Krajcova 2018 MiPschool Tromso E2
Event: MiPschool Tromso-Bergen 2018
Impaired myocardial bioenergetics is a hallmark of many cardiac diseases and adverse effects of commonly used drugs. Several methods are currently used for investigation of mitochondrial metabolism of human heart muscle using high-resolution respirometry, but these are limited either by need of a large amount of tissue biopsy (isolated mitochondria) or lengthy and time-consuming process of sample preparation (permeabilized muscle fibers). In this study we aimed to develop simple, reliable and reproducible method of assessment of mitochondrial function from small tissue samples by adoption of high-resolution respirometry technique to homogenates of native human cardiac muscle.
We used atrial appendages resected during cardiac surgery (N=18) and atrial and left ventricular muscle biopsies from brain-dead organ donors (N=10). In a series of experiments, we have optimised and tested the technique. Final protocol consists of two-step homogenization and exposure of 2.5% cardiac muscle homogenate in the high-resolution respirometry (Oroboros O2k) 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 and 4 µM antimycin. The technique requires only 20 mg of myocardium and sample preparation takes less than 20 min.
Mitochondria in the homogenate are intact with preserved integrity of outer mitochondrial membrane (increase of respiration after addition of cytochrome c = 15±2%) and very minor uncoupling of inner mitochondrial membrane (Respiratory Control Ratio = 3.00±0.37). Results are reproducible with coefficient of variation between two duplicate measurements <8% for all indices, and tissue samples can be stored on ice for up to 12 hours. We found that despite atrial myocardium contains less mitochondria compared to ventricle, atrial bioenergetic profiles are representative for left ventricle.
In conclusion, high-resolution respirometry has been adopted to homogenates and shown to be simple, reliable and reproducible method.
Labels: MiParea: Respiration, Instruments;methods
Organism: Human Tissue;cell: Heart Preparation: Homogenate
Coupling state: LEAK, OXPHOS, ET Pathway: N, NS, ROX HRR: Oxygraph-2k Event: E2
Affiliations and support
- Krajčová A(1), Megvinet D(1), Urban T(1), Waldauf P(1), Hlavička J(2), Budera P(2), Janoušek L(3), Pokorná E(4), Duška F(1)
- OXYLAB – Lab Mitochondrial Physiology, Dept Anaesthesia Intensive Care, Third Fac Medicine
- Dept Cardiac Surgery, Third Fac Med; Charles Univ and FNKV Univ Hospital
- Transplantation Surgery Dept, Inst Clinical Experimental Medicine
- Dept Organ Recovery Transplantation Databases, Inst Clinical Experimental Medicine; Prague, Czech Republic. - [email protected]
- The work was supported by PROGRES Q36/7, UNCE, AZV 16-28663 A.