Over-expression of microRNA-1 causes arrhythmia by disturbing intracellular trafficking system

Figures

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  Figure 1. Heatmap of hierarchical clustering of differentially expressed targets of miR-1 in miR-1 Tg mice (TG) and wild type mice (WT). Red denotes high expression levels, whereas green depicts low expression levels.

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  Figure 2. GO biological process terms with significant enrichment of differentially expressed miR-1 targets.

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  Figure 3. Network of miR-1 regulated trafficking-related genes. Round rectangle depicts miR-1 and octagon depicts targets of miR-1. The targets which were significantly down-regulated in the mice with over-expression of miR-1 (FDR < 0.05) were marked with green color and the targets with FDR < 0.01 were marked with dark green.

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  Figure 4. The dysregulation of trafficking-related genes in miR-1 Tg mice. (A) The expression of miR-1 in miR-1 Tg mice. qRT-PCR detection (B) and Western blot analysis (C) for trafficking-related genes in WT and miR-1 Tg mice. n = 6, **p < 0.01 vs.WT.

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  Figure 5. Stx6 was down-regulated in CMs subjected to ischemic or hypoxia stress. (A) Western blot detected Stx6 protein level in the hearts of mice under ischemic infarction for 12 hours. MI: myocardial infarction. (B) The mRNA expression level of Stx6 was down-regulated in the heart of mice under ischemic stress. Western blot (C) and qRT-PCR (D) were applied to detect the expression of Stx6 in the neonatal mice CMs which subjected to 12 hours of hypoxia with 1% O2. n = 6, *p < 0.05, **p < 0.01 vs. Sham or Control.

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  Figure 6. Stx6 is one of the direct targets of miR-1. (A) Conservation of the Stx6 3′UTR binding site for miR-1 among different species and the mutated binding sites of the Stx6 3′UTR. (B) Luciferase activity assay was used to measured the directly regulation of miR-1 on Stx6. Western blot (C) and qRT-PCR (D) were adopted to examine the effect of miR-1 on the protein and mRNA levels of Stx6. n = 6, *p < 0.05 vs.NC; #p < 0.05 vs.miR-1.

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  Figure 7. Stx6 participated in the regulation of intracellular Ca2+ in cultured CMs. (A) Western blot analysis for the protein levels of PLM and Cav1.2 in myocardial infarcted mice heart tissues. (B) The neonatal mice CMs were subjected to 12 hours of hypoxia with 1% O2, and western blot analyzed the protein level of PLM and Cav1.2. (C) Western blot analysis for Stx6 protein level at 48 hours after transfection with siRNA. (D) qRT-PCR detection for Stx6 mRNA level at 36 hours after transfection with siRNA. (E) Western blot analysis of the protein levels of Cav1.2 and PLM in Stx6-deficient neonatal mice CMs. (F) Resting [Ca2+]i in empty plasmid and Stx6 knockdown neonatal mice CMs. (G) The linear graph showed confocal analysis for SR Ca2+ content changes induced by KCl (10 μM) in scramble and Stx6 knockdown neonatal mice CMs, F/F0 values represent Ca2+ transient. n = 6–9. *p < 0.05, **p < 0.01vs. MI or Control or si-Scr.

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  Figure 8. Over-expression of miR-1 broke the balance of Ca2+, and induced cardiac arrhythmia. Neonatal mice CMs were transfected with Stx6 during overexpression of miR-1 (A) or hypoxia (B), and western blot was applied to analyze the protein levels of PLM and Cav1.2. (C) Immunofluorescent localization of Stx6 and PLM in neonatal mice CMs. Green represent staining of Stx6, red represent staining of PLM, blue represent staining of nuclei. (D) Western blot analysis for the protein levels of PLM and Cav1.2 in WT and miR-1 Tg mice. (E) Schematic depiction of the results and proposed hypothesis. n = 4−6. **p < 0.01 vs.Control, WT or NC; #p < 0.05 vs.miR-1 or Hypoxia.