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. 2022 Jun 11;25(7):104582. doi: 10.1016/j.isci.2022.104582

Figure 5.

Figure 5

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-β-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-β-Gal positive cells in the heart sections (J). Mean ±SEM (n = 4). ∗∗p < 0.01.