Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy

Abstract

In response to stress, the heart undergoes extensive cardiac remodeling that results in cardiac fibrosis and pathological growth of cardiomyocytes (hypertrophy), which contribute to heart failure. Alterations in microRNA (miRNA) levels are associated with dysfunctional gene expression profiles associated with many cardiovascular disease conditions; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes. Surprisingly, evaluation of the miRNA content of cardiac fibroblast-derived exosomes revealed a relatively high abundance of many miRNA passenger strands ("star" miRNAs), which normally undergo intracellular degradation. Using confocal imaging and coculture assays, we identified fibroblast exosomal-derived miR-21_3p (miR-21*) as a potent paracrine-acting RNA molecule that induces cardiomyocyte hypertrophy. Proteome profiling identified sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) as miR-21* targets, and silencing SORBS2 or PDLIM5 in cardiomyocytes induced hypertrophy. Pharmacological inhibition of miR-21* in a mouse model of Ang II-induced cardiac hypertrophy attenuated pathology. These findings demonstrate that cardiac fibroblasts secrete star miRNA-enriched exosomes and identify fibroblast-derived miR-21* as a paracrine signaling mediator of cardiomyocyte hypertrophy that has potential as a therapeutic target.

Figure 1. Cardiac fibroblasts produce and secrete exosomes.

(A and B) Electron microscopy images of rat cardiac fibroblasts. (A) Cytoplasm of rat cardiac fibroblasts with MVB. MVB membrane invaginated inward (arrows), forming intraluminal vesicles (inset: higher magnification of intraluminal vesicles; scale bar: 100 nm). (B) MVB fusing with the cell membrane. (A and B) Scale bar: 200 nm (n = 3). (C) Electron microscopy image of rat cardiac fibroblast–derived exosomes, showing a size of approximately 50 to 100 nm in diameter. Scale bar: 100 nm (n = 4). (D) Western Blot of fibroblast-derived exosomes for CD63 (60 kDa) and GAPDH (34 kDa). (E) Flow cytometry analysis of CD63 of fibroblast-derived exosomes. Fibroblast-derived exosomes were immunostained against CD63 (red curve) and compared with the appropriate isotype control (gray curve). (F and G) Total RNA from (F) rat cardiac fibroblasts and (G) fibroblast-derived exosomes was analyzed by bioanalyzer. Gels and electropherograms are shown. The left gel lane is the ladder standard and the right lane is the total RNA from (F) cardiac fibroblasts or (G) exosomes. Y axis of the electropherogram is the arbitrary fluorescence unit intensity (FU) and x axis is migration time in seconds (s). (H) Western blot analysis of NSMASE2 (84 kDa) and GAPDH (34 kDa) in neonatal rat cardiac fibroblast lysates (n = 3).

Figure 2. miRNAs are enriched in fibroblast-derived exosomes.

(A) miRNA profiling assays were performed with rat cardiac fibroblast–derived exosomes and cardiac fibroblasts (n = 2). Fold change of the ratio exosomes/cells is shown as log₁₀. Only reproducibly detectable miRNAs, having a Ct <35 were considered. miR-21* is shown in red. Other miRNA star strands are shown in green. Data are normalized to U1. The fold changes of miR-9-star, miR-135a, miR-181c, miR-547 are not shown in the graph because their fold change is more than 100. Mean ratios of exosomes/cells are shown in Supplemental Table 1. (B) Ratio of exosomes/cells of miR-21 and miR-21* expression level using TaqMan qRT-PCR. (C) Expression levels of miR-21 and miR-21* using TaqMan qRT-PCR in rat cardiac fibroblasts and (D) in secreted fibroblast-derived exosomes (n = 3–4). Data are normalized to a C. elegans miRNA (cel-miR-54). (E) Differential expression analysis of miR-21 and miR-21* in cardiac fibroblasts and fibroblast-derived exosomes using next-generation deep sequencing. Normalized read counts were used for fold change of the ratio cells/exosomes, which is shown as log₂. n = 3 for fibroblast-derived exosomes and cardiac fibroblasts. (F) Immunofluorescence staining of control and NSMASE2 inhibitor–treated rat cardiac fibroblasts. Treatment with 10 μM NSMASE2 inhibitor for 48 hours resulted in accumulation of CD63-positive microvesicles (green) in rat cardiac fibroblasts. Actin cytoskeleton is stained with Phalloidin-TRITC (red), and nuclei are stained with DAPI (blue). Scale bar: 50 μm. (G) Level of miR-21* in fibroblast-derived exosomes treated with NSMASE2 inhibitor (10 μM) for 48 hours and after treatment with (H) Ang II (1 nM) for 24 hours. Data are normalized to U1. (n = 3). Data are mean ± SEM. ^#P = 0.07, *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 3. miR-21* is transported to cardiomyocytes, leading to cellular hypertrophy.

(A) Exosome uptake experiment. Purified fibroblast-derived exosomes were labeled with green fluorescent dye and incubated with cardiomyocytes. (B) Murine cardiomyocytes were incubated with PKH67-labeled (green) exosomes from mouse fibroblasts and fixed for confocal imaging. Cardiomyocytes were incubated with PKH67-labeled exosomes for 30 minutes, 2 hours, and 24 hours (3 independent experiments; n = 3–7). Scale bar: 5 μm. (C and E) Cardiomyocytes were incubated with PKH67-labeled exosomes for 2 hours (4°C) or (D and E) treated with cytochalasin D (Cyt D, 0.5 μM) for 30 minutes and then incubated with PKH67-labeled fibroblast-derived exosomes for 2 hours (37°C). (C and D) Exosome uptake was quantified as percentage of fluorescence intensity. Control is exosome uptake for 2 hours (37°C). HL-1 cardiomyocytes were stained with Phalloidin-TRITC (red) and DAPI (blue). Scale bar: 5 μm (n = 3). (F) A transwell coculture assay was used, with cardiac fibroblasts in the top well, cardiomyocytes in the bottom well, and a 0.4-μm porous membrane between the 2 wells inhibiting cell-cell contact. (G) Cardiac fibroblasts were transfected with a precursor of miR-21* (pre-21*) or a control precursor miRNA (scr), cocultured with cardiomyocytes for 72 hours, and expression of miR-21* was measured in cardiomyocytes (n = 3). (H and I) Cardiomyocyte size was measured after incubation with scrambled (scr) or pre-miR-21*–transfected cardiac fibroblast–derived exosomes (n = 63–87). (J) Experimental setup of cardiomyocyte transfection. (K and L) Cardiomyocytes were transfected with a precursor of miR-21, miR-21*, and miR-132 (pre-132) or a control miRNA (scr) for 72 hours. Cardiomyocytes were stained, and cell size was measured (n = 62–82). (I and L) Cardiomyocytes were stained with α-actinin (red) and nuclei with DAPI (blue). Scale bar: 50 μm. *P < 0.05, ***P < 0.005.

Figure 4. miR-21* regulates SORBS2 and PDLIM5 in cardiomyocytes.

(A) Principal component analysis of protein expression patterns obtained by a proteomics approach from rat cardiomyocytes 72 hours after transfection with a scrambled control (blue dots), a miR-21 precursor (red dots), or a miR-21* precursor (green dots). (B) Pathway analysis of regulated protein patterns. GO term enrichment graph of regulated proteins in cardiomyocytes using functional annotation cluster tool DAVID 6.7. The top 11 gene groups that are regulated in cardiomyocytes after transfection of miR-21* are shown. (C) Protein expression of SORBS2, PDLIM5, and GAPDH in cardiomyocytes transfected with control miRNA (Scr) and precursor of miR-21 and miR-21*. Quantification of (D) SORBS2 protein expression and (E) PDLIM5 protein expression (n = 3 per transfection group). (F) Activity of luciferase reporter construct containing the 3′UTR of Sorbs2 mRNA relative to β-gal control plasmid after transfection of scrambled miRNA (Scr) or precursor of miR-21*. (G) Measurement of cardiomyocyte cell size and (H) immunofluorescence staining after transfection of control, SORBS2, and PDLIM5 siRNA in cardiomyocytes. Scale bar: 25 μm. *P < 0.05, ***P < 0.005.

Figure 5. Antagonism of miR-21* attenuates Ang II–induced cardiac hypertrophy.

(A) Overview of the experimental setup. (B) Pericardial fluid was collected from 4-week-old TAC and sham mice, and miR-21* expression level was measured by qRT-PCR (n = 5 for sham group, n = 6 for TAC group). (C) Overview of the experimental in vivo procedure. (D) Heart weight/body weight ratio of sham mice and Ang II minipump–implanted mice treated either with scrambled (Scr ant) or miR-21* antagomir (miR-21* ant). (E) Immunofluorescence staining and (F) measurement of cardiomyocyte diameter in sham mice or mice with Ang II minipumps treated either with scrambled or miR-21* antagomir. Three different heart section areas from each animal were selected, and at least 50 cells were measured per area. Scale bar: 25 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005. WGA, wheat germ agglutinin (membrane stain).

Figure 6. miR-21* acts as a paracrine signaling mediator during fibroblast-derived cardiomyocyte hypertrophy.

During cardiac stress, cardiac fibroblasts secrete exosomes enriched with miR-21*, which are transported to cardiomyocytes. Fibroblast-derived miR-21* regulates the expression of the targets SORBS2 and PDLIM5 in cardiomyocytes, leading to cardiomyocyte hypertrophy.