Assessment of mitochondrial viability under calcium Stress: Insights for mitochondrial transplantation

Mitochondrial transplantation has emerged as a promising cardioprotective strategy for ischemia-reperfusion injury, aiming to restore bioenergetic function by delivering healthy mitochondria to damaged tissue. However, conflicting reports exist regarding whether mitochondria can survive exposure to the calcium-rich extracellular environment, such as the bloodstream, prior to cellular uptake. Resolving this question is essential for advancing the therapeutic use of mitochondria in clinical settings. Isolated mitochondria from L6 rat skeletal muscle cells were incubated with physiologic (1.3  mM), sub-physiologic (0.65  mM), and supraphysiologic (2.6  mM) concentrations of calcium. Mitochondrial membrane potential was assessed using MitoTracker™ Red FM fluorescence, and structural integrity was evaluated using impedance-based Coulter counter analysis over a 12-hour time course. Mitochondria exposed to 1.3  mM calcium retained 90-95 % membrane potential by 12 h, while 2.6  mM calcium caused progressive loss of function and integrity, approaching levels seen in freeze-thawed controls. Coulter counter measurements revealed more extensive mitochondrial loss across all calcium-treated groups than fluorescence assays alone, suggesting that dye-based methods may underestimate structural damage. Nonetheless, a substantial proportion of mitochondria remained both structurally and functionally intact at physiologically relevant calcium levels. These findings demonstrate that a substantial number of mitochondria can retain membrane potential and structural integrity after exposure to extracellular calcium concentrations approximating those found in blood. This supports the feasibility of intracoronary mitochondrial transplantation and underscores the need for further in vivo studies to optimize survival and efficacy of mitochondria delivered in calcium-rich environments.