Comparative analysis of mitochondrial CRC in permeabilized cells and isolated cell mitochondria

Mitochondria undergo permeability transition (PT) resulting in the opening of the non-selective PT pores (PTPs) in the inner mitochondrial membrane in response to energy and oxidative stresses associated with Ca2+ overload and ROS accumulation. The mitochondrial PTPs are permeable to ions and solutes with a molecular mass <1.5 kD that increases the colloidal osmotic pressure in the matrix leading to mitochondrial swelling. Calcium retention capacity (CRC) reflects the maximum amount of Ca2+ mitochondria can uptake to provoke the PTP opening. Quantification of CRC is important to study the effects of various pathological stimuli and the efficacy of pharmacological agents on the metabolism and function of mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the advantages/disadvantages of the fluorescent technique in isolated mitochondria vs. permeabilized cells. The cells were permeabilized using digitonin or saponin, and the CRC was assessed using the Ca2+ -sensitive fluorescence probe Calcium Green-5N. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. The CRC of saponin-permeabilized cells was higher than the CRC of digitonin-permeabilized cells. In addition, the mitochondrial CRC of saponin-permeabilized cells was higher than isolated mitochondria using the same number of cells. In conclusion, this study demonstrates that the fluorescent technique for CRC analysis in saponin-permeabilized cells has more advantages than isolated mitochondria.