Figure 8. Inhibition of mitochondrial fission in MCU^−/− VSMCs augments mitochondrial Ca²⁺ uptake, dynamics, and respiration.

A) Oxygen consumption rate (OCR) in WT VSMCs after treatment with P110 (2 μM for 16 h) and PDGF (20 ng/ml for 1 h). n = 3 independent experiments. B) OCR in MCU^−/− VSMCs after treatment with P110 and PDGF. n=3 independent experiments. C) Confocal microscopic images of mitochondria (mito-GFP, green) in WT and MCU^−/− VSMCs after treatment with P110 for 16 h and PDGF or control for 20 min. Scale bar = 20 μm (larger image) or 5 μm (inset). D) Summary histogram of form factor in control WT and MCU^−/− VSMCs in D). Number of cells analyzed in 3 independent experiments indicated in bars. E) Average tracing of mitochondrial Ca²⁺ by mtPericam in WT or MCU^−/− VSMCs after treatment with P110 (2 μM) for 16 h. Arrow indicates addition of PDGF (20 ng/ml). n = 3 independent experiments. F) Summary histogram of form factor in control WT VSMCs with adenoviral overexpression of constitutively active CaMKII (CaMKII T287D, MOI 100) or control. Samples pretreated with P110 as indicated. n = 3 experiments, number of cells analyzed indicated in bars. G) Confocal microscopic images of mitochondria (mito-GFP, green) in WT and MCU^−/− VSMCs after overexpression of OPA1 and MFN1/2 at the baseline and after PDGF treatment. Scale bar = 20 μm (larger image) or 5 μm (inset). H) Summary histogram of form factor in baseline WT and MCU^−/− before and after overexpression of OPA1 and MFN1/2 before and after PDGF treatment. n = 3 independent experiments, number of cells analyzed indicated in bars. I) Immunoblots for OPA-1, Myc (myc-tagged MFN1), MFN2 and GAPDH in WT VSMCs and VSMCs with overexpression of OPA1 and MFN1/2. n = 2 independent experiments. J) Graphical summary, indicating that MCU deletion increases CaMKII activation, DRP1 phosphorylation and mitochondrial fission. * p<0.05, ** p<0.01, *** p<0.005, **** p<0.001 by Kruskal-Wallis test (D, F, H).