Phosphorylation of Hsp20 Promotes Fibrotic Remodeling and Heart Failure

Abstract

Cardiomyocyte-specific increases in phosphorylated Hsp20 (S16D-Hsp20) to levels similar to those observed in human failing hearts are associated with early fibrotic remodeling and depressed left ventricular function, symptoms which progress to heart failure and early death. The underlying mechanisms appear to involve translocation of phosphorylated Hsp20 to the nucleus and upregulation of interleukin (IL)-6, which subsequently activates cardiac fibroblasts in a paracrine fashion through transcription factor STAT3 signaling. Accordingly, treatment of S16D-Hsp20 mice with a rat anti-mouse IL-6 receptor monoclonal antibody (MR16-1) attenuated interstitial fibrosis and preserved cardiac function. These findings suggest that phosphorylated Hsp20 may be a potential therapeutic target in heart failure.

Keywords: Ccl2, C-C motif chemokine ligand 2; Ccl3, C-C motif chemokine ligand 3; Col1a1, collagen 1A1; Col3A1, collagen 3A1; ECM, extra-cellular matrix; Hsp, heat shock protein; Hsp20; I/R, ischemia/reperfusion; IL, interleukin; IL-6; Postn, periostin; SMA, smooth muscle actin; STAT3, signal transducer and activator of transcription 3; TG, transgenic; TGF, transforming growth factor; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WT, wild type; fibroblast; heart failure; remodeling.

Graphical abstract

Figure 1

S16D-Hsp20 Transgenic Mice Exhibit Reduced Survival, Left Ventricular Remodeling, and Dysfunction (A) Kaplan-Meier survival curve of S16D and WT control mice. Longitudinal echocardiographic assessment of S16D and WT mice at 2, 4, and 6 months of age: (B) LVEDV; (C) LVESV; and (D) EF. Gravimetric analysis of S16D and WT control mice at 2, 4, and 6 months of age. Ratios of (E) heart weight/body weight and (F) lung weight/body weight in S16D and WT control mice. Values are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 versus WT. The number of samples (n) per group is indicated on the bar graphs. EF = ejection fraction; LVEDV = left ventricular end diastolic volume; LVESV = left ventricular end systolic volume; S16D = phosphorylated Hsp20; WT = wild type.

Figure 2

Extensive Interstitial Fibrosis Evident in S16D-Hsp20 Hearts (A) Representative confocal images (original magnification: ×20) for TUNEL staining (green) counterstained with actin (red) and DAPI to visualize nuclei (blue). Arrows indicate TUNEL-positive cells; scale bar: 50 μm. (B) Quantitative analysis of TUNEL-positive nuclei (expressed as a percentage of total nuclei) in S16D and WT ventricular sections at 2, 4, and 6 months of age. (C) Representative images (original magnification: ×20) of ventricular sections stained with Picrosirius red; scale bar: 50 μm. (D) Quantitative analysis of ventricular fibrosis (expressed as a percentage of total area) in S16D and WT hearts at 2, 4, and 6 months of age. The number of samples (n) per group is indicated on the bar graphs. DAPI = 4′,6-diamidino-2-phenylindole; TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; other abbreviations as in Figure 1.

Figure 3

S16D-Hsp20 Cardiomyocyte-Conditioned Medium Activates Myofibroblast Differentiation Adult mouse cardiac fibroblasts were cultured in conditioned medium from Ad.GFP, Ad.S16D, Ad.S16A. Fibroblast proliferation and markers of myofibroblast differentiation were assessed. (A) Quantification of fibroblast proliferation was determined using the MTT assay. Values are fold changes relative to those of Ad.GFP. Relative gene expression levels are shown for (B)Col1a1; (C)Col3a1; (D)TGFβ1; and (E)IL-6. Values are fold-changes relative to those of Ad.GFP. (F) Representative confocal images (original magnification: ×40) of fibroblasts stained with αSMA (green) plus DAPI to visualize nuclei (blue); scale bar: 50 μm. (G) Quantification of αSMA fluorescence intensity. The number of samples (n) per group is indicated on the bar graphs. SMA = smooth muscle actin; Ad.GFP = adenovirus-infected green fluorescent protein; Ad.S16D = adenovirus-infected phosphorylated Hsp16D; Ad.S16A = adenovirus-infected dephosphorylated Hsp16A; Col1a1 = collagen 1a1; Col3a1 = collagen 3a1; IL = interlukin-6; TGF = transforming growth factor; other abbreviations as in Figure 2.

Figure 4

Acute Overexpression of S16D-Hsp20 in Cardiomyocytes Promotes Upregulation of the IL-6 Gene Expression and Secretion (A) Assessment of the following cytokine gene expression levels in Ad.GFP and Ad.S16D cardiomyocytes Ccl2, Ccl3, TNF-α, and IL-6. TGFβ1 and IL-1β were not detectable in either group. Values were normalized to those of the internal control, 18 S, and expressed as fold-changes relative to those of Ad.GFP. (B) Quantification of IL-6 secretion into the conditioned medium from Ad.GFP and Ad.S16D cardiomyocytes was determined by using an ELISA assay. The number of samples (n) per group is indicated on the bar graphs. Ccl2 = C-C motif chemokine ligand 2; Ccl3 = C-C motif chemokine ligand 3; ELISA = enzyme-linked immunosorbent assay; MR16-1 = rat anti-mouse IL-6 receptor monoclonal antibody; TNF = tumor necrosis factor; WT = wild type; other abbreviations as in Figure 3.

Figure 5

MR16-1 Treatment Attenuates Fibrosis and Improves Cardiac Function (A) Quantification of IL-6 gene expression levels in S16D hearts and WT control hearts. Values were normalized to those of the internal control GAPDH and expressed as fold-changes relative to those of WT mice. (B) Quantification of IL-6 serum levels in S16D and WT control mice was determined by using an ELISA assay. (C) Schematic presentation of S16D and WT control mice treated with MR16-1 or PBS (control) starting at 1 month of age. Following the baseline echocardiographic assessment, mice were injected with 2 mg/body weight MR16-1 or PBS. Subsequently, mice received 0.5 mg/body per week (2 injections of 0.25 mg/body) during weeks 1, 2, and 3. (D) Left ventricular ejection fraction at baseline and at 2 and 4 weeks; *p < 0.05 versus WT-PBS; #p < 0.05 versus S16D-MR16-1. (E) Representative images (original magnification: ×20) of Picrosirius red staining following 4 weeks of treatment; scale bar: 50 μm. (F) Quantification of percent of ventricular fibrosis (fibrotic area/total ventricular area). (G) Representative Western blots and (H) quantification of P-STAT3 (Y705)/total STAT3 protein levels of the indicated groups. Values are fold-changes relative to those of WT-PBS. The number of samples (n) per group is indicated on the bar graphs. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; I.P. PBS = intraperitoneal phosphate buffered saline; P-STAT3 = phosphorylated STAT3; other abbreviations as in Figures 3 and 4.