Cardiac fibroblast–derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy
Claudia Bang, … , Jan Fiedler, Thomas Thum
Claudia Bang, … , Jan Fiedler, Thomas Thum
Published April 17, 2014
Citation Information: J Clin Invest. 2014;124(5):2136-2146. https://doi.org/10.1172/JCI70577.
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Research Article

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.

Authors

Claudia Bang, Sandor Batkai, Seema Dangwal, Shashi Kumar Gupta, Ariana Foinquinos, Angelika Holzmann, Annette Just, Janet Remke, Karina Zimmer, Andre Zeug, Evgeni Ponimaskin, Andreas Schmiedl, Xiaoke Yin, Manuel Mayr, Rashi Halder, Andre Fischer, Stefan Engelhardt, Yuanyuan Wei, Andreas Schober, Jan Fiedler, Thomas Thum

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Figure 2

miRNAs are enriched in fibroblast-derived exosomes.

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miRNAs are enriched in fibroblast-derived exosomes. (A) miRNA profiling ...
(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.