SkMs reduced the myocardial infarct area and improved cardiac function in rat hearts with MI
The beneficial effect of SkMs on MI was investigated using three randomized groups of experimental rats: a sham group, a MI group, and a SkM group. To evaluate the functional consequences of transplantation of SkMs in infarcted myocardium, echocardiography was performed in the three groups (n = 6 per group). MI rendered cardiac dysfunction, as indicated by reduced LVEF (51.03 ± 1.69% for the MI group vs. 80.38 ± 1.78% for the control group; p < 0.01) and LVFS (22.2 ± 0.95% for the MI group vs. 43.58 ± 1.64% for the control group; p < 0.01). Quantitative analyses showed that rats that received transplantation of SkMs following MI had a significantly better ejection fraction when compared to MI (64.67 ± 4.14% for the SkM group vs. 51.03 ± 1.69% for the MI group p < 0.01). Furthermore, SkM-transplanted rats also had significantly improved fractional shortening, post-MI, compared to the MI group (31.08 ± 2.98% for the SkM group vs. 22.2 ± 0.95% for the MI group p < 0.01). These data are shown in Fig. 1a, b.
After imaging, animals were euthanized and hearts were explanted for histological analysis. Using a confocal laser-scanning microscope, we found a population of PKH26 positive cells in the explanted hearts of the SkM group and no PKH26 positive cells in the control groups (Fig. 1c, d). The heart infarction area in the MI group was associated with fibrosis, which was attenuated in the SkM group. At 4 weeks, Masson trichrome staining indicated a reduced infarction size in the SkM group compared to the MI group (Fig. 2a–c, g) (15.39 ± 1.04% for the SkM group vs. 20.52 ± 0.91% for the MI group P < 0.01). The rate of cardiomyocyte apoptosis was increased in MI rats compared to the sham group (38.25 ± 2.81% for the MI group vs. 2.25 ± 0.75% for the sham group; p < 0.01), whereas SkM transplantation substantially inhibited MI-induced cardiomyocyte apoptosis, as determined by TUNEL staining (26.59 ± 2.28% for the SkM group vs. 38.25 ± 2.81% for the MI group p < 0.01) (Fig. 2d–f, h).
Global miRNA expression profiling in MI treated with SkMs from rat heart
The data showed a distinct miRNA expression signature (Additional file 4: Table S4). Compared to the sham group, the expression of 160 miRNAs significantly changed, with 60 miRNAs being down-regulated and 100 miRNAs being up-regulated at 4 weeks post-MI. In contrast, in the SkM group, differential expression of 78 miRNAs was observed at 4 weeks compared to the MI group, in which 46 miRNAs were down-regulated and 32 miRNAs were up-regulated. At 4 weeks, the expression of 164 miRNAs significantly changed in the SkM group compared to the sham group, and of the miRNAs, 66 miRNAs were down-regulated and 98 miRNAs were up-regulated. We also found that 68 miRNAs were reversed, thirteen miRNAs had reversal expression of downregulation, and 55 miRNAs showed reversal expression of upregulation after SkM treatment, in contrast to the sham group and MI group (Additional file 5: Table S5). It is therefore likely that these miRNA are functionally important in regulating SkM cell therapy for MI.
Differentially expressed mRNA profiling in MI treated with rat heart SkMs
We analyzed mRNA arrays to compare changes in gene expression for the three groups. Similar to the miRNAs, the intersection of significantly up- or down-regulated mRNAs in the three groups’ tissue samples was determined using the Rat 4 × 44K Gene Expression Array. The expression of 1,627 genes was significantly altered between the MI group and the sham group at 4 weeks post-MI. Compared to the sham group, we found that 759 genes were significantly up-regulated, whereas the remaining 868 genes were significantly down-regulated in the MI group. In addition, the differential expression of 1,579 genes was observed at 4 weeks post-MI in the SkM group compared to the MI group, of which 632 were down-regulated and 947 were up-regulated (Additional file 6: Table S6). In contrast to the miRNA array data, there were more mRNAs altered (up-regulated or down-regulated) in the heart tissue samples.
Analysis of significant trends in miRNA and mRNA expression
We observed a significant trend in the expression of some apoptosis-related miRNA and mRNA, including the expression of miR-30a-5p, miR-30c-5p, miR-145-5p, miR-143-3p, and miR-140-3p (Fig. 3). The horizontal axis represents different groups, and the vertical axis represents the logarithm of the ratio of miRNA and mRNAs expressing signal values to the control. When the P value was small, the impacts by the analysis on the trends of miRNA or mRNA expression were more remarkable.
Identification of apoptosis-related miRNAs in rat MI’s treated with SkMs
To validate the miRNA array results, we used 2-cut off criteria and screened the microRNAs which were related to Wnt signal pathway and related to apoptosis. The expression of miR-30a-5p, miR-30c-5p, miR-145-5p, miR-143-3p, and miR-140-3p in the infarcted border zone region was measured by real-time analysis 4 weeks after MI. Interestingly, as shown in Fig. 3, at 4 weeks post-MI, with the exception of miR-143-3p, changes in miR-30a-5p, miR-30c-5p, miR-145-5p, and miR-140-3p expression tended to increase compared to the control group, whereas the expression of these four miRNAs decreased and the expression of miR-143-3p increased in the SkM +MI group, suggesting that these miRNAs may be associated with SkM therapy in myocardial injury. The results from real-time qPCR analysis showed high concordance with our microarray results for all investigated transcripts, as shown in Fig. 4.
Pathway analysis and GO analysis of targeted mRNA changes
In total, among the target genes, KEGG pathways were overrepresented to help us obtain useful information about the function of these targets, as shown in Additional file 7: Figure S1. Several genes implicated in MI are involved in adrenergic signaling in cardiomyocytes, the TNF signaling pathway, the TGF-beta signaling pathway, and metabolic pathways.
To further explore the relationship between miRNAs and gene function in the three groups at 4 weeks, we built a miRNA-GO-network in heart tissue. The five key miRNAs in the network were identified as miR-30a-5p, miR-30c-5p, miR-145-5p, miR-143-3p, and miR-140-3p. GO biological processes were also observed to be associated with up- and down-regulated genes, respectively, as shown in Additional file 8: Figure S2 and Additional file 9: Figure S3. Genes involved in positive regulation of cell death, response to hypoxia, and negative regulation of apoptotic process were found, as shown in Additional file 10: Table S7.
Differential expression of mRNAs targeted by regulated miRNAs post-MI
To examine the potential mRNA targets of regulated miRNAs, mRNA and miRNA array data sets were analyzed using a microRNA and mRNA integrated analysis software that integrates mRNA and miRNA expression data based on miRNA-predicted targets (Additional file 11: Table S8). We constructed a miRNA-Gene-network of these differential genes and miRNAs in heart tissue. The five key miRNAs in the network were identified as miR-30a-5p, miR-30c-5p, miR-145-5p, miR-143-3p, and miR-140-3p, as shown in Additional file 12: Figure S4.
miRNA target prediction and validation
MIRANDA, MICROCOSM, and MIRDB programs were employed to predict potential targets of five key miRNAs, miR-30a-5p, miR-30c-5p, miR-145-5p, miR-143-3p, and miR-140-3p. The targets of each miRNA predicted by three different programs were graphed in Venn’s diagram as shown in Additional file 13: Figure S5, among which seven anti-apoptotic genes: Angpt14, Eif5a, Egr1, Irs2, Cebpb, Tsc22d3, and Dpep1 were selected out for further validation. qRT-PCR results showed that the expression of the above seven target genes was negatively correlated to the levels of five miRNAs we identified (Fig. 5). Furthermore, qPCR validation results showed high concordance with our microarray results for all investigated transcripts, as shown in Fig. 5.