Mesaconine alleviates doxorubicin-triggered cardiotoxicity and heart failure by activating PINK1-dependent cardiac mitophagy

Figures

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  FIGURE 1. Mesaconine attenuated DOX-induced cardiomyocytes injury. (A) Chemical structure of mesaconine. (B) The cell viability of DOX and/or mesaconine-treated cardiomyocytes were detected by CCK-8 assay. (C) Representative images of cardiomyocytes stained with trypan blue (n = 5). (D) Quantitative analysis of trypan blue positive cardiomyocytes. (E, F) Treatment mesaconine showed resistance to DOX-induced LDH release (E) and reduced ATP production (F). (G) Representative images of MitoSOX (mitochondrial ROS) levels were measured by immunofluorescence staining. (H) Quantification of MitoSOX levels. Data are represented as means ± SEM of at least 3 biologically independent replicates assays. *, # p < 0.05; **, ## p < 0.01.

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  FIGURE 2. Mesaconine protected mice from acute DIC by reducing cardiac dysfunction and remodeling. (A) Flowchart for preparing acute DIC model and corresponding treatments. (B, C) The dose-dependent serum activity of LDH and CK. (D) Representative images of left ventricular M-mode echocardiograms (n = 6–8/group). Left ventricular ejection fraction [EF, (E)], fractional shortening [FS, (F)], and diastolic anterior wall thickness [LVAWd, (G)], posterior wall thickness [LVPWd, (H)] were measured by echocardiography. (I) RT-PCR detected mRNA expression of ANP and BNP. (J) Representative images of Sirius red stained heart sections and a summary of collagen deposition. Data are represented as means ± SEM of at least 3 biologically independent replicates assays. * p < 0.05; **, ## p < 0.01.

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  FIGURE 3. Transcriptome profiles of the myocardium and protective effects of mesaconine on mitochondrial damage. (A) Volcano plot of the gene expression values in DOX versus normal hearts (left panel) or DOX + mesaconine versus DOX hearts (right panel). Red, gene expression upregulated; green, gene expression downregulated in myocardial tissues. (B) Gene Set Enrichment Analysis (GSEA) plots of the mitochondrion-related gene modules in the myocardium of mice following the treatment of DOX or DOX and mesaconine. NES, normalized enrichment score; FDR q, false discovery rate q value. (C) Representative TEM images of mitochondria (upper panel) and autophagosomes (lower panel) in heart tissues. (D) The numbers of mitochondrial contents were determined by quantitative analysis. (E) The ATP content in myocardial tissues. Data are represented as means ± SEM of 3 biologically independent replicates assays. *, # p < 0.05.

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  FIGURE 4. Mesaconine alleviated DOX-induced mitochondrial damage through activating mitophagy. (A) Levels of SQSTM1, LC3-II/LC3-I and insoluble SQSTM1 (Inso-SQSTM1) in heart tissues were detected using western blotting (n ≥ 3). (B) Cardiomyocytes expressing RFP-GFP-LC3 were treated with DOX and/or mesaconine. Autophagic flux was detected with live-cell imaging microscopy. Representative images showing red-colored autolysosomes or red/green double-colored autophagosomes. (C) Levels of PINK1, SQSTM1, and LC3-II/LC3-I in isolated mitochondria form the cardiomyocytes of DOX and/or mesaconine treated mice. (D, E) Primary cardiomyocytes were immunostained with an anti-COX-IV Ab and an anti-LC3 Ab to detect the colocalization of autophagosomes with mitochondria. (F) Mesaconine-induced restoring of autophagic flux could be turnovered by CQ treatment in mitochondria isolated from the cardiomyocytes of DOX-treated mice. Representative immunoblots and the ratio of the indicated protein to TOM20 are presented. Data are represented as means ± SEM of 3 biologically independent replicates assays. *, # p < 0.05; **, ##, ^^ p < 0.01.

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  FIGURE 5. PINK1 or SQSTM1 deficiency restricted the cardioprotective effects of mesaconine by inhibiting mitophagy. (A) Immunoblotting analysis for the expressions of PINK1, SQSTM1 and LC3II/I in mitochondria isolated primary cardiomyocytes. Representative immunoblots and the ratio of the indicated protein to TOM20 are presented. (B) Cardiomyocytes expressing Mito-Keima were used to visualize mitophagy following the treatment of DOX or/and mesaconine under a confocal microscope. (C) Representative trypan blue staining images of primary cardiomyocytes. Quantitative analysis of (D) trypan blue staining positive cardiomyocytes, (E) LDH release, and (F) ATP contents of primary cardiomyocytes. (G) Representative images of mitochondrial ROS are indicated by MitoSOX red which can be oxidized by superoxide in mitochondria and then produce red fluorescence. (H) Quantification of MitoSOX levels. (I–K) Trypan blue staining images (I) LDH release (J) and ATP contents (K) of primary cardiomyocytes were assayed and showed that depletion of SQSTM1 reversed the cardioprotective effects of mesaconine. Data are represented as means ± SEM of at least 3 biologically independent replicates assays. *, ^ p <…

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  FIGURE 6. Mesaconine treatment improved left ventricular dysfunction and abnormal myocardial remodeling in DOX-induced chronic heart failure mice. (A) The schematic diagram for preparing the heart failure model and corresponding treatments. (B) Survival rate curves were analyzed by the Kaplan-Meier method and compared by the log-lank test. (C) The TUNEL assay and quantitative analysis in the myocardium. (D) Representative images of left ventricular (LV) M-mode echocardiograms. (E–H) The LV ejection fraction (E), fractional shortening (F), diastolic anterior wall thickness (LVAWd, (G) and posterior wall thickness (LVPWd, (H) were measured to evaluate the cardiac systolic function and structure. (I, J) Representative images of H&E (I) and Sirius Red (J) staining of the heart sections. (K, L) Quantitative analysis of myocardial histopathological scores (K) and collagen deposition (L). (M, N) The serum activity of LDH (M) and CK (N). (O) Levels of PINK1 in myocardial tissues were analyzed by immunostaining. (P) Colocalization of LC3 and COX-IV was examined by confocal analysis. Data are represented as means ± SEM (n≥7). *, # p < 0.05.

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  FIGURE 7. The schematic diagram illustrates the role of mesaconine in restoring energy metabolism disorder in the myocardium through activating PINK1-SQSTM1-mediated mitophagy, which makes mesaconine an attractive therapeutic agent for the treatment of heart failure and other diseases of myocardial energy metabolism.