Krajcova 2020 PLOS ONE
|Krajčová A, Urban T, Megvinet D, Waldauf P, Balík M, Hlavička J, Budera P, Janoušek L, Pokorná E, Duška F (2020) High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle. PLOS ONE 15:e0226142.|
Abstract: 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 compared 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 dependence 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.
- Iglesias-Gonzalez et al (2021) Proficiency test in mt-respiration: A necessary tool for reliable and reproducible results. MitoFit Preprints 2021 (in prep).
Labels: MiParea: Respiration
Stress:Cryopreservation Organism: Human Tissue;cell: Heart Preparation: Homogenate, Isolated mitochondria
Regulation: Oxygen kinetics Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS, ROX HRR: Oxygraph-2k
Alert2020, MitoFit 2021 PT