MicroRNA Expression in the Infarcted Heart Following Neonatal Cardiovascular Progenitor Cell Transplantation in a Sheep Model of Stem Cell-Based Repair

Abstract

Myocardial infarctions affect approximately 735,000 people annually in the United States and have a substantial impact on quality of life. Neonates have an enhanced capability of repairing cardiovascular damage, while adults do not. The mechanistic basis for this age-dependent difference in regenerative capacity remains unknown. Recent studies have shown that microRNAs (miRNAs) play a significant role in regulating the regenerative ability of cardiovascular cells. This report defines the alterations in miRNA expression within the cardiovascular repair zone of infarcted sheep hearts following intracardiac injection of neonatal islet-1+ cardiovascular progenitor cells. Sheep were infarcted via left anterior descending coronary artery ligation. After 3 to 4 weeks of infarction, sheep neonatal islet-1+ cardiovascular progenitor cells were injected into the infarcted area for repair. Cell-treated sheep were euthanized 2 months following cell injection, and their hearts were harvested for the analysis of miRNA and gene expression within the cardiovascular repair zone. Ten miRNAs were differentially regulated in vivo, including miR-99, miR-100, miR-302a, miR-208a, miR-665, miR-1, miR-499a, miR-34a, miR-133a, and miR-199a. These miRNAs promote stemness, cell division, and survival. Several signaling pathways are regulated by these miRNAs, including Hippo, Wnt, and Erythroblastic Leukemia Viral Oncogene B (ERBB). Transcripts encoding Wnt, ERBB, and Neuregulin 1 (NRG1) were elevated in vivo in the infarct repair zone. Wnt5a signaling and ERBB/NRG1 transcripts contribute to activation of Yes-Associated Protein 1. MiRNAs that impact proliferation, cell survival, and signaling pathways that promote regeneration were induced during cardiovascular repair in the sheep model. This information can be used to design new approaches for the optimization of miRNA-based treatments for the heart.

Keywords: cardiac; cardiac progenitor cells; miRNAs; myocardial infarction.

Figure 1.

The cardiovascular repair zone of infarcted, islet-1+ cell–injected sheep hearts demonstrates ongoing presence of stem cells at the site of infarction and activation of select microRNA (miRNA) transcripts. Labeled CFSE+ cells (green) were quantified within the cardiovascular repair zone. The black arrows in the Masson’s trichrome–stained tissue section identify the cardiovascular repair zone where viable tissue (red) borders nonviable tissue (blue) in the infarcted heart (A). MiRNAs involved in various aspects of cardiovascular repair, including proliferation, differentiation, and survival, were identified using TargetScan and existing literature (B). Quantitative real-time polymerase chain reaction was done to identify the relative expression of miRNAs involved in cardiovascular repair. The heatmap represents the changes in miRNA expression in the cardiovascular repair zone following neonatal islet-1+ cardiovascular progenitor cell treatment in infarcted sheep relative to the non-infarcted area (C). CFSE: carboxyfluorescein succinimidyl ester.

Figure 2.

Predicted pathways targeted by miR-302a, miR-99a, miR-100, miR-199a, miR-92a, miR-208a, miR-499a, miR-1, miR-133a, and miR-665. Prediction is based on the results from DIANA-mirPath v.3 software.

Figure 3.

Differentially expressed miRNAs in the cardiovascular repair zone and activation of ERBB signaling and NRG1 in the CPC-treated infarcted region. A decrease in miR-99a and miR-100 transcripts (A, B) and an increase in miRNA-199a compared with controls that included infarct only or the control/non-infarcted area of infarcted, cell-treated sheep support cell cycle re-entry and cardiovascular regeneration (C). ERBB signaling pathway transcripts are elevated as predicted based on the miRNA profile (D) and NRG1 transcripts are activated in the cardiovascular repair zone (E). Fold changes are displayed as mean ± SEM. ERBB: Erythroblastic Leukemia Viral Oncogene B; NRG1: Neuregulin 1; CPC: cardiovascular progenitor cell. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure 4.

MicroRNAs (miRNAs) associated with proliferation are upregulated in the infarct repair zone when compared with controls that included infarct only or the control/non-infarcted area of infarcted, cell-treated sheep. MiRNA-133a (A), miR-208a (B), miR-499a (C), miR-665 (D), and miR-302a (E) expression is positively correlated with an induction of proliferation. Fold changes are displayed as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure 5.

Signaling pathways responsible for driving proliferation are regulated by miRNAs. An induction of miRNAs that promote YAP1 activation was observed (A). Elevated levels of KI-67 are shown by real-time polymerase chain reaction in the islet-1+ CPC-treated infarcted tissue region compared with the control/non-infarcted normal tissue (B). Fold changes are displayed as mean ± SEM. CPC: cardiovascular progenitor cell. **P ≤ 0.01.

Figure 6.

Wnt signaling is induced after islet-1+ neonatal cardiovascular progenitor cell transplantation as shown by RT-qPCR. Noncanonical Wnt ligands Wnt5a and Wnt11 are significantly elevated in the islet-1+ CPC-treated infarcted region by 2.3-fold and 6.9-fold, respectively, compared with the control/non-infarct normal tissue (A). Canonical Wnt ligands Wnt3a and Wnt9a are also significantly increased in the islet-1+ CPC-treated infarcted region compared with the control/non-infarcted area (B); fold changes are displayed as mean ± SEM. RT-qPCR: quantitative reverse transcription polymerase chain reaction; CPC: cardiovascular progenitor cell. **P ≤ 0.01; ****P ≤ 0.0001.

Figure 7.

ERBB/YAP1/NRG1 signaling forms an autocrine loop. NRG1 binding to the ERBB3/ERBB2 receptor stimulates downstream signaling to the PI3K-AKT pathway to promote cell survival. Activation of PDK1 indirectly inhibits the Hippo kinase cascade which activates YAP1. An autocrine loop interaction may occur when the expression of intranuclear YAP1 is elevated, resulting in the upregulation of NRG1 downstream of YAP1. NRG1 is transported through the cell membrane to rebind with ERBB3/ERBB2 receptors on the cell surface (A). If YAP1 protein levels are induced as shown by Western blot following exposure to Wnt5a (B, C), transcripts encoding NRG1 and ERBB3 are also induced as shown by RT-qPCR (D–F). Fold changes are shown as mean ± SEM. PCR samples were run in triplicates and were normalized to actin. ERBB: Erythroblastic Leukemia Viral Oncogene B; NRG1: Neuregulin 1; CPC: cardiovascular progenitor cell; YAP1: Yes-Associated Protein 1; RT-qPCR: quantitative reverse transcription polymerase chain reaction.