Inhibition of the Cardiac Fibroblast–Enriched lncRNA Meg3 Prevents Cardiac Fibrosis and Diastolic DysfunctionNovelty and Significance
Rationale: Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after pressure overload, leading to fibrosis and diastolic dysfunction. Recent studies described the role of long noncoding RNAs (lncRNAs) in cardiac pathologies. Nevertheless, detailed reports on lncRNAs regulating CF biology and describing their implication in cardiac remodeling are still missing.
Objective: Here, we aimed at characterizing lncRNA expression in murine CFs after chronic pressure overload to identify CF-enriched lncRNAs and investigate their function and contribution to cardiac fibrosis and diastolic dysfunction.
Methods and Results: Global lncRNA profiling identified several dysregulated transcripts. Among them, the lncRNA maternally expressed gene 3 (Meg3) was found to be mostly expressed by CFs and to undergo transcriptional downregulation during late cardiac remodeling. In vitro, Meg3 regulated the production of matrix metalloproteinase-2 (MMP-2). GapmeR-mediated silencing of Meg3 in CFs resulted in the downregulation of Mmp-2 transcription, which, in turn, was dependent on P53 activity both in the absence and in the presence of transforming growth factor-β I. Chromatin immunoprecipitation showed that further induction of Mmp-2 expression by transforming growth factor-β I was blocked by Meg3 silencing through the inhibition of P53 binding on the Mmp-2 promoter. Consistently, inhibition of Meg3 in vivo after transverse aortic constriction prevented cardiac MMP-2 induction, leading to decreased cardiac fibrosis and improved diastolic performance.
Conclusions: Collectively, our findings uncover a critical role for Meg3 in the regulation of MMP-2 production by CFs in vitro and in vivo, identifying a new player in the development of cardiac fibrosis and potential new target for the prevention of cardiac remodeling.
Cardiac fibroblasts (CFs) are the main orchestrators of extracellular matrix (ECM) remodeling after left ventricular (LV) pressure overload and produce different types of matrix metalloproteinases (MMPs), calcium-dependent zinc-containing endopeptidases capable of cleaving several components of the cardiac ECM.1 Recent studies have uncovered the paradox that higher levels of specific members of the MMP family, far from leading to matrix degradation and improvement of fibrosis, are associated to worse outcomes, with increased stiffness and diastolic dysfunction.1 Besides, some roles of long noncoding RNAs (lncRNAs) in cardiomyocyte hypertrophy have been recently uncovered.2 Nevertheless, the existence of functional lncRNAs in CFs remains largely unexplored, and it is unknown whether they undergo dysregulation in vivo during cardiac remodeling, playing a role in the development of fibrosis and diastolic dysfunction. Characterization of CF-lncRNAs is currently based on few in vitro evidences.3
Editorial, see p 486
In This Issue, see p 469
Meet the First Author, see p 470
Here, we report data about an lncRNA regulating Mmp-2 transcription in CFs in vitro and in vivo and present a preventive strategy to inhibit the development of cardiac fibrosis and diastolic dysfunction after LV pressure overload by modulation of the CF-enriched lncRNA maternally expressed gene 3 (Meg3).
A detailed description of all used methods is provided in the Online Data Supplement.
Transverse aortic constriction (TAC) was performed as previously described,4 using a 26-G needle to achieve a moderate degree of pressure overload.
For lncRNA profiling, the labeled complementary RNAs obtained from CFs of 13-week sham and TAC mice were hybridized onto the Mouse LncRNA Array v2.0 (8×60K) from Arraystar.
In vitro experiments were performed in CFs isolated from the heart of 8-week-old C57BL/6 N mice.
In vivo grade GapmeRs purchased from Exiqon were injected intraperitoneally at a dose of 20 mg/kg.
LncRNA Meg3 Is Downregulated in CFs During Late Cardiac Remodeling
We first aimed at characterizing the expression pattern of lncRNAs in CFs during pressure overload–induced cardiac remodeling. A global profiling of lncRNAs was, therefore, performed in CFs isolated from mice undergoing 13 weeks of TAC or sham surgery (Online Figure I).
Microarray analysis of the CF lncRNome revealed the dysregulation of 1425 lncRNAs in CFs between mice after TAC and those after sham operation (Figure 1A and 1B). Specifically, 940 transcripts were upregulated after TAC, whereas 485 lncRNAs were downregulated. Bioinformatic filtering of the array data set was performed as explained in Figure 1C and in the Online Data Supplement. Among the 617 filtered transcripts (Online Table I), 22 lncRNAs were found to be expressed in the adult murine heart, including the known and conserved lncRNA Meg3 (Online Figure II A). Specifically, Meg3 transcript variants 1 and 2 were hardly detectable in DNase-treated whole heart and CF RNA samples, whereas transcript variant 3 (NR_027652) showed a much stronger expression (Online Figure IIB through IIE). We, therefore, focused on Meg3 transcript variant 3 for the subsequent validation of the array data set and for the following quantitative polymerase chain reaction–based measurements. After validation, 17 lncRNAs were confirmed to be upregulated or downregulated in CFs from 13-week TAC-operated mice compared with sham controls. Seven of these dysregulated transcripts reached statistical significance (Figure 1D).
To identify fibroblast-enriched lncRNAs, the expression of those 7 transcripts was measured in CFs, cardiomyocytes, and cardiac endothelial cells (ECs). Three lncRNAs, namely Ak138321, Ak158004, and Meg3, were enriched in CFs in comparison with other cardiac cell types (Figure 1E). No significant dysregulation of such transcripts was observed in cardiomyocytes or ECs (Figure 1F).
Among the 3 fibroblast-enriched transcripts, the lncRNA Meg3 was the most abundant both in freshly isolated and cultured CFs (Figure 1G). Sequencing of the Meg3 amplicon after gene-specific reverse transcription and quantitative polymerase chain reaction confirmed the identity of the quantitative polymerase chain reaction product.
Given the strong expression in adult CFs and the previous reports hinting at a possible role of Meg3 as a fibrosis regulator,5 the transcript was selected for further studies.
Meg3 Regulates the Expression of Mmp-2 in CFs in a P53-Dependent Manner
We found Meg3 to be expressed in CFs almost exclusively as a chromatin-associated RNA (Online Figure IIF).
Even though expressed in the heart and enriched in CFs, Meg3 is also present in other murine tissues and is particularly abundant in the brain (Online Figure III).
In CFs, transfection with a Meg3-antisense GapmeR led to an 80% reduction of the transcript levels (Figure 2A).
A transcriptome analysis after Meg3 silencing was performed to obtain first insights into the genetic changes induced by Meg3 dysregulation. Three thousand and twenty-six dysregulated genes (fold change ≥1.3 or ≤0.7; P≤0.01; normalized intensity >400) were identified (Figure 2B; Online Table II). Gene ontology term enrichment analysis highlighted the regulation of metal ion–binding proteins (Figure 2C), a broad class of proteins, including MMPs.
In particular, knockdown of Meg3 led to transcriptional downregulation of Mmp-2 (Figure 2D). Additionally, Mmp-2 was transcriptionally induced by transforming growth factor (TGF)-β I, a well-characterized fibrogenic factor (Figure 2E). However, silencing of Meg3 prevented the induction of Mmp-2 by TGF-β I, indicating that Meg3 is required for the induction of Mmp-2 expression (Figure 2E).
Because cleavage of pro-MMP-2 is inhibited in the extracellular environment when TIMP-2 (TIMP metallopeptidase inhibitor 2) is maintained at relatively high levels,6 we measured Timp-2 expression after Meg3 silencing and found that it increased both in the absence and in the presence of TGF-β I (Online Figure IV). Consequently, we quantified active MMP-2 in the CF supernatant via gel zymography and observed an increased intensity of the MMP-2 gelatinolytic band after TGF-β I stimulation and a decreased intensity after Meg3 silencing (Figure 2F).
Previous studies have described the regulation of Mmp-2 by P53 in human cancer cell lines,7 but whether P53 can regulate the expression of Mmp-2 in CFs is currently unknown. Additionally, Meg3 was previously shown to regulate P53 levels and the transcription of a subset of known P53 targets.8 However, the function of Meg3 in the heart remains unexplored.
We hypothesized that the negative regulation of Mmp-2 after Meg3 silencing in CFs was because of the inhibition of P53 activity on the Mmp-2 promoter. To verify this hypothesis, we first assessed whether induction or inhibition of P53 under basal conditions could affect transcription of Mmp-2. Treatment with the P53 inducer Nutlin-3 induced Mmp-2 transcription, while a decrease in Mmp-2 was observed after treating cells with the P53 inhibitor Pifithrin-α (Figure 2G), indicating that P53 positively regulates Mmp-2 in CFs. Additionally, treatment with TGF-β I led to induction of P53 at a transcriptional level, while the concomitant siRNA-mediated silencing of P53 hindered Mmp-2 induction, indicating that P53 mediates induction of Mmp-2 by TGF-β I (Figure 2H).
Using the Jaspar database, we predicted the presence of a P53 binding site on the Mmp-2 promoter between the -843 and -825 positions (Figure 2I). To assess whether Meg3 could affect the binding of P53 to such site, we performed chromatin immunoprecipitation after TGF-β I stimulation with or without silencing of Meg3. The results confirmed binding of P53 to the Mmp-2 promoter and showed that TGF-β I increased binding only in the presence of Meg3 (Figure 2J). Interestingly, neither TGF-β I nor Meg3 levels affected the binding of P53 to the cell cycle regulator and positive control promoter Cdkn1a. As a result, expression of Cdkn1a was found to be not affected by Meg3 silencing (Online Figure VA). Additionally, apoptosis levels and cell cycle distribution did not change after transfection of CFs with GapmeR Meg3 (Online Figure VB and VC).
To determine how Meg3 affects the transcriptional activity of P53, we performed RNA immunoprecipitation in adult mouse CFs. Immunoprecipitation of P53 with a specific antibody followed by quantitative polymerase chain reaction to detect Meg3 in the immunoprecipitated RNA samples confirmed the direct binding of Meg3 to P53 (Figure 2K).
These results indicate that production of MMP-2 by CFs is under the control of the P53/Meg3 couple and that P53 is a downstream effector of TGF-β I in CFs.
Targeting Meg3 In Vivo Prevents Induction of Cardiac Mmp-2 and Decreases Fibrosis After TAC Ameliorating Diastolic Dysfunction
Given the relationship between Meg3 and Mmp-2 expression in vitro, we measured both Meg3 and Mmp-2 levels in the heart of mice undergoing TAC surgery. Both transcripts showed a similar expression pattern with an initial increase, followed by downregulation in advanced cardiac remodeling (Online Figure VI). This has been also observed for Mmp-2 by others.9 Furthermore, similarly to Meg3, Mmp-2 is mainly expressed by fibroblasts in the heart (Figure 3A).
To verify whether Meg3 silencing would prevent Mmp-2 induction during the initial phase of pressure overload–induced cardiac remodeling, characterized by Mmp-2 upregulation and not yet by Meg3 downregulation, achieving a beneficial effect on cardiac fibrosis and diastolic dysfunction, we first tested whether Meg3 could be efficiently knocked down in CFs in vivo via a single GapmeR dosing in adult mice, followed, 5 days later, by heart fractionation. Expression of Meg3 was significantly reduced in the CF fraction, whereas a lower silencing efficiency was observed in the cardiomyocyte or EC fraction (Figure 3A). Also, cardiac Mmp-2, which is mostly expressed by fibroblasts compared with other cardiac cell types, resulted to be downregulated after GapmeR injection (Figure 3A).
Consistently with these results, Meg3 silencing 1 week after TAC, with follow-up injections every 10 days and up to 6 weeks (Figure 3B), prevented the TAC-induced increase in Mmp-2 expression (Figure 3C). Furthermore, Meg3 myocardial levels correlated significantly with those of Mmp-2 in TAC mice (Figure 3D). Timp-2 expression was affected neither by TAC nor by Meg3 silencing (Figure 3E). As a result, active MMP-2 in the heart was significantly increased after TAC and reduced to sham levels in TAC mice injected with GapmeR Meg3 (Figure 3F).
It has been previously reported that Mmp-2 ablation protects mice from pressure overload–induced cardiac fibrosis and diastolic dysfunction.10 We, therefore, assessed the percentages of LV fibrosis via picrosirius red staining and observed a significant improvement in TAC-operated mice injected with GapmeR Meg3 (Figure 4A). In line with this observation, expression of the fibrosis marker Ctgf was decreased, although not significantly, after Meg3 silencing and correlated with Meg3 levels in TAC mice (Online Figure VII).
Hypertrophic growth of cardiomyocytes was also impaired by blocking of Meg3 (Online Figure VIIIA), with a concomitant reduction in hypertrophy markers (Online Figure VIIIB). To investigate the presence of a paracrine mechanism mediating the effect of Meg3 silencing on cardiomyocyte hypertrophy, we isolated neonatal rat cardiomyocytes and incubated them with conditioned medium from CFs transfected with either GapmeR control or GapmeR Meg3. Cell size measurement after 72 hours revealed a significant reduction of hypertrophy in neonatal rat cardiomyocytes incubated with conditioned medium from CFs transfected with GapmeR Meg3 (Online Figure VIIIC and VIIID).
The reduced ECM deposition and cardiomyocyte hypertrophy was reflected in a lower wall thickness and LV mass, as measured by echocardiography (Figure 4B and 4C). Hearts did not show prominent increases in end-diastolic diameters after 6 weeks of TAC, and Meg3 silencing did not affect this parameter either (Online Figure IX A). However, because of aortic banding and increased end-systolic volumes and diameters, ejection fraction and fractional shortening were decreased in the TAC+GapmeR control group, as well as in the TAC+GapmeR Meg3 group (Online Figure IXB). Despite changes in echocardiographic parameters, LV contractility was not severely compromised 6 weeks after TAC, as shown by the nonsignificant changes in end systolic pressure–volume relationship, a load-independent measure of contractile function (Online Figure IXC and IXD). Additionally, Meg3 inhibition did not significantly influence LV contractility (Online Figure IXC and IXD).
Conversely, diastolic function, represented by the dP/dTmin value, was significantly impaired by TAC surgery and showed an improvement with Meg3 inhibition (Figure 4D). A similar effect was observed for the LV myocardial performance index, measured by Doppler echocardiography (Figure 4E). The shorter isovolumic relaxation time indicated a decreased myocardial stiffness and an improved diastolic function (Figure 4E). Finally, the end-diastolic pressure–volume relationship, a load-independent measure of LV relaxation, was strongly affected by TAC surgery and showed a significant improvement with Meg3 inhibition (Figure 4F). All measured echocardiographic and hemodynamic parameters are listed in Online Table IV.
In summary, after 6 weeks of TAC, mice developed cardiac hypertrophy, fibrosis, and diastolic dysfunction without significant systolic impairment. Silencing of Meg3, and the resulting inhibition of MMP-2, prevented the development of cardiac fibrosis and hypertrophy, leading to an improved performance of the heart in the diastolic phase. At a cellular level, GapmeR-mediated targeting of Meg3 disrupts the interaction between Meg3 and P53, preventing the binding of P53 to the promoter of Mmp-2, leading to decreased Mmp-2 expression and MMP-2–mediated remodeling of the ECM. In parallel, cardiomyocyte hypertrophy is reduced potentially through the decreased production by CFs of prohypertrophic paracrine factors (Figure 4G).
This study represents the first detailed report on the dysregulation of lncRNAs in CFs during chronic cardiac remodeling and identifies the lncRNA Meg3 to be a crucial regulator of cardiac MMP-2, promoting cardiac fibrosis and impairment of diastolic function after pressure overload.
Meg3 is a conserved lncRNA11 known to have oncosuppressive properties12 and to undergo downregulation in several types of cancer because of promoter hypermethylation.13,14 In cancer cells, as well as in other cell types,15 Meg3 induces apoptosis by stabilizing and activating P53, resulting in increased expression of a subset of known P53 targets.8,16,17
More recently, Meg3 has been described as a transcriptional regulator, recruiting components of the polycomb repressor complex 2 on their genomic target sites.5,18 In particular, Meg3 has been described to guide chromatin remodeling complexes to their action site on the genome because of the formation of sequence-specific DNA/RNA triplex structures.5
In line with the previous reports, and with the noncoding nature of the transcript, Meg3 is expressed in CFs as a chromatin-associated lncRNA (Online Figure IIF) and regulates, by direct interaction, the transcriptional activity of P53 on the promoter of Mmp-2. It may be speculated that the effect of Meg3 silencing on the expression of Mmp-2, but not on that of Cdkn1a, could be because of a sequence-specific transcription factor guiding activity of the lncRNA. Even though the direct interaction of Meg3 with P53 has been confirmed by us in CFs, it remains to be elucidated whether the target-specific activity of the Meg3/P53 couple is because of a DNA–RNA triplex-forming ability of Meg3, directly interacting with P53 and guiding it, at the same time, to specific genomic sites.
In vivo, we found out that preventive inhibition of Meg3 is cardioprotective, leading to decreased MMP-2 expression and activity, decreased fibrosis and hypertrophy, and better diastolic function. Besides regulating expression of Mmp-2, it is likely that Meg3 influences the expression of other genes that are involved in cardiac fibrosis development and diastolic dysfunction. For instance, it has been described that Meg3 inhibition upregulates expression of TGF-β genes in BT-549 human epithelial cells via epigenetic mechanisms.5 Interestingly, we observed, in contrast with this previous report, that inhibition of Meg3 in vitro led to downregulation of TGF-β isoforms in CFs, an effect that was confirmed by our in vivo study (Online Figure X). These effects of Meg3 inhibition on TGF-β expression might further enhance the preventive action on cardiac fibrosis development. Future studies will deepen our understanding of the CF-specific events leading to regulation of TGF-β expression by Meg3 and whether they involve epigenetic mechanisms.
Previously, it has been shown that targeted deletion of Mmp-2 protects mice from hypertrophy and fibrosis induced by TAC.10 In a separate study, cardiac overexpression of Mmp-2 was sufficient alone for induction of remodeling and fibrosis without superimposed injury,19 providing a strong evidence for the primary role of MMP-2 in cardiac ECM remodeling. Because CFs can produce additional MMP types, we assessed the expression and activity of MMP-9 after Meg3 silencing, given the proven role of MMP-9 in cardiac disease.20 However, no changes in active MMP-9 could be observed after Meg3 silencing (data not shown).
The administration of antisense GapmeRs in vivo has been performed in this study without the use of selective delivery agents. Nevertheless, we observed a greater silencing of Meg3 in CFs, compared with cardiomyocytes and ECs. This effect, similarly to the one achieved on Mmp-2, could be because of the much higher basal expression of Meg3 in CFs compared with cardiomyocytes and ECs. Additionally, because the design of the performed preventive in vivo study includes repeated injections of GapmeR Meg3, we cannot exclude that a greater Meg3 silencing efficiency is finally achieved in the cardiomyocyte and EC fractions, compared with the one observed in Figure 3A.
In parallel with the reduced fibrosis and improved diastolic function, we observed a decreased cardiomyocyte size after Meg3 silencing. This effect could be because of the inhibition of MMP-2–mediated degradation of basal membrane components and of the preservation of ECM properties. Furthermore, our in vitro experiment using neonatal rat cardiomyocytes (Online Figure VIIIC and VIIID) suggested that prohypertrophic paracrine factors produced by CFs might be reduced after Meg3 silencing, leading to decreased cardiomyocyte cell size. What are the specific factors regulated by Meg3 and involved in this cross talk between CFs and cardiomyocytes remains to be understood.
Meg3 was shown to regulate EC biology and exert antiangiogenic functions,21–23 whereas Mmp-2 was described to have proangiogenic functions during compensated hypertrophy.9 We detected Meg3 in murine cardiac endothelial cells with a mean Ct value of 28, which intrinsically suggests a good level of expression. However, expression of Meg3 resulted to be even higher in fibroblasts isolated from adult murine hearts. Given the previous reports on Meg322,24 and Mmp-29 regulating EC functions, we measured capillary density in TAC mice injected with GapmeR control or GapmeR Meg3. No difference was observed between the 2 groups, suggesting a lack of either direct or indirect effects of Meg3 silencing and Mmp-2 downregulation on EC functions in the pressure overload model (Online Figure XIA). This, however, does not exclude a higher impact of Meg3 silencing on endothelial cell behavior in other disease settings.
Finally, because 2 microRNAs overlap with the Meg3 primary transcript and expression of these microRNAs could be affected by GapmeR-mediated targeting of Meg3, we measured the expression of the mature microRNAs in sham and TAC hearts with or without inhibition of Meg3. All were expressed at low levels (Ct values ≥31). Most importantly, none of them was affected by either TAC or Meg3 silencing (Online Figure XIB). Of note, the Meg3 locus contains additional microRNA clusters whose expression was reported to be knocked out concomitantly with the deletion of Meg3 and of part of its promoter in Meg3 knockout mice.25 These observations suggest that posttranscriptional silencing might be a more indicated approach to study Meg3 functions in vivo.
In summary, we could show that preventive inhibition of Meg3 during the early phase of cardiac remodeling impedes the induction of cardiac MMP-2 normally occurring during the first weeks after TAC, leading to decreased cardiac fibrosis and improved diastolic function. Therefore, our study identifies a potential new target for the prevention of ECM remodeling in heart diseases.
Microarray data for mRNA profiling after long noncoding RNA (lncRNA) modulation used in this publication were generated by the Research Core Unit Transcriptomics of the Hannover Medical School. We thank Dr Arnold Kloos, Dr Amit Sharma, and Dr Nidhi Jyotsana for providing instrumentation and advice for chromatin immunoprecipitation (ChIP) experiments and Dr Xiao Ke for advising on bioinformatics.
Sources of Funding
This work was supported by the 7th Framework EU Grant Fibrotarget, the ERC Consolidator grant LongHeart, the IFB-TX (BMBF), and the REBIRTH Excellence Cluster. S.K. Gupta is funded by the Deutsche Forschungsgemeinschaft (DFG; GU 1664-1-1 grant).
M.T. Piccoli, S.K. Gupta, J. Viereck, and T. Thum filed a patent about the diagnostic and therapeutic use of Meg3. T. Thum is founder and CSO of Cardior Pharmaceuticals GmbH. The other authors report no conflicts.
In May 2017, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.28 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.117.310624/-/DC1.
- Nonstandard Abbreviations and Acronyms
- cardiac fibroblast
- endothelial cell
- extracellular matrix
- long noncoding RNA
- left ventricle
- maternally expressed gene 3
- matrix metalloproteinase
- transverse aortic constriction
- transforming growth factor-β
- Received January 12, 2017.
- Revision received June 13, 2017.
- Accepted June 18, 2017.
- © 2017 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Long noncoding RNAs (lncRNAs) are important regulators of cellular functions and have been recently implicated in the development of cardiovascular diseases.
The conserved lncRNA maternally expressed gene 3 (Meg3) is known to interact with chromatin remodeling complexes and to stabilize and activate P53, thereby, blocking proliferation and inducing apoptosis in several noncardiac cell types.
What New Information Does This Article Contribute?
The first cardiac fibroblast (CF)–specific global expression profiling of lncRNAs in an in vivo murine model of cardiac remodeling and the identification of Meg3 among the lncRNAs undergoing downregulation during late cardiac remodeling.
Evidence that the conserved lncRNA Meg3 is highly expressed in CFs and interacts with P53 to regulate the expression of matrix metalloproteinase-2 (Mmp-2) without significant effects on cell proliferation and apoptosis.
Evidence that the inhibition of Meg3 hinders the induction of Mmp-2 in the murine heart after cardiac stress, leading to decreased myocardial fibrosis and ameliorated diastolic dysfunction.
Understanding the molecular basis of the pathological responses involving CFs after cardiac stress might lead to the identification of new therapeutic targets for the treatment of cardiac fibrosis and diastolic dysfunction in heart failure. Despite recent evidence on the role of lncRNAs in regulating the functions of endothelial cells and cardiomyocytes, less is known on lncRNAs expressed by CFs and participating in the remodeling of the cardiac extracellular matrix. In this study, we identified Meg3 to be a conserved CF-enriched lncRNA undergoing downregulation during late cardiac remodeling. In vitro, we found that Meg3 inhibition is accompanied by Mmp-2 downregulation. Mechanistically, Meg3 interacts with P53, facilitating its binding to the promoter of Mmp-2. In vivo, the inhibition of Meg3 results in reduced Mmp-2 expression after left ventricular pressure overload and impairs the development of cardiac fibrosis and diastolic dysfunction. The results of this study suggest the inhibition of Meg3 as a potential strategy to prevent the development of cardiac fibrosis and diastolic dysfunction after cardiac stress.