Fig 2: Mdivi-1 restored the vulnerability of MITOL-KO hearts(A) Study design for determining cardiac stress resistance in MITOL-cKO mice. Mice were intraperitoneally (i.p.) administered five consecutive days of treatment with tamoxifen (30 mg/kg) once daily. One month after administration of tamoxifen, mice were administered with isoproterenol (60 mg/kg) via subcutaneous infusion from implanted osmotic pumps. Seven days after infusion of isoproterenol, mice were then assessed for cardiac function. For Drp1 inhibition, mice were i.p. administered seven consecutive days of treatment with Mdivi-1 (50 mg/kg) once daily.(B and C) Myocardial fibrosis in MITOL-cKO mice under isoproterenol (ISO) stress was attenuated by Mdivi-1. Representative photographs show Masson’s trichrome staining for collagen (B). Bar, 200 µm. The fibrotic areas were measured (C) Mean ± SEM (n = 3). Analysis was performed with two-way ANOVA followed by Bonferroni post hoc analysis. ***p < 0.001..(D and E) Cardiomyocyte hypertrophy in MITOL-cKO mice under isoproterenol stress was attenuated by Mdivi-1. Frozen heart tissue sections were also stained with FITC-conjugated wheat germ agglutinin (WGA) to detect cardiomyocyte borders. The areas of cardiomyocytes were measured from 100 cells. Mean ±SEM (n = 3) (D). Heart-to-body and weight ratio in control and MITOL-cKO mice. Mean ±SEM (n = 3) (E). *p < 0.05, ***p < 0.001.(F) Mdivi-1 attenuates Drp1 accumulation in MITOL-cKO mouse cardiomyocytes. Lysates from total heart cells of control mice, and MITOL-cKO mice treated with or without Mdivi-1 under isoproterenol (ISO) stress were subjected to immunoblotting with respective antibodies. Cardiac mitochondrial fractions of MITOL-cKO mice treated with or without Mdivi-1 (described in A) were subjected to immunoblotting with respective antibodies.(G) Decreased size of mitochondria in MITOL-cKO was recovered by Mdivi-1 administration. Cardiac mitochondrial areas of control mice, and MITOL-cKO mice treated with or without Mdivi-1 (described in A) were measured. Mitochondrial fractions isolated from cardiomyocytes were stained with MitoTracker, followed by flow cytometric analysis. Mean ±SEM (n = 3). Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. ***p < 0.001.(H) Attenuation of mitochondrial ROS in MITOL-cKO by Mdivi-1 administration. Cardiac mitochondrial fractions of control mice, and MITOL-cKO mice treated with or without Mdivi-1 (described in A) were stained with MitoSOX and mitochondrial-derived superoxide generation was measured by flow cytometric analysis. Bar graphs show the mean fluorescence intensity of MitoSOX. Mean ±SEM (n = 4). Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. ***p < 0.001..(I and J) Increased senescent cardiomyocytes in MITOL-cKO were recovered by Mdivi-1 administration. Senescent cells were detected by SA-ß-Gal staining in the heart for the indicated periods after MITOL deletion by tamoxifen (I). Bar, 200 µm. Bar graph shows the percentage of SA-ß-Gal positive cells in the heart sections (J). Mean ±SEM (n = 4). **p < 0.01.

Fig 3: Downregulation of MITOL in infarct cardiomyocytes and transduction of AAV-MITOL ameliorates cardiac fibrosis under myocardial infarction(A and B) Downregulation of MITOL in infarct rat cardiomyocytes. MI was induced by ligation of the left coronary artery in rats. After two weeks of MI, IB assay of MITOL and Drp1 was performed on lysates of whole and mitochondrial fractions isolated from rat hearts. Anti-VDAC and anti-tubulin antibodies were used as a mitochondrial marker and a cytosolic marker, respectively. Data are standardized to VDAC levels and are expressed relative to sham operated rats. Mean ±SEM (n = 4). ***p < 0.001, Student’s t-test.(C) MITOL decreased in infarct cardiomyocytes of humans. Lysates from total heart cells of normal and infarct left ventricle were immunoblotted with anti-MITOL, anti-Drp1 and anti-tubulin antibodies. Age: 67–70.(D) Study design for the transduction of MITOL in MI-induced cardiomyocytes. Two days after the induction of MI, rats were introduced with AAV vectors (GFP: AAV-GFP and GFP-P2A-MITOL: AAV-MITOL) packaged in AAV-9 capsids via intrathoracic injection. Two weeks after virus injection, hearts were dissected and the expression of GFP and MITOL was assessed by immunoblotting.(E and F) MITOL was upregulated in cardiomyocytes of rats transduced with AAV-MITOL. IB assay of MITOL, GFP and tubulin was performed on lysates of total rat heart cells (E). The relative protein levels of MITOL and GFP were quantified by densitometry (F). Data are standardized to tubulin levels and are expressed relative to AAV-GFP injected rats. Mean ± SEM (n = 3). **p < 0.01.(G) MI-induced MITOL downregulation was rescued by AAV-MITOL. After MI or sham operation, rats were injected with indicated viruses (as described in D). Two weeks after injection of viruses, IB assay of MITOL, Drp1 and VDAC were performed on lysates of whole and mitochondrial fractions of rat cardiomyocytes. Data are standardized to VDAC levels and are expressed relative to AAV-GFP injected rats.(H and I) Mitochondria damage induced by MI was rescued by AAV-MITOL. Representative electron microscopic images of mitochondria from the hearts of sham-operated, MI treated, and MI +AAV-MITOL-treated mice. 27,600-fold magnification (H). Bar, 500 nm. The distribution of mitochondrial size is shown (I).(J and K) Myocardial fibrosis was attenuated in MI-treated mice transduced with AAV-MITOL. Representative photographs show Masson’s trichrome staining for collagen(J). Mean ±SEM (n = 4) (K). **p < 0.01.(L) Cardiac malfunction was restored in MI-treated mice transduced with AAV-MITOL. Echocardiographic analysis of left ventricular dimensions and cardiac function in mice. FS, fractional shortening of left ventricular diameter. Mean ±SEM (n = 3). Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. *p < 0.05.(M) Gene expression of markers of cardiac function and hypertrophy. Relative mRNA abundance of Tumor Necrosis Factor a (TNF-a), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP; B), a-myosin heavy chain (a-MHC), ß-MHC, collagen-1 (Col-1), MHC-ß, a-smooth muscle actin (a-SMA), transforming growth factor ß (TGF-ß), and fibronectin (FN) in the myocardium of control mice and MITOL cKO mice treated with AAV-GFP or with AAV-MITOL tested by real-time qPCR. Data are presented as mean ± SEM. *p < 0.05 vs. control. Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(N) Gene expression of markers of cardiac aging. Relative mRNA abundance of P21 and Interleukin-1 (IL-1b) in the myocardium of control mice and MITOL cKO mice treated with AAV-GFP or with AAV-MITOL tested by real-time qPCR. Data are presented as mean ± SEM. *p < 0.05 vs. control. Analysis was performed with one-way ANOVA followed by Bonferroni post hoc analysis. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig 4: Mitochondrial morphological change by accumulated Drp1 and dysfunction in heart-specific MITOL-KO mice(A and B) Heart-specific MITOL KO enhances Drp1 accumulation in the heart. Samples were collected from the hearts of MITOLflox/flox (Control) and MITOLflox/flox;aMHC-CreMer (MITOL cKO) mice treated with tamoxifen for the indicated periods, followed by immunoblotting with indicated antibodies. The relative protein levels of Drp1 were quantified by densitometry. Data are standardized to tubulin levels and are expressed relative to control mice. Mean ±SEM (n = 3). Analysis was performed with two-way ANOVA followed by Bonferroni post hoc analysis. ***p < 0.001, **p < 0.01.(C and D) Abnormal morphologies of mitochondria in MITOL-KO mouse cardiomyocytes. Representative electron microscopic images of mitochondria from the hearts of MITOLflox/flox (Control) and MITOLflox/flox;aMHC-CreMer (MITOL cKO) mice treated with tamoxifen for the indicated periods. 27,600-fold magnification (C). Mitochondrial size was represented as median surface area, and frequency distributions of mitochondrial surface were calculated from mitochondria imaged by TEM (D). Kruskal-Wallis test, ****p < 0.0001. Data are median values. Bar, 500 nm.(E) Decreased size of mitochondria in MITOL-KO mouse cardiomyocytes. Cardiac mitochondrial areas of MITOLflox/flox (Control), and MITOLflox/flox;aMHC-CreMer (MITOL cKO) mice treated with tamoxifen for the indicated periods were measured. Mitochondrial fractions isolated from cardiomyocytes of MITOLflox/flox and MITOLflox/flox;aMHC-CreMer mice treated with tamoxifen for the indicated periods were stained with MitoTracker, followed by flow cytometric analysis. Bar graphs show the relative levels of mean fluorescence intensity of forward-scatter (FSC) and MitoTracker to MITOLflox/flox mice. Mean ±SEM (n = 3). ***p < 0.001.(F) Mitochondrial ROS was upregulated in MITOL-KO mouse cardiomyocytes. Cardiac mitochondrial fractions of MITOLflox/flox (Control), and MITOLflox/flox;aMHC-CreMer (MITOL cKO) mice treated with tamoxifen for the indicated periods were stained with MitoSOX and mitochondrial-derived superoxide generation was measured by flow cytometric analysis. Bar graph shows the relative levels of mean fluorescence intensity of MitoSOX to MITOLflox/flox mice. Mean ± SEM (n = 3). Analysis was performed with two-way ANOVA followed by Bonferroni post hoc analysis. **p < 0.01.(G and H) MITOL knockout induces mitochondrial oxidative damage in cardiomyocytes. Protein carbonyl contents of cardiac mitochondrial extracts were determined by protein carbonyls western blot detection kit (G). The levels of carbonylated proteins were quantified by densitometry. Data are standardized to VDAC levels and are expressed relative to MITOLflox/flox (Ctrl) mice prior to tamoxifen treatment. Mean ±SEM (n = 3). Analysis was performed with two-way ANOVA followed by Bonferroni post hoc analysis. *p < 0.05, ***p < 0.001. (H).(I) Reduced oxygen consumption rate of mitochondria isolated from MITOL-KO mouse cardiomyocytes. The respiratory control ratio (RCR) represents the mitochondrial coupling state. Mean ±SEM (n = 3). *p < 0.05, Student’s t-test.(J) Reduced ATP content in the MITOL-KO heart. ATP content was measured by luciferase assay. Mean ±SEM (n = 4). **p < 0.01, Student’s t-test.