Paracrine regulation of cardiac fibrosis by miR-378

Understanding the regulation of cardiac fibrosis is critical for controlling adverse cardiac remodeling during heart failure. Previously we identified miR-378 as a cardiomyocytes-abundant miRNA downregulated 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 LNA-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 conditionedmedia 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 NRas 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 presence in cardiomyocyte of miR-378 plays a critical role in the protection of http://www.jbc.org/cgi/doi/10.1074/jbc.M114.580977 The latest version is at JBC Papers in Press. Published on August 7, 2014 as Manuscript M114.580977 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 2 neighboring fibroblasts from activation by profibrotic 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, . 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 via acting through paracrine mechanisms, promote hypertrophy of neighboring cardiomyocytes, and via 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 are highly conserved small (18 – 22nt) non-coding RNA molecules. They regulate gene expression post-transcriptionally by partially pairing with the 3′-untranslated region of the target mRNAs, repressing their translation and / or accelerating mRNA decay . 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 patho-physiological 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, reviewed in reference. 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, miR133 and miR-30 are known to suppress expression of collagens and ECM proteins. While miR-29 is expressed in fibroblasts, miR-133 is in myocytes, and miR-30 is present both in myocytes and fibroblasts. 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. 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. Over-expression of miR-378 protected cardiomyocytes from undergoing hypertrophy and prevented cardiac dysfunction induced by pressure overload in mice. Based on these results, miR-378 is considered as an anti-hypertrophic miRNA. In contrast to the anti-hypertrophic effects of miR378 over-expression, whole body knockout of miR-378 also showed beneficial results against high-fat diet induced changes in systemic energy metabolism. 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 miR378 alone led to increased expression and by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 3 secretion of profibrotic cytokine, TGFβ1, by involving Ras-signaling dependent mechanisms and AP-1 transcription factor activities. Our study thus demonstrates a new protective role of miR378 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 Figure 1A. The LNA control (SCR) was a Celegans–specific miRNA. Adult C57BL/6 mice were injected with LNA-antimiR or LNA-SCR (70 mg / kg, i.p.) 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 and by Angiotensin II (Ang II, 1mg/kg/day) for 1 week. Subcutaneously implanted osmotic mini pumps (ALZET model 1007D) were used to deliver Ang II at a flow rate 0.5μl / h / day. Echocardiography: Transthoracic echocardiography in mice was performed at UIC Center for Cardiovascular research core facility in a blinded fashion under isoflurane (~1%) anesthesia with a VisualSonics Vevo 770 instrument, using a 30 MHz high-frequency transducer as described . Cell culture, transfection and treatments: Primary cultures of cardiomyocytes and fibroblasts were prepared from neonatal Sprague Dawley rats as described before. Fibroblasts obtained during 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 2 or 3 passage were used all throughout the experiment. Cells were treated with phenylephrine (PE, 20 μM) or Ang II (100 nM) for 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 Inc) 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 Figure 1. All transfections were carried out in OptiMEM using Lipofectamine 2000 (Invitrogen) according to our published procedures . For adenovirus infection, cardiomyocytes were infected at a multiplicity of infection of 10 in complete growth medium. For knocking down Ras signaling and CJun expression, adenoviruses Ad-CMV-c-Ras (Dn) (N17; 1031; Vector biolabs) and Ad-CMV-cJun (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 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 pooled, and labeled as conditioned medium. This was centrifuged at low speed to remove cell debris, frozen in aliquots and was 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 NC membrane and hybridized with radiolabelled microRNA-specific 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 βMHC were analyzed by using Taqman assays, remaining mRNAs were analyzed by SYBR Green by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 4 based assays. Primer sequences are available upon request. Tissue histology and immuno-staining: Hearts were isolated and perfusion-fixed and processed for light microscopy. Tissue embedding and staining of heart sections was performed by the histology core facility at University of Chicago . Cell imaging was performed on a Bio-Rad Laser Sharp 2000 system (Bio-Rad) using a 40X objective (Zeiss). Hearts were excised, perfused with saline and fixed in formalin. Following dehydration in graded ethanol solutions, tissues were cleared with xylene and processed for embedding. The paraffin-embedded hearts were sectioned at 4 μm and subsequently stained with wheat germ agglutinin coupled to tetramethylrhodamine isothiocyanate (Sigma-Aldrich) for detection of cell size as described earlier. Left ventricular myocyte cross-sectional area (CSA) 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 image J software (http://rsbweb.nih.gov/ij/). 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. Collagen fraction (stained with aniline blue in Masson’s trichrome–stained 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 five replicates of each sample, and 10 visual fields measured in each replicate. Fibroblasts (10,000 to 20,000) were plated on coverslips and processed for immuno-staining for Collagen and Vimentin as per the protocols described previously 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, CA) as per manufacture’s protocol. Antibodies The following antibodies were used in this study: Akt1(C73H10; Cell Signaling), P-Akt (Ser473; Cell Signaling), P-p44/42MAPK(T202/Y204; Cell Signaling), P-GSK3-β (S9; Cell Signaling), P-p70S6Kinase(T389; Cell Signaling), IGF1R(3027; Cell Signaling), P-Glycogen Synthase (Ser641; Cell Signaling), Glycogen Synthase(15B1; Cell Signaling), GAPDH(sc-25778; Santa Cruz), TGFβ1 (sc-146; Santa Cruz), ERK-2(sc-154; Santa Cruz), GRB-2(sc-255; Santa Cruz), βactin(sc-1616; Santa Cruz), P-c-Jun(sc-822; Santa Cruz), c-Jun(sc-1694; Santa Cruz), Fibronectin(sc9068; Santa Cruz), c-Fos(sc-253; Santa Cruz), PSmad2/3(Ser423/ Ser425; sc-11679; Santa Cruz), HRP-conjugated anti-mouse (A2304; Sigma), HRP-conjugated anti-goat (sc-2020; Santa Cruz), HRP-conjugated anti-rabbit (7074; Cell Signaling), Anti-atrial natriuretic factor Rabbit (T4014; Peninsula Laboratories), Anti-Collagen Type I Rabbit (234167; Calbiochem), AntihTGFβ1 Mouse (MAB240; R&D Systems), AntiVimentin Mouse (V5255; Sigma) donkey antigoat IgG-Alexa Fluor 594 (A11058; Invitrogen) and donkey anti-rabbit IgG-Alexa Fluor 594 (A21207; Invitrogen). Statistical analysis: Data are expressed as mean ± S.D. Student’s t-test and 1-way ANOVA were used for statistical analysis, Echocardiography data were analyzed with ANOVA followed by the Bonferroni post hoc test in Graphpad Prism. Differences with a p value of <0.05 were considered statistically significant. RESULTS 1. Inhibition of cardiac miR-378 by antimiR: To understand the role of miR-378 in cardiac pathophysiology, we used a locked nucleic acid (LNA) modified oligonucleotide to knockdown endogenous miR-378 in mice. LNA-based oligonucleotide modification has been shown to by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 5 impart resistance to nuclease degradation in vivo with minimal biological toxicity. This approach has been used by several investigators for the therapeutic inhibition of microRNAs in vivo. 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 data base. 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, i.p.), and analyzing the expression of miR-378 on the 8 day. Control animals injected were 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 antimiRmediated targeting of miR-378 was validated by probing the same membrane for expression of miR-1 and miR-208a, which remained unchanged (Fig. 1B, 1C, data not shown for miR-208a). For subsequent experiments, we used 70 mg / kg dose of antimiR which consistently decreased miR-378 expression by about 70% (Fig. 1C, 1D). 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. 1E, 1F). As compared to the heart, the kidney and liver had much lower basal expression of miR-378, consistent with previous findings , however in our study the higher molecular weight antimiRmiR-378 duplex was observed only in the kidney, the tissue that exhibited least expression levels of miR-378. 2. 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 , a finding also confirmed by others. 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 heart-to-body weight ratio, cardiac chamber dimensions, cardiomyocyte size, and expression of hypertrophy markers (ANF, BNP, α-skeletal actin, β-MHC). Fibrotic response of the heart was evaluated by measuring expression levels of collagen isoforms (Col1α1 and Col3α1), 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 / BW, and HW / TL showed significantly 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 to pre-injection measurements. These parameters did not change with SCR (Table 2). AntimiR administration also resulted in increased cardiomyocyte cross-sectional area (Fig. 2A, 2B), and increased expression of hypertrophy markers, ANF, BNP and α-skeletal actin. However, there was no change in β-MHC mRNA levels (Fig. 2C). These effects of AntimiR were associated with increased phosphorylation of AKT and GSK3β, which correlated with reduced phosphorylation of GSK3β substrate, glycogen synthase, thus suggesting increased activity of pro-hypertrophic AKT and reduced anti-hypertrophic GSK3βsignaling (Fig. 2D, 2E). We also observed derepression of previously identified 10-13 direct targets of miR-378, IGF1R and GRB-2 in antimiR treated hearts (Fig. 2F, 2G). 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 AKTGSK3β-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 by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 6 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’sTrichrome staining of serial heart sections which showed increased formation of collagen fibers in the antimiR treated hearts, compared to SCR controls (Fig. 3A and 3B). Since we and others have previously shown that miR-378 is primarily expressed in cardiomyocytes , observing changes in the expression of fibrotic markers in antimiR treated hearts with mostly preserved cardiac function was an unexpected finding. For further confirmation we compared mRNA levels of fibrotic markers Col1α1, Col3α1, FN and CTGF by real-time PCR and found significant induction in all 4 gene transcripts in antimiR treated than SCR control hearts. Similarly, there was increased protein expression of FN and Collagen1 in antimiR treated hearts (Fig. 3C – 3E). These data thus demonstrated that miR-378 depletion induces cardiac fibrosis in mouse hearts. 3. 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 the hypertrophy agonist, angiotensin II (Ang II). In our previous studies, 3 mg /kg/day infusion of Ang II for 2 wks produced extensive fibrosis and severe cardiac dysfunction with reduced fractional shortening. In this study, we used a low dose of Ang II (1 mg /kg/day) for 1 wk to avoid severe adverse remodeling, and to study possible additive effects of miR-378 depletion. We found that at this dose, Ang II produced almost 15 20% increase in HW / TL 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 . We have previously reported downregulation of miR-378 during isoproterenolinfusion and pressure-overload induced hypertrophy of the heart. In the current study we found that Ang II infusion also reduced miR-378 levels by ~40% when administered alone, and about 85% when combined with antimiR (Fig. 4A, 4B). In antimiR+AngII animals there was a larger increase in HW / TL ratio, cross-sectional area of cardiomyocytes and mRNA levels of hypertrophy markers, ANF, BNP, α-skeletal actin and β-MHC, than in SCR+AngII treated controls (Fig. 4C – 4E). We also measured elements in the Ang IIinduced signaling cascade, and found higher activation of pro-hypertrophic molecules such as pS6K, pERK1/pERK2 and pAKT in antimiR treated hearts. The kinase activity of the antihypertrophic signaling molecule, GSK3β was reduced in antimiR+Ang hearts, as measured by increased levels of pGSK3β and reduced phosphorylation of the substrate, glycogen synthase (Fig. 5A and 5B). 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, antimiR+AngII 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 SCR+AngII hearts (Table 3). By trans-mitral flow Doppler measurements, as compared to their baseline values, SCR+AngII hearts showed reduced E/A ratio and prolonged E wave deceleration time, whereas antimiR+AngII hearts showed increased E-wave amplitude with shortened E wave deceleration time and significantly higher E/A ratio. Collectively Echo data suggested impaired myocardial relaxation in SCR+AngII, which deteriorated further to a by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 7 restrictive diastolic dysfunction in antimiR+AngII group (Table 3). The pro-fibrotic activity of Ang II was significantly exaggerated when combined with antimiR than with SCR. This was reflected in (1) significantly larger collagen fiber staining area (2) higher induction of Col1α1, Col3α1, FN and CTGF mRNAs and, (3) higher protein levels of fibronectin and collagen1 in antimiR+AngII treated hearts than the corresponding control hearts (Fig. 6A – 6E). 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 wks. Again we found greater induction of fibrotic markers in antimiR+TAC hearts than SCR+TAC hearts (Fig. 6F). From these data it became apparent that miR-378 inhibition enhances cardiac fibrosis induced by Ang II as well as by pressure overload. 4. MiR-378 depletion induces TGFβ 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 (TNFα, TGFβ, IL-6, IL-1α, IL-1β, MCP1, VEGF and IL15) in the conditioned media by using a multiplex ELISA assay. Results showed significantly higher levels of TGFβ1 in the conditioned media prepared from antimiR transfected myocytes as compared to that prepared from SCR control myocytes (Fig. 7A). The enhanced secretion of TGFβ1 from cardiomyocytes involved a corresponding increase in TGFβ1 mRNA levels in antimiR-treated mouse hearts and in cardiomyocytes (Fig. 7B and 7C). We next examined antimiR treated mouse hearts for the expression of different components of TGFβ-signaling. These included TGFβ receptors (TGFβR1, TGFβR2), and ligands (TGFβ1, TGFβ2 and TGFβ3). Results showed a significant increase only in TGFβ1 mRNA in antimiR-treated hearts than SCR-treated control group (Fig. 7C, data not shown for other TGFβsignaling components). More importantly TGFβ1 protein levels were also increased in antimiRtreated mouse hearts as compared to SCR controls (Fig. 7D and 7E). To gain further support for these findings, we performed a complementary experiment where cardiomyocytes were transfected with a doublestranded synthetic precursor of miR-378 (378mimic), a random sequence (mimic-C) was used as a control. After 72 h of transfection we measured TGFβ mRNA in cardiomyocytes and its release in the media. We found that expression of 378-mimic reduced both TGFβ1 mRNA as well well its release into the media (Fig. 7F and 7G). Additionally, we examined mRNAs of TGFβ1, Col1, Col3, CTGF and FN in other tissues such ass 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. 8A – 8D). These observations collectively indicated that miR-378 is a negative regulator of TGFβ1 in cardiomyocytes and its depletion results in the increased synthesis and release of TGFβ1 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 conditioned medium (CM) from miR378 depleted cardiomyocytes (antimiR-CM) following 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 TGFβ 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 by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 8 with SCR-CM, triggered phosphorylation of SMADs (Fig. 9A). We also found that treatment of fibroblasts with antimiR-CM alone was sufficient to induce mRNAs of fibrotic markers (FN, Col1α1, Col3α1 and CTGF, Fig. 9B) as well as it promoted collagen and fibronectin protein expression induced by Ang II (Fig. 9C – 9E) To gain further support for these findings, we took advantage of the ability of adrenergic stimulation of cardiomyocytes to induce TGFβ1 expression. We tested the combined effect of miR-378 inhibition and adrenergic stimulation of cardiomyocytes with phenylephrine (PE) on cardiac fibroblast activation. The results showed that antimiR-CM of PE stimulated cardiomyocytes further enhanced fibronectin expression, than the SCR-CM of similarly stimulated myocytes (Fig. 9D and 9E), thus again supporting pro-fibrotic activity of antimiR-CM involving TGFβ1. To further confirm involvement of TGFβ1 in antimiRmediated fibrotic response, we included a TGFβ1 neutralizing antibody during cardiac fibroblasts incubation with antimiR-CM. We found that TGFβ1-neutralizing 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. 9F and 9G). These data collectively established that inhibition of miR-378 in cardiomyocytes caused increased synthesis and release of TGFβ1, which consequently led to the activation of cardiac fibroblasts and induction of fibrotic gene expression. TGFβ1 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. In our published report we demonstrated that in cardiomyocytes, inhibition of miR-378 led to induction of Ras activity. In this study we also observed increased Ras activity in antimiR treated mouse hearts (data not shown). We therefore asked whether 378-antimiR-stimulated TGFβ1 expression in cardiomyocytes could be mediated by activation of AP-1 and / or induced Rassignaling. To this end, we first investigated the effect of antimiR on c-Fos and c-Jun expression. 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 TGFβ1 by using adenovirus vectors expressing either dominant negative N17-Ras or dominant negative c-Jun and measuring pc-Jun. The results showed that antimiR-mediated activation of c-Jun was abolished by expression of Dn-Ras as well as by Dn-c-Jun. AntimiR-mediated induction of TGFβ1 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, Tanshinone, which blocked antimiR-induced c-Jun activation, also inhibited antimiR-mediated TGFβ1 induction (Fig. 10B and 10C). These data thus demonstrated that activation of Ras-signaling and c-Fos and c-Jun activities significantly contribute to the induction of TGFβ1 resulting from miR-378 depletion in cardiac myocytes. DISCUSSION Data presented in the current study defines 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 miR378 levels are depleted, cardiomyocytes synthesize and release TGFβ1 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 Figure 11. Several lines of experimental evidences are presented here to support that miR-378 is a negative regulator of TGFβ1 and cardiac fibrosis involving a paracrine mechanism. (1) Systemic inhibition of miR-378 by a LNA-modified antimiR alone led to induction of TGFβ1 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 cultured cardiomyocytes by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 9 induced TGFβ1 expression, whereas replenishment of miR-378 by a miR-378-mimic counteracted this effect. (4) Conditioned-media of miR-378-depleted cardiomyocytes exhibited higher levels of TGFβ1. (5) Cardiac fibroblasts when treated with conditioned-media of miR-378 depleted cardiomyocytes showed activated phenotype, which was suppressed by a TGFβneutralizing antibody. (6) We show that induction of TGFβ1 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 cardiomyocytespecific microRNA to regulate activation of cardiac fibroblasts by indirect modulation of TGFβ1 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 non-muscle 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 TGFβ. TGFβ1 is a prominent example of a secreted cytokine which is produced both by cardiomyocytes and cardiac fibroblasts, and has been considered a central mediator of tissue fibrosis in the heart . Its expression and signaling activity are significantly increased in experimental models of cardiac hypertrophy and human heart disease . In our study we found that the conditioned medium of miR-378 depleted cardiomyocytes had higher TGFβ levels and induced fibroblast gene expression which was blocked by a TGFβ1neutralizing antibody. Another cardiomyocyte specific miRNA, miR-133 has also shown antifibrotic properties. Genetic deletion of miR-133 resulted in the induction of cardiac fibrosis. These effects involved direct targeting of TGFβ1 and also of collagen mRNA 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 3’UTR of TGFβ mRNA. We found that miR-378-antimiR increased TGFβ1 expression and release from cardiomyocytes indirectly by activating Rassignaling 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 TGFβ1 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 . Of note, activation of the Ras signaling pathway is also known to induce c-Jun phosphorylation and its transcriptional activity. A role of Ras GTPases in inducing cardiac hypertrophy is fairly established. It has been shown that besides controlling cell growth, Ras GTPases also regulate TGFβ / SMAD signaling, but in a cell-context dependent manner. In epithelial cells, over-expression of constitutively active Ras blocked nuclear accumulation of SMADs, whereas in kidney mesangial cells, it promoted Smad3-dependent processes 38, . In our study, besides heart, we also found higher upregulation of TGFβ1 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 by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 10 absolute expression level of miR-378 rather tissuespecific factors that must also be playing a role in the induction of tissue-specific fibrotic response. In previous studies, we and others have identified several members of receptor tyrosine kinase (RTK) / Ras / ERK-signaling pathway as direct targets of miR-378 in cardiomyocytes 12, . 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 RTK/Ras/ERK signaling pathway Grb2 is an adapter protein which is essential for the recruitment of SOS (son of sevenless) to the cell membrane and for the activation of Ras-signaling cascade. 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 TGFβ-signaling, and that both c-Fos and c-Jun also serve as substrates for ERKs (see reference for an excellent review). Therefore, by direct targeting of several members of RTK/Ras/ERK signaling pathway, miR-378 plays a significant role in the regulation of TGFβ synthesis and the activity of TGFβ-signaling in the heart. There are ten miR-378 isomiR sequences that are described to originate from different genomic loci in the most recent available microRNA database (www.mirbase.org) and as illustrated in our previous publication. 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 PGC1β 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 PGC1β gene locus showed a metabolic role of miR-378 and miR-378* in the control of mitochondrial metabolism and maintenance of systemic energy homeostasis. These mice were found resistant to high fat diet induced obesity, which correlated with direct targeting of carnitine-O-acetyltransferase (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 / miR378* 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 with spared miR378*, we found evidence of cardiac hypertrophy and fibrosis. Whether cardiac hypertrophy and fibrosis is a feature of combined deletion of miR378a 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 cardio-protective 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 should be exercised with caution as it could have deleterious consequences particularly in preexisting myocardial diseases. by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Paracrine regulation of cardiac fibrosis by miR-378 11 Acknowledgements: Authors thank Jose J. Vargas for technical assistance.