Mitochondrial Respiratory Kinetics Assay

High-resolution O₂ consumption measurements were conducted in 2 mL of buffer Z using the OROBOROS Oxygraph-2k (OROBOROS INSTRUMENTS, Corp., Innsbruck, AT) with stirring at 750 rpm. Buffer Z contained 20 mM creatine hydrate to saturate creatine kinase, which facilitates mitochondrial ADP transport [4 (link), 10 (link), 23 (link)-25 (link)], with the exception of specific experiments on human PmFBs which were conducted in the presence of 24 mM phosphocreatine and 12 mM creatine hydrate (described below). 5 mM pyruvate and 2 mM malate were added as complex I substrates. ADP was titrated in step-wise increments and all experiments were completed before oxygraph chamber [O₂] reached 150 μM. At the conclusion of each experiment, PmFBs were washed in double-distilled H₂O to remove salts, frozen at -20°C, and dried via lyophilization (Labconco Corp., Kansas City, MO). Polarographic oxygen measurements were acquired in 2-second intervals, with the rate of respiration derived from 40 data points, and expressed as pmol • s^-1 • mg^-1 dry weight. Dry and wet bundle weights were consistently between 0.2 - 0.6 mg and ∼1.0 to 2.5 mg, respectively. Cytochrome c was added to test for mitochondrial membrane integrity as partial loss of cytochrome c during sample preparation may limit active respiration. A cytochrome c response was dectected in <5% of all experiments and no response generated >10% increase in respiration. No relationship was observed between the relative cytochrome c response and Km when grouping all human and rodent data (R² = 0.013, p>0.05). Additionally, no significant relationship was observed in humans when using a paired t-test to compare the Km for those experiments showing 0-5% cytochrome c response relative to those few samples exhibiting a 5-10% cytochrome c response. Four PmFBs from each rat or human were run simultaneously in four separate oxygraph chambers. Two of the chambers contained either 100 μM BTS or 25 μM BLEB. A third chamber contained 1.25% DMSO (vehicle, +V) to match the content of DMSO added in the BTS and BLEB conditions with the remaining chamber serving as the control (minus vehicle) condition.
The Km for ADP was determined through the Michaelis-Menten enzyme kinetics - fitting model (Y = Vmax*X/(Km + X)), where X = [free ADP; ADP_f] and Y = JO₂ at [ADP_f], using Prism (GraphPad Software, Inc., La Jolla, CA). This equation was also used to calculate the fraction of maximal mitochondrial respiration in resting human skeletal muscle in vivo. This calculation was performed using the experimentally determined Km values assuming resting [ADP_f] to be ∼14.6 μM in human skeletal muscle [6 (link)].