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 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.
Highlights
Human heart muscle has very high and continuous energy demand, mostly due to its contractile work when propelling blood through the circulatory system [1,2,3]
Minced tissue fragments were diluted in MiR05 buffer to obtain a 10% homogenate and manually homogenized by 10–12 strokes up and down using a Dounce tissue grinder set with a clearance 0.114 ± 0.025 mm
We adopted high resolution respirometry to homogenates of human atrial myocardium to enable measurement of mitochondrial function from a sample of less than 20 mg of fresh muscle. We demonstrated that this simple technique has better variability and the same or better preservation of outer membrane integrity and OXPHOS coupling efficiency than the established techniques of respirometry in isolated mitochondria [11,13,24,42]
Summary
Human heart muscle has very high and continuous energy demand, mostly due to its contractile work when propelling blood through the circulatory system [1,2,3]. Intracellular stores of ATP and creatine phosphate can be exhausted within a few seconds [4] and any defect in mitochondrial metabolism leading to decreased ATP production results in rapid contractile dysfunction [4,5]. Cardiac function depends on the continuous ability of myocardium to generate ATP at a high rate [3,4]. A range of congenital and acquired heart diseases are associated with altered mitochondrial metabolism and insufficient ATP production[8,9], as is cardiac aging [10]. Most data are obtained by studying cell lines or animal models [12], but their applicability to humans is questionable
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
