Increased level of phosphorylated desmin and its degradation products in heart failure

Abstract

Although several risk factors such as infarct size have been identified, the progression/severity of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of ischemic HF and phosphoproteomic technology, we found an increased level of phosphorylated desmin in the left ventricle (LV) of HF-rats. The purpose of the present work is to assess whether desmin is a circulating or only a tissue biomarker of HF. We used several antibodies in order to detect desmin, its proteolytic fragments and its phosphorylated form in LV and plasma by western blot, phosphate affinity electrophoresis, mass spectrometry and immunofluorescence. Plasma was treated with combinatorial peptide ligand library or depleted for albumin and immunoglobulins to increase the sensitivity of detection. We found a 2-fold increased serine-desmin phosphorylation in the LV of HF-rats, mainly in the insoluble fraction, suggesting the formation of desmin aggregates. Desmin cleavage products were also detected in the LV of HF rats, indicating that the increased phosphorylation of desmin results in more susceptibility to proteolytic activity, likely mediated by calpain activity. The native desmin and its degradation products were undetectable in the plasma of rat, mouse or human. These data suggest the potential of serine-phosphorylated form of desmin and its degradation products, but not of desmin itself, as tissue but not circulating biomarkers of HF.

Keywords: Desmin; Heart failure; Mass spectrometry; Phosphorylation; Plasma; Western blot.

Fig. 1

Phosphoproteomic analysis of LV of HF rats. A: Representative 2D-electophoresis gel of LV proteins from HF (n=4) and sham-rats (n=4) stained with ProQDiamond®. The two polypeptidic spots identified to be desmin were enlarged and data of modulation of phosphorylated polypeptidic spots (5 and 45) are expressed as mean of normalized spot's volume±SEM of normalized volume of total spots from sham (white box) and MI (black box)rats at 2 months.*P, 0.05. B: Quantification of desmin (soluble and insoluble fractions) levels in LV (50 µg) of sham- (n=11) (white box) and HF- (n=11) (black box) rats at 2 months after MI. Soluble and insoluble desmin were respectively quantified in LV proteins extracted in RIPA buffer (50 µg proteins) and Urea/Thiourea buffer (20 µg proteins). C: Ser-phosphorylated, Thr-phosphorylated and Tyr-phosphorylated desmin were quantified in LV (50 µg) of sham- (n=16) (white box) and HF- (n=19) (black box) rats at 2 months after MI. Data are presented as ratio of Ser-, Thr- and Tyr-phosphorylated desmin/total soluble desmin. The positions of Mw are indicated on the left. An internal standard was loaded in each gel for the standardization and quantification. Data are expressed as means of an arbitrary unit (AU)±SEM.*P, 0.05.

Fig. 2

A: Structural molecular organization of the rat desmin molecule (P48675) (adapted from and desmin sequence recognized by the three desmin antibodies (clones Y66 (Abcam, ab32362), DE-U-10 (Sigma-Aldrich, D1033) and D33 (Dako, M0760) used for western blot. A dashed line is shown for the D33 antibody as no information on the epitope recognized is available. B, C: Western blot analysis of desmin in plasma from rat, mouse and human species with the three desmin antibodies. Plasma was either depleted for albumin and IgG (B) or treated by combinatorial peptide ligand library (CPLL) (C). M: size marker, C: 50 µg of treated plasma, IP: immunoprecipitated treated plasma with 2 µL of DE-U-10 desmin antibody, LV: left ventricle (10 µg), S: supernatant of LV IP (10 µg), Ab: antibody (2 µL). The antibody used for the western blot is indicated on each blot. Arrows indicate the specific desmin band recognized by the antibodies and ns the non-specific bands.

Fig. 3

Mass spectrometry analysis of rat, mouse and human plasma (A) and rat LV (B) samples. A: Venn diagram of identified proteins in crude plasma (upper panel) and plasma immunoprecipitated with DE-U-10 desmin antibody (lower panel). For plasma, 4 samples from 2 HF- (HF1, HF2) and 2 sham- (C1, C2) rats, 3 different samples from mouse (M1, M2, M3) and human (H1, H2, H3) were analysed. B: Quantification of desmin in LV samples corresponding to the rats tested for plasma (HF1, HF2, C1, and C2). Levels of the 3 replicates injection of each sample show the reproducibility (left panel). Average levels of desmin in LV rats with standard deviation less than 10% (middle panel). Ratio levels of desmin in LV rats with the sham-C1 rats considered as the lower level for the ratio calculation (right panel). Detailed information's are presented in Table 1.

Fig. 4

Biochemical characterization of desmin. A: Western blot analysis of desmin in LV proteins (50 µg) extracted from sham- and HF-rats in RIPA and Urea/Thiourea buffer. The ponceau red staining gel (left panel) and the western blot images (right panel) with desmin DE-U-10 (upper panel) and Y66 (lower panel) antibodies at short exposure are presented. B: Western blot analysis of desmin in LV proteins separated using Phos-Tag (8%, 50 µmol/L) affinity electrophoresis performed with the DE-U-10 antibody. LV proteins are extracted in RIPA (50 µg of proteins) (upper panel) or Urea/Thiourea (20 µg of proteins) (lower panel) buffer. The western blot images presented are at long exposure (>1000 s) for RIPA extraction and short exposure (12 s) for Urea/Thiourea extraction. Arrows indicate the specific desmin bands recognized. M: size marker.

Fig. 5

Representative immunofluorescence stainings of desmin (green) in primary culture of neonate rat cardiomyocytes (NCM) (A, B, C) and in left ventricle from control rat (D, E, F) with the three desmin antibodies. Nuclei are stained in blue (DAPI).