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This study is aiming to find the signal transduction underlying calcium overloading caused cell apoptosis phenomenon post myocardial treatments.
For the information of Wnt5a/frizzled-2 signal pathway in mammalian cardiac cells were hardly seen before, first we needed to confirm the gene and protein expression of the effectors (wnt5a;frizzle-2;p-camkii, the marker presents the activation of this signal) which consisting this signal within myocardial cells, through q-rtpcr, western blot and some specific methods in detail.
Second, we transfected the cells with frizzled-2 gene in order to activate this pathway, and recorded the intracellular and cellular effects like cell apoptosis. Third, we used Stealth RNAi to conduct frizzled-2 gene suppression, then observed the following effects. Results indicated the role of Wnt5a/Frizzled-2 pathway played in calcium overloading process and cell apoptosis. Next, we analyzed the expression of the effectors of this pathway after the cells were conducted through hypoxia/reoxygenation treatment. In this part, the same testing objects and methods were used to consistent with different treatment groups.
First, results from simple cell interference test showed that wnta5a, frizzled-2 and p-camkii is stable expressing in cardiac cells.
Second, High expression of p-CamkII followed by intracellular accumulation of calcium post frizzled-2 transfection indicates the activation of wnt5a/Frizzled-2 pathway. This was proved by gene suppression of frizzled-2 on the membrane: down regulation of frizzled-2 gene caused down expression of Frizzled-2 protein and p-CamkII marker, also the calcium accumulation and apoptosis.
Third, data from hypoxia/reoxygenation treatment group is found have the same trend echoes to part 1. Whatever in hypoxia group or in reoxygenation group, expression of Frizzled-2 on the cell membrane determines the character of wnt5a and downstream effectors, and eventually affects the intracellular calcium accumulation and apoptosis.
We hypothesis that activation of wnt5a/Frizzled-2 pathway post myocardial treatments could be a main reason causing calcium overloading and cell apoptosis. Results have proved our hypothesis, and this is the first paper in this area in evaluating this signal pathway within mammalian cardiac cells and raising a possible concept that activation of this signal pathway might be one of the mechanisms underlying cell apoptosis post myocardial treatments.
- 2013 American College of Cardiology Foundation