Figure 8

The hypertrophic effect of KLK8 is dependent on EGF signaling.

(A) Primary cultured neonatal cardiomyocytes were infected with KLK8 adenovirus, 24 h later control siRNA, kinin B₁R siRNA or B₂R siRNA was transfected into the cardiomyocytes. Twenty four hours later, all the media of cells were replaced with fresh media, then serine protease inhibitors (antipain or ZnSO4), kinin B₁R antagonist R715, or kinin B₂R antagonist HOE140 was added into the culture media, After incubation for 24 h, culture media were collected for determination of EGF. (B–F) Cells were infected with KLK8 adenovirus, 24 h later control siRNA or EGFR siRNA was transfected into the cardiomyocytes. Twenty four hours later, all the media of cells were replaced with fresh media, then EGFR antagonist AG1478 was added into the culture media. The hypertrophic effects were detected after additional 48 h of incubation. B showed KLK8 protein expression. Total protein content was determined by BCA assay (C). (D) Cardiomyocytes were stained with FITC-conjugated phalloidin as described in “Methods”. One hundred cells from randomly selected fields in three independent expreriments were evaluated for cell size using the Image-Pro Plus cell area measurement software. Transcripts of cardiac hypertrophy markers including ANP (E), BNP (F) and Myh7 (G) were determined by quantitative real-time RT-PCR. Values are presented as mean ± SEM (n = 3). **P < 0.01 vs cells treated with Ad-vector and control siRNA; ^##P < 0.01 vs cells treated with Ad-KLK8 and control siRNA.