Frequency-dependent shortening of cardiomyocytes isolated from control-TAC and MITOL-cKO-TAC mice (J). gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI. analysis. ???p? ?0.001, ????p? ?0.0001. (B) MITOL ubiquitinates Drp1. MITOLF/F MEFs (control) or MITOL-KO MEFs were co-transfected with/without indicated expression vectors for FLAG-tagged Drp1, HA-tagged ubiquitin and non-tagged MITOL for 24?h and lysates were immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting with anti-HA antibody or anti-FLAG antibody. Cells were treated with MG132 (10?M) for 10?h before harvest. Whole lysates were immunoblotted with anti-MITOL and anti-tubulin antibodies. (C) Accumulation of Drp1 in mitochondrial fraction. IB assay was performed on lysates of whole (WCL), cytosolic (Cyt/Micro), and mitochondrial (mito) fractions isolated from MITOLF/F MEFs (control) or MITOL-KO (KO) MEFs with Drp1 antibody. Anti-VDAC and anti-tubulin antibodies were used as a mitochondrial marker and a cytosolic marker, respectively. Mitochondrial Drp1 was normalized by the intensity of VDAC. Error bars represent?SEM (n?= 3). ???p? ?0.001 (Students analysis. ????p? ?0.0001 (F). (G) Mdivi-1 attenuates mitochondrial ROS production in MITOL-KO MEFs. MITOLflox/flox MEFs (control) or MITOL-KO (KO) MEFs treated with or without 5?M Mdivi-1 for 12?h were stained with MitoSOX and mitochondria-derived superoxide generation was measured by flow cytometric analysis. Bar graphs show relative levels of mean fluorescence intensity of MitoSOX compared with that of MITOLflox/flox MEFs (control). Mean? SEM (n?= 3). Analysis was performed with one-way ANOVA followed by Bonferroni analysis. ?p? ?0.05, ??p? ?0.01. (H and I) Accumulation of senescent cells in MITOL knockout MEFs was restored by Mdivi-1 treatment. Cytochemical staining of SA–gal activity in MITOLflox/flox MEFs (control), or MITOL-KO MEFs treated with or without 5?M Mdivi-1 for six hours. Bar, 50?m (H). The bar graph shows the percentages of SA–gal positive cells (100 cells/experiment, n?= 3). Mean? SEM. Analysis was performed with one-way ANOVA followed by Bonferroni analysis. ????p? ?0.0001 (I). (J) MITOL-KO MEFs promote age-related hypertrophy. The areas of MITOLflox/flox MEFs (control) and MITOL-KO MEFs were measured. Percentages of cells showing each cell size were calculated from 100 SMARCB1 cells of each MEFs. Mean? SEM (n?= 3). ?p? ?0.05. (K) Mitochondrial fragmentation correlates ROCK inhibitor-2 with age-related hypertrophy in MITOL-KO MEFs. MITOL-KO MEFs were stained by anti-Tom20 and anti-Actin antibodies. Percentages of cells showing each mitochondrial morphology were calculated from 100 cells of each MEFs. Mean? SEM (n?= 4). ?p? ?0.05. To assess the toxicity of Drp1 in MITOL-KO MEFs, we used a Drp1 inhibitorMdivi-1or siRNA targeting Drp1 to complement the effect of Mdivi-1 (Cassidy-Stone et?al., 2008). We first confirmed that Mdivi-1 blocked the accumulation of Drp1 to mitochondria in MITOL-KO MEFs (Physique?1D). The introduction of siRNA targeting Drp1 suppressed the expression of endogenous Drp1 by 80% compared with that of scramble siRNA (Physique?S1A). Mitochondrial fragmentation was induced in MITOL-KO MEFs and restored by Mdivi-1 treatment (Figures?1E and 1F) or siRNA transfection (Figures?S1B and S1C). Mitochondrial fragmentation is usually often associated ROCK inhibitor-2 with enhanced reactive oxygen species (ROS) production (Kluge et?al., 2013; Shenouda et?al., 2011). In line with the accumulation of Drp1 to mitochondria, mitochondrial ROCK inhibitor-2 ROS production was enhanced in MITOL-KO MEFs and was restored by Mdivi-1 treatment (Physique?1G) or siRNA (Physique?S1D). These data suggest that ROS production in MITOL-KO MEFs was induced, at least in part, by Drp1 accumulation because of the OMMAD abnormalities. It is widely ROCK inhibitor-2 reported that intracellular ROS accelerates cellular senescence (Miquel et?al., 1980). We, therefore, monitored cellular senescence by -gal staining. -gal positive cells accumulated in MITOL-KO MEFs, whereas these cells decreased following Mdivi-1 treatment (Figures?1H and 1I) or siRNA targeting Drp1 (Figures?S1E and S1F). These data indicate that cellular senescence observed in MITOL-KO MEFs was.