HuR inhibition reduces post‐ischemic cardiac remodeling by dampening myocyte‐dependent inflammatory gene expression and the innate immune response

Figures

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  FIGURE 1. HuR activation and expression is increased following I/R injury. HuR cytoplasmic translocation, indicative of activation, is increased at 2 h post‐I/R (N and C represent nuclear and cytosolic fractions, respectively) (A). Cytoplasmic translocation of HuR is quantified in panel B. Total HuR protein expression is increased in hearts at 7 days post‐I/R (C, D). Treatment with the HuR inhibitor KH‐3 (50 mg/kg) at the time of reperfusion reduces expression of pro‐inflammatory gene expression of IL‐6 (E) and TNF‐⍺ (F) at 2 h post‐reperfusion. *p ≤ .05. **p ≤ .01.

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  FIGURE 2. HuR inhibition reduces post‐ischemic cardiac remodeling and preserves cardiac function. HuR inhibition significantly reduced post‐ischemic cardiac remodeling as assessed by LV end‐diastolic volume (A) and end‐systolic volume (B) and preserved LV function (ejection fraction; C) at 2 weeks post‐I/R. Treatment with HuRi also reduced total cardiac fibrosis as determined by picrosirius red staining (D; quantified in E) and myofibroblast activity as assessed by total expression of the myofibroblast marker periostin (F; quantified in G). *p ≤ .05. **p ≤ .01.

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  FIGURE 3. HuR mediates inflammatory gene expression following TLR stimulation in cardiomyocytes. Principle component analysis (PCA) (A) and heat map analysis (B) show a distinct and consistent differential gene expression profile in NRVMs treated with LPS alone or LPS plus HuRi (KH‐3). Volcano plots of differentially expressed genes show a predominant increase in gene expression in NRVMs following LPS treatment (C) that is largely reversed in the presence of HuR inhibition (D). Gene labels in C and D represent genes present in the Immune System Process Gene Ontology class (GO0002376) that was subsequently identified as the most strongly enriched HuR‐dependent GO Term (H). Donut plot analysis of all LPS‐dependent significant upregulated (E) and downregulated (F) gene expression changes grouped by gene ontology (GO) term. GO grouping of all HuR‐dependent gene expression changes among those upregulated by LPS shows that HuR overwhelmingly regulates immune and inflammation related GO term‐associated genes (G). The top 10 HuR‐dependent GO terms from panel E are graphed out by p‐value and fold enrichment (H).

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  FIGURE 4. HuR inhibition reduces macrophage infiltration to the post‐ischemic myocardium. Immunofluorescent staining of CD68+ cells shows a reduction in macrophage infiltration to the heart at 7 days post‐I/R upon HuR inhibition (A; total area of CD68+ stain is quantified in B). *p ≤ .05.

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  FIGURE 5. TLR signaling in myocytes induces inflammatory gene expression in macrophages in a HuR‐dependent endocrine manner. Schematic representation of NRVM treatment and conditioned media transfer to BMDMs (A). HuR inhibition reduces mRNA expression of IL‐1β (B) and TNF‐α (C) directly in NRVMs. Conditioned media from LPS‐treated NRVMs induces expression of TNF‐α in BMDMs that is blunted when NRVMs are treated with HuR inhibitor (D). siRNA‐mediated knockdown of HuR in NRVMs inhibits the LPS‐mediated increase in TNF‐α (E), IL‐1β (F), and IL‐6 (G) in BMDMs treated with conditioned media from NRVMs. Flow cytometric analysis of BMDMs shows an increase in the percentage of F4/80hi, CD11chi BMDMs following treatment with conditioned media from LPS treated NRVMs that is blunted when NRVMs are treated with a HuR inhibitor (H, I). Conditioned media transfer from NRVMs treated with LPS induces monocyte (PBMC) migration that is lost when NRVMs are pre‐treated with HuR inhibitor (J). All experimental groups in panels D–H received conditioned media. *p ≤ .05. **p ≤ .01. ***p ≤ .001.