Author + information
- Peng Zhang,
- Chunxiao Hu,
- Ying Wang,
- Yongyong Li,
- Dewei Wang,
- Shu Qin and
- Dongying Zhang
The Wnt/Planar Cell Polarity (Wnt/PCP) pathway, responsible for tissue polarity in cardiogenesis in vertebrates, has been shown to play multiple roles during cardiac differentiation and development. Van Gogh-like-2 (Vangl2) is a core PCP component that regulates the induction of polarized cellular and tissue morphology during animal development. However, little is known about Wnt/PCP signaling pathway in the process of myocardial remodeling.
Primary cultures of ventricular cardiomyocytes were prepared from 1 to 3-day-old Sprague-Dawlay rats. Cells were divided into the following four groups: (1) Control group: cardiomyocytes transfected with scramble-siRNA (scr-siRNA) were cultured for 5h with 10% fetal bovine serum-DMEM/F12; (2) Wnt5a group: cardiomyocytes transfected with scr-siRNA were exposed to 100ng/ml Wnt5a for 5 h; (3) Vangl2-siRNA group: cardiomyocytes transfected with Vangl2-siRNA were cultured for 5 h with 10% fetal bovine serum-DMEM/F12; (4) Wnt5a+Vangl2-siRNA group: cardiomyocytes transfected with Vangl2-siRNA were were exposed to 100ng/ml Wnt5a for 5h. Cell surface and arrangement of cytoskeleton were determined by immunofluorence. Atriopeptin (ANP), JNK activity, Voltage-Dependent Anion Channel (VDAC) and apoptosis protein were detected by western blot. Changes in mitochondrial transmembrane potential were measured with dye 5,5′, 6,6′-tetrachloro-1,1′, 3,3′-tetraethylbenzimidazocarbocyanine iodide (JC-1). The apoptotic rate was measured by flow cytometry.
Compared to control cells we found an increase of myocyte surface area (2.18±0.91 vs 0.97±0.36,p<0.001) and ANP protein synthesis(1.70±0.34 vs 1.00 ±0.21,p<0.005) in Wnt5a treated cells. However, the hypertrophic effect of Wnt5a was inhibited in Vangl2 depleted cells. Meanwhile, stimulation with Wnt5a induced strong JNK activation(1.64±0.34 vs 1.00±0.13,p<0.001) and cytoskeleton rearrangement(72.3±5.4% vs 11.8±1.9%,p<0.001) in control cells but failed to activate these effects in cells lacking Vangl2. Expression of VDAC and mitochondrial membrane potential in Wnt5a group was significantly less than those in control group. After administration of Vangl2-siRNA, the expression of VDAC (0.89±0.08 vs 0.59±0.11,p<0.01) and mitochondrial membrane potential (15.28±0.99 vs 8.25±0.97,p<0.05) was higher than those in Wnt5a group. Moreover, the apoptotic rate, protein expression of cleaved caspase-3 and -9, Bax were increased in Wnt5a group compared with those from control group, whereas depletion of Vangl2 decreased the apoptotic rate(11.26±1.02% vs 41.55±2.78%, p<0.005), expression of cleaved casepase-3(1.07±0.12 vs 1.85±0.43,p<0.001) and cleaved casepase-9 (1.29±0.27 vs 1.78±0.32,p<0.05), Bax (0.95±0.14 vs 1.89±0.32, p<0.001).
These results suggest that activation of non-canonical Wnt/PCP signaling by Wnt5a stimulates myocardial remodeling (structure and mitochondrial function), in which Vangl2 may play an important regulatory effect.