| Objectives:To clarify if mechanical stretch aggravates cardiomyocyte injuries caused by ischemia/ hypoxia and if cytoskeleton is involved.Methods: 1. A mechanical stretch model using rat cardiomyocytes, which were cultured on silicone membrane in vitro, was established. LDH leaking, PI staining positive ratio and cardiomyocyte morphology were investigated to test the effects of mechanical stretch.2. Cultured cells were divided into three groups: 10% static axial stretch group (S), ischemia/hypoxia simulation group (I) and S and I combined group (IS). Samples were collected at 1,3,6,12h and immunofluoresecence histochemistry, LDH leaking, TNF-αmRNA, MHCmRNA, PI staining positive ratio were investigated.3. Western Blotting and laser confocal microscopy were used to detect proteins and morphology of microtubule and microfilament in cultured cells of different group.4. Cytoskeleton stabilizer taxol (for microtubule) or (and) phalloidin (for non-sarcomeric microfilament) was (were) administered to evaluate the effects of cytoskeleton on cardiac myocytes injury in IS group. Immunofluoresecence histochemistry, LDH leaking, TNF-αmRNA expression, MHCmRNA expression, PI staining positive ratio were investigated.Results:1. After modification, a uniaxial static mechanical stretch device was successfully manufactured. It is easy to make and observe during stretch, and also it is convenient for ischemia/hypoxia exposure. Cells cultured in this stretch model were not obviously damaged according to the results of LDH leaking and PI staining. 2. Compared to S and I group, cells of IS group differ greatly: LDH leaking from cultured cardiomyocytes increased sharp; TNF-αmRNA expression increased; β-MHC mRNA expression reached its peak at 3h, which is earliest and highest among three groups and then decreased quickly, which was the lowest; PI staining positive ratio increased greatly.3. Changes of the structure of non-sarcomeric microfilament and microtubule varied according to different stimulus: 10% stretch led non-sarcomeric microfilament to reorganize and reorientate. This phenomenon could be observed from the enhanced parallel filament by immunofluoresecence histochemistry staining. Disruption of microfilament occurred when cells were exposed to ischemia/hypoxia and the situation deteriorated as time went by. In contrast to the former, in IS group, the cytoskeleton suffered even more damages: protein content decreased quickly as detected by Western Blotting, and the interlaced network was rapidly bankrupted. The cells demonstrated by non-sarcomeric microfilament fluoresecence staining lost their typical cardiac myocyte shape. Changes of microtubule were drastic but similar to non-sarcomeric microfilament. 4. When taxol or (and) phalloidin was (were) added into the culture medium of IS group, LDH leaking and PI staining positive ratio decreased, down-regulation ofα-MHCmRNA expression became slightly and the extent of fluctuation of β-MHCmRNA expression diminished, the elevation of TNF-αmRNA expression decreased.Discussions:1. We designed a simple, economical and feasible uniaxial static mechanical stretch device and imposed 10% static stretch on cells cultured on silicone sheet. Relevant experiments were carried out to testify that 10% stretch intensity is a physiologically relational stimulus and it is not an acute damage factor for cell. Furthermore, some results revealed that cell become more active in many ways after stretch.2. Mechanical stretch aggravates cardiomyocyte injury caused by ischemia/hypoxia. It can be inferred from the following: aggravation of morphological changes and deterioration of cell membrane rupture, up-regulation of inflammation relevant gene expression, enhancement of the negative regulation of ischemia/hypoxia on cardiac contractile protein gene expression.3. The damages of cytoskeleton caused by ischemia/hypoxia can be exaggerated by 10% stretch, which was evidenced by decreased protein content and disappearance of filament-like structure. The resistance of c...
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