Loss of Miro1 blocks neointima formation.

(A) Immunoblots for MIRO1 in lysates from brain and aorta isolated from WT and SM-Miro1-/-mice. (B) Verhoeff-Van Gieson staining in the unligated and ligated common carotid arteries of wild type and SM-Miro1-/- mice, at 21 days post ligation. Scale bar = 100 µm. (C) Neointimal area in ligated carotid arteries at 250 µm from the bifurcation. Neointimal area was determined by subtracting the luminal area from the area defined by the internal elastic lamina. (D) Cumulative neointimal area, calculated from all neointimal areas within 1000 µm of the bifurcation. (E) Neointimal area at the indicated distances from the site of ligation. (F) Medial area at the indicated distances from the site of ligation. The medial area was determined by subtracting the area defined by the internal elastic lamina from the area defined by the external elastic lamina. (G) Immunofluorescence of MIRO1 expression in the mouse carotid artery. MIRO1, green; SM-actin, red; Nuclei, DAPI, blue. Scale bar: 20 µm in upper images; 50 µm in lower images. Upper images are magnifications of the areas labeled with a box in the lower images. (H) Immunofluorescence of MIRO1 in the left anterior descending artery of a healthy subject and that of a patient with coronary artery disease. MIRO1, green; SM-actin, red; Nuclei, DAPI, blue. Scale bar: 80 µm in upper, 20 µm in lower images. Statistical analyses were performed using Mann-Whitney test (C, D) and two-way ANOVA (E, F).

Loss of Miro1 reduces G1/S transition and VSMC proliferation.

(A) MIRO1 levels in mitochondrial fractions of VSMCs from Miro1fl/fl mice transduced with adenovirus expressing Cre recombinase or control adenovirus, as determined by immunoblotting. (B) Number of VSMCs from Miro1fl/fl mice following transduction with adenovirus expressing cre recombinase (MIRO1-/-) or control (WT) adenovirus, at 72 h after incubation with PDGF (20 ng/ml) or control (saline). (C) DNA content of synchronized WT and MIRO1-/- VSMCs, as assessed by fluorescence-activated cell sorting (FACS). Times are 0, 24, and 48 h after release from growth arrest for 48h in FBS-free media, and then at 24 and 48 h after release from arrest with media containing 10% FBS. Analysis compared differences between genotypes. (D-F) Quantification of cell-cycle phase distribution of WT and MIRO1-/- VSMCs shown in C. (G) Immunoblots for cyclin E and D1 as markers of the indicated cell cycle phases in whole-cell lysates of WT and Miro1-/- VSMCs following synchronization by serum starvation, at G0 (after growth arrest for 48 h in FBS-free media), and G1/S (after release from growth arrest with media containing 10% FBS for 24 h). (H, I) Quantification of Cyclin D1 and E levels on immunoblots like those shown in panel G. Statistical analyses were performed using Kruskal-Wallis test (B) and two-way ANOVA (D-F), and Friedman test (H, I).

Loss of EF hands in MIRO1 reduces mitochondrial motility and cell proliferation.

(A, B) Immunoblots for MIRO1 and c-MYC in WT and Miro1-/- VSMCs transduced with adenovirus expressing MIRO1-WT (A) and MIRO1-KK (B). (C) Number of WT and Miro1 -/- VSMCs transduced with adenovirus expressing MIRO1-WT (Ad WT), MIRO1-KK (Ad KK), or control adenovirus (empty vector, Ad EV) and treated with PDGF (20 ng/ml) for 72 h. (D) Representative confocal images of WT and Miro1-/- VSMCs grown on Y-shaped adhesive micropatterns (CYTOOchipsTM), with VSMCs synchronized by serum starvation for 24 h (0 htimepoint), followed by change to medium containing 10% FCS and PDGF (20 ng/ml) for 6h (Phalloidin, red; mitochondrial GFP, green; To-Pro-3, blue). (E) Mitochondrial probability map. The cumulative distribution of mitochondria was assessed for images as in (D) by modified Sholl’s analysis. Data are plotted by growth conditions (as in A). (F) Mito95 values, defined as the distance from the center of the CYTOOchipsTM at which 95% of the mitochondrial signal is found, under the conditions used in (A). (G) Representative confocal images of WT and Miro1-/- VSMCs grown on CYTOOchipsTM. Miro1-/- VSMCs were transduced with adenovirus expressing MIRO1-WT or MIRO1-KK for 72 h before being seeded onto CYTOOchipsTM. The cell cycle was synchronized by serum starvation for 24 h (not shown); the cells were subsequently treated with medium containing 10% FCS and PDGF (20 ng/ml) for 6 h. (H) Mitochondrial probability map. The cumulative distribution of mitochondria was assessed for images shown in (G) by modified Sholl’s analysis. Data are plotted by growth conditions (as in B). (I) Mito95 values, as defined in (F) but under the conditions used in (B). (J) Immunoblots for MIRO1 and c-Myc in WT and Miro1-/- VSMCs transduced with adenoviruses expressing MIRO1-WT, MIRO1-KK, or with control adenovirus. Statistical analyses were performed by one-way ANOVA (C, F) and Kruskal-Wallis test (I).

Inhibition of mitochondrial ATP production reduces VSMC proliferation, but inhibiting mitochondrial motility does not affect ATP levels.

(A) Number of WT VSMCs after 72-h treatment with either PDGF (20 ng/ml) and/or oligomycin (1 µM). (B, C) ATP levels in WT VSMCs after treatment with PDGF (20 ng/ml) or with PDGF in addition to oligomycin (1 µM) for 72 h (B) or 16 h (C). (D) Representative confocal images of WT VSMCs on CYTOOchipsTM with Y-shaped micropatterns, following 16-h treatment with PDGF (20 ng/ml) and oligomycin (1 µM). Mitochondria, green; phalloidin, red; DAPI, blue. (E) Mitochondrial probability map. The cumulative distribution of mitochondria was assessed for images as in (D) by modified Sholl’s analysis. (F) Mito95 values, defined as the distance from the center of the Y-shaped pattern at which 95% of the mitochondrial signal is found for growing cells like in (D). (G) Representative confocal images of WT VSMCs infected with mitoGFP and stained with phalloidin. Cells were synchronized by serum starvation for 48 h and then released from starvation by replacing the medium with one containing 10% FBS and PDGF (20 ng/ml). Some samples were treated with nocodazole (1 µM). Images were taken immediately after growth media (Control) or growth media with nocodazole was added (Nocodazole) and after incubation for 16 h (Control + PDGF, Nocodazole + PDGF). (H) ATP levels in WT VSMCs after 16-h treatment in growth media with PDGF or growth media supplemented with PDGF and Nocodazole (1 µM). Statistical analyses were performed by one-way ANOVA (A, B) or Mann-Whitney test (C, F, H).

Loss of Miro1 impairs mitochondrial metabolic activity and is associated with decreased proliferative capacity.

(A) Intracellular ATP levels in WT and Miro1-/- VSMCs after treatment with PDGF (20 ng/ml for 16 h) or control (no PDGF), normalized to levels in control WT VSMCs (no PDGF). (B, C) ATP/ADP (B) and ATP/AMP (C) ratios assessed in WT and Miro1-/- VSMCs.(D) Intracellular ATP levels in WT and Miro1-/- VSMCs transduced with adenovirus expressing MIRO1-WT (Ad WT) or MIRO1-KK (Ad KK) or control adenovirus (empty vector, Ad-EV) after treatment with PDGF (20 ng/ml for 16 h) or control (no PDGF), normalized to ATP levels in control WT VSMCs (no PDGF). (E) Immunoblot of lysates from WT and Miro1-/- VSMCs for markers of the metabolic state (phosphorylated (p-Thr172) and total AMPKα), and cell cycle progression (p53, p21 and cyclin D1) at 48 h after incubation in serum-free medium (cell-cycle arrest). (F-J). Quantification of immunoblot signal in samples shown in (E) for (F) phosphorylated (p-Thr172) AMPKα normalized to actin, (G) phosphorylated (p-Thr172) AMPKα normalized to AMPKα, (H) p53, (I) p21, and (J) cyclin D1, all normalized to actin. (K) Oxygen consumption rate (OCR), as determined by Seahorse, for WT and Miro1-/- VSMCs with and without PDGF treatment (20 ng/ml for 16 h). (L) Quantification of basal OCR for WT and Miro1-/- VSMCs treated with PDGF (n=5). (M) Extracellular acidification rate (ECAR), as determined by Seahorse, for WT and Miro1-/-VSMCs with and without PDGF treatment (20 ng/ml for 16 h). Statistical analyses were performed by Friedman test (A, D), and Mann-Whitney (B, C, F-J, L).

Loss of Miro1 leads to reduced ETC activity under growth conditions.

(A) Transmission electron microscopy images of WT and Miro1-/- VSMCs. (B, C) Quantification of (B) the number of mitochondrial cristae and (C) the volume density of the mitochondrial cristae in WT and Miro1-/- VSMCs. (D) Levels of c-Myc tagged MIRO1-WT (Ad WT), c-Myc tagged MIRO1-KK (Ad KK), or MIRO1-ΔTM (Ad ΔTM) and proteins of the MIB/MICOS complex in HEK cells following pull-down assay, as determined by immunoblotting. C-Myc tagged Miro1 constructs were expressed in HEK cells for 72 h before cell lysis and pull-down were performed. (E-H) Quantification of (E) MIC60, (F) MIC19, (G) SAM 50, and (H) NDUFA9 from (D), adjusted for immunoprecipitated c-Myc-tagged MIRO1 as in (D). (I) Levels of mitochondrial supercomplex and ETC subunits in WT and Miro1-/- VSMCs, as determined by blue-native poly-acrylamide gel electrophoresis (BN-PAGE). (J) Quantification of supercomplex 2 in (I). (K) Quantification of activity of ETC complex 1 in WT and Miro1-/- VSMCs, as determined by the decrease in the rate of absorbance at 340 nm with and without rotenone incubation for 10 min. (L) Quantification of activity of ETC complex 1, plotted as the difference between absorbance curve slopes with and without rotenone (as in K). Statistical analyses were performed by unpaired t-test (B, C), Kruskal-Wallis (E-H), Mann-Whitney (J, L).

MIRO1 knockdown in human coronary artery smooth muscle cells inhibits mitochondrial motility, mitochondrial bioenergetics, and proliferation.

(A) Immunoblot for MIRO1 and COX IV in mitochondrial fractions of lysates from human coronary artery smooth muscle cells transfected with siControl or siMiro1 for 72 h. (B) Quantification of immunoblot signal in samples as shown in (A). (C) Number of human coronary artery smooth muscle cells transfected with siControl or siMiro1 at 72 h after incubation with growth media in addition to PDGF (20 ng/ml) or control. (D) Confocal images of human coronary artery smooth muscle cells grown on CYTOOchipsTM. Cells were transfected with siControl or siMiro1 for 72 h before being seeded onto CYTOOchipsTM. The cell cycle was synchronized by serum starvation for 24 h (not shown); the cells were subsequently treated with medium containing 10% FCS and PDGF (20 ng/ml) for 6 h. (E) Mitochondrial probability map. The cumulative distribution of mitochondria was assessed for images as in (D) by modified Sholl’s analysis. (F) Mito95 values, defined as the distance from the center of the CYTOOchipsTM at which 95% of the mitochondrial signal is found, under the conditions used in (D). (G) ATP levels assessed by luminescence assay in human coronary artery smooth muscle cells transfected with siControl or siMiro1 at 72 h. (H) Images of human coronary artery smooth muscle cells transfected with siControl or siMiro1 at 72 h after transduction with an adenovirus expressing a cytoplasmic ATP sensor with red reference protein (cyto-Ruby3-iATPSnFR1.0) for 24 h. (I) Quantification of GFP/RFP ratios, indicating cytosolic ATP levels in cells shown as in (G). (J) Quantification of activity of ETC complex 1 in human coronary artery smooth muscle cells following transfection with siControl or siMiro1 as determined by the decrease in the rate of absorbance at 340 nm with and without rotenone incubation for 10 min. (K) Quantification of activity of ETC complex 1, plotted as the difference between absorbance curve slopes with and without rotenone (as in I). Statistical analyses were performed by Mann-Whitney (B, F, H, J) and Kruskal-Wallis (C).

MIRO1 reducer inhibits VSMC proliferation.

(A) Immunoblots for MIRO1, ATP5α and COX IV in lysates of VSMCs treated for 72 h with MIRO1 reducer (MR, concentrations as indicated). Ponceau-stained membrane as loading control. (B) Representative confocal images of wild type VSMCs treated with DMSO (control) or MIRO1 reducer (MR, 10 μM) for 48 h. (mtGFP, green; phalloidin, red). (C) Quantification of mtGFP fluorescence in experiments like those shown in (B) (n=4). (D) Cell counts of wild type VSMCs treated with DMSO or MIRO1 reducer (10 μM), following a 72-h incubation in medium containing 10% FBS with or without PDGF (20 ng/ml). (E) Cell counts of wild type VSMCs treated with DMSO or MIRO1 reducer (10 and 20 μM), (n=3). (F) ATP levels in wild type VSMCs treated with DMSO or MIRO1 reducer (10 μM), following 72-h incubation in medium containing 10% FBS with or without PDGF (20 ng/ml). Analysis by Mann-Whitney (C), one-way ANOVA (D, F), and Kruskal-Wallis test (E).

MIRO1 expression in a transgenic model of Miro1 deletion in VSMCs (SM-Miro1-/-).

(A) Representative images of MIRO1 expression in sections of the mouse carotid artery, as determined by immunofluorescence. MIRO1 green, SM-actin red, DAPI blue. Scale bar = 20 µm. (B) Levels of the Miro1 mRNA in the murine SM-Miro1-/- aorta vs wild type counterparts, as determined by quantitative real-time PCR. Analysis by Wilcoxon test.

MIRO1 expression in smooth muscle cells in the human neointima.

Immunofluorescence of MIRO1 in coronary arteries (left anterior descending artery, LAD; right coronary artery, RCA) of heart transplant recipients with a diagnosis of coronary artery disease. MIRO1, green; phalloidin, red; TOPRO3, blue. Scale bar: 80 µm in left images; 20 µm in right images.

Ki67 colocalizes with MIRO1 in the human neointima.

Immunofluorescence of MIRO1 and Ki67 in coronary arteries of heart transplant recipients with non-ischemic cardiomyopathy (left) and coronary artery disease (right). MIRO1, green; phalloidin, red; Ki67, blue. Same magnifications as in Figure 1-Figure supplement 2 were used.

Deletion of MIRO1 inhibits VSMC proliferation.

(A) Cell counts of aortic VSMCs explanted from wild type and SM-Miro1-/- mice, following a 72-h incubation in medium containing 10% FBS with or without PDGF (20 ng/ml). (B) Immunoblot of MIRO1 expression in mitochondrial fractions of Miro1fl/fl VSMCs transduced with adenovirus expressing cre recombinase (Ad CRE, denoted as Miro1-/-VSMCs) or empty vector control adenovirus (AD EV, denoted as WT VSMCs). ATP5A and VDAC1 serve as loading controls. (C, D) Quantification of experiments shown in (B). Analysis by unpaired t-test (C, D).

Deletion of MIRO1 alters mitochondrial dynamics without altering mitochondrial DNA copy number.

(A) Confocal microscopy images of WT and MIRO1-/- VSMCs and quantification of mitochondrial morphology as a form factor at 0, 24, and 48 h after release from growth arrest for 48 h in FBS-free media. (B) Quantitative polymerase chain reaction for genes encoded by mitochondrial DNA normalized to NDUFV. Statistical analyses were performed by Kruskal-Wallis (A), and Mann-Whitney (B).

Nocodazole treatment does not impair cellular bioenergetics.

(A, B) Oxygen consumption rate (OCR), as determined by Seahorse, for WT, Miro1-/-, and Miro1-/- VSMCs transduced with adenovirus expressing MIRO1-WT (Ad WT), MIRO1-KK (Ad KK), or MIRO1-ΔTM (Ad ΔTM) VSMCs without (A) and with (B) Nocodazole treatment (1 µM for 16 h). (C, D) Extracellular acidification rate (ECAR), as determined by Seahorse, for WT, Miro1-/-, and Miro1-/- VSMCs transduced with AdWT, AdKK, or Ad ΔTM without (C) and with (D) Nocodazole treatment (1 µM for 16 h).

Miro1 deletion does not affect levels of subunits of MIB/MICOS or ETC complexes.

(A) Immunoblot of MIB/MICOS proteins in whole cell lysates of wild type and Miro1-/- VSMCs. (B) Immunoblot of subunits of ETC complexes in mitochondrial fractions of wild type and Miro1-/-VSMCs. (C-G) Quantification of (C) ATP5A, (D) UQCRC2, (E) MTCO1, (F) MTCO2, and (G) MIRO1 normalized to VDAC1 levels. (n=12 independently isolated samples per genotype). (H-J) Complex V in gel-activity assay in mitochondrial samples from wild type and Miro1-/-VSMC (H), normalized to ATP5F1 expression levels (I). Miro1-/- knockout validation (J). (K) Quantification of Complex V activity as in (H). Statistical analyses were performed by unpaired t-test (C-G, K).

Complex I activity is regulated by the wild type form of MIRO1.

(A-D) Quantification of activity of ETC complex 1 in WT and Miro1-/- VSMCs (A), WT, Miro1-/-, and Miro1-/- VSMCs transduced with adenovirus expressing with MIRO1-WT (Ad WT) (B), MIRO1-KK (Ad KK) (C), or MIRO1-ΔTM (Ad ΔTM) (D) as determined by the decrease in the rate of absorbance at 340 nm. (E) Quantification of activity of ETC complex 1, plotted as the difference between absorbance curve slopes (as in A-D). (F) Quantification of activity of ETC complex 1 in WT and Miro1-/- VSMCs, WT, Miro1-/-, and Miro1-/- VSMCs infected with Ad WT, Ad KK or Ad ΔTM with rotenone. Statistical analyses were performed by Kruskal-Wallis (E).