Figure 3

Mechanical stretch up-regulates BNP and natriuretic peptide receptor A (NPRA) in human cardiac fibroblasts. Human primary cardiac fibroblast cells underwent either biaxial mechanical stretch for 72 h or were left un-stretched (control) in the presence or absence of 10 ng/mL recombinant TGFβ. Changes in gene expression of b-type natriuretic peptide (BNP) and its receptor NPRA were investigated using quantitative real-time PCR (A). Further evidence that mechanical stretch has the ability to up-regulate NPRA is demonstrated at the protein level in the absence of TGFβ stimulation. Increased NPRA protein on stretched cardiac fibroblasts is evident, as shown by immunofluorescent microscopy (B). The intensity of NPRA immunofluorescence (red) is greater in cells mechanically stretched for 72 h compared to un-stretched (control) cells. The nuclei of cells were counterstained using DAPI (blue). Images were captured (Zeiss Axio Imager M1), at the same exposure time for comparison. Stretch-induced (72 h) up-regulation of NPRA protein was measured by western blotting and quantified using densitometry (C). Protein extracted from HEK-293-T cells over-expressing NPRA were used as a positive control (+ve). Bar graphs represent mean fold change ± SD. **P < 0.01, ***P < 0.001.