Fig. 3.

Mfn2 ablation decreases mitochondrial Ca²⁺ uptake in situ but not in vitro. (A) Mean ± SEM of peak cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in response to ATP (0.2 mM) in Ca²⁺-free Krebbs Ringer buffer (KRB) from in three independent experiments (n = 10 recordings per experiment) where Mfn2^flx/flx MEFs were infected with mtYFP or CRE-2A-mtYFP (CRE) adenoviruses and after 48 h transfected with cytAEQ. *P < 0.05 vs. mtYFP. (B) Mean ± SEM of mitochondrial Ca²⁺ ([Ca²⁺]_mit) in Ca²⁺-free KRB from in three independent experiments (n = 10 recordings per experiment) where Mfn2^flx/flx MEFs were infected with the indicated adenoviruses and after 48 h transfected with mtAEQ. Where indicated, cells were perfused with 0.2 mM ATP and with 0.2 mM ATP + 2 mM Ca²⁺. (C) Average ± SEM of peak [Ca²⁺]_mit from in three independent experiments (n = 10 recordings per experiment) performed as in B. Where indicated, cells were perfused with (+Ca²⁺) or without (−Ca²⁺) extracellular Ca²⁺. *P < 0.05 vs. mtYFP. (D) Mean ± SEM of [Ca²⁺]_i from three independent experiments (n = 10 recordings per experiment) where Mfn2^flx/flx MEFs were infected with the indicated adenoviruses and after 48 h transfected with cytAEQ. Cre infected cells were incubated for 30 min in Ca²⁺ free media. Where indicated, cells were perfused with 0.2 mM ATP. (E) Average ± SEM of peak [Ca²⁺]_i in three independent experiments performed as in D. (F) Experiments were performed as in D except that [Ca²⁺]_mit was recorded in in Mfn2^flx/flx MEFs infected with the indicated adenoviruses and after 48 h transfected with mtAEQ. (G) Average ± SEM of three independent peak [Ca²⁺]_mit in experiments performed as in F. *P < 0.05 vs. the corresponding mtYFP bar. (H) Expanded scale of G. The Ca²⁺ uptake rate is indicated. (I) Control (WT) and liver-specific Mfn2 knockout mice (Mfn2^LKO) mitochondria (0.5 mg/mL) were incubated in experimental buffer supplemented with 1 µM CaGreen-5N and 2 µM cyclosporine A. Where indicated, 50 µM Ca²⁺ pulses were added. (J) Expanded scale of I. (K) Average ± SEM of [Ca²⁺]_mit uptake rate recorded from three couples of littermates as in I.