MITOCHONDRIAL RESPIROMETRY IN FROZEN BIOLOGICAL SAMPLES

AbstractImpaired mitochondrial respiration plays a key role in metabolic, cardiovascular, and aging-related
diseases. However, mitochondrial respirometry analysis currently requires processing of the living tissue
sample within an hour after being taken from the patient. This requirement makes respirometry analysis largely
unfeasible to standard clinical practice and clinical studies. We invented a new technology to assess maximal
mitochondrial respiratory capacity in previously-frozen biological samples, which thus far been considered
impossible by the scientific community. Side-by-side comparison confirmed that our assay accurately reflects
results measured in fresh samples. Until this point, we had not considered techniques to measure Complex V
since we know that previously frozen mitochondria do not synthesize ATP due to disruption in the inner
mitochondrial membrane and loss of mitochondrial membrane potential.
Enspire Bio will further develop this patented Frozen Mitos technology to enable respirometry measurements
and ATP synthase (Complex V) activity for clinical applications and frozen samples, which currently is not
available. We will achieve our goals by Aim 1: Establishing standard protocols for ATP synthase Complex V
from frozen tissue samples derived already stored cell lines and mouse tissue samples. We will further improve
throughput by enabling measurements of oxygen consumption from tissue homogenate instead of isolated
mitochondria. This approach allows for retrospective studies to examine changes in mitochondrial function and
has potential utilization as a diagnostic tool in minimally and non-invasive clinical samples. While this technique
can be used to determine changes in electron transport chain Complexes I, II, and IV, we have not yet
assessed the mitochondrial Complex V in previously frozen samples.Narrative
Our original application described a technology to measure the respiratory function of mitochondria in frozen
tissue samples. Here we propose to develop an approach to simultaneously measure the activity of ATP
synthase in the same sample. This addition will cut the cost, time and size of tissue sample by half.