Myocardial knockdown of mRNA-stabilizing protein HuR attenuates post-MI inflammatory response and left ventricular dysfunction in IL-10-null mice

Abstract

Prolonged inflammatory response is associated with left ventricular (LV) dysfunction and adverse remodeling following myocardial infarction (MI). IL-10 inhibits inflammation by suppressing HuR-mediated mRNA stabilization of proinflammatory cytokines. Here we report that following MI, IL-10^−/− mice showed exaggerated LV dysfunction, fibrosis, and cardiomyocyte apoptosis. Short-hairpin RNA (shRNA)-mediated knockdown of HuR in the myocardium significantly reversed MI-induced LV dysfunctions and LV remodeling. HuR knockdown significantly reduced MI-induced cardiomyocyte apoptosis concomitant with reduced p53 expression. Moreover, HuR knockdown significantly reduced infarct size and fibrosis area, which in turn was associated with decreased TGF-β expression. In vitro, stable knockdown of HuR in mouse macrophage cell line RAW 264.7 corroborated in vivo data and revealed reduced mRNA expression of TNF-α, TGF-β, and p53 following LPS challenge, which was associated with a marked reduction in the mRNA stability of these genes. Taken together, our studies suggest that HuR is a direct target of IL-10, and HuR knockdown mimics anti-inflammatory effects of IL-10.—Krishnamurthy, P., Lambers, E., Verma, S., Thorne, T., Qin, G., Losordo, D. W., Kishore, R. Myocardial knockdown of mRNA-stabilizing protein HuR attenuates post-MI inflammatory response and left ventricular dysfunction in IL-10-null mice.

Prolonged inflammation characterized by enhanced production of proinflammatory cytokines during myocardial infarction (MI) leads to left ventricular (LV) dysfunction and adverse remodeling changes such as LV dilation and fibrosis (1,2,3,4,5). Inflammatory cells that infiltrate the injury site secrete a number of proinflammatory (IL-1β, IL-6, TNF-α, IP-10, MCP-1, etc.) and anti-inflammatory (IL-4, IL-10) cytokines that mediate homeostasis within the heart in response to injury (5,6,7). IL-10, a potent anti-inflammatory cytokine, is a strong deactivator of monocytes and suppressor of various proinflammatory mediators (8, 9). IL-10 inhibits the synthesis of a number of proinflammatory cytokines implicated in LV dysfunction, including TNF-α, IL-1β, and IL-6 following MI in mice (10). Previous reports suggest that the IL-10 genotype influences the risk for cardiovascular events in hemodialysis patients. The quantitative production of IL-10 is subject to genetic variation based on polymorphisms in the promoter of its gene. IL-10 “low-producer” patients have a significantly enhanced risk of death due to cardiovascular disease compared to IL-10 “high-producers” (11). IL-10 inhibits inflammatory response by suppressing HuR-mediated mRNA stabilization of proinflammatory cytokines and attenuates post-MI LV dysfunction and remodeling (10). An important mechanism of posttranscriptional gene regulation of proinflammatory cytokines is the rapid degradation of messenger RNAs (mRNAs) signaled by AU-rich elements (AREs) in their 3′ untranslated regions. HuR, an ubiquitously expressed member of the Hu family of RNA-binding proteins related to Drosophila ELAV, selectively binds AREs and confers stability to ARE-containing mRNAs (12,13,14). IL-10-knockout (KO) mice display an exaggerated inflammatory syndrome including elevated levels of circulating proinflammatory cytokines including TNF-α (15) and develop symptoms of Crohn’s-like disease (16). IL-10 inhibits superoxide anion (O₂⁻) production by down-regulation of the gp9l-phox and p47-phox genes in human monocytes (17). We have earlier reported that systemic administration of IL-10 inhibits a panel of proinflammatory cytokine/chemokine mRNA expression in the LV after MI via inhibition of cytokine mRNA-stabilizing protein HuR and suppression of p38 MAP kinase (10). Following MI, IL-10-KO mice showed increased infiltration of inflammatory cells in the border zone of the infarct with LV dysfunction, fibrosis, and cardiomyocyte apoptosis. We tested whether HuR knockdown attenuates these undesirable effects in IL-10-KO mice. The therapeutic effects of targeting IL-10-sensitive protein HuR on post-MI inflammation, LV dysfunction, and remodeling and the molecular signaling that governs these effects remain to be studied.

Although HuR expression increased in LV post-MI, much remains to be understood concerning the HuR protein-mediated mechanisms controlling mRNA stability of various genes and their effect on the pathogenesis of MI. Given the strong association between proinflammatory cytokines and LV dysfunction and remodeling, the potential role of targeted anticytokine treatment strategies in MI needs to be fully evaluated.

Ischemia-induced cardiomyocyte loss plays a prominent role in the pathophysiology of cardiac remodeling after MI (18). Ischemia stimulates p53, leading to apoptosis of cardiac cells, including myocytes both in vivo (MI) (18) and in vitro (19). Apoptosis leads to the disruption of normal myocardial structures, resulting in replacement of dead cells with excessive deposition of extracellular matrix (ECM; fibrosis) (20). p53 is a well-known proapoptotic factor, and TGF-β promotes fibrogenesis through connective tissue growth factor (CTGF) and SMAD3 signaling during heart failure (21). However, the underlying signaling mechanisms that may mediate crosstalk between IL-10 and HuR, in the context of HuR-mediated modulations in either TGF-β or p53 signaling, need to be explored. We hypothesize that shRNA-mediated knockdown of HuR attenuates post-MI LV dysfunction and adverse LV remodeling in IL-10-deficient mice, and therefore mimics IL-10 effects. This study was undertaken to elucidate the effects of HuR knockdown in modulating post-MI inflammation, LV dysfunction and remodeling, and the signaling mechanisms that regulate HuR mediated effects such as apoptosis and fibrosis during LV remodeling.

METHODS AND MATERIALS

Vertebrate animals

All experiments conform to the protocols approved by the Institutional Animal Care and Use Committee. Six-week-old wild-type (WT) and IL-10-KO (IL10^tm1Cgn) mice of C57BL/6J background were procured from Jackson Research Laboratory (Bar Harbor, ME, USA). Although the development of chronic enterocolitis and other abnormalities have been reported in the IL-10-KO mice, we did not observe any phenotypic or behavioral abnormalities in these mice before experimentation. The mice were allowed to acclimatize for 10 d under sterile animal management conditions.

Cell culture and reagents

RAW 264.7 cells (mouse mononuclear/macrophage cell line) were cultured in DMEM (Clonitech, Palo Alto, CA, USA) with 10% FCS. Cells were stimulated with LPS and/or IL-10 at a dose of 10 ng/ml concentration unless otherwise indicated. Recombinant murine and human IL-10 was obtained from R&D Systems (Minneapolis, MN, USA). LPS was obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse HuR shRNA (HshRNA) and a nontarget control shRNA (CshRNA) lentiviral particles were purchased from Sigma-Aldrich.

MI and study design

Mice were subjected to MI by ligation of the left anterior descending coronary artery (LAD) as described previously (22). HuR-specific shRNA (4×10⁶ viral particles/mice, HshRNA group) or control, nonspecific shRNA (4×10⁶ viral particles/mice, CshRNA group), was injected intramyocardially into the LV wall (border zone) at 5 different locations on d 0 immediately after LAD ligation. Subsequently shRNA was intravenously injected on d 1, 2, 3, 4, 5, and 7 post-MI. The mice in the sham group underwent the same procedure except for the LAD ligation. Inflammatory response and cardiomyocyte apoptosis was assessed at 3 d, LV functional changes at 14 and 28 d, and structural remodeling at 28 d post-MI.

Echocardiography

Transthoracic 2-dimensional M-mode echocardiogram was obtained using the Vevo 770 (VisualSonics, Toronto, ON, Canada) equipped with a 30-MHz transducer. Echocardiographic studies were performed before MI (baseline) and at 14 and 28 d post-MI on mice anesthetized with a mixture of 1.5% isoflurane and oxygen (1 L/min). M-mode tracings were used to measure LV wall thickness, end-systolic diameter (LVESD), and end-diastolic diameter (LVEDD). The mean value of 9 measurements was determined for each sample. Percentage fractional shortening (%FS) and ejection fraction (%EF) were calculated as described previously (23).

Morphometric studies

The hearts were perfused with 30% KCl followed by fixation with 4% paraformaldehye. Hearts were cut into 3 slices (apex, mid-LV, and base), and frozen sections were made. The morphometric analysis including infarct size and percentage fibrosis area (%LV area) was performed on Masson’s trichrome-stained tissue sections using ImageJ 1.30 software (U.S. National Institutes of Health; http://rsb.info. nih.gov/ij/).

Immunohistochemistry

Immunohistochemical detection of HuR was carried out using the avidin-biotin-DAB complex method on frozen sections as described previously (24). In brief, after an overnight incubation at 4°C with primary monoclonal antibodies against HuR (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), a biotin-conjugated goat anti-rabbit second antibody (1:250; Vector Laboratories, Burlingame, CA, USA) and subsequently streptavidin conjugated with horseraddish peroxidase (HRP, 1:250; Vector Laboratories) were applied. DAB peroxidase substrate (Vector Laboratories) was utilized for visualization, and specimens were counterstained with hemotoxylin (Vector Laboratories).

Immunofluorescent staining for CD68 on tissue sections was performed as described previously (25). Tissue sections were permeabilized and stained with anti-CD68 (Serotec, Raleigh, NC, USA) for inflammatory cell infiltration, followed by incubation with respective secondary antibodies. Staining without the primary antibodies was used as control for nonspecific fluorescence. Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, 1:5000; Sigma-Aldrich), and sections were examined with a fluorescent microscope (Eclipse TE200; Nikon, Tokyo, Japan). Inflammatory cell infiltration (CD68⁺) was assessed at 10 randomly selected high-power visual fields (HVFs) in the border zone of infarcted myocardium and expressed as number per HVF.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining

TUNEL staining was carried out on 4-μm-thick frozen sections as per the manufacturer’s instructions (Cell Death Detection Assay; Roche, Indianapolis, IN, USA). Cardiac myocytes were identified using α-sarcomeric actinin antibodies (Sigma Chemicals, St. Louis, MO, USA). DAPI staining was used to count the total number of nuclei. The index of apoptosis was calculated as the percentage of apoptotic myocyte nuclei/total number of nuclei.

Quantitative real-time PCR (Q-PCR)

Gene expression levels of TNF-α, IL-1β, TGF-β, and p53 were quantified in the border zone of infarct as described previously (26). RNA was collected from heart tissue or RAW 264.7 cells with RNA STAT-60 (TEL-TEST, Friendswood, TX, USA). Total RNA was reverse transcribed with the iScript cDNA synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA), and amplification was performed using the Taqman 7300 (Applied Biosystems, Foster City, CA, USA). Relative mRNA expression of target genes was normalized to the endogenous 18S control gene (Applied Biosystems) and represented as fold change vs. control untreated cells.

Generation of stable HuR-knockdown RAW 264.7 clones by puromycin selection

RAW 264.7 cells were cultured in DMEM with 10% FBS. Cells were treated with short-hairpin lentiviral particles against HuR [5 MOI (multiplicity of infection)] in the presence of hexadimethrine bromide (8 μg/ml; increases transduction efficiency). After 24 h transduction, the cells were selected using puromycin (6 μg/ml; dose determined by titration). Puromycin-resistant HuR-knockdown cell clones were grown, analyzed, and frozen for future use.

RNA protein-binding assay

Immunoprecipitation (IP) of endogenous HuR-mRNA complexes, used to assess the association of endogenous HuR with endogenous p53 and TGF-β mRNA, was performed as described previously (27). Cells were subjected to 254-nm UV light exposure for 20 min in a stratalinker to cross-link cellular mRNA-protein complexes. Total cell lysate from UV-cross-linked cells was used for IP at 4°C, overnight in the presence of excess (30 μg) IP antibody (IgG1 or anti-HuR; Santa Cruz Biotechnology). RNA bound to immunoprecipitated HuR in IP material was isolated and used in Q-PCR reactions to detect the presence of TGF-β or p53 mRNA. Data are represented as fold change vs. control.

Assessment of mRNA stability

The mRNA stability of TGF-β and p53 mRNA was determined by actinomycin D chase experiments, following a standard protocol described elsewhere (28). Briefly, WT and HuR-knockdown RAW 264.7 cell clones were stimulated with LPS (10 ng/ml) in DMEM culture medium. Actinomycin D was added to a final concentration of 5 μg/ml to block further transcription. At 0, 30, 60, and 120 min after actinomycin D treatment, the cells were harvested, and mRNA was quantified by Q-PCR as described above. The mRNA decay was recorded as the percentage of mRNA remaining over time compared with the amount before the addition of actinomycin D.

Statistical analyses

Data are presented as means ±se. Between 2 groups of mice, an unpaired Student’s t test was performed to determine statistical significance. When >2 groups were involved, ANOVA with the Tukey’s post hoc test was used to analyze the data. Values of P < 0.05 were considered significant.

RESULTS

HuR knockdown inhibits post-MI LV inflammation

All animal experiments were carried out in accordance with the Northwestern University Institutional Animal Care and Use Committee (IACUC)-approved protocols. MI was induced in WT and IL-10-deficient mice and followed immediately by intramyocardial injections of either lentiviral-control scrambled shRNA (CshRNA) or HuR shRNA (HshRNA). HuR expression was assessed by immunoperoxidase staining for HuR on LV tissue sections at 3 d after MI and shRNA injection (Fig. 1A). The inflammatory cells infiltrated at the border zone of the infarct were strongly positive for HuR. However, the numbers of HuR⁺ cells were higher in the KO mice as compared to WT mice in the CshRNA-treated group (Fig. 1A, top panels). HuR shRNA significantly knocked down the expression of HuR in mice of both genotypes (Fig. 1A, bottom panels). HuR staining was observed mostly in inflammatory cells (Fig. 1A, arrows) and in a few degenerating cardiomyocytes (data not shown). mRNA expression of HuR in the myocardium (border zone) at 3 d post-MI was assessed by Q-PCR. HuR mRNA expression significantly increased in IL-10-KO mice as compared to WT mice, post-MI (P<0.01; Fig. 1B). HshRNA significantly knocked down HuR mRNA as compared to CshRNA (P<0.01; Fig. 1B).

Figure 1.

A) Immunoperoxidase staining of HuR in the LV tissue sections at 3 d post-MI. HuR knockdown (HshRNA) reduced HuR⁺ cells (brown) in border zone of infarct as compared to CshRNA-treated mice. n = 4/group. B) Quantitative real-time PCR analysis of mRNA expression of HuR in the border zone of LV infarct at 3 d post-MI. mRNA expression normalized to 18S expression and depicted as fold change vs. WT-CshRNA. n = 5/group. C) Immunofluorescent staining of inflammatory cells (CD68⁺, green) in the border zone of infarct at 3 d post-MI. D) Quantitative analysis of infiltrating CD68⁺ cells per HVF at 3 d post-MI. n = 5/group. shRNA-mediated HuR knockdown inhibited CD68⁺ cell infiltration as compared to CshRNA-treated hearts. WT-CshRNA, IL-10 WT mice treated with control shRNA post-MI; WT-HshRNA, IL-10 WT mice treated with HuR-specific shRNA post-MI; KO-CshRNA, IL-10-KO mice treated with control shRNA post-MI; KO-HshRNA, IL-10-KO mice treated with HuR-specific shRNA post-MI. *P < 0.01 vs. CshRNA groups; #P < 0.01 vs. HshRNA group.

Immunofluorescence staining of CD68⁺ cells on cardiac tissue sections was carried out to study the inflammatory cell infiltration at 3 d post-MI (Fig. 1C). IL-10-KO mice treated with control shRNA (CshRNA) showed increased infiltration of CD68⁺ cells (macrophage and monocyte) in the border zone of LV infarct as compared to WT mice (P<0.01; Fig. 1C, D). However, ShRNA-mediated HuR knockdown significantly inhibited infiltration of inflammatory cells in the border zone of infarct (P<0.01 vs. CshRNA group; Fig. 1C, D). WT mice receiving HshRNA showed reduced infiltration of inflammatory cells as compared with KO mice (P<0.01 vs. KO; Fig. 1C, D).

Resolution of inflammatory cell infiltration by HuR knockdown was corroborated by a significant repression in the expression of a panel of inflammatory cytokines including TNF-α, IL-1β, and monocyte chemoattractant protein-1 (MCP-1, data not shown). Q-PCR for TNF-α and IL-1β showed that the increased inflammatory cell infiltration was associated with increased mRNA expression of TNF-α and IL-1β in the myocardium, post-MI (P<0.01 vs. sham; Fig. 2). HuR knockdown significantly reduced mRNA expression of TNF-α and IL-1β as compared to CshRNA-treated mice (P<0.01; Fig. 2).

Figure 2.

Quantitative real-time PCR analysis of mRNA expression of proinflammatory cytokines such as TNF-α (A) and IL-1β (B) in the border zone of LV infarct at 3 d post-MI. mRNA expression normalized to 18S expression and depicted as fold change vs. WT-CshRNA. n = 5/group. *P < 0.01 vs. CshRNA groups; #P < 0.01 vs. HshRNA group.

HuR knockdown reduces MI-induced myocardial apoptosis and p53 mRNA expression

Heart sections were stained with the TUNEL method to detect cardiac apoptosis at 3 d post-MI (Fig. 3A). MI increased the number of apoptotic cardiomyocytes at the border zone of infarct as compared to the sham group (WT sham, 0.30±0.02; KO sham, 0.39±0.05; P<0.01 MI+CshRNA vs. sham; Fig. 3A, B). However, HuR knockdown significantly reduced the number of apoptotic cells in the border zone of LV infarct (P<0.01 vs. CshRNA; Fig. 3A, B). It was interesting to note that cardiomyocyte apoptosis was higher in IL-10-KO mice as compared to WT mice (P<0.01; Fig. 3A, B); however, HuR knockdown significantly reduced exaggerated post-MI apoptosis in IL-10-KO mice.

Figure 3.

A) TUNEL staining for cardiomyocyte apoptosis (red nuclei) in border zone of LV infarct at 3 d post-MI. HuR knockdown attenuated cardiomyocyte apoptosis after MI. B) Quantitative analysis of TUNEL⁺ cardiomyocytes at 3 d post-MI. shRNA-mediated HuR knockdown reduced cardiomyocyte apoptosis as compared to CshRNA-treated hearts. n = 5/group. C) Quantitative analysis of mRNA expression of p53 in border zone of LV infarct at 3 d post-MI. mRNA expression normalized to 18S expression and depicted as fold change. Q-PCR data shows that HuR knockdown reduced p53 expression as compared to CshRNA-treated mice. n = 5/group. D) Western blot for p53 protein expression in LV at 3 d post-MI. Equal loading of proteins in each lane is shown by β-actin. n = 3/. *P < 0.01 vs. CshRNA groups; #P < 0.01 vs. HshRNA group.

p53 plays an important role in cardiomyocyte apoptosis following MI (18). We examined p53 mRNA (Fig. 3C) and protein expression (Fig. 3D) in the border zone of infarct at 3 d post-MI. Cardiomyocyte apoptosis was associated with increased p53 mRNA expression and protein levels after MI. HuR knockdown reduced p53 mRNA and protein expression levels in the LV (P<0.01, CshRNA vs. HuR shRNA; Fig. 3C, D). These data suggest that the HuR-knockdown-mediated decrease in p53 expression was directly associated with the suppression of post-MI apoptosis. LPS-induced apoptosis was studied in control (Csh) and HuR knockdown (Hsh) RAW 264.7 cell clones. We observed reduced apoptosis in HuR knockdown clones as compared to control cells treated with LPS (Supplemental Fig. 1).

HuR knockdown attenuates post-MI LV dysfunction and reduces the infarct size

We have earlier reported that IL-10 treatment rescues post-MI LV functions in mice (10). To assess whether HuR knockdown in the myocardium may mimic IL-10 effects, LV functions were determined by echocardiography in both WT and IL-10-KO mice injected with either control or HuR-shRNA, 14 and 28 d after the induction of MI. M-mode tracings analyzed at 14 (data not shown) and 28 d post-MI showed similar changes in LV functions (Fig. 4). MI increased LVESD and LVEDD (P<0.01 vs. baseline; Fig. 4A, B) and reduced %FS and %EF in CshRNA-treated mice at 28 d post-MI (P<0.05 vs. baseline; Fig. 4C, D), suggesting LV contractile dysfunctions. HuR knockdown attenuated LV dysfunction, with significantly lowered LVESD and LVEDD (P<0.05; Fig. 4A, B), and increased %FS and %EF as compared to the CshRNA-treated group (P<0.05; Fig. 4C, D). Heart rates were not significantly different among the groups. Infarct size was measured as percentage of the LV circumference from trichrome-stained sections at 28 d post-MI. There was no difference in the infarct size/fibrosis in WT and KO mice treated with CshRNA (Fig. 5A, B). HuR knockdown resulted in a significant reduction in the infarct size as compared to CshRNA-treated mice (P<0.01; WT-CshRNA, 41.57±1.02; WT-HshRNA, 25.89±0.62; KO-CshRNA, 43.13±0.65; KO-HshRNA, 30.86±1.24; Fig. 5A, B).

Figure 4.

Echocardiography: analysis of M-mode tracing of LVEDD (A) and LVESD (B), and %FS (C) and %EF (D) calculations. HuR knockdown improved LV function with significantly lowered LVESD and LVEDD and increased %FS and %EF. n = 10/group. *P < 0.05 vs. CshRNA groups; #P < 0.05 vs. HshRNA group.

Figure 5.

A) Trichrome-stained heart sections (28 d post-MI). B) Infarct size analysis at 28 d post-MI. C) Quantitative analysis of fibrosis area (%LV area) at 28 d post-MI. shRNA-mediated HuR knockdown attenuated fibrosis when compared to CshRNA-treated group. n = 5/group. *P < 0.05 vs. CshRNA groups; #P < 0.05 vs. HshRNA group. D) Quantitative analysis of mRNA expression of TGF-β in border zone of LV infarct post-MI. mRNA expression normalized to 18S expression and depicted as fold change. Q-PCR data show that HuR knockdown reduced TGF-β expression as compared to CshRNA-treated mice. n = 5/group. *P < 0.01 vs. CshRNA groups; #P < 0.01 vs. HshRNA group.

HuR knockdown inhibits TGF-β mRNA expression and attenuates LV fibrosis post-MI

TGF-β plays an important role in post-MI ECM remodeling. TGF-β expression increases rapidly in the heart post-MI (7). Quantitative analysis of trichrome-stained sections indicated increased fibrosis in the LV after 28 d post-MI in IL-10-KO mice compared to WT mice both treated with CshRNA (P<0.01 vs. sham; Fig. 5C). It was interesting to note that fibrosis was significantly reduced in the LV following HuR knockdown (P<0.05 vs. CshRNA; Fig. 5C). We assessed mRNA expression of TGF-β in the myocardium in the border zone post-MI (Fig. 5D). Q-PCR analysis indicated that the mRNA expression of TGF-β was increased following MI (P<0.01 vs. sham, not shown). However, HshRNA-treated mice showed significantly reduced TGF-β mRNA expression levels in the LV (P<0.01 vs. CshRNA; Fig. 5D). These data indicate that HuR knockdown leads to decreased expression of TGF-β mRNA, which in turn correlates with decreased post-MI fibrosis.

HuR protein physically associates with TGF-β and p53 mRNAs in vivo

Both p53 and TGF-β mRNA harbors ARE elements in their respective 3′UTRs, sequences to which HuR binds and stabilizes posttranscription mRNA stability. Real-time PCR analysis for HuR mRNA expression showed that IL-10 significantly reduced LPS-mediated increases in HuR mRNA expression (P<0.01 vs. LPS-treated cells; Supplemental Fig. 2). To assess the endogenous association of HuR with TGF-β and p53 mRNA, RNA-protein immunoprecipitation experiments were performed on whole-cell extracts obtained from cells treated with LPS and/or IL-10 for 2 h and were UV cross-linked. RNA cross-linked to HuR was immunoprecipitated by HuR antibodies, isolated from HuR-RNA complexes, and assessed for TGF-β and p53 mRNA by Q-PCR. Figure 6A, B shows the association of TGF-β and p53 mRNAs with endogenous HuR. LPS increased the association of TGF-β and p53 mRNA to HuR, while IL-10 inhibited amount of p53 and TGF-β mRNA bound to HuR (Fig. 6A, B). Importantly, the p53 or TGF-β mRNAs were undetectable in nonspecific IgG1 IPs (not shown). A similar trend was observed for other IL-10-sensitive targets TNF-α and MMP-9 (data not shown).

Figure 6.

HuR physically binds to TGF-β and p53 mRNAs in vivo. A, B) Cells treated with LPS and/or IL-10 for 2 h were UV cross-linked and lyzed, and RNA-protein complexes were immunoprecipitated in the presence of anti-HuR antibody or nonspecific IgG1. IL-10 inhibited LPS-mediated increases in TGF-β (A) and p53 (B) mRNA association to HuR. n = 3/group. *P < 0.01 vs. LPS. C) Western blotting on LPS-treated puromycin-selected HuR-knockdown RAW 264.7 cell clones using different target constructs (lanes 1 and 2). HuR knockdown (HshRNA) abrogated LPS-stimulated HuR protein expression. β-actin signals served to assess the equal protein loading in all lanes. D, E) Quantitative analysis of mRNA expression of TGF-β (D) and p53 (E) following HuR knockdown. Q-PCR data show that TGF-β and p53 expression increased on LPS treatment as compared to untreated cells (Unt). HshRNA reduced TGF-β and p53 expression as compared to CshRNA-treated cells. n = 3/group. *P < 0.05 vs. CshRNA. F, G) mRNA stability of TGF-β (F) and p53 mRNA (G), assessed in HuR-null RAW264.7 cell clones by using actinomycin D (5 μg/ml) chase assays. Total cellular RNA was isolated at times shown; percentage remaining levels of TGF-β and p53 mRNAs were measured by Q-PCR analysis. Values are means ± se from triplicate experiments. *P < 0.05 at 120 min.

HuR knockdown inhibits mRNA expression of TGF-β and p53 in RAW 264.7 cells

To determine whether HuR mimics IL-10 inhibitory effect on p53 and TGF-β mRNA expression, we generated stable HuR-knockdown clones from mouse monocyte-macrophage cell line RAW264.7. Cells infected with lentiviral HuRshRNA were selected with puromycin, and the stable knockdown of HuR was confirmed in 2 of the generated clones by Western blotting, which showed complete abrogation of HuR expression as compared to control shRNA-treated cells (Fig. 6C). HuR binds and stabilizes a number of proinflammatory cytokines under various inflammatory conditions. To determine whether inflammation-mediated TGF-β and p53 mRNA expression is mediated by HuR, LPS-treated RAW 264.7 cells (100 ng/ml for 2 h; WT and HuR-knockdown clones) were analyzed by Q-PCR. As shown in Fig. 6D, E, LPS increased mRNA expression of the above genes as compared to untreated cells (P<0.05, Fig. 6D, E). HuR knockdown significantly inhibited LPS-mediated increases in TGF-β and p53 mRNA expression (Fig. 6D, E). Note that silencing of HuR gene mimicked the effects of IL-10 (Fig. 6A, B), therefore suggesting HuR is a downstream target of IL-10. Similar effects were observed with TNF-α and MMP-9 expression (data not shown).

HuR knockdown inhibits mRNA stability of TGF-β and p53 in LPS-treated RAW 264.7 cells

The mRNA half-life of TGF-β and p53 mRNA was determined by actinomycin D chase experiments to assess their mRNA stability in the HuR-null RAW264.7 cells (Fig. 6F, G). Interestingly, in HuR-knockdown RAW 264.7 cell clones, mRNA decay of both these genes was significantly accelerated compared to CshRNA-treated RAW 264.7 cells, suggesting that HuR is required for stabilization of TGF-β and p53 mRNAs. Similar effects were observed with TNF-α and MMP-9 mRNA expression and degradation (data not shown). Also, the relative protein expression levels of p53 and TGF-β following LPS treatment for 16 h was confirmed in control (CshRNA) and HuR-knockdown cells (HshRNA), as shown in Supplemental Fig. 3.

DISCUSSION

Homeostasis of proinflammatory and anti-inflammatory cytokines play a prominent role in cardiac pathophysiological conditions such as MI and reperfusion injury (29). Increased circulating proinflammatory cytokines have been associated with LV dysfunction and remodeling. We and others have previously shown that strategies targeting proinflammatory cytokines in the myocardium resulted in attenuation of LV dysfunction and remodeling (3, 30,31,32,33). However, targeting HuR, an mRNA-stabilizing protein of various proinflammatory cytokines and their effect on inflammatory response, LV dysfunction, and remodeling has not been explored so far. In the present study, we inhibited inflammatory response in the myocardium with knockdown of HuR using lentivirus shRNA, thereby enhancing mRNA degradation of inflammatory mediators. We also assessed the effect of proinflammatory cytokine mRNA destabilization on LV function, cardiomyocyte apoptosis, and remodeling post-MI. The important findings of this study are that HuR knockdown mimicked IL-10 treatment effects and attenuated myocardial inflammation, cardiomyocyte apoptosis, and expression of TGF-β and p53 at 3 d post-MI, followed by attenuation of LV dysfunction and remodeling with effects on fibrosis at 28 d post-MI. The above effects were suggested to be partly due to HuR-knockdown-mediated mRNA destabilization of the proinflammatory cytokines TGF-β and p53.

Inflammatory cytokines have been implicated in post-MI LV remodeling and cardiomyocyte hypertrophy (1) with alterations in fetal gene expression and contractile abnormalities (2, 3, 31). At baseline, IL-10-KO mice did not show any phenotypical differences in LV function and remodeling changes as compared to WT mice. However, following MI, infiltration of CD68⁺ monocyte/macrophages in the border zone of the myocardium was higher in IL-10-KO mice as compared to WT mice, at 3 d post-MI. Consistent with this finding, MI-induced increases in HuR expression were higher in the KO mice vs. WT mice. Inflammatory cell infiltration was associated with an increase in mRNA expression of various proinflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, IP-10, MCP-1) after MI (10). These “stress-activated” cytokines are produced by various cell types in the myocardium, including cardiomyocytes, or by the resident/infiltrating inflammatory cells. The mRNAs encoding most proinflammatory cytokines are short lived, with instability conferred by an ARE in the 3′ noncoding region. HuR selectively binds AREs and stabilizes mRNAs. IL-10, a potent anti-inflammatory cytokine, has been shown to limit the infiltration of inflammatory cells in MI (10) and vascular injury models (13). In agreement with the above studies, HuR knockdown inhibited inflammatory cell infiltration and proinflammatory cytokines and chemokines (P<0.05 vs. MI) in the myocardium. This suggests that HuR could be a downstream molecule of IL-10 signaling. An earlier report suggests that IL-10 knockout in an ischemia reperfusion model after LAD ligation increases mortality in mice (34). Our laboratory has shown that IL-10 injection reduced inflammation-mediated adverse cardiac remodeling and dysfunction (10). The present data targeting HuR (a downstream molecule) mimic IL-10 effects. We presume that IL-10-KO-induced deleterious effects on the left ventricle, taken together, can be significantly reduced, if not completely. In addition, ongoing experiments in our laboratory involving IL-10 WT bone marrow transplantation in IL-10-KO mice will provide critical insights that will further confirm the above effects.

Myocardial expression of proinflammatory cytokines contributes to depression of contractile performance and adverse LV remodeling (2, 29). In the present study, echocardiography showed increase in LVEDD and LVESD and decrease in %FS and %EF after MI and HuR knockdown attenuated these effects at 28 d post-MI. Proinflammatory cytokine-induced depression of contractile performance might be a direct result of interference with myocardial calcium handling (35, 36), myoD degradation (37), cardiac fibrosis (38), or cardiomyocyte apoptosis (18). Although HuR-mediated effects on myoD, MMP-9, and p53 have been reported (27, 37, 39), its effects on calcium signaling are not clear. The significant up-regulation of proinflammatory cytokines (at 3 d) could trigger a second phase of elevated cytokines levels in the noninfarcted myocardium that promotes interstitial fibrosis and collagen deposition leading to ventricular dysfunction (29). Most important, a recent study (40) has shown that transplantation of bone marrow mononuclear cells (BM-MNCs) in infarcted mouse hearts led to a significant improvement in cardiac function. These BM-MNCs secreted significant amounts of IL-10, and the cardiac protection was associated with decreased T-lymphocyte accumulation, reactive hypertrophy, and myocardial collagen deposition. Also, various studies have reported that activation of MMPs and p38, and reduced angiogenesis and STAT-3, have affected remodeling and cardiac dysfunction (32, 41,42,43).

Ischemic-oxidative stress stimulates p53 (proapoptotic factor), leading to apoptosis of cardiac cells including myocytes both in vivo (MI) (18) and in vitro (19). Ischemia-induced cardiomyocyte cell death (apoptosis) plays a prominent role in the pathophysiology of cardiac remodeling after MI (18). Several studies have shown involvement of inflammatory mediators in progressive myocytes loss due to necrosis and/or apoptosis, suggesting that these cytokines are involved in the progression of cardiac remodeling (33, 44). In the present study, MI increased the number of apoptotic cells in the border zone of infarction, which was significantly higher in IL-10-KO mice as compared to WT mice. Consistent with the above finding, p53 mRNA and protein expression was increased in KO mice vs. WT mice. HuR knockdown attenuated cardiomyocyte apoptosis associated with decreased p53 mRNA expression. In addition to the various proinflammatory cytokines and MMP-9 mRNA, HuR protein also binds and stabilizes p53 mRNA (27). Immunoprecipitation of HuR proteins in LPS-treated RAW 264.7 cells showed increased association of p53 mRNA with the endogenous HuR protein. However, HuR knockdown significantly enhanced decay of p53 mRNA. Taken together, our data suggest that HuR knockdown attenuates cardiomyocyte apoptosis by significantly reducing p53 mRNA stability. Also, in addition to HuR, other p53 regulators, such as minute double minute 2 and herpes virus-associated ubiquitin-specific protease (45) might also play an important role in the remodeling process.

Apoptosis leads to disruption of normal myocardial structures, resulting in replacement of dead cells with excessive deposition of ECM (fibrosis) (20). An underlying morphological correlate of LV dysfunction is cardiomyocyte apoptosis and cardiac fibrosis, which leads to increased stiffness of the heart. Homeostasis of ECM (degradation and accumulation) mediates pathogenesis of LV remodeling. Increased TGF-β production associated with sustained inflammatory response may lead to excessive extracellular matrix deposition, leading to cardiac fibrosis, resulting in adverse remodeling changes (2, 7, 43).

Our study shows that MI increased fibrosis associated with increased mRNA expression of TGF-β in the LV. These findings corroborate well with the previous finding that TGF-β promotes fibrogenesis through CTGF and SMAD3 signaling during heart failure (21). An interesting finding of the present study is that HuR knockdown significantly inhibited fibrosis. Also, IL-10 suppressed HuR expression and had similar effects on fibrosis (10). Therefore, silencing of HuR mimics the effects of IL-10 on LV function and remodeling.

Recent studies have suggested that fibrosis might result from proliferation of resident fibroblasts, bone marrow-derived fibroblasts, and epithelial-mesenchymal transition (EMT). TGF-β1, a promoter of cardiac fibrosis, induced EMT in adult coronary endothelial cells and was mediated through transcription factor Smad3 (20). A synergy of TNF-α and TGF-β signaling promotes a rapid morphological conversion of the epithelial cells to the mesenchymal phenotype, and this process is dependent on enhanced p38 MAPK activity (46). Also, earlier reports have suggested that TGF-β expression in mesangial cells is mediated by mRNA-stabilizing factor, HuR (39). In the present study, mRNA decay of TGF-β was accelerated in HuR-knockdown RAW cell clones as compared to CshRNA-treated RAW 264.7 cells, suggesting that HuR stabilizes mRNA of TGF-β. These findings, along with our previous reports of IL-10-mediated inhibition of HuR and p38 MAPK, suggest that inflammation might be playing an important role in endothelial to mesenchymal transition, and the mechanism is yet to be determined from our ongoing experiments.

IL-10 regulation of HuR might be occurring through various signaling pathways. A previous publication from our laboratory (13) has reported that anti-inflammatory cytokine IL-10 inhibits a panel of proinflammatory cytokines through suppression of p38 MAPK. Our data in this work suggest that IL-10 exhibits its anti-inflammatory effects by inhibiting the mRNA-stabilizing protein HuR. Our previous reports have also shown that IL-10 inhibits inflammation and attenuates LV remodeling after MI via activation of STAT3 and suppression of HuR. The inflammatory effects were also associated with p38 MAPK expression in the left ventricle (10). There is increasing evidence that the p38 MAPK cascade is crucial for the control of its mRNA-destabilizing activity, particularly for the zinc finger protein TTP (another mRNA-stabilizing protein) (47, 48). However, it is not clear whether IL-10-mediated reduction in HuR expression is regulated through p38 MAPK and/or STAT3. Recently, Abdelmohsen et al. (49) has reported that microRNA (miR-519) regulates HuR translation in several human carcinoma cell lines. However, the role of miR-519 in IL-10-mediated reduction in HuR levels is not yet explored.

In summary, the data presented here suggest that HuR knockdown reduces severity of proinflammatory responses and contributes to improved LV function and remodeling by inhibition of TGF-β-associated fibrosis and p53-associated cardiomyocyte apoptosis after MI. The effects are due to HuR-knockdown-mediated destabilization of TGF-β and p53 mRNA. These results define HuR as a critical player in proinflammatory cytokine-induced LV dysfunction and remodeling and therefore establishing mRNA stability of cytokines as a potential therapeutic target that could attenuate inflammation-mediated fibrosis and apoptosis. Also, understanding the effects of inflammation on transplanted progenitor cells function and survival in the heart could enhance cell-based therapeutic approaches in cardiac interventions.