Cryopreservation of heart mitochondria: Effect on respiration and regulation of the permeability transition pore by nitric oxide

Studies on respiration and the mitochondrial permeability transition pore (MPTP) need to be performed on freshly-isolated mitochondria, since an intact inner membrane is required. However, it would be desirable to establish a protocol to store frozen heart mitochondria in a way that maintains their integrity and bioenergetic function for future studies. Cryopreservation of heart mitochondria has not been previously reported, to the best of our knowledge. In this study we compared the respiratory control ratio and opening of the MPTP in freshly isolated and frozen mitochondria isolated from rat hearts. Half the sample was used immediately, and half snap-frozen with 10% DMSO. We found that frozen mitochondria retained their ability to couple ATP synthesis to electron transport (measured using an oxygen electrode) albeit with a reduced coupling ratio: 71.4 ± 2.9% of that of fresh mitochondria (p <0.001). Frozen mitochondria were also slightly more sensitive to Ca2+-induced MPTP opening (measured as decrease in absorbance at 520 nm), but nitric oxide was able to inhibit the MPTP in both fresh and frozen mitochondria at similar concentrations. In conclusion we believe that cryopreserving heart mitochondria using 10% DMSO gives a sufficiently well-coupled mitochondria which can be used to study the regulation of respiration and MPTP opening, for example by nitric oxide. In the future this will allow large preparations of mitochondria from cow or pig hearts to be stored for subsequent studies, thus reducing the use of laboratory animals and enabling large numbers of experiments to be performed on the same sample of mitochondria. Critical results would then be confirmed on freshly-isolated mitochondria.