Laminin-derived peptide drives the cardiomyogenic potential and cardiac cells functionality

Abstract

Laminin represents a major component of the basement membrane in cardiac tissue. Through integrins binding, laminin can sustain cell adhesion, proliferation, differentiation and mechanotransduction. The role of a small bioactive peptide (KKGSYNNIVVHV) derived from laminin in cardiac differentiation and functionality has been demonstrated. Briefly, results showed how the presence of laminin-derived peptide enhanced the differentiation of mesenchymal stem cells into cardiomyocytes-like phenotype, by modulating not only the cytoskeletal apparatus but also enhancing the cardiac and adhesion markers. Moreover, neonatal mouse cardiomyocytes in presence of the peptide showed well organized cytoskeletal rearrangements and regulated contractility. Therefore, in this study the role of laminin-derived peptide in guiding cardiomyogenesis has been characterized, opening innovative approaches in functional cardiac tissue engineering and regenerative medicine.

Keywords: bioactive peptides; cardiac tissue engineering; cardiomyogenesis; integrins; laminin.

FIGURE 1

Morphometrical evaluation. (A) Cell morphology obtained after Phalloidin (green) staining and (B) anti-Vinculin (red) staining, nuclei were stained with DAPI (blue). Images represent control and differentiated cells, seeded onto G2 coated surfaces. Images are representative of all results obtained in three independent experiments. Scale bar: 50 μm (phalloidin staining), 20 μm (vinculin staining). (C,D) morphometric analyses of focal adhesion length (μm) and focal adhesion number of MSCs treated with different concentrations of G2, in control and differentiative medium. Data are represented as Gaussian curves with n ≥ 100. Measures were obtained from fluorescence microscopy images. Results are significant with respect to control, p ≤ 0.05.

FIGURE 2

Evaluation of cardiac differentiation markers. Gene expression profiling of (A) NKX 2.5, (B) GATA-4 and (C) Troponin I. All results were normalized with respect to the control (CTR). Western blot analyses with (D) anti-Connexin 43 and (E) anti-Troponin I. GAPDH expression was used to normalize the results. Proteins were revealed on total lysates and all results were normalized with respect to control. Densitometry obtained represents results from three different experiments, expressed as mean ± SD. *Statistical significance with respect to control with p ≤ 0.05.

FIGURE 3

Western blot analyses with anti-Integrin β1 and anti-Integrin α7 antibodies. Tubulin expression was used to normalize the results. Proteins were revealed on total lysates and all results were normalized with respect to control. Densitometry obtained represents results from three different experiments, expressed as mean ± SD. *Statistical significance with respect to control was indicated with p ≤ 0.05.

FIGURE 4

Western blot analyses using anti-Integrin β1 and anti-Integrin α7 antibodies. Tubulin expression was used to normalize the results. Proteins were revealed on fractionated lysates and all results were normalized with respect to control cytoplasmic portion. C=Cytoplasm, M = Membrane. Densitometry obtained represents results from three different experiments, expressed as mean ± SD. *Statistical significance with respect to the relative cytoplasmic portion with p ≤ 0.05.

FIGURE 5

Morphological evaluation of Y201 cells after 7 days of culture with anti-integrin β1 as a neutralizing antibody. Cell morphology obtained after Phalloidin (green) staining, nuclei were stained with DAPI (blue). Images represent control and differentiated cells, previously seeded onto peptide coating surfaces. Images are representative of all results obtained in the three different experiments. Scale bar 50 μm.

FIGURE 6

Gene expression profiling of NKX 2.5 and GATA-4 in (A–C) normal condition and with (B–D) anti-integrin β1 as a neutralizing antibody. All results were normalized with respect to the control (CTR). Results obtained from three different experiments, expressed as mean ± SD. *Statistical significance with respect to control with p ≤ 0.05.

FIGURE 7

Morphometrical evaluation. (A) Cell morphology obtained after Phalloidin (green) staining anti-Vinculin (red) staining, nuclei were stained with DAPI (blue). Images represent control and treated cells with G2. Images are representative of all results obtained in the three different experiments. Scale bar: 50 μm (phalloidin staining), 20 μm (vinculin staining). (B,C) morphometric analyses of focal adhesion length (μm) and focal adhesion number of primary cardiomyocytes treated with different concentrations of G2. Data are represented as Gaussian curves with n ≥ 100. Measures were taken from fluorescence microscopy images. Results are significant with respect to control (CM), p ≤ 0.05.

FIGURE 8

Evaluation of cardiac differentiation markers. Gene expression profiling of (A) GATA-4, (B) Troponin I and (C) Desmin. All results were normalized with respect to the control (CTR). Western blot analyses with (D) anti-Connexin 43 and anti-Troponin I antibodies and (E) anti-Integrin β1 and anti-Integrin α7 antibodies. The GAPDH and Tubulin expressions was used to normalize the results. Proteins were revealed on total lysates and all results were normalized with respect to control and differentiated not treated. Densitometry obtained represents results from three different experiments, expressed as mean ± SD. *Statistical significance with respect to control with p ≤ 0.05.

FIGURE 9

(A) Costameres formation revealed with anti-α-Actinin (green), anti-Integrin α7 (red) staining on cardiomyocytes (CM) and CM seeded onto G2 coating (G2). Nuclei were stained with DAPI (blue). Scale bar: 20 μm. (B) anti-Integrin α7 fluorescence quantification. The corrected total cell fluorescence (CTCF) is represented as whisker plot with n = 8. *Statistical significance with respect to CM with p ≤ 0.05.

FIGURE 10

Contracting frequency of cardiomyocytes measured over a 60-s recording period. Images obtained from MYOCYTER software as dataplot.