miR-145 transgenic mice develop cardiopulmonary complications leading to postnatal death

Abstract

Background: Both downregulation and elevation of microRNA miR-145 has been linked to an array of cardiopulmonary phenotypes, and a host of studies suggest that it is an important contributor in governing the differentiation of cardiac and vascular smooth muscle cell types.

Methods and results: To better understand the role of elevated miR-145 in utero within the cardiopulmonary system, we utilized a transgene to overexpress miR-145 embryonically in mice and examined the consequences of this lineage-restricted enhanced expression. Overexpression of miR-145 has detrimental effects that manifest after birth as overexpressor mice are unable to survive beyond postnatal day 18. The miR-145 expressing mice exhibit respiratory distress and fail to thrive. Gross analysis revealed an enlarged right ventricle, and pulmonary dysplasia with vascular hypertrophy. Single cell sequencing of RNA derived from lungs of control and miR-145 transgenic mice demonstrated that miR-145 overexpression had global effects on the lung with an increase in immune cells and evidence of leukocyte extravasation associated with vascular inflammation.

Conclusions: These data provide novel findings that demonstrate a pathological role for miR-145 in the cardiopulmonary system that extends beyond its normal function in governing smooth muscle differentiation.

Keywords: miR-145; cardiovascular; pulmonary; smooth muscle differentiation.

FIGURE 1

Overexpression of miR‐145 in the cardiovascular system leads to premature death in mice. Kaplan–Meier survival curve to illustrate the range in which miR‐145 transgenic mice (miR‐145Tg;MCC) succumb. Genotype of control mice were +/+;+/+, miR‐145Tg/+;+/+, or +/+;MCC/+ (n = 18) (a). LacZ expression demonstrates expression of the Myocardin‐Cre transgene (MCC) in the hearts and lungs of mice at embryonic day (E)16.5 (b), and in the lungs in postnatal day 14 mice (c) (n = 3)

FIGURE 2

miR‐145 transgenic mice exhibit right ventricular hypertrophy. Dissected hearts from control and miR‐145Tg;MCC mice show miR‐145 overexpressing mice have an enlarged right ventricle (arrow) (n = 7) (a). Fulton index show weight ratio of right ventricle (RV) to left ventricle and septum is significantly greater in the miR‐145Tg;MCC hearts compared to controls (b). H&E staining of cross‐sectioned hearts demonstrate increased thickness of the RV wall in miR‐145Tg;MCC mice (n = 7) (c). Genotype of control mice were +/+;+/+, miR‐145Tg/+;+/+, or +/+;MCC/+ (n = 7)

FIGURE 3

Hearts of miR‐145Tg;MCC mice show evidence of heart injury. Increased mRNA expression of BNP (Nppb), Postn, and Col1A in transgenic mice suggest cardiac injury, with a concomitant decrease in cardiac markers, cTNT (Tnnt2), Myh6, and Myl2, in miR‐145Tg;MCC (Tg, n = 11) hearts compared to control (Con, n=9) hearts (a). ** p < 0.05, ns =not significant. Histological analysis using pentachrome staining of the aortic and pulmonary valves indicates no obvious defects in the valves of the transgenic mice, (n = 6) scale bar = 100µm (b). Genotype of control mice were +/+;+/+, miR‐145Tg/+;+/+, or +/+;MCC/+

FIGURE 4

Lungs of miR‐145Tg;MCC mice exhibit alveolar remodeling and increased vessel muscularization. H&E staining of lung sections of control and miR‐145 transgenic mice show alterations in lung architecture and alveolar remodeling (a) (n = 10). Quantification of structural differences by mean linear intercept (MLI) indicates transgenic mice are significantly different from control mice (b). Degree of blood vessel muscularization was determined by measuring wall thickness (c), counting cell number (d), and staining for smooth muscle α‐actin (Acta2) (e), which shows an increase in smooth muscle surrounding small caliber arteries in the lung (n = 6)

FIGURE 5

miR‐145 induces the expression of smooth muscle marker genes in pulmonary artery smooth muscle cells. Human pulmonary artery smooth muscle cells (hPASMCs) were transfected with control (Con), miR‐145‐3p (3p), or miR‐145‐5p (5p) mimics followed by RNA isolation and qPCR to detect marker gene expression as indicated (n = 6). *p < 0.05, relative to control

FIGURE 6

Single cell RNA‐seq of wild‐type and miR‐145Tg P13 lungs. t‐SNE plot of cell clusters from wild‐type and miR‐145‐Tg;MCC lungs combined (a). Overlap of cells from wild‐type (CTRL) and miR‐145‐Tg;MCC (MIR145) lungs (b). Cell type identification of clusters (c) and violin plots of smooth muscle marker genes reveals vascular (cluster 10) and airway (cluster 15) smooth muscle cell subpopulations (d)

FIGURE 7

miR‐145Tg alters cell number and cell cycle of distinct cell types. Graph of cell number in each cluster from wild‐type (CTRL) and miR‐145Tg (MIR145) P13 lungs (a). Significance P value determined by Fisher exact test. Reactome pathway table identified by differential gene expression shows pathways that are most affected by overexpression of miR‐145Tg (b). FDR (false discovery rate)