Desmoplakin is important for proper cardiac cell-cell interactions

Abstract

Normal cardiac function is maintained through dynamic interactions of cardiac cells with each other and with the extracellular matrix. These interactions are important for remodeling during cardiac growth and pathophysiological conditions. However, the precise mechanisms of these interactions remain unclear. In this study we examined the importance of desmoplakin (DSP) in cardiac cell-cell interactions. Cell-cell communication in the heart requires the formation and preservation of cell contacts by cell adhesion junctions called desmosome-like structures. A major protein component of this complex is DSP, which plays a role in linking the cytoskeletal network to the plasma membrane. Our laboratory previously generated a polyclonal antibody (1611) against the detergent soluble fraction of cardiac fibroblast plasma membrane. In attempting to define which proteins 1611 recognizes, we performed two-dimensional electrophoresis and identified DSP as one of the major proteins recognized by 1611. Immunoprecipitation studies demonstrated that 1611 was able to directly pulldown DSP. We also demonstrate that 1611 and anti-DSP antibodies co-localize in whole heart sections. Finally, using a three-dimensional in vitro cell-cell interaction assay, we demonstrate that 1611 can inhibit cell-cell interactions. These data indicate that DSP is an important protein for cell-cell interactions and affects a variety of cellular functions, including cytokine secretion.

Figure 1

Tight interactions between various cells of the heart. TEM of murine left ventricle demonstrating cell-cell interactions between (A) myocytes and fibroblasts, (B) myocytes and ECs, and (C) fibroblasts and ECs. High magnification of inset is shown in panels D, E, and F. Red arrows indicate regions of tight cell-cell interactions. Scale bars are shown in individual panels.

Figure 2

Desmoplakin staining co-localizes with 1611 antibody staining. A–D: Confocal micrograph of murine left ventricle showing 1611 staining (red) of cardiac fibroblasts, myocytes with phalloidin (green), and nuclei with DAPI (blue). E–I: Confocal micrograph demonstrating partial co-localization of 1611 (red) with DSP (white) in the left ventricle of the mouse heart. The section was also stained with phalloidin (green) and DAPI (blue). Scale bars are shown in individual panels.

Figure 3

Two-dimensional electrophoresis profile of cardiac fibro-blast proteins recognized by 1611 antibody. Cardiac fibroblast protein lysates were immunoprecipitated with 1611 antibody and subjected to 2D gel electrophoresis. Samples were focused on 7 cm IPG strips (pH 3–10) and separated in 4–20% SDS-PAGE gels. The proteins were visualized with silver stain and subjected to MALDI-MS analysis. The protein spots were identified as DSP I, DSP II, plakoglobin, obscurin, and tropomyosin 2 as indicated in the table. N = 5.

Figure 4

IP analyses demonstrating that 1611 antibody recognizes DSP. A: Protein lysates from myocytes, cardiac fibroblasts, or whole heart were immunoprecipitated with 1611 antibody and then subjected to western blot analyses with either 1611 antibody (upper panel) or anti-DSP antibody (lower panel). B: Protein lysates from myocytes, cardiac fibroblasts, or whole heart were immunoprecipitated with anti-DSP antibody and then subjected to western blot analyses with either 1611 antibody (upper panel) or anti-DSP antibody (lower panel). N = 3.

Figure 5

Knockdown of DSP expression decreases cell-cell interactions between cardiac fibroblasts, myocytes, and ECs. A: Cardiac fibroblasts were left untreated or transfected with control or DSP siRNA (10 nM). Either at 24 or 48 h, the cells were collected and examined for DSP expression. Significant knockdown of DSP was observed at both 24 and 48 h (p < 0.05). Fibroblasts were collected at 48 h and used in cell-cell interaction assays. The effects of DSP knockdown or treatment with 1611 antibody were examined on cell-cell interactions between (B) myocytes and fibroblasts, (C) fibro-blasts and fibroblasts, or (D) ECs and fibroblasts. 50 μg of 1611 antibody were used in these studies. Cell-cell interactions were blocked in all three cultures treated with 1611 antibody (p < 0.05). DSP knockdown in fibroblasts also inhibited cell-cell interactions (p < 0.05). N = 3. * indicates a significant difference between experimental sample (1611 treated or DSP knockdown cells) and control cells. # indicates a significant difference between the two experimental groups (1611 treated versus DSP knockdown cells).

Figure 6

Disruption of cell-cell interactions results in reduced IL-6 expression. Cardiac fibroblasts were left untreated, treated with 1611 antibody (50 mg), or treated with DSP siRNA (10 nM). Fibroblasts were then co-cultured with either (A) myocytes or (B) ECs, and media were collected at indicated time points. Disruption of cell-cell interactions with either 1611 antibody (p < 0.05) or with DSP knockdown (p < 0.05)resulted in reduced IL-6 secretion. N = 3.