554250

High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle

Souhrn

Klíčová slova:

Zdroje

PLOS One

INSIGHTS from European Respiratory Congress

Současné pohledy na riziko v parodontologii

Svět praktické medicíny 3/2024 (znalostní test z časopisu)

Kardiologické projevy hypereozinofilií

Střevní příprava před kolonoskopií