Figure 1.

Schema of experimental timeline used to study the effect of Cyclosporin A (CsA) and adenine nucleotide (AdN) on mitochondrial Ca²⁺ handling and bioenergetics during repeated CaCl₂ pulses. (A) In Protocol A, at t = 0 s, mitochondria (mito, 0.5 mg) were added to the Na⁺-free experimental buffer solution. The mitochondrial suspension was exposed to 0.5 μM CsA, 250 μM ADP, 10 μM oligomycin (OMN), or a combination of OMN+ADP at t = 30 s. Pyruvic acid (PA, 0.5 mM), was added at t = 60 s to energize mitochondria (state 2). At t = 180 s, 20 μM of CaCl₂ was added, followed by sequential additions of 20 µM CaCl₂ at every 300 s intervals until mPTP (mitochondrial permeability transition pore) opened or no further Ca²⁺ uptake was observed. (B) In Protocol B, the mitochondrial suspension was exposed to similar treatments as in Protocol A, but given after the last consecutive CaCl₂ bolus preceding the imminent onset of mPTP opening.