Inhibition of HuR/ELAVL-1 attenuates fibrotic progression in Mdx mice with dilated cardiomyopathy

Abstract

Duchenne muscular dystrophy (DMD) arises from dystrophin deficiency, a crucial component of the dystrophin-glycoprotein complex (DGC) essential for maintaining cellular structural integrity by linking intracellular actin filaments to the basal lamina. Dysfunctions within this complex, coupled with increased inflammatory immune cell infiltration, contribute to the onset of dilated cardiomyopathy (DCM). This cardiac condition, characterized by necrosis and fibrosis, significantly impairs left ventricular function. Despite various treatment approaches, reliable effects on these pathogenic mechanisms remain elusive. RNA-binding proteins play pivotal roles in modulating pathways often dysregulated in cardiac pathology. Notably, HuR, which is upregulated in fibrotic cardiac regions and modulates innate immune system activation, emerges as a promising target. We investigated HuR expression in cardiac tissues of mdx murine model of DMD and assessed the impact of its inhibition with regards to DCM progression. Our findings reveal that HuR is indeed upregulated in mdx mice, and its inhibition leads to attenuation of cardiac fibrosis and improvement in heart function. These preclinical results underscore the potential of targeting HuR for therapeutic intervention to mitigate DCM-associated pathological changes, warranting further exploration for the development of effective treatments.

Keywords: Dilated cardiomyopathy; Duchenne muscular dystrophy; HuR; RNA binding proteins.

Fig. 1

HuR expression in cardiac muscles of mdx mice of different ages and the effects of its modulation through MS-444 on cardiac fibroblasts. (A) Cropped images of representative WB analysis of cardiac muscle of 3 m mdx and 9 m mdx and age-matched C57Bl (n = 3 each, two independent experiments) mice showing the expression of HuR. (B) Cropped images of representative WB analysis showing the expression of HuR in cardiac muscles of 9 m, 14 m and 18 m mdx and age-matched C57Bl (n = 2 each, two independent experiments). (C) Proliferation and (D) cell viability (MTT) of cardiac fibroblasts harvested from mdx and C57Bl mice. (E) Digital PCR evaluation of HuR expression in cardiac fibroblasts treated with different concentrations of MS-444. Cropped images of representative WB analysis of HuR expression in cytoplasmic (F) and nuclear (G) compartments from cardiac fibroblasts treated in vitro. Normalization was carried out using histone H3 as a nuclear marker and GAPDH for the cytoplasmic fraction, confirming the specificity and efficiency of the fractionation. (H) Cropped images of representative WB analysis of cardiac fibroblasts treated with different concentrations of MS-444 to assess the expression of HuR, IL6, TLR4, P38, TNF-α; HMGB1, P21, FOXO-1; collagen I, Cyclin D1, fibronectin according to total proteins loading. Data information: densitometric data were normalized on vinculin and expressed as mean ± SD for WB. Stain-free gels were used for total protein quantification and expressed as mean ± SD. (*p < 0.05, **p < 0.01, ***p < 0.001 with Brown-Forsythe and Welch ANOVA tests). ^#p < 0.05 and ^###p < 0.001 with unpaired t test with Welch’s correction

Fig. 2

Left and right ventricular function in mdx mice following MS-444 treatment. Representative whole-heart images (scale bar: 1 mm) (A) and transthoracic echocardiography analysis in 9 m mdx mice treated with MS-444 and vehicle control (n = 6 each) and age-matched C57Bl ones (n = 11) (B-D). Abbreviations: LV volume in diastole (LV vol D); LV volume in systole (LV vol S); left ventricular weight (LVW) mass; LVW normalized to body mass weight (LVW/BW); Left Ventricle Internal Diameter at diastole (LVIDD); Left Ventricle Internal Diameter at systole (LVIDS); isovolumic relaxation time (IVRT); Right ventricular outflow tract (RVOT); Right ventricular outflow tract – stroke volume (RVOT-SV); Right ventricular outflow tract – cardiac output (RVOT-CO); RVOT-Velocity Time Integral (RVOT-VTI). Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 with Brown-Forsythe and Welch ANOVA tests; ^# p < 0.05, ^## p < 0.01, with unpaired t-test with Welch’s correction (9 m mdx vs. 9 m mdx + MS-444)

Fig. 3

Fibrotic development in mdx cardiac tissues following MS-444 treatment. Representative images of cardiac tissues of 9 m wild type mice, mdx mice treated with MS-444 and vehicle control (n = 4 each) following the PicroSirus Red staining (A) and the relative quantification of the percentage of fibrotic tissue (B). Representative images of cardiac tissues of 9 m C57Bl, mdx and 9 m mdx + MS-444 (n = 4 each) showing the CD18 + cells (C) and the relative histogram representing the amount of Ki-67 per slice (D). Representative images of cardiac tissues of 9 m C57Bl, mdx and 9 m mdx + MS-444 (n = 4 each) showing the Sirius Red staining and – in higher magnification – the HuR + cells and the Ki-67 + cells (identified by black arrowheads) (E). Histogram representing the number of cells expressing Ki-67 (F) and HuR (G) per slice in 9 m wild type mice, mdx mice treated with MS-444 and vehicle control (n = 4 mice; n = 4 images per mice). All the images were taken at 20X. Scale bar: 100 μm. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 with Brown-Forsythe and Welch ANOVA tests

Fig. 4

MS-444 affects pro-fibrotic genes’ expression in mdx cardiac tissues. Digital PCR evaluation of genes commonly associated to cardiac dysfunctions in mdx cardiac tissues. Data were obtained from 3 independent experiments with the 3 animals/group each experiment. Abbreviations: collagen isoforms 3a1 and 1a1 (Col3a1 and Col1a1); connective tissue growth factor (CCN2/CTGF); fibrillin-1 (FBN1); Proline And Arginine Rich End Leucine Rich Repeat Protein (PRELP); Matrix Metallopeptidase 2 and 9 (MMP2 and MMP9); Thrombospondin-4 (thbs4); periostin (POSTN); fibronectin (FN1); WNT1-inducible signaling pathway protein 1 (WISP1/ccn4); Glutathione S-Transferase Omega 1 (Gsto1). Data are presented as mean ± SD. **p < 0.01; ***p < 0.001; ****p < 0.0001 one-way ANOVA Kruskal-Wallis test

Fig. 5

Proteomic evaluation of fibrotic and inflammatory mediators in mdx cardiac tissues following MS-444 treatment. Cropped images of representative WB analysis of cardiac muscle of 9 m mdx mice treated with MS-444 and vehicle control and age-matched C57Bl ones (n = 3 each, two independent experiments) showing the expression of cardiac fibrotic enhancers (A), alarmins and other proteins involved in anti-/pro-inflammatory signalling and fatty acid metabolism (B). Data information: densitometric data were normalized on vinculin and expressed as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ordinary one-way ANOVA, Tuckey multiple comparison test)

Fig. 6

Evaluation of proteins involved in mitochondrial functions, apoptosis and TCA cycle in mdx cardiac tissues following MS-444 treatment. Cropped images of representative WB analysis of cardiac muscle of 9 m mdx mice treated with MS-444 and vehicle control and age-matched C57Bl ones (n = 3 each, two independent experiments) showing the expression of proteins involved in (A) mitochondrial functions, oxidative phosphorylation and apoptosis/mitophagy and (B) TCA cycle. Data information: densitometric data were normalized on vinculin and expressed as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 ordinary one-way ANOVA, Tuckey multiple comparison test)