Fig. 2

Profibrotic stimuli alter mtCU gating to reduce _mCa²⁺ uptake. a MEFs plus or minus (+/−) 12 h TGFβ were loaded with Ca²⁺-sensitive dye Fura-2. Fluorescence was recorded and 1 mM ATP was delivered to initiate IP3R-mediated Ca²⁺ release. Cytosolic Ca²⁺ (_cCa²⁺) load was determined by calculating area-under-the-curve (AUC) of _cCa²⁺ transients; n = 28 cells. b MEFs +/− 12 h TGFβ were transduced with adenovirus encoding the mitochondrial calcium (_mCa²⁺) sensor, Mito-R-GECO. Fluorescence was recorded during 1 mM ATP treatment. _mCa²⁺ load was determined by calculating AUC; n = 24 cells. c–g MEFs +/− 12 h TGFβ were permeabilized with digitonin in the presence of thapsigargin (SERCA inhibitor) and CGP-37157 (NCLX inhibitor) and loaded with Ca²⁺ sensor Fura-2 and Δψ sensor JC-1 for ratiometric monitoring during Ca²⁺ additions. c Representative Ca²⁺ traces in untreated (black) and TGFβ-treated (blue) MEFs. d JC-1 derived Δψ in untreated (black) and TGFβ-treated (blue) MEFs. e, f Dose response curve of _mCa²⁺ uptake following different [Ca²⁺] boluses. g Kinetic parameters derived from data in panel e. h–j MEFs were treated with TGFβ and cell lysates were immunoblotted for components of the mtCU, including pore forming subunit MCU and regulatory subunits MICU1 (Mitochondrial Ca²⁺ Uptake 1), MCUR1 (Mitochondrial Ca²⁺ Uniporter Regulator 1), MCUB, and EMRE (Essential MCU Regulator), as well as OxPhos Complexes CV (ATP5A) and CIII (subunit-UQCRC2), VDAC (Voltage-dependent anion channel), and tubulin. Mitochondrial loading controls: VDAC and CIII; total lysate loading control: tubulin. Band density was normalized to CIII; n = 3. k, l MEFs and mouse adult cardiac fibroblasts (ACFs) were treated with TGFβ and Micu1 mRNA was analyzed by qPCR. n = 6. m–o MEFs were treated with AngII and cell lysates were immunoblotted for MCU, MICU1, MCUR1, MCUB, and EMRE, as well as OxPhos Complexes CV and CIII, VDAC, and tubulin. Mitochondrial loading controls: VDAC and CIII. Total lysate loading control: tubulin. Band density was normalized to CIII; n = 3. p, q MEFs and mouse ACFs were treated with AngII and Micu1 mRNA was analyzed by qPCR (n = 6). Data shown as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 vs. vehicle control analyzed by ANOVA. Also see Supplementary Fig. 2