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. 2008 Jul-Aug;1784(7-8):1068-76.
doi: 10.1016/j.bbapap.2008.04.003. Epub 2008 Apr 18.

Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and alpha-B-crystallin

Giulio Agnetti  1 , Karel Bezstarosti, Dick H W Dekkers, Adrie J M Verhoeven, Emanuele Giordano, Carlo Guarnieri, Claudio M Caldarera, Jennifer E Van Eyk, Jos M J Lamers
Affiliations

Proteomic profiling of endothelin-1-stimulated hypertrophic cardiomyocytes reveals the increase of four different desmin species and alpha-B-crystallin

Giulio Agnetti et al. Biochim Biophys Acta. 2008 Jul-Aug.

Abstract

We performed a proteomic investigation on primary cultures of neonatal rat cardiomyocytes after treatment with 10 nM endothelin-1 (ET1) for 48 h, an in vitro model for cardiac hypertrophy. Two-dimensional gel electrophoresis profiles of cell lysates were compared after colloidal Coomassie Blue staining. 12 protein spots that significantly changed in density due to ET1 stimulation were selected for in-gel digestion and identified through mass spectrometry. Of these, 8 spots were increased and 4 were decreased. Four of the increased proteins were identified as desmin, the cardiac component of intermediate filaments and one as alpha-B-crystallin, a molecular chaperone that binds desmin. All the desmins increased 2- to 5-fold, and alpha-B-crystallin increased 2-fold after ET1 treatment. Desmin cytoskeleton has been implicated in the regulation of mitochondrial activity and distribution, as well as in the formation of amyloid bodies. Mitochondria-specific fluorescent probe MitoTracker indicated mitochondrial redistribution in hypertrophic cells. An increase of amyloid aggregates containing desmin upon treatment with ET1 was detected by filter assay. Of the four proteins that showed decreased abundance after ET1 treatment, the chaperones hsp60 and grp75 were decreased 13- and 9-fold, respectively. In conclusion, proteomic profiling of ET1-stimulated rat neonatal cardiomyocytes reveals specific changes in cardiac molecular phenotype mainly involving intermediate filament and molecular chaperone proteins.

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Figures

Fig. 1
Fig. 1
Hypertrophic response of cardiomyocytes to ET-1. Protein/DNA ratio (A), 3H-leucine incorporation (B), and morphology of the cardiomyocytes after 48 h incubation without (C) or with ET-1 (D) were determined. Data plotted in A and B were obtained from various experiments (n ≥ 3), the values normalized as percentages of average control values (±SEM). A significant increase of protein/DNA ratio (60 ± 10%, mean ± SEM) by ET1 was observed (A). A similar increase in 3H-leucine incorporation (74 ± 14%, mean ± SEM) was seen by ET1 (B). Photographs of fixed cells stained with FITC–phalloidin were taken by means of an epifluorescence microscope (Olympus IX50) using a FITC filter. Control cells are shown in panel C whereas cells stimulated with 10 nM ET1 are shown in panel D. After ET1 stimulation cells appeared to show a slight increase in cell size together with different “star-shaped” arrangement of cell clusters.
Fig. 2
Fig. 2
2DE gel-based proteomic profile of control cardiomyocytes. A representative image of colloidal Coomassie Blue-stained 2DE gel (18 × 20 cm) is shown (A). Proteins of which the quantity is changed in ET1-stimulated cultures relative to control cultures are encircled and marked by a number assigned on the basis of direction (+/−) and –fold (n) changes (1–12). Differential display analysis is derived from statistical variation of spot density between classes of gels (n=4). The squared box highlights the “desmin area”. The same area is magnified in panels B and C from representative gel images obtained from control (B) and hypertrophic (C) cell lysates. Refer to Table 1 for spot description.
Fig. 3
Fig. 3
Graphical illustration of the quantitative protein changes in control versus ET1-stimulated cells. Results of statistical analysis (p<0.05) for spot density variation in ET1-stimulated cells with relative to control cells are plotted. Numeric values are reported in Table 1. Representative spot images are shown each on top of the relative bar and encircled when accompanied by other spots present in the same gel area.
Fig. 4
Fig. 4
Desmin identity and changes validation. A representative fragmentation spectrum (tandem MS) of the peptide FLEQNAALEVNR obtained from the tryptic digestion of spot 5 is shown in panel A. The sequence of the peptide ion selected in the first MS is shown at the bottom of the spectra. The masses of detected b and y ions after fragmentation are listed in the table next to the spectra. y° indicates additional loss of H2O; b*,y* indicate additional loss NH3. Representative images of two-dimensional western blotting analysis for desmin are displayed for control (B) and ET1-treated cells (C). A Coomassie stained 1D gel is shown in panel D showing equal protein load (60 μg) together with representative image of 1D western blotting analysis for desmin aside.
Fig. 5
Fig. 5
Intracellular localization of desmin (A and B) and mitochondria (C and D). Control (A and C) and ET1-stimulated cells (B and D) were stained either with anti-desmin antibody or MitoTracker. Pictures were taken at 1000× magnification using oil-immersion microscopy. Desmin signal appeared to be markedly increased in ET1-stimulated cells (B) compared to control conditions (A), showing aggregate-like signals especially in the perinuclear area. MitoTracker probing revealed a network spread throughout the whole cell in control cells (C) whereas the signal appeared to be accumulated in the perinuclear region, after ET1 stimulation (D).
Fig. 6
Fig. 6
Detection of amyloid-like aggregates by filter assay. A representative image is shown of a membrane filter (A), probed with anti-desmin antibody (upper row) or the general protein stain Direct Blue 71 (lower row). Direct blue staining shows equal protein load. Mean density values obtained from three independent experiments (n≥2) are plotted in B. Density values are expressed as percentage (±SD). Each value was normalized to the relative average of control values (100%). Desmin signal was significantly increased (81%; p=0.007) after ET treatment.
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