MicroRNA-221 Is Cardioprotective and Anti-fibrotic in a Rat Model of Myocardial Infarction

Figures

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  Figure 1. miR-221 Protects H9c2 against H/R through the Inhibition of Apoptosis H9c2 cells were transfected with miR-221 mimic or control (miR-221 and MC) and subjected to 16-h hypoxia followed by 2-h reoxygenation (H/R). (A) Phase-contrast images of H9c2 cells at the end of H/R. Scale bar, 100 μm. Cell injury was measured by cell number count and lactate dehydrogenase (LDH) release. (B) Cell apoptosis was assessed by Annexin V and 7-AAD double staining followed by flow cytometry measurements. Representative apoptosis profiles are shown for each group. Results are expressed as the percentage of cells in each quadrant. (C) Mitochondrial membrane potential (MMP) was measured by MMP-activated fluorescence dye staining and flow cytometry analysis. Results are expressed as the percentage of depolarized cells. (D) Western blot of P53 phosphorylated at Serine 46 (P-P53 Ser46), total P53 (T-P53), and the loading control β-actin. (E) Putative miR-221-binding sites in the p53 3′ UTR and their mutations. (F) Luciferase reporter assay using co-transfection of the psicheck2 dual luciferase reporter plasmid carrying the specified 3′ UTR inserts and miR-221 mimics or mimic control. Sensor, a miR-221…

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  Figure 2. miR-221 Protects cFBs against H/R Injury through the Inhibition of Autophagy Adult rat cardiac fibroblasts (cFBs) were transfected with miR-221 mimic or control (miR-221 and MC) and subject to 24-h hypoxia followed by 2-h re-oxygenation (H/R). (A) Phase-contrast images of cFBs at the end of H/R. Scale bar, 100 μm. Cell injury was measured by cell number count and lactate dehydrogenase (LDH) release. (B) Cell apoptosis was assessed by Annexin V and 7-AAD double staining followed by flow cytometry measurements. Representative apoptosis profiles are shown for each group. Results are expressed as the percentage of cells in each quadrant. Western blot to detect (C) P53 phosphorylated at Serine 46 (P-P53 Ser46) and total P53 (T-P53); (D) P62, LC3-I, and LC3-II; and (E) TP53INP1 and DDIT4. The expressions were normalized to their loading control of β-actin.*p < 0.05, **p < 0.01, ***p < 0.001 versus MC. n = 3 in triplicates.

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  Figure 3. miR-221 Inhibits myoFB Activation and Collagen Synthesis Adult rat cFB was serum starved for 24 h, then transfected with miR-221 mimic or control (miR-221 and MC) and incubated for a specified period of time. (A) qRT-PCR and (B) western blot measurements of α-SMA mRNA and protein expressions. (C) Collagen gel contraction was measured as the percentage reduction of gel area over the culture surface after 48-h pre-stress and 48-h free contraction. (D) qRT-PCR measurement of mRNA expression of collagen I (Col1a1 and Col1a2), collagen III (Col3a), fibronectin (Fn1), and its extra domain A isoform (Fn-EDA). (E) Sircol collagen assay of cFB supernatants 7 days after mimic transfection. (F) qRT-PCR and (G) western blot measurement of Smad2 and Smad3 mRNA and protein expressions. Antibodies recognized phosphorylated (P-SMAD2/3, Serine 423/425) and total (T-SMAD2/3), respectively. *p < 0.05, **p < 0.01, ***p < 0.001 versus MC. n = 3 in triplicates.

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  Figure 4. miR-221 Reduces Apoptosis and Attenuates Autophagy at Day 2 Post-MI Rats with myocardial infarction (MI) received 1 mg/kg miR-221 mimic or PBS injection (miR-221 or MI +miR-221 and MI-Ctrl) right after MI. Hearts were harvested 48 h later, and they were dissected according to infarct, border, and remote areas. RNA and protein were extracted for analysis. (A) miRNA Taqman qPCR measurement of miR-221 levels in different regions. Western blot and densitometry for (B) blots, (ii) cleaved caspase-3, (iii) P53 phosphorylated at Serine 46 (P-P53 Ser46) and total P53 (T-P53); (C) P62 and LC3-I and LC3-II; and (D) TP53INP1 and DDIT4. β-actin was used as a loading control. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham; #p < 0.05, ##p < 0.01 versus MI-Ctrl. n = 5 each group.

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  Figure 5. miR-221 Reduces Post-infarct Fibrosis and Inhibits myoFB Activation at Days 2 and 7 Post-MI Rats with MI received 1 mg/kg miR-221 mimic or PBS injection right after coronary artery ligation and were harvested at day 2 post-MI, or they received another injection after 72 h and were harvested at day 7 post-MI (miR-221 or MI +miR-221 and MI-Ctrl). qRT-PCR measurements of (A) Col1a1 mRNA and (B, i) Smad3 mRNA; (B, ii and iii) western blot measurement of SMAD2/3 at day 2 post-MI. (C) miRNA Taqman qPCR measurement of miR-221 levels at day 7 post-MI. (D) Picro-sirius red staining to quantify tissue collagen content at day 7 post-MI. Representative images of heart cross-sections are shown for each group. Results are expressed as the percentage of positive staining over the area of LV. (E) Immunohistochemistry with anti-α-SMA (green) and DAPI (blue) nuclei staining at day 7 post-MI. Scale bar, 150 μm. Results are expressed as the percentage of α-SMA+ cells over the total number of nuclei. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham; #p < 0.05 versus MI-Ctrl. n = 4, 6, and 4 for sham, MI-Ctrl, and MI +miR-221, respectively.

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  Figure 6. miR-221 Attenuates LV Remodeling and Improves LV Function at Day 7 Post-MI Rats with MI received 1 mg/kg miR-221 mimic or PBS injections right after and 72 h post-MI. Hearts were harvested at day 7 post-MI for histological analysis. LV function was measured by echocardiography. (A) Masson trichrome staining of heart cross-sections. Representative images are shown for each group. Infarct area was measured as the percentage of blue + purple staining over the area of LV. (B) Ejection fraction (EF), LV internal dimension (LVID) at systole (s), and infarct and septum thickness at diastole (d) and systole (s), as measured by echocardiography. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham; #p < 0.05, ##p < 0.01 versus MI-Ctrl. n = 4, 6, and 4 for histology; n = 10, 15, and 11 for echocardiography, for sham, MI-Ctrl, and MI +miR-221, respectively.

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  Figure 7. miR-221 Attenuates LV Remodeling and Improves LV Function at Day 30 Post-MI Rats with MI received 1 mg/kg miR-221 mimic or PBS injections right after and 72 h post-MI. At day 30 post-MI, LV function was measured terminally by LV catheterization and heart tissues were harvested for histology. (A) Picro-sirius red staining of collagen. (i) Complete cross-sections of representative hearts. (ii) Quantification of collagen content over the area of LV. (iii) Representative images of infarct, border, and remote areas. Scale bar, 200 μm. (iv) Quantification of collagen content in the remote areas. (B) (i) Representative Labchart data acquisition: upper panel, right carotid artery blood pressure and LV developing pressure; lower panel, ±dP/dt. (ii) LVDP, left ventricle developing pressure; EDP, end diastolic pressure (EDP); ±dP/dt, maximum and minimum pressure changes. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham; #p < 0.05 versus MI-Ctrl. n = 6, 7, and 7 for sham, MI-Ctrl, and MI +miR-221, respectively.