The effect of shear on the cytoskeleton remodeling and physiological performance of myocardium cells through Tmod1

Abstract

Objective: mechanical stimulation alters cell metabolism, but little is known about the effects of mechanical strain on the cytoskeleton of myocardium cells. This study was to investigate the changes of F-actin, a cytoskeleton protein of myocardium cells, and to provide a theoretical basis for further investigation of the mechanism of myocardium-remodeling. Methods: we examined the effects of fluid shear stress on the Tmod1 expression and F-actin cytoskeleton remodeling. Then, after myocardial cells, silenced by si-Tmod1, were treated by fluid shear stress, the change of intracellular calcium ion concentration, ROS in myocardial cells, cytochrome C, and the amount of F-actin, LDH and T-SOD MDA were evaluated with laser light confocal microscopy, western blot, and ELISA, respectively. Results: fluid shear stress can induce F-actin cytoskeleton remodeling and upregulate Tmod1 expression. After myocardial cells were under the conditions of Tmod1 inhibition, shear stress can significantly reduce the increase of ROS levels and calcium content, decrease the release of cells cytochrome C and LDH, decrease the MDA content, and increase the level of T-SOD. Conclusion: in conclusion, shear treatment can remodel the cytoskeleton through Tmod1, and its mechanism may be related to scavenging oxidative stress products, ROS and MDA, the increase of intracellular antioxidant enzyme activity of SOD and improvement in mitochondrial dysfunction.

Fig. 1. The expression level of Tmod1 protein in other tissues is very low, however the expression level in the heart tissue is high. (from NCBI gene).

Fig. 2. F-actin cytoskeleton remodeling observed by laser scanning confocal microscope was induced by shear stress in cardiac myocytes (A), and the F-actin content change was manifested in (B). Compared with static group, *: P < 0.05, n = 6.

Fig. 3. Tmod1 mRNA expression levels were upregulated in myocardial cells induced by fluid shear stress for 8 h (A), 16 h (B), as detected with RT-PCR. (C) Elisa experiment result indicated that Tmod1 expression was significantly up-regulated. *: P < 0.05, compared to static group, ^Δ: P < 0.05, compared to shear 8 h group n = 6.

Fig. 4. siTmod1 inhibited F-actin expression in myocardial cells. (A and B) Tmod1 protein expression level was decreased in myocardial cells transfected with si-Tmod1. (C) F-actin fluorescence intensity stained with FITC phalloidin was decreased in myocardial cells transfected with 100 nM si-Tmod1. The fold changes in F-actin expression amount were displayed in (D). *: P < 0.05, compared to static group, n = 6.

Fig. 5. Effect of fluid shear on the expression of mitochondrial ROS, MDA, T-SOD and mitochondrial calcium in myocardial cells by knockdown of Tmod1. (A) The production of mitochondrial ROS (DCFH-DA green fluorescent image) and mitochondrial calcium (Fluo-4AM green fluorescent) were estimated by confocal microscopy. (B) Quantification of the production of mitochondrial ROS and the level of mitochondrial calcium by analysis software of confocal microscopy. (C) Effect of fluid shear decrease the MDA content in cardiomyocyte undergoing si-Tmod1 treatment. (D) Effect of fluid shear increase the T-SOD content in cardiomyocyte undergoing si-Tmod1 treatment. (*P < 0.05, other groups vs. si-Tmod1 group, n = 6).

Fig. 6. (A) Effect of fluid shear on cytochrome-c release in myocardial cells suffering by knockdown of Tmod1. (B) Effect of fluid shear decrease the LDH content in cardiomyocyte undergoing si-Tmod1 treatment. (*P < 0.05, other groups vs. si-Tmod1 group, n = 6).