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 3

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

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miR-21* is transported to cardiomyocytes, leading to cellular hypertroph...
(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.