Fig 2: In mesenteric resistance arteries from female mice, inhibition of MCU (mitochondrial Ca2+ uniporter) by treatment with mtCaMKIIN reduces vasodilation to a larger extent than in male mice. A, Acetylcholine (ACh)‐induced vasodilation of second‐order mesenteric arteries from male and female mice with endothelium‐specific expression of mitochondria‐targeted CaMKIIN (e‐mtCaMKIIN) and in littermate control (wild‐type [WT]) mice. B, EC50 of vasodilation to Ach as in (A). C, EMax of vasodilation to ACh as in (A). D, Endothelium‐independent vasodilation to sodium nitroprusside (SNP). E, Vasoconstriction to KCl. Analysis by repeated‐measure 2‐way ANOVA (A, D, and E) and 2‐way ANOVA (B and C). No significant interactions between sex and genotype were seen for Figure 1B and 1C. P values for source of variations in treatment groups as shown. *P<0.05, **P<0.01, ***P<0.005. EC50 indicates half maximal effective concentration; EMax, maximal effect; mtCaMKIIN, mitochondrially targeted peptide inhibitor of CaMKII; KCl, potassium chloride.

Fig 3: Variation and composition of MCU-EMRE complexes in mitochondrial inner membranes based on the stoichiometry of MCU and EMRE. This study showed that, in all tissues and HeLa cells, 0-3 molecules of EMRE were assembled in a single MCU tetramer. Based on the probabilities of each complex (MCU : EMRE = 4 : 0, 4 : 1, 4 : 2, 4 : 3), which were obtained in Fig. 6, the ratios of each MCU-EMRE complex in the mitochondrial inner membrane were calculated. By use of these ratios, the breakdown of each complex in 10 MCU tetramers is illustrated. In this model, MCU tetramers without EMRE are shown as functionally inactive (grey). MCU tetramers with 1 EMRE, those with 2 EMREs, and those with 3 EMREs were assumed to be functionally active (yellow).

Fig 4: Mitochondrial calcium buffering capacity is reduced and MCU protein level is decreased in gba1-/- and gba1+/- neurons. a Mitochondria-target aequorin plate reader assay was used to measure mitochondrial Ca2+ uptake in mixed neuronal and astrocytic cultures from gba1+/+, gba1+/-, and gba1-/- in response to 10 µM glutamate. The data show that mitochondrial Ca2+ uptake was significantly reduced in both gba1+/- and gba1-/- cells compared with gba1+/+ (n = 3–5 cultures per genotype, one-way Anova, post-hoc Bonferroni). b Protein expression levels of MCU complex components and MCU regulatory proteins evaluated by western blot in gba1+/+, gba1+/-, and gba1-/- brains (data shown as scatter plots and mean ± SEM, n = 3–5 brains per genotype). MCU expression was significantly downregulated in gba1+/- and gba1-/- tissue (One-way Anova, post-hoc Bonferroni, *p < 0.05), while EMRE, MICU2, and MCUR1 expression levels were unchanged

Fig 5: Inhibition of MCU via the pharmacological inhibitor Ru360 or siMCU attenuated noise-induced increases in cleaved caspase 9 (CC9) in OHCs of the basal turn. (A) Representative images show an increase in immunoreactivity for CC9 (red) in OHCs stained with phalloidin 1 h after completion of the exposure (panel 2) compared to control mice without exposure (panel 1). Treatment with Ru360 attenuated noise-induced CC9 in OHCs (panel 3). Images were taken from the region of the surface preparations corresponding to sensitivity to 22–32 kHz using a Leica SP5 confocal microscope; scale bar = 10 µm. (B) Quantification of CC9 in OHCs confirmed a significant increase after noise exposure and attenuation of this increase with Ru360 treatment; n = 4 per group with one cochlea used per mouse. Data are presented as means + SD, **p < 0.01. (C) Representative images show that pretreatment with siMCU decreases immunoreactivity for CC9 (red) in OHCs stained with phalloidin (green) 1 h after completion of the exposure compared to siControl treatment. Images were taken from the region of the surface preparations corresponding to sensitivity to 22–32 kHz using a Zeiss confocal microscope; scale bar = 10 µm. (D) Pretreatment with siMCU also significantly reduced noise-increased immunolabeling for CC9 in OHCs compared to mice exposed to scrambled siRNA (siControl). Data are presented as means + SD, n = 4 per group with one cochlea used per mouse, *p < 0.05.