Functional Screening Identifies MicroRNAs as Multi-Cellular Regulators of Heart Failure

Abstract

Heart failure (HF) is the leading cause of death in the Western world. Pathophysiological processes underlying HF development, including cardiac hypertrophy, fibrosis and inflammation, are controlled by specific microRNAs (miRNAs). Whereas most studies investigate miRNA function in one particular cardiac cell type, their multicellular function is poorly investigated. The present study probed 194 miRNAs -differentially expressed in cardiac inflammatory disease - for regulating cardiomyocyte size, cardiac fibroblasts collagen content, and macrophage polarization. Of the tested miRNAs, 13%, 26%, and 41% modulated cardiomyocyte size, fibroblast collagen production, and macrophage polarization, respectively. Seventeen miRNAs affected all three cellular processes, including miRNAs with established (miR-210) and unknown roles in cardiac pathophysiology (miR-145-3p). These miRNAs with a multi-cellular function commonly target various genes. In-depth analysis in vitro of previously unstudied miRNAs revealed that the observed phenotypical alterations concurred with changes in transcript and protein levels of hypertrophy-, fibrosis- and inflammation-related genes. MiR-145-3p and miR-891a-3p were identified to regulate the fibrotic response, whereas miR-223-3p, miR-486-3p, and miR-488-5p modulated macrophage activation and polarisation. In conclusion, miRNAs are multi-cellular regulators of different cellular processes underlying cardiac disease. We identified previously undescribed roles of miRNAs in hypertrophy, fibrosis, and inflammation, and attribute new cellular effects to various well-known miRNAs.

Figure 1

Phenotypical screening using a HF-associated miRNA library was performed in parallel for multiple HF-underlying processes. Effects induced under control and HF-mimicking conditions by miRNA mimics on hypertrophy, fibrosis and inflammation were studied using nRCMs, nRCFs, and BMDMs. Using automated image analysis, quantified read-outs were cardiomyocyte size, cardiac fibroblast number and collagen area, and macrophage roundness and NFκB nuclear translocation.

Figure 2

Hypertrophy screen identifies HF-associated miRNAs that affect cardiomyocyte cell size. MiRNA mimic-induced changes in cardiomyocyte cell size without (panel A) or with (panel B) PE stimulation. Values represent cardiomyocyte cell size expressed as log₂ fold change, normalised to negative control mimic-transfected cells within the same condition, n = 6 replicates per miRNA mimic per condition. MiRNA mimics that induce a significant (Unpaired t-test, P < 0.05) effect on cell size, deviating more than 2x STDEV from negative control (indicated by vertical lines) are highlighted in red or blue. The PE-induced increase in cell size is represented in green in panel A. (C) Representative images of the miRNAs with pronounced effects on cells size, miR-486-3p and miR-125a-5p, under unstimulated conditions. (D) Quantification of cardiomyocyte cell size under control and PE-stimulated conditions after transfection of miR-486-3p and miR-125a-5p mimics (Unpaired t-test, P < 0.001). (E) The majority of miRNA mimics affect cardiomyocyte cell size irrespectively of stimulation. (F) Several miRNA mimics blunted PE-induced increase in cell size (Unpaired t-test, P < 0.05); presented values are expressed as means ± standard error, ***denotes P < 0.001 versus unstimulated negative control, ^#denotes P < 0.05, ^##denotes P < 0.01, ^###denotes P < 0.01 versus PE-stimulated negative control.

Figure 3

Effect of miRNA mimics on cardiac fibroblast proliferation and collagen production upon transfection. Vulcano plots showing collagen area and number of nuclei for individual miRNA mimics without (panel A and B) or with (panel C and D) TGFβ stimulation. Values represent log₂ fold change, normalised to negative control mimic-transfected cells within the same condition, n = 6 replicates per miRNA mimic per condition. MiRNA mimics that significantly (Unpaired t-test, P < 0.05) increased or reduced these readouts according to previously described criteria are highlighted in red or blue, respectively. As a point of reference, the effects that are reached by TGFβ stimulation of untransfected controls are represented as green triangles in both (panels A and B). (E) Representative images of miR-199a and miR-17-5p. (F) Quantification of cardiac fibroblast collagen area under control and TGFβ-stimulated conditions upon transfection of miR-199a and miR-17-5p mimics (Unpaired t-test, P < 0.05). (G) MiRNA-mimic induced changes in collagen area in the presence and absence of treatment. (H) Different miRNA mimics decreased collagen area only upon TGFβ stimulation (Unpaired t-test); presented values are expressed as means ± standard error, *denotes P < 0.05, ***denotes P < 0.001 versus unstimulated negative control, ^#denotes P < 0.05, ^##denotes P < 0.01 versus TGFβ-stimulated negative control.

Figure 4

Inflammation screen identifies HF-associated miRNAs affecting macrophage roundness and NFκB nuclear translocation. MiRNA mimic-induced changes in macrophage roundness (A) and NFκB nuclear translocation (B) under control conditions. Values are represented as log₂ fold change, normalised to negative control mimic-transfected cells within the same condition, n = 6 replicates per miRNA mimic per condition. MiRNA mimics that significantly (Unpaired t-test, P < 0.05) increased or reduced these readouts are highlighted in red or blue. In panel A, polarization effects on cell roundness in negative control mimic-transfected cells induced by IFNγ and IL-4 stimulation are represented as yellow and green triangles. In panel B, LPS-induced effect on NFκB nuclear translocation in negative control mimic-transfected cells is represented as a green triangle. (C) Representative images of miR-488-5p and miR-146a-3p with pronounced effects on macrophage roundness and NFκB nuclear translocation. (D) Quantification of macrophage roundness under control, INFγ, and IL-4-stimulated conditions upon transfection with miR-488-5p and miR-146a-3p mimics (Unpaired t-test, P < 0.001). The majority of miRNA mimics affect macrophage roundness to a comparable extent under control, IFNγ (E) or IL-4 (F) stimulated conditions. (G) MiR-143-3p prevents IFNγ-induced increase in roundness, while having no effect under basal conditions (Unpaired t-test, P < 0.01). (H) Different miRNA mimics increase roundness only in combination with IL-4 stimulation (Unpaired t-test, P < 0.01), presented values are expressed as means ± standard error, ***denotes P < 0.001 versus unstimulated negative control, ⁺⁺denotes P < 0.01 and ⁺⁺⁺denotes P < 0.001 versus IFNγ-stimulated negative control, ^##denotes P < 0.01 and ^###denotes P < 0.001 versus IL-4-stimulated negative control.

Figure 5

MiRNAs with a multi-cellular function control the expression of common target genes. (A) The majority of the miRNAs mimics affect at least one pathological process. Included read outs are cell size, collagen content, and cell roundness, for the hypertrophy, fibrosis, and inflammation screen, respectively. (B) The 17 miRNAs with a multi-cellular function target various common genes. (C) Overexpression of multi-cellular miR-145-3p decreases the transcript expression of various target genes in multiple cell types in parallel.

Figure 6

MiR-145-3p and miR-891a-3p control the fibrotic response while miR-223-3p, miR-486-3p, and miR-488-5p control the inflammatory response. Quantification of mRNA levels of (A) hypertrophic (ANF, BNP, and αSKA), (B) fibrotic (CTGF, COL1α1, and α-SMA), and (C) inflammatory markers (TNFα, ICAM and MR) in respectively nRCMs, nRCFs, or BMDMs after miRNA mimic transfection (Unpaired t-test). (D) Representative western blots of collagen type I (Col I) and normalizer α-Tubulin (α-Tub) performed with cell lysates of nRCFs transfected with negative control (NEG), miR-145-3p, miR-151a-5p, and miR-891a-3p mimics. Samples were run on the same gel and blot, different regions of the blot were cropped to quantify Col I and a-Tub, and every sample per protein was acquired under the same exposure conditions. (E) Western blot quantification reveals increased and decreased Collagen Type I protein expression in miR-145-3p and miR-891a-3p transfected nRCFs, respectively (One Way ANOVA). (F) Levels of cytokines and chemokines determined using a Mouse Cytokine Array in the supernatant of BMDMs transfected with miR-223-3p, miR-486-3p, or miR-488-5p mimics. In (A–C), bar graphs display mean log₂ fold change in comparison with negative control transfected cells ± standard error. In (E,F), bar graphs display mean fold change in comparison with negative control transfected cells ± standard error. *Denotes P < 0.05, **denotes P < 0.01, and ***denotes P < 0.001 versus unstimulated negative control.