Deficiency of Cardiomyocyte-specific MicroRNA-378 Contributes to the Development of Cardiac Fibrosis Involving a Transforming Growth Factor β (TGFβ1)-dependent Paracrine Mechanism*

Background: MicroRNA-378, a cardiomyocyte-specific miRNA, is down-regulated during heart failure. Results: miR-378 inhibition induced TGFβ1 expression, which correlated with the activity of c-Fos, c-Jun, and Ras. Conditioned media of miR-378-depleted myocytes induced fibroblast activation by utilizing TGFβ1-dependent paracrine mechanisms. Conclusion: miR-378 is a negative regulator of TGFβ1 and cardiac fibrosis. Significance: miR-378 offers therapeutic potential for the management of heart failure. Understanding the regulation of cardiac fibrosis is critical for controlling adverse cardiac remodeling during heart failure. Previously we identified miR-378 as a cardiomyocyte-abundant miRNA down-regulated in several experimental models of cardiac hypertrophy and in patients with heart failure. To understand the consequence of miR-378 down-regulation during cardiac remodeling, our current study employed a locked nucleic acid-modified antimiR to target miR-378 in vivo. Results showed development of cardiomyocyte hypertrophy and fibrosis in mouse hearts. Mechanistically, miR-378 depletion was found to induce TGFβ1 expression in mouse hearts and in cultured cardiomyocytes. Among various secreted cytokines in the conditioned-media of miR-378-depleted cardiomyocytes, only TGFβ1 levels were found to be increased. The increase was prevented by miR-378 expression. Treatment of cardiac fibroblasts with the conditioned media of miR-378-depleted myocytes activated pSMAD2/3 and induced fibrotic gene expression. This effect was counteracted by including a TGFβ1-neutralizing antibody in the conditioned-medium. In cardiomyocytes, adenoviruses expressing dominant negative N-Ras or c-Jun prevented antimiR-mediated induction of TGFβ1 mRNA, documenting the importance of Ras and AP-1 signaling in this response. Our study demonstrates that reduction of miR-378 during pathological conditions contributes to cardiac remodeling by promoting paracrine release of profibrotic cytokine, TGFβ1 from cardiomyocytes. Our data imply that the presence in cardiomyocyte of miR-378 plays a critical role in the protection of neighboring fibroblasts from activation by pro-fibrotic stimuli.

The heart responds to hemodynamic overload by promoting myocyte hypertrophy, where cardiomyocytes add sarcomeres, grow in size, and induce the expression of a set of genes that are normally expressed during fetal heart development (1,2). Initially, this phenomenon is considered compensatory to manage increased workload on the heart; however, prolonged hypertrophy results in ventricular remodeling leading to contractile dysfunction, apoptosis, and development of heart failure. There is ample evidence to indicate that cardiac fibroblasts play a pivotal role in the process of pathological remodeling. Cardiac fibroblasts are activated in response to a variety of stressors including mechanical stress and hypoxia. They secrete cytokines and growth factors, which by acting through paracrine mechanisms promote hypertrophy of neighboring cardiomyocytes and through autocrine mechanisms lead to the deposition of extracellular matrix, resulting in further deterioration of cardiac function. These changes are orchestrated by distinct alterations in intracellular signaling cascades and gene expression where microRNAs play critical roles. miRNAs 2 are highly conserved small (18 -22 nucleotides) non-coding RNA molecules. They regulate gene expression post-transcriptionally by partially pairing with the 3Ј-untranslated region of the target mRNAs, repressing their translation and/or accelerating mRNA decay (3). Because of their small size, miRNAs possess a unique ability to simultaneously target multiple mRNAs often of functionally related transcripts. From recent studies it has become clear that miRNAs act as powerful regulators of gene expression in almost every organ system and are involved in a variety of pathophysiological processes. In the heart the abnormal expression of these regulatory molecules is linked to various cardiovascular diseases including cardiac hypertrophy. Functional analysis using both gain and loss of function approaches have demonstrated that by targeting distinct mRNAs in myocytes and in fibroblasts, miRNAs either promote (pro-hypertrophic) or prevent (anti-hypertrophic) development of hypertrophy and fibrosis (for review see Ref. 4).
It is now apparent that within the heart some miRNAs specifically function to regulate fibroblast proliferation, differentiation, and induction of fibrosis. For example, miR-29, miR-133, and miR-30 are known to suppress expression of collagens and extracellular matrix proteins. Although miR-29 is expressed in fibroblasts, miR-133 is in myocytes, and miR-30 is present both in myocytes and fibroblasts (5)(6)(7)(8). Interestingly, there is evidence that miR-29 and miR-30 are secreted from cardiac fibroblasts and influence cardiomyocyte growth. Conditioned medium of miR-29-overexpressing cardiac fibroblasts attenuated cardiomyocyte growth, whereas media of miR-30-expressing fibroblasts resulted in the induction of cardiomyocyte growth (9).
We and others have recently demonstrated that miR-378 (also known as miR-378 -3p) is primarily expressed in cardiac myocytes, and its expression level is reduced in human failing hearts as well as in various experimental models of cardiac hypertrophy (10 -13). Overexpression of miR-378 protected cardiomyocytes from undergoing hypertrophy (12) and prevented cardiac dysfunction induced by pressure overload in mice (13). Based on these results, miR-378 is considered as an anti-hypertrophic miRNA. In contrast to the anti-hypertrophic effects of miR-378 overexpression, whole body knock-out of miR-378 also showed beneficial results against high fat dietinduced changes in systemic energy metabolism (14). The cardiac function and phenotype resulting from miR-378 depletion were, however, not described in this study.
In the current study we provide further evidence of a protective role of miR-378 in both cardiac myocytes as well as in fibroblasts. We demonstrate that inhibition of this miRNA by locked nucleic acid (LNA)-modified oligonucleotides promotes cardiomyocyte hypertrophy and exaggerates angiotensin II-induced adverse cardiac remodeling and dysfunction in mouse hearts. Depletion of miR-378 alone led to increased expression and secretion of profibrotic cytokine, TGF␤1, by involving Rassignaling-dependent mechanisms and AP-1 transcription factor activities. Our study thus demonstrates a new protective role of miR-378 in the process of cardiac remodeling during stress and that the presence of miR-378 is critical in maintaining cardiac cellular homeostasis.

EXPERIMENTAL PROCEDURES
All animal protocols were reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee.
Delivery of LNA AntimiR-The 378-antimiR oligonucleotides were synthesized at Exiqon Inc as fully phosphorothiolated oligonucleotides with LNA modifications as outlined in Fig. 1A. The LNA control (SCR) was a Caenorhabditis elegansspecific miRNA (15). Adult C57BL/6 mice were injected with LNA-antimiR or LNA-SCR (70 mg/kg, intraperitoneally) in a similar volume of saline on three consecutive days.
Cardiac Hypertrophy-Cardiac hypertrophy was induced in adult C57BL/6 mice by transverse aortic constriction for 4 weeks as described before (16) and by angiotensin II (Ang II, 1 mg/kg/day) for 1 week. Subcutaneously implanted osmotic mini pumps (ALZET model 1007D) were used to deliver Ang II at a flow rate 0.5 l/h/day.
Echocardiography-Transthoracic echocardiography in mice was performed at the University of Illinois Center for Cardiovascular research core facility in a blinded fashion under isoflurane (ϳ1%) anesthesia with a VisualSonics Vevo 770 instrument using a 30-MHz high frequency transducer as described (17,18).
Cell Culture, Transfection, and Treatments-Primary cultures of cardiomyocytes and fibroblasts were prepared from neonatal Sprague-Dawley rats as described before (10). Fibroblasts obtained during the pre-plating step of cardiomyocyte culture preparation were grown in Dulbecco's modified Eagle's medium supplemented with penicillin/streptomycin and 10% fetal bovine serum. Fibroblasts of 2nd or 3rd passage were used all throughout the experiment. Cells were treated with phenylephrine (20 M) or Ang II (100 nM) for the indicated time periods in serum-free DMEM medium.
For overexpression studies cells were transfected with 25 nM synthetic mimic of hsa-miR-378 -3p (Ambion Inc.). A sequence (mimic-ctrl, Ambion) was used as a control. For inhibition studies, cells were transfected with 10 nM LNA-modified 378-antimiR or scramble control. These sequences were the same as described in Fig. 1. All transfections were carried out in Opti-MEM using Lipofectamine 2000 (Invitrogen) according to our published procedures (10,12). For adenovirus infection, cardiomyocytes were infected at a multiplicity of infection of 10 in complete growth medium. For knocking down Ras signaling and C-Jun expression, adenoviruses Ad-CMV-c-Ras (Dn) (N17; 1031; Vector Biolabs) and Ad-CMV-c-Jun (Dn) (1046; Vector Biolabs) were used. For control, adenovirus Ad-CMV-GFP was used.
Preparation of Conditioned Medium-Cardiomyocytes were transfected with either LNA-modified miR-378-antimiR or scramble control (each at 10 nM). After 24 h of transfection, medium was changed to complete growth medium, and cells were incubated for an additional 48 -72 h. During this period, 25% of the medium was collected every 24 h and replaced with the fresh medium. At the end of the incubation period medium was collected, and all collected media were pooled and labeled as conditioned medium. This was centrifuged at low speed to remove cell debris, frozen in aliquots, and used as needed.
Real-time PCR and Northern Blot Analysis-Total RNA was extracted and resolved on a urea gel for Northern analysis using standard techniques. The RNA was transferred to nitrocellulose membranes and hybridized with radiolabeled microRNAspecific probes; U6 was used as a normalization control. For real-time PCR total RNA was reverse-transcribed using standard protocols. Expression of miR-378, U6, ANF, and ␤MHC were analyzed by using Taqman assays, and the remaining mRNAs were analyzed by SYBR Green-based assays. Primer sequences are available upon request.
Tissue Histology and Immunostaining-Hearts were isolated and perfusion-fixed and processed for light microscopy. Tissue embedding and staining of heart sections were performed by the histology core facility at University of Chicago. Cell imaging was performed on a Bio-Rad Laser Sharp 2000 system using a 40ϫ objective (Zeiss).
Hearts were excised, perfused with saline, and fixed in formalin. After dehydration in graded ethanol solutions, tissues were cleared with xylene and processed for embedding. The paraffin-embedded hearts were sectioned at 4 m and subsequently stained with wheat germ agglutinin coupled to tetramethylrhodamine isothiocyanate (Sigma) for detection of cell size as described earlier (19). Left ventricular myocyte crosssectional area was measured on sections of mid-free wall of the left ventricle. Suitable cross-sections were defined as having nearly circular capillary profiles and circular to oval myocyte cross-sections. The outer borders of the myocytes were traced, and myocyte areas were calculated with NIH ImageJ software (rsbweb.nih.gov). Approximately 200 cells were counted per sample, and the average was used for analysis.
Masson's trichrome staining was performed to detect collagen fiber density using standard protocols (19). The collagen fraction (stained with aniline blue in Masson's trichromestained sections) was calculated as blue-stained collagen fiber area divided by total area of the visual field. Analysis was performed in a minimum of 5 hearts for each experimental group with at least 5 replicates of each sample, and 10 visual fields were measured in each replicate. Fibroblasts (10,000 -20,000) Highlighted in gray is seed sequence, underlined nucleotides denote divergence from miR-378a, and the dotted line marks the complimentary sequences in antimiR. Asterisks identify LNA modified nucleotides in 378-antimiR (antimiR) and in the scramble control (SCR) oligonucleotides. B, dose-dependent inhibition of miR-378 by antimiR by Northern analysis using 5 g of total RNA. Mice were injected on three consecutive days, and analysis was performed after 7 days. The membrane was sequentially hybridized with miR-1 for testing specificity of inhibition and with U6 for loading control. C, Northern analysis of miR-378 expression using 5 g of total RNA of control (SCR) and antimiR (70 mg/kg)-treated mouse hearts. Each lane represents a biological replicate. D, measurement of miR-378 expression by real-time PCR. Data are presented as the mean Ϯ S.D. of 8 -10 hearts. *, significant compared with SCR control. E and F, Northern analysis using 20 g total RNA (skeletal muscle) and 50 g of RNA (for kidney and liver).
were plated on coverslips and processed for immuno-staining for collagen and vimentin as per the protocols described previously (10,12).
Western Blot Analysis-Western blotting was performed using standard protocols.
ELISA Assay-A multiplex ELISA sandwich assay was performed for measuring the levels of cytokines in the conditioned media by using Rat Oxidative ELISA Strip (Signosis Inc.) as per the manufacturer's protocol.

RESULTS
Inhibition of Cardiac miR-378 by AntimiR-To understand the role of miR-378 in cardiac pathophysiology, we used a LNAmodified oligonucleotide to knockdown endogenous miR-378 in mice. LNA-based oligonucleotide modification has been shown to impart resistance to nuclease degradation in vivo with minimal biological toxicity (20). This approach has been used by several investigators for the therapeutic inhibition of microRNAs in vivo (21). A 15-bp phosphorothioate oligonucleotide with 46% LNA-modified nucleotides was used to target miR-378. The antimiR is expected to target the seed sequence of all variant forms of miR-378 that are listed in the microRNA database. A control, scrambled (SCR) sequences were selected and LNA modified from C. elegans (Fig. 1A).
The effectiveness of antimiR-mediated inhibition of miR-378 was tested by injecting mice for 3 consecutive days with 3 different doses of antimiR (10, 35, and 70 mg/kg, intraperitoneal) and analyzing the expression of miR-378 on the 8th day. Control animals were injected similarly with SCR oligonucleotide. We found that antimiR caused a dose-dependent inhibition of miR-378 expression in the heart, whereas SCR control had no effect (Fig. 1B). The specificity of antimiR-mediated targeting

TABLE 2 Effect of miR-378 inhibition on cardiac function by echocardiography in mice before (basal) and 7 days after scramble or antimiR injection
The number of animals in each group is given in parentheses. LVAWd, LV anterior wall during diastole; LVAWs, LV anterior wall during systole; LVPWd, LV posterior wall during diastole; LVPWs, LV posterior wall during systole; LVIDs, LV internal diameter during systole; LVIDd, LV internal diameter during diastole; EF, ejection fraction; FS, fractional shortening; E wave (mm/s), velocity of blood flow across the mitral valve during early diastole; A wave (mm/s), velocity of blood flow across the mitral valve during atrial contraction; E DT, E wave deceleration time; RWT, relative wall thickness. of miR-378 was validated by probing the same membrane for expression of miR-1 and miR-208a, which remained unchanged ( Fig. 1, B and C; data are not shown for miR-208a).

Parameters
For subsequent experiments, we used a 70 mg/kg dose of anti-miR that consistently decreased miR-378 expression by ϳ70% ( Fig. 1, C and D). We also analyzed tissue samples from the skeletal muscle, liver, and kidney of antimiR-injected animals and found a significant inhibition of miR-378 expression in these tissues (Fig. 1, E and F). As compared with the heart, the kidney, and liver had much lower basal expression of miR-378, consistent with previous findings (10,14); however, in our study the higher molecular weight antimiR-miR-378 duplex was observed only in the kidney, the tissue that exhibited least expression levels of miR-378. MiR-378 Depletion Induces Cardiac Hypertrophy in Mouse Hearts-We have previously shown that miR-378 is down-regulated in experimental models of cardiac hypertrophy as well as in human failing hearts (11,12), a finding also confirmed by others (13). To investigate whether miR-378 knockdown alone is sufficient to modulate cardiac gene expression in vivo, mice were injected with antimiR as described above. After 7 days of injection, the animals were sacrificed, and the hypertrophic response was determined by measuring the heart-to-body weight ratio, cardiac chamber dimensions, cardiomyocyte size, and expression of hypertrophy markers (ANF, BNP, ␣-skeletal actin, ␤-MHC). Fibrotic response of the heart was evaluated by measuring expression levels of collagen isoforms (Col1␣1 and Col3␣1), fibronectin (FN), connective tissue growth factor (CTGF), and by Masson's trichrome staining for collagen fibers. The cardiac function was assessed by M-mode echocardiography and by Doppler flow imaging. We found that inhibition of miR-378 had no significant effect on the mouse body weight or on the gross morphology of the heart. Measurements of HW/body weight, and HW/tibia length showed significantly FIGURE 2. Characterization of hypertrophy response after miR-378 inhibition. A, representative wheat germ agglutinin-stained images of cross-sections of cardiac tissues (40ϫ). B, measurement of cardiac cell size obtained in ImageJ. C, real-time PCR analysis of stress markers in antimiR and scramble control (SCR) animals with n in parentheses. D, Western analysis of the hypertrophy signaling cascade in individual hearts of animals treated with antimiR or SCR; the same membrane was stripped and re-probed with the indicated antibodies. E, quantification of normalized expression from D (n ϭ 6). F, Western analysis of GRB-2 and IGF1R expression in mouse hearts after antimiR and SCR administration. GAPDH was used as the loading control. G, quantification of normalized expression of GRB-2 and IGF1R (n ϭ 6). *, significant difference from SCR control.
higher ratios in antimiR than in SCR controls (Table 1). M-mode echocardiography revealed increased LV mass, anterior wall thickness, and relative wall thickness in antimiR-injected animals compared with pre-injection measurements. These parameters did not change with SCR (Table 2). AntimiR administration also resulted in increased cardiomyocyte crosssectional area (Fig. 2, A and B) and increased expression of hypertrophy markers, ANF, BNP, and ␣-skeletal actin. However, there was no change in ␤-MHC mRNA levels (Fig. 2C). These effects of AntimiR were associated with increased phosphorylation of AKT and GSK3␤, which correlated with reduced phosphorylation of GSK3␤ substrate, glycogen synthase, thus suggesting increased activity of pro-hypertrophic AKT and reduced anti-hypertrophic GSK3␤-signaling (Fig. 2, D and E). We also observed de-repression of previously identified (10 -13) direct targets of miR-378, IGF1R, and GRB-2 in antimiRtreated hearts (Fig. 2, F and G). These results thus indicated that miR-378 depletion induces cardiac hypertrophy and that miR-378 is critically involved in maintaining lower expression levels of IGF1R, GRB-2, and in the regulation of AKT-GSK3␤ signaling under basal conditions. We next examined the effects of miR-378 depletion on systolic and diastolic function of the mouse heart. The results showed that systolic function is preserved in antimiR-injected animals as there was no difference in fractional shortening or ejection fraction before and after antimiR treatment and between antimiR-injected and SCR control animals. Measurements of diastolic function by flow Doppler of trans-mitral flow velocities showed that antimiR treatment produced relatively faster early ventricular filling with higher E-wave amplitude and reduced deceleration time (E DT). The late ventricular filling A-wave amplitude or E/A ratios were, however, not significantly altered by antimiR (Table 2). Thus, miR-378 depletion over a 7-day treatment period caused only mild diastolic abnormalities.
Another consequence of induction of pathologic hypertrophy is the development of fibrosis. We, therefore, performed Masson's trichrome staining of serial heart sections, which showed increased formation of collagen fibers in the antimiR treated hearts compared with SCR controls (Fig. 3, A and B). Because we and others have previously shown that miR-378 is primarily expressed in cardiomyocytes (10,13), observing changes in the expression of fibrotic markers in antimiRtreated hearts with mostly preserved cardiac function was an unexpected finding. For further confirmation we compared mRNA levels of fibrotic markers Col1␣1, Col3␣1, FN, and CTGF by real-time PCR and found significant induction in all four gene transcripts in antimiR-treated than SCR control hearts. Similarly, there was increased protein expression of FN and Collagen1 in antimiR-treated hearts (Fig. 3, C-E). These data thus demonstrated that miR-378 depletion induces cardiac fibrosis in mouse hearts.

MiR-378 Depletion Exaggerates Angiotensin II-induced Cardiac Hypertrophy, Fibrosis, and Cardiac Dysfunction in Mice-
To further evaluate the role of miR-378 in cardiac remodeling, we studied the effect of miR-378 depletion in combination with  (20ϫ). B, quantification of collagen fiber area. A total of 10 -15 fields was evaluated from each heart (n ϭ 5), and the blue-stained collagen fiber area was measured using ImageJ and expressed as % of total field area analyzed. C, cardiac expression of fibrosis markers, CTGF, collagens (Col1␣1, Col3␣1), and FN by real-time PCR in antimiR and control (SCR) animals with n shown in parentheses. D, Western analysis of FN and collagen 1 in cardiac samples of two groups of animals. GAPDH was used as a loading control. The same membrane was re-probed after stripping. E, normalized expression from C; each bar represents the mean Ϯ S.D. of (n ϭ 5). *, significant difference from control. the hypertrophy agonist, Ang II. In our previous studies, 3 mg/kg/day infusion of Ang II for 2 weeks produced extensive fibrosis and severe cardiac dysfunction with reduced fractional shortening (22). In this study we used a low dose of Ang II (1 mg/kg/day) for 1 week to avoid severe adverse remodeling and to study the possible additive effects of miR-378 depletion. We found that at this dose, Ang II produced almost 15-20% increase in HW/tibia length ratio accompanied with significant induction of hypertrophy and fibrotic markers but without altering ejection fraction and fractional shortening, consistent with other reports using a similar dose (23,24).
We previously reported down-regulation of miR-378 during isoproterenol infusion and pressure-overload-induced hyper-trophy of the heart (12). In the current study we found that Ang II infusion also reduced miR-378 levels by ϳ40% when administered alone and ϳ85% when combined with antimiR (Fig. 4, A  and B). In antimiRϩAng II animals there was a larger increase in HW/tibia length ratio, cross-sectional area of cardiomyocytes, and mRNA levels of hypertrophy markers, ANF, BNP, ␣-skeletal actin, and ␤-MHC, than in SCRϩAng II-treated controls (Fig. 4, C-E). We also measured elements in the Ang IIinduced signaling cascade and found higher activation of prohypertrophic molecules such as pS6K, pERK1/pERK2, and pAKT in antimiR-treated hearts. The kinase activity of the antihypertrophic signaling molecule, GSK3␤, was reduced in antimiRϩAng hearts, as measured by the increased levels of pGSK3␤ and reduced phosphorylation of the substrate, glycogen synthase (Fig. 5, A and B). The de-repression of miR-378 targets, IGF1R and GRB-2, was also exacerbated by combined treatment of Ang II with antimiR than Ang II with SCR (Fig.  5C). Overall our findings indicated that miR-378 inhibition augments adverse cardiac remodeling induced by Ang II in mouse heart.
By M-mode echocardiography, antimiRϩAng II hearts showed a significantly larger increase in LV wall thickness particularly of the anterior wall, reduced LV cavity dimensions, and a larger increase in the calculated LV mass than SCRϩAng II hearts (Table 3). By trans-mitral flow Doppler measurements, as compared with their base-line values, SCRϩAng II hearts showed reduced E/A ratio and prolonged E-wave deceleration time, whereas antimiRϩAng II hearts showed increased E-wave amplitude with a shortened E wave deceleration time and significantly higher E/A ratio. Collectively echocardiography data suggested impaired myocardial relaxation in SCRϩ Ang II, which deteriorated further to a restrictive diastolic dysfunction in antimiRϩAng II group ( Table 3).
The pro-fibrotic activity of Ang II was significantly exaggerated when combined with antimiR than with SCR. This was reflected in a 1) significantly larger collagen fiber staining area, 2) higher induction of Col1␣1, Col3␣1, FN, and CTGF mRNAs, and 3) higher protein levels of fibronectin and collagen1 in antimiRϩAng II-treated hearts than the corresponding control hearts (Fig. 6, A-E). For further confirmation of pro-fibrotic effects of miR-378 inhibition, we tested fibrotic marker expression in another model of cardiac hypertrophy induced by transverse aortic constriction (TAC) for 4 weeks. Again we found greater induction of fibrotic markers in antimiRϩTAC hearts than SCRϩTAC hearts (Fig. 6F). From these data it became FIGURE 5. Hypertrophy signaling cascade and expression of direct targets of miR-378 in Ang II-stimulated and antimiR treated mouse hearts. A, Western analysis and quantification of phosphorylated S6 kinase (normalized to GAPDH) and pERK1/2 (normalized to ERK2) in mouse hearts of the indicated experimental groups. B, Western analysis and quantification of pAKT (normalized to total AKT) and pGSK3␤ and p-glycogen synthase (normalized to GAPDH) in heart samples of various experimental groups. C, Western analysis of miR-378 targets GRB-2 and IGF1R after angiotensin II infusion in control and antimiRtreated mouse hearts. GAPDH was used as a loading control. In A-C, the same membrane was sequentially used to probe with indicated antibodies after stripping the membrane. Each bar represents the mean Ϯ S.D. of n ϭ 5 -7 animals. The p prefix denotes the phosphorylated form. *, significant (p Ͻ 0.05) when compared SCR ϩ saline control; #, significant compared with SCR ϩ angiotensin group. apparent that miR-378 inhibition enhances cardiac fibrosis induced by Ang II as well as by pressure overload.

MiR-378 Depletion Induces TGF␤ Expression and Enhances Fibroblast Differentiation in Mouse Hearts by Involving a Paracrine
Signaling-To probe into the mechanism of fibrotic response with mostly preserved cardiac function, we posited that inhibition of miR-378, which is a cardiomyocytes-specific miRNA, modulates the expression of a secretory factor(s) within cardiomyocytes, which then enhances fibroblast differentiation and induces fibrosis.
To test our hypothesis we prepared conditioned media from miR-378-depleted cardiomyocytes after transfection of primary cultures of cardiomyocytes with antimiR. We measured levels of various cytokines (TNF␣, TGF␤, IL-6, IL-1␣, IL-1␤, MCP1, VEGF, and IL-15) in the conditioned media by using a multiplex ELISA assay. Results showed significantly higher levels of TGF␤1 in the conditioned media prepared from antimiR transfected myocytes as compared with that prepared from SCR control myocytes (Fig. 7A). The enhanced secretion of TGF␤1 from cardiomyocytes involved a corresponding increase in TGF␤1 mRNA levels in antimiR-treated mouse hearts and in cardiomyocytes (Fig. 7, B and C).
We next examined antimiR-treated mouse hearts for the expression of different components of TGF␤ signaling. These included TGF␤ receptors (TGF␤R1, TGF␤R2) and ligands (TGF␤1, TGF␤2, and TGF␤3). Results showed a significant increase only in TGF␤1 mRNA in antimiR-treated hearts than SCR-treated control group (Fig. 7C, data are not shown for other TGF␤-signaling components). More importantly TGF␤1 protein levels were also increased in antimiR-treated mouse hearts as compared with SCR controls (Fig. 7, D and E).
To gain further support for these findings, we performed a complementary experiment where cardiomyocytes were trans-fected with a double-stranded synthetic precursor of miR-378 (378-mimic); a random sequence (mimic-C) was used as a control. After 72 h of transfection we measured TGF␤ mRNA in cardiomyocytes and its release in the media. We found that a 378-mimic at higher concentrations (75 nM) reduced both TGF␤1 mRNA as well its release into the media (Fig. 7, F and  G). Additionally, we examined mRNAs of TGF␤1, Col1, Col3, CTGF, and FN in other tissues such as liver, kidney, and skeletal muscle tissues of antimiR-injected animals. We found higher levels of these mRNAs in kidneys of antimiR-injected animals; the induction of these transcripts in the liver and skeletal muscle tissues was not as robust and consistent as observed in kidneys (Fig. 8, A-D). These observations collectively indicated that miR-378 is a negative regulator of TGF␤1 in cardiomyocytes and its depletion results in the increased synthesis and release of TGF␤1 from cardiomyocytes and that the impact of miR-378 inhibition could be observed in remote tissues such as kidneys.
In the next series of experiments we examined whether conditioned media (CM) from miR-378-depleted myocytes is capable of modulating cardiac fibroblast activation. We prepared CM from miR-378-depleted cardiomyocytes (antimiR-CM) after transfection with 378-antimiR. CM of SCR-transfected myocytes (SCR-CM) was used as a control. The effect of CMs was tested on the activation of TGF␤ signaling in cardiac fibroblasts by measuring SMAD2/3 phosphorylation, a critical step for transcriptional activation of fibrotic gene program. We observed that treatment of fibroblasts with antimiR-CM, but not with SCR-CM, triggered phosphorylation of SMADs (Fig.  9A). We also found that treatment of fibroblasts with anti-miR-CM alone was sufficient to induce mRNAs of fibrotic markers (FN, Col1␣1, Col3␣1, and CTGF; Fig. 9B) as well as it

Effect of miR-378 inhibition on angiotensin-induced changes in echocardiographic measurements of left ventricular wall thickness, chamber dimensions, and systolic and diastolic function by two-dimensional, M-mode, conventional Doppler, and tissue Doppler imaging
LVAWd, LV anterior wall during diastole; LVAWs, LV anterior wall during systole; LVPWd, LV posterior wall during diastole; LVPWs, LV posterior wall during systole; LVIDs, LV internal diameter during systole; LVIDd, LV internal diameter during diastole; EF, ejection fraction; FS, fractional shortening; E wave (mm/s), velocity of blood flow across the mitral valve during early diastole; A wave (mm/s), velocity of blood flow across the mitral valve during atrial contraction; E DT, E wave deceleration time; RWT, relative wall thickness. promoted collagen and fibronectin protein expression induced by Ang II (Fig. 9, C-E).

Parameters
To gain further support for these findings, we took advantage of the ability of adrenergic stimulation of cardiomyocytes to induce TGF␤1 expression (25). We tested the combined effect of miR-378 inhibition and adrenergic stimulation of cardiomyocytes with phenylephrine on cardiac fibroblast activation. The results showed that antimiR-CM of phenylephrinestimulated cardiomyocytes further enhanced fibronectin expression than the SCR-CM of similarly stimulated myocytes ( Fig. 9, D and E), thus again supporting pro-fibrotic activity of antimiR-CM involving TGF␤1. To further confirm involvement of TGF␤1 in antimiR-mediated fibrotic response, we included a TGF␤1-neutralizing antibody during cardiac fibroblasts incubation with antimiR-CM. We found that TGF␤1neutralizing antibody, but not a control antibody, counteracted antimiR-CM-stimulated induction of FN, collagens, and CTGF mRNAs as well as it reduced protein expression of FN (Fig. 9, F  and G). These data collectively established that inhibition of miR-378 in cardiomyocytes caused increased synthesis and FIGURE 6. Increased cardiac fibrosis by antimiR after angiotensin II and transverse aortic constriction in mouse hearts. A, sections of cardiac muscle tissue with Masson's trichrome stain for collagen (blue) fibers. Magnification scale is shown at the lower right. B, quantification of the collagen fiber area measured as described for Fig. 3B. C, representative Western blots showing increased expression of collagen 1 and of FN by angiotensin in antimiR-treated hearts than in scramble control. D, quantification of FN and collagen expression (n ϭ 5 in each group). E, real-time PCR analysis of mRNA levels of fibrotic markers in antimiR and scramble control (SCR) animals in combination with saline (Sal) or angiotensin (Ang) infusion. Data are presented as the mean Ϯ S.D. of 8 -10 hearts. *, significant as compared with SCR ϩ Sal group; #, significant when compared with SCR ϩ Ang group. F, real-time PCR analysis of fibrotic marker expression in sham control (SCR sham), pressure overload control (SCR ϩ TAC), and pressure overload antimiR (AntimiR ϩ TAC) hearts. Each bar represents the mean Ϯ S.D. of n ϭ 4 -6 animals in each group. *, significant as compared with sham SCR control; #, significant (p Ͻ 0.05) when compared with SCR ϩ TAC group.
release of TGF␤1, which consequently led to the activation of cardiac fibroblasts and induction of fibrotic gene expression.
TGF␤1 is known to stimulate its own gene promoter via enhancing transcriptional activity of AP-1 (c-Fos-c-Jun complex) and Ras-signaling in a positive feed-forward regulatory mechanism (26 -28). In our published report we demonstrated that in cardiomyocytes inhibition of miR-378 led to induction of Ras activity (12). In this study we also observed increased Ras activity in antimiR-treated mouse hearts (data not shown). We, therefore, asked whether 378-antimiR-stimulated TGF␤1 expression in cardiomyocytes could be mediated by activation of AP-1 and/or induced Ras-signaling. To this end we first investigated the effect of antimiR on c-Fos and c-Jun expres-sion. We found that in cells where miR-378 was depleted, there was increased expression of both of these factors. This was further confirmed when the same membrane was probed for c-Jun phosphorylation, an indicator of c-Jun activity (Fig. 10A). We next tested involvement of Ras or c-Jun in the antimiR-stimulated induction of TGF␤1 by using adenovirus vectors expressing either dominant negative N17-Ras or dominant negative c-Jun and measuring pc-Jun. The results showed that antimiRmediated activation of c-Jun was abolished by expression of Dn-Ras as well as by Dn-c-Jun. AntimiR-mediated induction of TGF␤1 mRNA was also abrogated in cardiomyocytes expressing these Dn-adenoviruses but not in those infected with a control adenovirus. Furthermore, a chemical inhibitor of AP-1, FIGURE 7. miR-378-dependent regulation of TGF␤1 expression and release. A, multiplex ELISA assay of secreted cytokines in the conditioned media of miR-378-depleted cardiomyocytes (AntimiR cond med) and control myocytes (SCR cond med). ODU, optical density units. B, cytokine mRNAs in myocytes (n ϭ 5) analyzed by real-time PCR. C, real-time PCR analysis of TGF␤1 mRNAs in mouse hearts in response to miR-378 inhibition. D, Western analysis of TGF␤1 expression in individual heart lysates of control (Scramble) and antimiR-treated mice. E, quantification of TGF␤1 protein from D in mouse hearts (n ϭ 6 samples in each group). F, real-time PCR analysis of TGF␤1 mRNAs in cardiomyocytes transfected with precursor of miR-378 (378-mimic) or a control (mimic-C). G, ELISA measurement of TGB␤ release in the conditioned-media by miR-378 overexpressing (378-mimc) or (control Mimic-C) myocytes. *, significant as compared with SCR control.
Tanshinone, which blocked antimiR-induced c-Jun activation, also inhibited antimiR-mediated TGF␤1 induction (Fig. 10, B  and C). These data thus demonstrated that activation of Rassignaling and c-Fos and c-Jun activities significantly contribute to the induction of TGF␤1 resulting from miR-378 depletion in cardiac myocytes.

DISCUSSION
Data presented in the current study define a novel regulatory role of miR-378 in the maintenance of cardiac cellular homeostasis and control of cardiac fibroblast activation. We believe that miR-378 is a cardio-protective miRNA. In pathological conditions, when miR-378 levels are depleted, cardiomyocytes synthesize and release TGF␤1 and sensitize cardiac fibroblasts to pathological stimuli. Based on our findings reported here, a working model illustrating miR-378-mediated paracrine regulation of cardiac fibrosis is outlined in Fig. 11.
Several lines of experimental evidences are presented here to support that miR-378 is a negative regulator of TGF␤1 and cardiac fibrosis involving a paracrine mechanism. 1) Systemic inhibition of miR-378 by a LNA-modified antimiR alone led to induction of TGF␤1 mRNA and protein levels and stimulated fibrotic gene expression in mouse hearts. 2) When miR-378 depletion was combined with pro-fibrotic stimuli such as angiotensin II or transverse aortic constriction, a synergistic induction of cardiac fibrosis was observed in mouse hearts. 3) Depletion of miR-378 in cardiomyocytes induced TGF␤1 expression, whereas replenishment of miR-378 by a miR-378mimic counteracted this effect. 4) Conditioned media of miR-378-depleted cardiomyocytes exhibited higher levels of TGF␤1. 5) Cardiac fibroblasts when treated with conditioned media of miR-378-depleted cardiomyocytes showed activated phenotype, which was suppressed by a TGF␤-neutralizing antibody. 6) We show that induction of TGF␤1 expression in miR-378-depleted cardiomyocytes largely depended on the activation of Ras signaling and on the expression of AP-1 transcription factors. To the best of our knowledge, our study is the first report demonstrating the ability of a cardiomyocyte-specific microRNA to regulate activation of cardiac fibroblasts by indirect modulation of TGF␤1 synthesis and release from cardiomyocytes.
Because induction of fibrosis is a common characteristic of many diseases, understanding the molecular regulation of fibroblast activation is of intense research interest. In the heart, development of fibrosis is a pathological feature associated with a variety of cardiomyopathies including myocardial infarction, hypertension, cardiac hypertrophy, and aging. Many studies suggest that cardiac muscle and nonmuscle cells communicate through exchange of a variety of secreted proteins, growth factors, and hormones, which together form a complex regulatory network. Perturbations in this regulatory network by pathological factors lead to activation and differentiation of fibroblasts into myofibroblasts, which synthesize excessive extracellular matrix proteins leading to the development of tissue fibrosis.
In experiments with primary cultures of cardiomyocytes as well as in in vivo in mouse hearts, we found that inhibition of miR-378 led to increased expression of TGF␤. TGF␤1 is a prominent example of a secreted cytokine that is produced both by cardiomyocytes and cardiac fibroblasts and has been considered a central mediator of tissue fibrosis in the heart (29,30). Its expression and signaling activity are significantly increased in experimental models of cardiac hypertrophy and human heart disease (30,31). In our study we found that the conditioned medium of miR-378-depleted cardiomyocytes had higher TGF␤ levels and induced fibroblast gene expression that was blocked by a TGF␤1 neutralizing antibody. Another cardiomyocyte-specific miRNA, miR-133 has also shown anti-fibrotic properties (32). Genetic deletion of miR-133 resulted in the induction of cardiac fibrosis. These effects involved direct targeting of TGF␤1 and also of collagen mRNA (33)(34)(35), although mechanistic details on targeting of fibroblast gene by a cardiomyocyte-specific miRNA were not addressed in these studies. In our study we observed no binding site of miR-378 in the 3Ј-UTR of TGF␤ mRNA. We found that miR-378-antimiR increased TGF␤1 expression and release from cardiomyocytes indirectly by activating Ras signaling and AP1 transcription factors, c-Fos, and c-Jun within cardiomyocytes. These findings were also supported from our data obtained from the use of dominant-negative adenoviruses against N-Ras and c-Jun. The 378-antimiR-mediated induction of TGF␤1 mRNA was not only blocked by c-Jun inhibition but also by the inhibition of Ras, suggesting Ras as an upstream regulator in this process. Induction of Ras in antimiR-treated hearts is consistent with our previously published studies, where we reported miR-378 depletion in cardiomyocytes-induced Ras activation (10,12).
Of note, activation of the Ras signaling pathway is also known to induce c-Jun phosphorylation and its transcriptional activity (36). A role of Ras GTPases in inducing cardiac hypertrophy is fairly established (37). It has been shown that besides controlling cell growth, Ras GTPases also regulate TGF␤/SMAD signaling but in a cell-context dependent manner. In epithelial cells, overexpression of constitutively active Ras blocked nuclear accumulation of SMADs, whereas in kidney mesangial cells, it promoted Smad3-dependent processes (28,38,39). In our study, besides heart, we also found higher up-regulation of TGF␤1 and fibrotic markers in kidneys than the liver or skeletal muscles of antimiR-treated mice. In this regard it should be noted that kidney and liver exhibit considerably lower expression levels of miR-378 than skeletal muscle and heart tissues. Yet, antimiR treatment induced fibrotic gene expression only in kidneys and in cardiac tissues, suggesting that it is not the absolute expression level of miR-378 but, rather, tissue-specific factors that must also play a role in the induction of tissue-specific fibrotic response.
In previous studies we and others have identified several members of the receptor-tyrosine kinase/Ras/ERK-signaling pathway as direct targets of miR-378 in cardiomyocytes (10,12,13). These included IGF1R, GRB-2, KSR-1, and ERK1. In the present study we found activation of downstream Ras effectors, AKT, S6K, and ERKs, and de-repression of IGF1R and GRB-2 in antimiR-treated mouse hearts. In the receptor-tyrosine kinase/ Ras/ERK signaling pathway GRB-2 is an adapter protein that is essential for the recruitment of SOS (son of sevenless) to the cell membrane and for the activation of the Ras-signaling cascade (40). KSR-1 is a scaffold protein required for binding of the Raf to Ras and for the activation of downstream MAPK cascade that includes MEK and ERK. Research has shown that PI3K-AKT as well as MAPK-ERK signaling cascades activate and also get activated by TGF␤ signaling and that both c-Fos and c-Jun also serve as substrates for ERKs (see Ref. 41 for an excellent review). Therefore, by direct targeting of several members of the receptor-tyrosine kinase/Ras/ERK signaling pathway, miR-378 plays a significant role in the regulation of TGF␤ synthesis and the activity of TGF␤ signaling in the heart.
There are 10 miR-378 isomiR sequences that are described to originate from different genomic loci in the most recent available microRNA database and as illustrated in our previous pub-  . Proposed model of miR-378 dependent paracrine signaling from cardiac myocytes in the regulation of fibroblast activation. Pathological stimuli down-regulate miR-378, which results in the de-repression of its targets, IGF1R (receptor-tyrosine kinase (RTK)) and GRB-2, and induction of Ras activity. The pathway shown in green demonstrates that inhibition of miR-378 induces cardiac fibrosis by inducing Ras and AP-1 transcription factors (c-Jun, c-Fos). This then induces synthesis and release of TGF␤ from cardiomyocytes leading to fibroblast activation and development of fibrosis.
The pathway marked in red shows that inhibition of miR-378 induces the PI3K-AKT-GSK3␤ pro-hypertrophic signaling cascade, as demonstrated in in vitro in cardiomyocytes in our earlier publication (12) and verified here in the present study in vivo in mouse hearts.
lication (12). These isomiRs share the exact same seed sequence and differ at most by only two nucleotides outside the seed region. The isoform miR-378a (also known as miR-378, miR-378a-3p, and miR-422b) originates from the first intron of the PGC1␤ gene, which also co-transcribes miR-378* (also known as miR-378 and miR-378 -5p). These two miRNAs possess distinct seed sequences and, accordingly, target distinct sets of mRNAs. An earlier study involving whole body knockout of miR-378 from its PGC1␤ gene locus showed a metabolic role of miR-378 and miR-378* in the control of mitochondrial metabolism and maintenance of systemic energy homeostasis (14). These mice were found resistant to high fat diet-induced obesity, which correlated with direct targeting of carnitine-Oacetyltransferase (CRAT) by miR-378 and of mediator complex subunit 13 (MED-13) by miR-378*. Intriguingly these targets were found to be de-repressed only in the liver tissues of the animals fed with high fat diet but not in the skeletal or cardiac tissues of miR-378 KO mice irrespective of normal chow or high fat diet, again suggesting a role of tissue-specific factors in miR-378-mediated targeting. Although the effects of genetic deletion of miR-378/miR-378* on cardiac hypertrophy and fibrotic markers were not analyzed in that study, the H&E staining of cardiac tissue of KO animals on high fat diet revealed no abnormalities in myofiber structure or organization. In our study short term treatment with antimiR, which was designed to target all forms of miR-378 but that spared miR-378*, we found evidence of cardiac hypertrophy and fibrosis. Whether cardiac hypertrophy and fibrosis is a feature of combined deletion of miR-378a and miR-378* and/or the possibility that miR-378* acts as a pathological molecule in the absence of miR-378 remains to be addressed in future studies.
Based on our findings, we conclude that miR-378 is a cardioprotective miRNA, and its presence is required to resist adverse ventricular remodeling during cardiac stress. Our study implicates that previously suggested therapeutic targeting of miR-378 in metabolic disorders (14) should be exercised with caution as it could have deleterious consequences particularly in pre-existing myocardial diseases.