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. 2015 Mar;65(3):241-51.
doi: 10.1097/FJC.0000000000000183.

MicroRNA-133a engineered mesenchymal stem cells augment cardiac function and cell survival in the infarct heart

Duaa Dakhlallah  1 , Jianying Zhang, Lianbo Yu, Clay B Marsh, Mark G Angelos, Mahmood Khan
Affiliations

MicroRNA-133a engineered mesenchymal stem cells augment cardiac function and cell survival in the infarct heart

Duaa Dakhlallah et al. J Cardiovasc Pharmacol. 2015 Mar.

Abstract

: Cardiovascular disease is the number 1 cause of morbidity and mortality in the United States. The most common manifestation of cardiovascular disease is myocardial infarction (MI), which can ultimately lead to congestive heart failure. Cell therapy (cardiomyoplasty) is a new potential therapeutic treatment alternative for the damaged heart. Recent preclinical and clinical studies have shown that mesenchymal stem cells (MSCs) are a promising cell type for cardiomyoplasty applications. However, a major limitation is the poor survival rate of transplanted stem cells in the infarcted heart. miR-133a is an abundantly expressed microRNA (miRNA) in the cardiac muscle and is downregulated in patients with MI. We hypothesized that reprogramming MSCs using miRNA mimics (double-stranded oligonucleotides) will improve survival of stem cells in the damaged heart. MSCs were transfected with miR-133a mimic and antagomirs, and the levels of miR-133a were measured by quantitative real-time polymerase chain reaction. Rat hearts were subjected to MI and MSCs transfected with miR-133a mimic or antagomir were implanted in the ischemic hearts. Four weeks after MI, cardiac function, cardiac fibrosis, miR-133a levels, and apoptosis-related genes (Apaf-1, Caspase-9, and Caspase-3) were measured in the heart. We found that transfecting MSCs with miR-133a mimic improves survival of MSCs as determined by the MTT assay. Similarly, transplantation of miR-133a mimic transfected MSCs in rat hearts subjected to MI led to a significant increase in cell engraftment, cardiac function, and decreased fibrosis when compared with MSCs only or MI groups. At the molecular level, quantitative real-time polymerase chain reaction data demonstrated a significant decrease in expression of the proapoptotic genes; Apaf-1, caspase-9, and caspase-3 in the miR-133a mimic transplanted group. Furthermore, luciferase reporter assay confirmed that miR-133a is a direct target for Apaf-1. Overall, bioengineering of stem cells through miRNAs manipulation could potentially improve the therapeutic outcome of patients undergoing stem cell transplantation for MI.

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Conflict of interest statement

Disclosure and Conflicts of Interest: None.

Figures

Figure 1
Figure 1. Measurement of miR-133a levels by qRT-PCR in an in vivo rat MI Model
qRT-PCR data from rat hearts subjected to ischemia and reperfusion showed a significant decrease in miR-133a at one and four weeks following MI. p<0.05 compared to untreated control (n=3 hearts/group).
Figure 2
Figure 2
(A) Measurement of miR-133a levels in MSCs transfected with miR-133a mimic or antagomir by qRT-PCR. MSCs transfected with miR-133a (100 nM) showed significant increase in miR-133a levels and MSCs transfected with miR-133a antagomir (100 nM) completely blocked miR-133a levels (B) Phase contrast images of MSCs following glucose and serum deprivation at 12 hours show increased cell death (yellow arrows) in untreated, scramble and miR-133a antagomir MSCs On the other hand decreased cell death was observed in MSCs transfected with miR-133a. (C) MTT assay showed significant increase in cell survival in miR-133a mimic transfected group compared to scramble or mock transfected groups. A value of p<0.05 was considered to be significant (n=3/group).
Figure 3
Figure 3. Measurement of Apaf-1, Caspase 9 and Caspase3 levels in MSCs with miR-133a mimic or antagomir
qRT-PCR data showed a dose dependent decrease in fold expression of APAF-1, Caspase-9 (CASP9) and Caspase-3 (CASP3) in MSCs transfected with 50 nM & 100 nM of miR-133a mimic under simulated ischemic conditions. In contrary, there was a significant increase in these genes in MSCs transfected with miR-133a antagomir. A value of p<0.05 was considered to be significant (n=3/group).
Figure 4
Figure 4. Assessment of cardiac function at 1 week after MI in miR-133a mimic + MSCs transplanted hearts
(A) Representative M-mode ultrasound echocardiography images in different groups at one-week after MI. (B) Significant improvement in ejection fraction was observed in miR-133a mimic + MSCs treated group compared to MSC-alone group. Values are expressed as mean ± SD (n=6 hearts/group). A value of p<0.05 was considered to be significant.
Figure 5
Figure 5. Assessment of MSCs engraftment in the heart
(A) MSCs labeled with SPIO, were identified in the heart sections at one week after transplantation by Prussian blue staining. Prussian-blue staining showing engraftment of SPIO-labeled cells (Blue color, Black arrows) in the infarct heart. The bar graph indicates a quantification of engrafted MSCs. There was a significant increase in MSCs engraftment in the miR-133a + MSCs group, when compared to the MSCs or miR-133a antagomir + MSCs groups (p<0.05 vs MSCs group, n=4/group). (B) Masson-trichrome staining was performed for measurement of infarct size or fibrosis and (C) LV % fibrosis and left ventricular wall thickness (LV-WT) are shown for infarcted hearts; MI, MSCs and MSCs+miR-133a treated groups. Infarct size, % fibrosis and LV wall thickness were all improved in the miR-133a + MSCs group. All values are expressed as mean ± SD (n=3 hearts/group). A value of p<0.05 was considered to be significant.
Figure 6
Figure 6. Measurement of miR-133a levels and gene expression in the heart following MI
(A) qRT-PCR data shows increase in miR-133a levels in the infarcted heart at one week after MI in miR-133a mimic MSCs transfected group. (B) qRT-PCR data shows repression of Apaf-1, Caspase-9 and Caspase-3 gene expression at one week following MI in miR-133a mimic MSCs transplanted hearts. All values are expressed as mean ± SD (n=3 hearts/group). A value of p<0.05 was considered to be significant.
Figure 7
Figure 7. Luciferase reporter assay confirming miR-133a as a direct target for Apaf-1
Luciferase assay was done by co-transfecting 293 cells with miR-133a mimic, antagomir or scramble and 3′UTR of Apaf-1 or mutated Apaf-1-3′UTR (Del1 and Del2; represented by Δ 1 & 2) and then subjected to luciferase reporter assay to measure gene activity. The intensity of the firefly luciferase in the cells co-transfected with miR-133a mimic and Apaf-1-3′UTR was lower when compared with scramble miRNA transfection. On the other hand, co-transfection with miR-133a antagomir lead to increased luciferase activity. However, miR-133a mimic or antagomir has no effect on the luciferase activity in the mutated Apaf-1- 3′UTR (Del-1 and Del-2; represented by Δ 1 & 2) transfection groups. All values are expressed as mean ± SD (n=3/group), p<0.05 vs scrambled or miR-133a antagomir groups.
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