Figure 6.
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 α (TNF-α), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP; B), α-myosin heavy chain (α-MHC), β-MHC, collagen-1 (Col-1), MHC-β, α-smooth muscle actin (α-SMA), transforming growth factor β (TGF-β), 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.