Cardiomyocyte mitochondrial dynamic-related lncRNA 1 (CMDL-1) may serve as a potential therapeutic target in doxorubicin cardiotoxicity

Figures

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  Figure 1. Microarray profiling identified differentially expressed lncRNAs and mRNAs between doxorubicin (DOX)-exposed and control cardiomyocytes (A and B) Boxplot of all of the tested samples to quickly visualize the distribution for the lncRNA (A) and the mRNA (B) profiles. The samples in the dataset were normalized, and the medians of lncRNAs were close to 5 (A) and the medians of mRNA were close to 8 (B). (C and D) lncRNA and mRNA expression profiles between the untreated control and DOX-treated H9c2 cardiomyocytes. Hierarchical clustering analysis of gene expression levels between the two samples is shown. Cluster analysis indicated 2,190 lncRNAs (C) and 4,683 mRNAs (D). In the heatmap, red indicates upregulated expression and green indicates downregulated expression. (E and F) Scatterplots summarize lncRNA (E) and mRNA (F) expression variation between the two samples. The values of the x and y axes in the scatterplot are the averaged normalized signal values of the group (log2 scaled). The green lines represent fold change (the default fold change given is 2.0).

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  Figure 2. KEGG analyses indicate that DOX can induce dysregulation of MAPK signaling and hypertrophic pathways in cardiomyocytes (A and B) KEGG pathways analysis of differentially expressed lncRNAs. The top 10 enrichment scores (−log10[p value]) of the significant enrichment pathway of downregulated (A) and upregulated pathways (B) are shown. A p value denotes the enrichment p value of the pathway ID used the Fisher’s exact test. The enrichment score value of pathway ID is defined by (−log10[p value]). The lower the p value, the higher the enrichment score, and the more significant is the pathway enrichment.

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  Figure 3. CMDL-1 is the most significantly downregulated lncRNA in cardiomyocytes after DOX exposure (A and B) Top 10 differentially expressed lncRNAs analyzed by microarray analysis. Comparison of the consistently downregulated (A) and upregulated (B) lncRNAs in untreated control and DOX-treated samples (n = 3) is shown. (C and D) Quantitative real-time PCR was used to validate downregulated (C) and upregulated (D) lncRNAs. (E and F) Fold change expression levels of quantitative real-time PCR validated downregulated (E) and upregulated (F) lncRNAs. Data are expressed as the mean ± SEM. ∗p < 0.05 compared to control samples as calculated by a two-sided Wilcoxon signed-rank test.

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  Figure 4. Sequence structure of CMDL-1 (A) Full-length cDNA sequence of CMDL-1 (2,890 bp). (B) UCSC BLAST search program. CMDL-1 contained 39 exons and 38 introns, located at 3q36 and of 96% (query cover 49%) with transient receptor potential cation channel, subfamily M, member 7 (Trpm-7). (C) Secondary structure of CMDL-1 predicted with minimum free energy (MFE) and minimum total base-paired distance to all structures in thermodynamics ensemble (centroid) models using the RNAfold web server (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3&ID=UhzKa0PEie).

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  Figure 5. Overexpression of CMDL-1 prevents the DOX-induced mitochondrial fission and apoptosis in cardiomyocytes (A) Dynamics of CMDL-1 expression at indicated durations of DOX exposure. ∗p < 0.05, ∗∗p < 0.01 versus 0 h. (B) qPCR analysis of CMDL-1 expression. H9c2 cells were infected with CMDL-1 overexpressed virus and CMDL-1 expression was analyzed. (C and D) Overexpression of CMDL-1 ameliorates DOX-induced mitochondrial fission. Mitochondrial morphology (C) and quantification of mitochondrial fission (D) were analyzed after 6 h of DOX treatment. The disintegration levels of mitochondria (mitochondrial fission) in 250–300 cells were counted in 30–40 random fields using a confocal microscope. (E and F) Overexpression of CMDL-1 prevents DOX-induced apoptosis. (E) shows a representative flow cytometry plot, and (F) shows quantification of the dynamics of apoptosis in control (CT), DOX exposure, empty vector (EV) treated with DOX (DOX + EV), and CMDL-1 overexpression treated with DOX (DOX + CMDL-1) as analyzed by flow cytometry. Data presented are representative of at least three independent experiments. Data are expressed as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗p < 0.001, one-way ANOVA.

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  Figure 6. CMDL-1 regulates apoptosis via Drp1 (A and B) CMDL-1 is predicted to interact with Drp1. A positive interaction score indicates the predicted binding between CMDL-1 and Drp1 (A) and maps the CMDL-1 interaction domain to nucleotides 1500–2000 (B). RNA-protein interaction scores were generated using catRAPID software. (C) Drp1 and Trpm-7 expression levels. H9c2 cells were treated with DOX at the indicated time points, and cells were harvested for immunoblot analysis. (D) Immunoblot analysis of Drp1 expression level. Cells were transfected with siDrp1 to knock down Drp1 expression and with scrambled Drp1 (scDrp1) as CT for 24–72 h and harvested for immunoblot analysis (upper panel). Cells were infected with lentiviral overexpressing CMDL-1, and after 24 h they were harvested for immunoblot analysis (low panel). (E and F) CMDL-1 regulates mitochondrial fission via Drp1. Cells were transfected with EV, CMDL-1, and siDrp1 and exposed to DOX, and mitochondrial morphology was analyzed by confocal microscopy. (E) shows the quantification of mitochondrial fission. (F) shows representative mitochondrial morphology stained with MitoTracker red. ∗p < 0.05, ∗∗p < 0.01 versus DOX. Scale bars,…

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  Figure 7. In vivo analysis of CMDL-1 expression in DOX-induced cardiac injury (A) Schematic presentation of DOX-induced cardiac injury model. (B) Kaplan-Meier survival curves in CT and DOX-exposed rats. (C–G) The M-mode ultrasonic cardiography change of rat hearts detected by echocardiography in CT and test adult rats after 6 weeks of exposure of saline and DOX, respectively. The quantitative analyses of left ventricular internal diameter at end systole (LVESd; D), left ventricular internal diameter at end diastole (LVEDd; E), left ventricular ejection fraction (LVEF; F), and left ventricular fractional shortening (LVFS; G) are shown; n = 10 rats for CT and n = 16 rats for DOX. (H) TUNEL staining for apoptosis analysis. The left panel shows TUNEL staining, and the right panel shows quantification of apoptosis. (I) CMDL-1 expression between CT versus DOX-treated groups was analyzed by quantitative real-time PCR. Data presented are representative of at least three independent experiments. Data are expressed as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, two-tailed Student’s t test.