Mfn1 depletion–induced perturbation of RyR1-mediated [Ca²⁺]_c oscillations, ΔΨm, and CSMDH activation. (A) Caffeine-induced (10 mM) [Ca²⁺]_c oscillations in individual RyR1-transfected WT and Mfn1KO cells (n = 39 and 86 cells, respectively). (B) Mean time-course traces and mean [Ca²⁺]_c (200–250 s). P < 0.001. (C) Evaluation of the number of oscillatory peaks and oscillation amplitude decay ([peak1 − peak 3]/peak1 × 100). (D) Mitochondrial membrane potential loss 200 s after caffeine stimulation (six experiments; ***, P < 0.001). (E and F) MEFs were transfected with mtDsRed and RyR1 plus NTsiRNA or Mfn1 siRNA (WT), or Mfn1KO with and without Mfn1myc overexpression. Cells were stimulated with 10 mM caffeine and [Ca²⁺]_c and NAD(P)H transients were simultaneously measured. Bar charts show (E) the amplitude decay for the [Ca²⁺]_c oscillations ([peak1 − peak 3]/peak1 × 100); cell numbers: WT+NTsiRNA, n = 31; WT+Mfn1siRNA, n = 25; KO, n = 34 cells; and KO+Mfn1myc, n = 38; *, P < 0.05) and (F) NAD(P)H levels at 200–250 s after stimulation normalized to the baseline (cell numbers: WT+NTsiRNA, n = 95; WT+Mfn1siRNA, n = 72; KO, n = 55; and KO+Mfn1myc, n = 63; **, P < 0.001); n.s., not significant. Data are from four experiments.