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


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: 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


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