polyP depletion prevents opening of the permeability transition pore induced by mitochondrial Ca²⁺ overload. (A) Original recordings of TMRM fluorescence in permeabilized cells upon elevation of the [Ca²⁺]_em from 0.1 to 2 µM, and subsequent addition of 1 µM FCCP in control (black) and polyP-depleted (PPX-expressing) myocytes (gray). (B) Average values reflecting the basal levels of TMRM fluorescence in control and PPX-expressing cells. (C) Average TMRM fluorescence decrease, measured at the end of exposure to 2 µM Ca²⁺ as percent decrease from initial levels in control and PPX-expressing cells. (D) Original recordings of calcein red fluorescence from permeabilized control (black), PPX-expressing (dark gray) and CsA-treated control (medium gray), and CsA-treated, PPX-expressing (light gray) cells. After permeabilization, cells were exposed to 2 µM Ca²⁺ in the presence or absence of CsA. To normalize calcein fluorescence, 10 µM alamethicin was added at the end of the experiment to achieve the maximal calcein red release from mitochondria. (E) Summary of calcein red release (as percentage of total release obtained after alamethicin addition) from mitochondria measured at the end of 2-µM Ca²⁺ exposure in control, PPX-expressing, CsA-treated control cells, and CsA-treated, PPX-expressing cells.