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. 2010 Sep 14;122(11 Suppl):S124-31.
doi: 10.1161/CIRCULATIONAHA.109.928424.

MicroRNA-210 as a novel therapy for treatment of ischemic heart disease

Shijun Hu  1 , Mei Huang, Zongjin Li, Fangjun Jia, Zhumur Ghosh, Maarten A Lijkwan, Pasquale Fasanaro, Ning Sun, Xi Wang, Fabio Martelli, Robert C Robbins, Joseph C Wu
Affiliations

MicroRNA-210 as a novel therapy for treatment of ischemic heart disease

Shijun Hu et al. Circulation. .

Abstract

Background: MicroRNAs are involved in various critical functions, including the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. We hypothesize that microRNA-210 can rescue cardiac function after myocardial infarction by upregulation of angiogenesis and inhibition of cellular apoptosis in the heart.

Methods and results: Using microRNA microarrays, we first showed that microRNA-210 was highly expressed in live mouse HL-1 cardiomyocytes compared with apoptotic cells after 48 hours of hypoxia exposure. We confirmed by polymerase chain reaction that microRNA-210 was robustly induced in these cells. Gain-of-function and loss-of-function approaches were used to investigate microRNA-210 therapeutic potential in vitro. After transduction, microRNA-210 can upregulate several angiogenic factors, inhibit caspase activity, and prevent cell apoptosis compared with control. Afterward, adult FVB mice underwent intramyocardial injections with minicircle vector carrying microRNA-210 precursor, minicircle carrying microRNA-scramble, or sham surgery. At 8 weeks, echocardiography showed a significant improvement of left ventricular fractional shortening in the minicircle vector carrying microRNA-210 precursor group compared with the minicircle carrying microRNA-scramble control. Histological analysis confirmed decreased cellular apoptosis and increased neovascularization. Finally, 2 potential targets of microRNA-210, Efna3 and Ptp1b, involved in angiogenesis and apoptosis were confirmed through additional experimental validation.

Conclusions: MicroRNA-210 can improve angiogenesis, inhibit apoptosis, and improve cardiac function in a murine model of myocardial infarction. It represents a potential novel therapeutic approach for treatment of ischemic heart disease.

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Conflict of interest statement

Conflict of Interest Disclosures: None

Figures

Figure 1
Figure 1
(A) Schematic highlighting the microRNA microarray experimental design. HL-1 cells were subjected to hypoxia for 48 hours. Following FACS, apoptotic cells and live cells were collected for miRNA microarray analysis. T-test analysis demonstrates statistically significant differential miRNA expression across the two samples. miRNAs with P<0.05 were selected for cluster analysis. (B) Quantitative RT-PCR showed miR-210 expression was 5.3±0.9 fold higher in live cells than in apoptotic cells. Welch’s t-test was used. (C) Quantitative RT-PCR showed miR-1187 expression was 8.4±0.9 folds higher in apoptotic cells compared to in live cells. Welch’s t-test was used. (D) Time course regulation of miR-210 by hypoxia in HL-1 cells. Induction of miR-210 was discernible at 12 hours, becoming significant at 24 hours and increased progressively at 48 and 72 hour time points. 1-way ANOVA was used. *P<0.01 and **P<0.05.
Figure 2
Figure 2
In vitro characterization of therapeutic potential for miR-210. (A) Angiogenesis antibody array indicated miR-210 can release several angiogenic factors in HL-1 cells. Welch’s t-test was used for statistical analysis. (B) Caspase 3/7 activity assay demonstrated that miR-210 overexpression could inhibit caspase activity whereas inhibition of miR-210 with anti-210 abrogated the favorable effect. Student’s t-test was used. (C) FACS analysis confirmed that miR-210 transduced group had more live cells (71.95±1.69% vs 63.39±0.95%; P<0.05) and less apoptotic cells (22.13±0.48% vs 32.14±1.52%; P<0.05). Student’s t-test was used. *P<0.01 and **P<0.05.
Figure 3
Figure 3
Transfection efficiency of minicircle in vitro and in vivo. (A) HL-1 cells were transfected with MC-Fluc-eGFP (MC-LG) in 6-well plate. (B) A robust correlation exists between minicircle dosage and bioluminescence signals (r2=0.96). Each data point is from an individual observation. Pearson’s correlation was used. (C) Bioluminescence imaging and (D) quantitative analysis indicate minicircle plasmid-mediated gene expression was stable for at least 8 weeks in the heart compared to <4 weeks using regular plasmid (data not shown).
Figure 4
Figure 4
Evaluation of cardiac function following MI after miR-210 treatment. (A) Representative echocardiogram of mice with LAD ligation after injection of MC-210, MC-Scr, or sham group at week 8. (B) Quantitative analysis of left ventricular fractional shortening (FS) among the 3 groups. Compared to MC-Scr control, animals injected with MC-210 had significant improvements in FS values at both week 4 and week 8. 2-way ANOVA was used for statistical analysis. (C) Representative Masson trichrome staining of explanted heart at week 8 showed increased wall thickness for the MC-210 group, confirming the positive functional imaging data seen in echocardiography. (D) TUNEL staining of explanted heart demonstrated significantly reduced apoptotic cells in MC-210 group compared to MC-Scr control group. (E) Immunofluorescence staining of CD31 endothelial marker (green) demonstrated increased neovascularization in the myocardium after MC-210 delivery compared to MC-Scr control. Cardiomyocyte staining is identified by α-sarcomeric actin (red) and nuclear staining is identified by DAPI (blue).
Figure 5
Figure 5
Confirmation of the target gene of miR-210. (A) The binding segments of mouse Efna3, Ptp1b, Dapk1, and Ctgf interacting with miR-210 was amplified and inserted downstream of firefly luciferase reporter gene in the pGL3 control vector for dual-luciferase assay (see Supplemental Figure 3). pRL-TK containing renilla luciferase was co-transfected for data normalization. Precursor miR-210 mimic (Pre-210) significantly reduced the luciferase activities of the wild-type Efna3, Ptp1b, Dapk1, and Ctgf reporters between 35%–60% compared to the Pre-miR scramble control (Pre-Scr). However, mutant reporters (Pre-210 + Mut) with non-complementary seed binding site were not repressed by miR-210 precursor as expected. The blank vector (PGL3-control) has no seed binding site and therefore the firefly luciferase activity was not affected by miR-210 precursor mimic. 1-way ANOVA was used. (B) miR-210 targets were enriched in miR-210 containing RISC. Compared to cells transfected with a scramble sequence, immune-precipitates of the miR-210 loaded RISC highly enriched its targets, including Efna3, Ptp1b, Dapk1, and Ctgf. Student’s t-test was used. *P<0.05 and **P<0.01.
Figure 6
Figure 6
Endogenous regulation of Efna3 and Ptp1b by miR-210. (A) Quantitative RT-PCR indicate miR-210 can inhibit Efna3 but not Ptp1b at the RNA level. Student’s t-test was used. (B) However, Western blotting showed Ptp1b can be inhibited at the protein level instead. Student’s t-test was used. (C) Immunofluorescence confirmed miR-210 can strongly diminish Efna3 and Ptp1b expression in HL-1 cardiomyocytes. (D) Western blot data show that Efnas and Ptp1b from the peri-infarct regions of explanted hearts are significantly lower in the MC-210 group compared to MC-Scr group. Student’s t-test was used. *P<0.05.
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