Enriched miRNA star strands in fibroblast-derived exosomes.

To analyze the miRNA content of fibroblast-derived exosomes, a miRNA profiling assay (388 rat miRNAs) was performed. Only miRNAs, which were detected in all samples and showed a Ct value <35, were considered for further analysis. When comparing the miRNA ratio derived from exosomes and fibroblast cells, 50 miRNAs were found to be enriched in fibroblast-derived exosomes (Figure ​(Figure2A2A and Supplemental Table 1). Interestingly, 25.5% of fibroblast-derived exosome-enriched miRNAs were so called “star” miRNAs, representing the passenger (star) strand that often is degraded within a cell. In a previous study, our group showed that miR-21 is predominantly expressed in cardiac fibroblasts and plays a major role in fibroblast biology (17). The present miRNA profiling in exosomes identified miR-21* to be enriched in fibroblast-derived exosomes, suggesting that miR-21* might be transported out of the cardiac fibroblast via exosomal transfer, whereas miR-21 remains inside. Indeed, TaqMan qRT-PCR validation experiments revealed miR-21 levels to be higher in cardiac fibroblasts when compared with those of miR-21* (Figure ​(Figure2,2, B and C), whereas, in exosomes, miR-21* expression was significantly higher compared with miR-21 expression (Figure ​(Figure2,2, B and D). In agreement with the qRT-PCR data, RNA sequencing from cardiac fibroblasts and fibroblast-derived exosomes confirmed an enrichment of miR-21 (5.2 log₂ fold change) in cardiac fibroblasts, whereas, in cardiac fibroblast–derived exosomes, miR-21* was significantly enriched (–2.1 log₂ fold change) (Figure ​(Figure2E).2E). To clarify whether exosome-generating NSMASE2 is also involved in the secretion of miRNA-enriched fibroblast-derived exosomes, we treated cardiac fibroblasts with a chemical NSMASE2 inhibitor (GW4869). Treatment with this NSMASE2 inhibitor for 48 hours resulted in a strong accumulation of CD63-positive vesicles within cardiac fibroblasts (Figure ​(Figure2F).2F). Inhibition of NSMASE2 also led to a significant reduction of miR-21* content in fibroblast-derived exosomes (Figure ​(Figure2G).2G). Reduced levels were also observed for many other but not all miRNAs (see Supplemental Figure 2A). This finding indicates that the secretion of miRNAs in cardiac fibroblasts is regulated at least in part by NSMASE2. In contrast, treatment with the profibrotic and prohypertrophic molecule Ang II increased the levels of miR-21* in fibroblast-derived exosomes, suggesting disease-specific regulation of miRNA-enriched fibroblast-derived exosomes (Figure ​(Figure2H).2H). Treatment with Ang II also led to higher levels of several other miRNAs (see Supplemental Figure 2B). Expression and secretion of miR-21* in cardiac fibroblasts was not changed by adding a miR-21 inhibitor (data not shown). We next focused on potential functional roles of secreted miR-21*.

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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.

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

Conditioned supernatant of cardiac fibroblasts is known to induce cellular hypertrophy in cardiomyocytes (7, 8). To investigate whether fibroblast-derived exosomes are crucially involved in this paracrine action, we first treated cardiomyocytes with conditioned medium of cardiac fibroblasts; surprisingly, depletion of exosomes by sequential ultracentrifugation completely abolished the prohypertrophic effects (Supplemental Figure 3). Further studies were performed to test whether miRNAs are transported via exosome transfer between cardiac fibroblasts and cardiomyocytes. To study exosome uptake mechanisms by cardiomyocytes, we labeled secreted fibroblast-derived exosomes with a green fluorescent marker, PKH67, and incubated cultured cardiomyocytes with the labeled exosomes (Figure ​(Figure3A).3A). Analysis of exosome uptake performed in the cardiomyocyte cell line HL-1 by confocal microscopy revealed an increasing uptake of labeled exosomes over time (Figure ​(Figure3B).3B). This was further confirmed in cultures of primary cardiomyocytes (Supplemental Figure 4A). To confirm that exosomes are taken up into cardiomyocytes and that they do not simply attach to the cell surface, we performed 3D reconstructions of the confocal image z-stacks, which confirmed the cytoplasmatic localization of internalized exosomes (Supplemental Video 1). Incubation of labeled exosomes with cardiomyocytes either at 4°C for 2 hours or in the presence of cytochalasin D (an inhibitor of actin polymerization) at 37°C at 2 hours resulted in decreased exosome uptake by cardiomyocytes (Figure ​(Figure3,3, C–E), indicating a temperature- and actin-dependent uptake mechanism.

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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.

Next, a coculture assay system was used to study the miRNA crosstalk between cardiac fibroblasts and cardiomyocytes (Figure ​(Figure3F).3F). To visualize whether miRNAs released from cardiac fibroblasts are transported to cardiomyocytes, we transfected cardiac fibroblasts with a Cy3-labeled precursor miRNA and cocultured the transfected fibroblasts with cardiomyocytes for 24 hours. Confocal imaging of cardiomyocytes demonstrated that Cy3-labeled miRNAs derived from fibroblasts were detectable in separated cardiomyocytes during the coculture period (Supplemental Figure 4B). As cardiac fibroblasts are not able to migrate through the 0.4-μm porous membrane filter, the Cy3-labeled miRNA must have been transported and taken up into cardiomyocytes. We next transfected cardiac fibroblasts with a precursor of miR-21* and measured miR-21* expression in cardiomyocytes 72 hours after coculturing. Indeed, fibroblast-derived miR-21* was enriched in cardiomyocytes after the coculture period (Figure ​(Figure3G).3G). In contrast, transfecting fibroblasts with an inhibitor of miR-21* resulted in a decrease in miR-21* concentration in cardiomyocytes (Supplemental Figure 4C). In line, transfection of fibroblasts with a miRNA that is only present in Caenorhabditis elegans confirmed a miRNA transport mechanism from fibroblasts to cardiomyocytes (Supplemental Figure 4D).

We previously showed, by in vitro and in vivo experiments, that miR-21 has no significant effect in cardiomyocyte hypertrophy (17). However, the role of the passenger strand miR-21* in cardiomyocytes is unknown so far. To investigate this, we first confirmed that miR-21* binds to Ago2 in cardiac fibroblast cells by applying RNA immunoprecipitation, which revealed that miR-21* may have functional effects (Supplemental Figure 5). We next analyzed the role of exosome-derived miR-21* on cardiomyocytes; exosomes were isolated from the conditioned medium of miR-21*–transfected fibroblasts and then incubated with cardiomyocytes, followed by analyses of cardiomyocyte cell size changes. Fibroblast exosomal–derived miR-21* led to a significant increase in cardiomyocyte cell size (Figure ​(Figure3,3, H and I). In line with this, transfection of a miR-21* precursor directly to cardiomyocytes (Figure ​(Figure3J)3J) similarly resulted in cardiomyocyte hypertrophy, which was not seen after transfection of miR-21 (Figure ​(Figure3,3, K and L). Transfection of miR-132 served as a positive control (26).

In contrast, inhibiting miR-21* in cardiomyocytes led to a decrease in cardiomyocyte size (Supplemental Figure 6A). Inhibition of miR-21* in cardiomyocytes treated with Ang II to induce cellular hypertrophy rescued the Ang II–induced prohypertrophic effect, suggesting a potential therapeutic use (Supplemental Figure 6B). Ang II treatment of cardiomyocyte did not have direct effects on miR-21* levels (data not shown).

SORBS2 and PDLIM5 are targets of miR-21* in cardiomyocytes.

We next applied a proteome profiling approach to identify miR-21*–regulated targets in cardiomyocytes. Proteome profiling in cardiomyocytes transfected either with miR-21 or miR-21* identified differently regulated protein candidates (Figure ​(Figure4A4A and Supplemental Table 2), many of them involved in important pathways in cardiomyocyte biology (Figure ​(Figure4B).4B). Because miRNAs normally bind to the 3′ untranslated region (3′UTR) of their target mRNAs and lead to their repression, we were interested in targets that were downregulated after transfection of miR-21*. Among them, sorbin and SH3 domain-containing protein 2 (SORBS2) was strongly silenced and a very interesting candidate (Table ​(Table1),1), as SORBS2 was predicted to have 2 miRNA recognition sites in the 3′UTR for miR-21*. Indeed, SORBS2, also known as Arg-binding protein 2 (ARGBP2), is expressed at high levels in the heart and is repressed during cardiac pathologies (27, 28). We revealed that transfection of miR-21* in cardiomyocytes led to repression of SORBS2 protein expression, which was not observed after transfection of miR-21 (Figure ​(Figure4,4, C and D). Consistent with this finding, incubation of cardiomyocytes with fibroblast-derived exosomes transfected with miR-21* resulted in reduced SORBS2 expression in cardiomyocytes (Supplemental Figure 7A). Mechanistically, we found that miR-21* directly targeted SORBS2 3′UTR, as shown by luciferase gene reporter assays (Figure ​(Figure44F).

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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.

Table 1

Deregulated proteins in cardiomyocytes after transfection of miR-21/miR-21*

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Based on the results from the proteome profiling, we also identified PDZ and LIM domain 5 (PDLIM5, also known as Enigma homolog [Enh]) as a further silenced target of miR-21* (Table ​(Table1).1). This is of interest, as cardiomyocyte-specific PDLIM5 knockout mice develop cardiomyopathy, suggesting PDLIM5 to play a major role in cardiac muscle structure and function (29). Validating Western blot experiments showed transfection of miR-21* in cardiomyocytes to reduce PDLIM5 protein expression (Figure ​(Figure4,4, C and E). Consistently, PDLIM5 was repressed in cardiomyocytes after incubation with miR-21*–transfected fibroblast-derived exosomes (Supplemental Figure 7B). However, using bioinformatic prediction tools, we did not find a predicted miR-21* recognition element in the 3′UTR, indicating PDLIM5 to be an indirect miR-21* target or to have putative binding sites in the 5′UTR or coding sequence.

To investigate whether SORBS2 and PDLIM5 are involved in the hypertrophic phenotype mediated by miR-21* in cardiomyocytes, we silenced either SORBS2 or PDLIM5 in cardiomyocytes using siRNA approaches. Silencing of the miR-21* targets SORBS2 and PDLIM5 in cardiomyocytes significantly induced cellular hypertrophy (Figure ​(Figure4,4, G and H).

Several other proteins were also regulated by miR-21*, as depicted by proteome profiling analysis (Figure ​(Figure4A,4A, Table ​Table1,1, and Supplemental Table 2), some of which may additionally contribute to the observed phenotype. In conclusion, we revealed that SORBS2 and PDLIM5 are regulated by miR-21*, which are involved in the development of cardiomyocyte hypertrophy.

miRNA secretion.

Cardiac fibroblasts produced and secreted exosomes enriched with miRNA passenger strands (star miRNAs), which are usually degraded during miRNA biogenesis (14). We identified miR-21* to be enriched in fibroblast-derived exosomes compared with the intracellular compartment of these cells. In contrast, the mature strand miR-21 was abundant in fibroblast cells compared with exosomes, indicating that there might be a selective packaging process of miRNAs into exosomes. Our findings further indicate that miR-21* is exported from the cardiac fibroblast via exosomes and is transported to neighboring cells. Consistent with our results, several studies showed that the miRNA content can vary in exosomes and their parental cells, as depicted in mast cell exosomes as well as B cell– and T cell–derived exosomes (12, 30). Interestingly, miR-21* was found to be abundant in T cell– and B cell–derived exosomes, but miR-21* was more abundant in cells than in exosomes (30). Our finding that about one-quarter of the miRNAs found in fibroblast-derived exosomes represent miRNAs derived from the passenger strand suggests the existence of a general transport mechanism of star miRNAs. This notion is further supported by the presence of natural antisense RNAs in exosomes from human colorectal cancer cell lines that were transported to donor cells (31).

With regard to the mechanism underlying the secretion of miRNAs, it was shown that the secretion of miRNAs via exosomes in HEK293 cells is ceramide-dependent and is regulated by NSMASE2, the rate-limiting enzyme of ceramide biosynthesis (25). In this study, inhibition of NSMASE2 in HEK293 cells resulted in a dose-dependent reduction of extracellular miR-146a and miR-16 and subsequent reduction of exosome secretion. In accordance, inhibition of NSMASE2 in cardiac fibroblasts resulted in a significant decrease of miR-21* levels in fibroblast-derived exosomes and in accumulation of CD63-positive vesicles. NSMASE2 inhibition led to the reduction of some other but not all miRNAs, suggesting that miRNA secretion via exosomes in cardiac fibroblasts is in part but not totally regulated by NSMASE2. miR-21* secretion from cardiac fibroblasts was increased after stimulation with the profibrotic and prohypertrophic molecule Ang II, indicating a disease-specific regulation of miRNA-enriched fibroblast-derived exosomes. However, other molecules, such as components of the endosomal sorting complex required for transport (ESCRT) complex and RAB and SNARE proteins, which have been shown to be involved in exosome secretion (32, 33), may also play a role in regulating miRNA-exosome release in cardiac fibroblasts but have not been investigated.

miRNA uptake.

Although several studies showed that exosomes can interact with their recipient cells, the underlying mechanism of how exosomes are taken up and exert their function in the recipient cells is still unclear. Possible mechanisms of exosome uptake by target cells include specific binding to surface receptors (34, 35), fusion to the plasma membrane (36), or internalization (37). In our study, we demonstrated that fibroblast-derived exosomes are taken up by cardiomyocytes in an actin- and temperature-dependent manner. In accordance to our findings, the involvement of temperature and actin during exosome uptake was also shown in PC12 cells (38). We also provide evidence that exosome uptake by cardiomyocytes is mediated either via internalization, such as endocytosis, or via a receptor-mediated mechanism, because extensive PBS washing steps could not remove fibroblast-derived exosomes from cardiomyocytes, indicating the effective internalization. However, we can exclude macropinocytosis (distinct pathway of endocytosis) as a possible uptake mechanism, because incubation of fibroblast-derived exosomes with the macropinocytosis marker FITC-dextran showed no colocalization (data not shown). Our present study shows that exosomes released by cardiac fibroblasts are major components triggering cardiomyocyte hypertrophy.

miRNA function.

We showed previously that miR-21 has an essential role in fibroblast biology and is increased in human and murine failing hearts (17). However, neither increase nor decrease of miR-21 expression levels in cardiomyocytes influenced morphology or size, suggesting that miR-21 plays no functional role in cardiomyocytes (17). In the current study, we provide the first evidence that miR-21* acts as a paracrine signaling mediator and is involved in the development of cardiomyocyte hypertrophy. Specifically, fibroblast-derived miR-21* induced cellular hypertrophy in cardiomyocytes but not miR-21, which indicates that miRNA passenger strands can act as mature functional miRNAs. Consistent with this, miR-21* has been shown recently to have a pivotal immunopathological function in controlling apoptosis in granulocyte eosinophils in vitro (39). Another study found that miR-21* expression was differentially expressed in the aging mouse heart (40). Of note, extensive deep RNA sequencing in failing human hearts identified miR-21* to be expressed in the human heart and increased during heart failure (41). Others have shown functional roles for star miRNAs also in other pathologies, such as colorectal and prostate cancer or obesity/metabolic syndrome (42–44). RNA deep sequencing studies in Drosophila melanogaster identified that a large number of miRNA* sequences are not degraded but are able to bind to Ago2-RISC complexes (45–47). Moreover, the expression of miRNA guide and miRNA* strand is controlled either by pri-miRNA transcription or Ago1/Ago2 proteins during adult aging (40). In accordance to these studies, we found that miR-21* binds to Ago2 in cardiac fibroblasts, revealing that miR-21* is not degraded in cardiac fibroblasts and might act as a functional miRNA. However, the exact mechanism of miRNA loading into exosomes was not further addressed in this study and remains to be investigated. It is also possible that other currently unknown factors/proteins are involved in the miRNA-loading process.

miRNA targets.

miRNAs can regulate dozens to hundreds of targets simultaneously. In our study, we thus performed proteome profiling in cardiomyocytes to identify miR-21*–regulated protein candidates. We demonstrated that miR-21* regulates the expression of SORBS2 and PDLIM5 in cardiomyocytes, which were further shown to be the main mediators of the prohypertrophic phenotype of miR-21*. We identified SORBS2 as a major direct target of miR-21*. In line, SORBS2 was detected recently in human heart tissue and was found to be repressed in cardiac pathologies (28). SORBS2 is localized in the Z-bands of mature myofibrils and regulates important processes in cardiomyocytes, including the assembly of myofibrils or regulation of signaling at the Z-band level (27). SORBS2 can interact with a number of binding partners, including signaling molecules such as tyrosine kinases and cytoskeletal molecules (27). Importantly, we found SORBS2 silencing to induce cardiomyocyte hypertrophy. Our proteome results showed that PDLIM5 is also silenced by miR-21* in cardiomyocytes and PDLIM5 knockout mice develop dilated cardiomyopathy (29), suggesting PDLIM5 to play a major role in cardiac muscle structure and function.

Exosome purification, analysis, and labeling.

Exosomes were purified from conditioned medium of neonatal rat cardiac fibroblasts cultured in DMEM supplemented with 1% exosome-depleted FBS (FBS is depleted of contaminating bovine exosomes by ultracentrifugation for at least 6 hours at 100,000 g). Conditioned medium of rat cardiac fibroblasts was collected for 48 hours, and exosomes were purified by several centrifugation and filtration steps as described previously (50). Briefly, the supernatant was centrifuged at 300 g for 10 minutes, 2,000 g for 10 minutes, and 10,000 g for 30 minutes, followed by filtration through a 0.22-μm filter to eliminate cells, dead cells, and cellular debris. For exosome purification, the supernatant was ultracentrifuged at 100,000 g for 70 minutes, followed by an additional washing step of the exosome pellet with PBS at 100,000 g for 70 minutes (Ultracentrifuge, Beckman Coulter, L8-70M). The exosome pellet was resuspended in 100 μl PBS and stored at –80°C. The protein content was measured using MicroBCA protein assay (Thermo Scientific). Fibroblast-derived exosomes were analyzed for the presence of the exosomal marker protein CD63 by Western blot and flow cytometry as previously described (50). For exosome uptake experiments, fibroblast-derived exosomes were labeled with the PKH26 Red Fluorescent Cell Linker Kit or the PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich) according to the manufacturer’s protocol with minor modifications. Exosomes diluted in PBS were added to 1 ml Diluent C (Sigma-Aldrich). In parallel, 4 μl PKH26/PKH67 dye was added to 1 ml Diluent C and incubated with the exosome solution for 4 minutes. To bind excess dye, 2 ml 0.5% BSA/PBS was added. The labeled exosomes were washed at 100,000 g for 1 hour, and the exosome pellet was diluted in 100 μl PBS and used for uptake experiments.

RNA sequencing.

Sample preparation for sequencing was performed as described in the Illumina TruSeq small RNA library preparation protocol. Briefly, approximately 200 ng total RNA was used to enrich for the small RNA fraction using native polyacrylamide gel electrophoresis. Both 3′ and 5′ adaptors were ligated along with the unique index sequence in each sample. The adaptor-ligated RNA fragments were then reverse transcribed and pooled together. The sequencing (for 50-base read length) was performed on a HiSeq 2000 (Illumina Inc.). Demultiplexing (generating individual sample groups) of the reads after sequencing was performed by sorting the different index sequences. For data analysis, the 3′ adapter sequence was removed from the reads using cutadapt ( http://code.google.com/p/cutadapt/). Truncated reads less than 10 bases were removed from the analysis data set. Reads were first mapped to all known miRNA sequences (miRBase; http://www.mirbase.org). For this purpose, a suffix tree approach for string mapping was used (51). Briefly, a minimum of 7 bases is required to be mapped to the miRNA sequence in order for the read to belong to the miRNA, with maximum of 2 mismatches allowed. For the reads that could be mapped to multiple miRNAs using the above criteria, the read was dedicated to the miRNA where it was mapped with maximum length and with least mismatches. The count table for each miRNA was prepared using a custom-made perl script. For differential miRNA expression analysis, the DeSeq package (52) from Bioconductor ( http://bioconductor.org) was applied with default settings, except in the case of dispersion estimation, for which the blind option was used.

Animal experiments.

Animal studies were performed in accordance to the relevant guidelines and regulations of the responsible authorities. The model of left ventricular pressure overload–induced cardiac hypertrophy (transverse aortic constriction [TAC]) in C57BL/6N mice is described in the Supplemental Methods. Pericardial fluid was collected 4 weeks after TAC or sham operation. In the Ang II model of cardiac stress, C57BL/6N mice were implanted with osmotic minipumps according to the manufacturers’ instructions (Alzet). Briefly, an incision was made in the midscapular region, and an osmotic minipump (Alzet, model 2002; 0.5 μl/h for 14 days) was implanted subcutaneously. The implanted minipumps contained Ang II (Sigma Aldrich) dissolved in water, and the infusion rate was 1.5 mg/kg/d. Sham-operated mice underwent an identical surgical procedure. sham-operated mice and Ang II minipump–implanted mice were either treated with a scrambled antagomir or a miR-21* antagomir (Integrated DNA Technology) at day 0 and day 2 (each dose 80 mg/kg) via retroorbital injection. After 2 weeks, cardiac function and dimension were measured and the hearts were removed for biochemical analysis.

Statistics.

Data are presented as mean ± SEM. Statistical analysis was carried out using Prism software (Graph Pad). For statistical analysis of 2 groups, unpaired, 2-tailed Student’s t test was used; for comparison of 3 or more groups, ANOVA followed by Bonferroni’s post-test was applied.

This work was supported by the Fondation Leducq (MIRVAD to T. Thum and M. Mayr), the IFB-Tx (01EO1302 to T. Thum), the DFG (TH903/10-1 to T. Thum), REBIRTH Excellence Cluster, the internal Hannover Biomedical Research School program Molecular Medicine (to C. Bang and T. Thum), EU grant FP7-CIG-294278, FIBROTARGET (7th Framework Program of the European Commission), and the German Federal Ministry of Education and Research (PtJ: 0315690D to A. Zeug),