GzmC directly causes mitochondrial swelling and depolarization in isolated mitochondria.

(A-B) Mitochondrial volume and membrane potential changes of purified murine liver mitochondria (MLM, 0.5 mg/mL) were monitored as described.16 In both panels, where indicated by the arrows, 40 μM Ca²⁺ and 4 μM rGzmC (black and gray traces) were added. Where noted (gray trace), 1 μM CsA was present from the beginning. In panel A, 400 μM Ca²⁺ (light gray trace) was added where indicated to induce complete swelling. In all the experiments depicted in panel B, complete depolarization was achieved by adding 400 nM FCCP where indicated. (C) Dose dependence of GzmC-induced mitochondrial swelling. The experiment was performed as in panel A, except that the indicated concentrations of rGzmC were added. (D) Ca²⁺ dependence of GzmC-induced swelling. The experiment was carried out as in panel A, except that the indicated Ca²⁺ concentrations were added before 4 μM rGzmC. Ca²⁺ concentrations of 50 μM or less did not cause detectable mitochondrial swelling. (E) Differential release of fluorescent dyes entrapped in the mitochondrial matrix by GzmC. Mitochondria loaded with 10 μM rhod-2-am or calcein-am were left untreated or were treated with 200 μM Ca²⁺ in the presence of 1 μM CsA, 2 μM mastoparan, or 4 μM rGzmC. After 30 minutes, mitochondria were pelleted, and calcein and rhod-2 fluorescence levels were measured in the pellet and in the supernatant. Data are normalized for the untreated mitochondria.