| Lipopolysaccharide (LPS), one of main cytoderm components of Gram-negative bacteria, is known as a critical harmful factor to organism. It has been found that release of LPS into blood flow by bacteria in sepsis can activate immune system and inflammatory reaction which would injure organism seriously, leading to shock, systemic inflammatory response syndrome and multiple organ dysfunctions. Our previous studies have found that:(1) LPS can damage myocardial function and reduce the contractile function; (2) LPS can damage myocardial tissue, cause capillary hemorrhage and destroy the muscle fibers significantly; (3) LPS can reduce the density of the F-Actin and Desmin in cardiomyocytes and directly induce the stress fiber formation; (4) LPS can also decrease the gene expression of myocardial cytoskeleton protein such as Actin and Tublin.Undoubtedly, LPS can injure the heart seriuosly. However, it is still unclear whether LPS could directly damage the cardiomyocytes and how the damages of cardiomyocytes induced by LPS could display in morphology.Atomic force microscope (AFM), based on the interaction between the scanning probe and the atom of the sample’s surface, is a new kind of surface analysis instrument, characterized by higher resolution compared with traditional electron microscope and the comparatively simple procedure of sample-preparation for AFM without embedding, covering and staining. The AFM can not only scan the surface topography of the samples and give the three-dimensional images, but also measure the length, width and height of the samples. Thus, the AFM has been playing an increaseingly important role in biological research because the application of the AFM makes the image of the cell junction and intracellular cytoskeleton available.Therefore, in this study we applied AFM to observe the changes of cardiomyocytes and the intracellular cytoskeleton stimulated by LPS in order to investigate the mechanism of LPS effects on cardiomyocytes and myocardial cytoskeleton. This study can be divided into following three parts.Part I:The neonatal rat cardiomyocytes, which had been cultured in vitro and stimulated by LPS for 1 h,4 h, and 8 h, respectively, were scanned by AFM, by which projected area, surface area and volume of each cell was measured.Part II:The cardiomyocytes, of which soluble proteins and organelles had been removed from the membrane by the nonionic detergent Triton X-100 in a low concentration, were stimulated in vitro by LPS. And then the remaining cytoskeleton was directly visualized by AFM. Meanwhile we quantitated the observed cytoskeleton by its density and volume. Furthermore, we used fluorescence microscopy to observe the changes of the F-Actin to investigate the effects of LPS on the intracellular cytoskeleton.Part III:The mechanism of the cardiomyocytes hypertrophy induced by LPS was performed by the investigation of Na+-K+-ATPase activity of the cardiomyocytes stimulated by LPS. The diameter and area of single cardiomyocyte and the levels of total proteins were measured after the neonatal rat cardiomyocytes were stimulated by Ouabain for 8 h. Also, the changes of cardiomyocytes in vitro stimulated by LPS and EGDA were observed in order to investigate the role of Ca2+ on cardiomyocyte hypertrophy.The results were as followed:In the first part:(1) The effects of LPS on the cardiomyocyte membrane cytoskeleton:We got the AFM images of cardiomyocyte membrane cytoskeleton and found that there were a lot of uneven bumps and caves on the surface of the cardiomyocyte membrane cytoskeleton. We also made the height curves of the membrane surface which were different in height and density. While we could not identify the differences between the LPS treatment groups and the normal one just based on the AFM images. We then measured the roughness of cardiomyocyte membrane cytoskeleton and found that it was not significantly different among the the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group (F=2.020, P=0.113)(2) The projected area of single cardiomyocyte was significantly different among the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group (F=5.683, P=0.001). The projected area increased significantly in the group of LPS 8 h treatment compared with the normal group (P=0.004), while it did not change significantly in the groups of LPS 1 h and LPS 4 h compared with the normal one (P values were 1.000 and 0.322 respectively)The surface area of single cardiomyocyte was significantly different among the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group(F=5.748,P=0.001). The surface area increased significantly in the group of LPS 8 h treatment compared with the normal group (P=0.004), while it did not change significantly in the groups of LPS 1 h and LPS 4 h compared with the normal one (P values were 1.000 and 0.336 respectively)The volume of single cardiomyocyte was significantly different among the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group (F=15.961, P=0.000). The volume increased significantly in the group of LPS 8 h treatment compared with the normal group (P=0.000), while it did not change significantly in the groups of LPS 1 h and LPS 4 h compared with the normal one (P values were 1.000 and 0.316 respectively)In the second part:(1) The surface fiber density index (SFDI) of intracellular cytoskeleton was significantly different among the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group(F=39.735, P=0.000). The SFDI increased significantly in the groups of LPS 1 h, LPS 4 h and LPS 8 h treatment compared with the normal one (All of the P values are 0.000). The result indicated that the distribution of the intracellular cytoskeleton gradually became looser with the time extension of the LPS treatment.(2) The volume/area of intracellular cytoskeleton was significantly different among the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group (F=26.658,P=0.000). The volume/area enlarged significantly in the group of LPS 8 h compared with the normal one (P=0.000), while it did not change significantly in the groups of LPS 1 h and LPS 4 h compared with the normal one (P values were 0.870 and 0.081 respectively).The result suggested that LPS may induce the increase of volume of the intracellular cytoskeleton in comparatively long time (8 h)(3) The width of single fiber of intracellular cytoskeleton was significantly different among the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group (F=16.128, P=0.000).The width of single fiber increased significantly in the groups of LPS 1 h, LPS 4 h and LPS 8 h treatment compared with the normal one (P values are 0.033,0.000 and 0.000, respectively)(4) The distribution of F-Actin became looser when the cardiomyocytes were stimulated by LPS and the LPS can directly induce the formation of stress fiber. (5) The fluorescence intensity of F-Actin was significantly different among the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group (F=7.216, P=0.000). The fluorescence intensity of F-Actin increased significantly in the group of LPS 8 h treatment compared with the normal one (P=0.001), while it did not change significantly in the groups of LPS 1 h and LPS 4 h compared with the normal one (P values were 1.000 and 0.279, respectively)In the third part:(1) The total proteins inside the cardiomyocytes were significantly different among the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group (F=530.828, P=0.000). The total proteins increased significantly in the group of LPS 8 h treatment compared with the normal one (P=0.000)(2) The activity of Na+-K+-ATPase in the cardiomyocytes was significantly different among the normal group, LPS 1 h group, LPS 4 h group and LPS 8 h group (F=79.710, P=0.000). The activity of Na+-K+-ATPase decreased significantly in the groups of LPS 4 h and LPS 8 h treatment compared with the normal group (Both the P values were 0.000), while it did not change significantly in the group of LPS 1 h compared with the normal one (P= 1.000)(3) The area of single cardiomyocyte was significantly different among the normal group, LPS group and Ouabain group (F=68.098, P=0.000). The area increased significantly in the group of Ouabain 8 h treatment compared with the normal group (P=0.000) and it was also significantly different from that in LPS group (P=0.014)The diameter of single cardiomyocyte was significantly different among the normal group, LPS group and Ouabain group (F=113.702, P=0.000). The diameter increased significantly in the group of Ouabain 8 h treatment compared with the normal group (P=0.000) and it was also significantly different from that in LPS group (P=0.000)The total proteins inside the cardiomyocytes were significantly different among the normal group, LPS group and Ouabain group (F=273.387, P=0.000). The total proteins increased significantly in the group of Ouabain 8 h treatment compared with the normal group (P=0.000) and they wre also significantly different from that in LPS group (P=0.004)(4) The area of single cardiomyocyte was significantly different among the normal group, EGTA group, LPS group and LPS+EGTA group(F=10.340, P=0.000). The area increased significantly in the group of LPS treatment compared with the normal group (P=0.000) and it was also significantly different from that in LPS+EGTA group (P=0.002)The diameter of single cardiomyocyte was significantly different among the normal group, EGTA group, LPS group and LPS+EGTA group(F=28.507, P=0.000). The diameter increased significantly in the group of LPS treatment compared with the normal group (P=0.000) and it was also significantly different from that in LPS+EGTA group (P=0.001)The total proteins inside the cardiomyocytes were significantly different among the normal group, EGTA group, LPS group and LPS+EGTA group (F=34.449, P=0.000). The total proteins increased significantly in the group of LPS treatment compared with the normal group (P=0.000) and they were also significantly different from that in LPS+EGTA group (P=0.001)Conclusions:(1) There is no remarkable change on the membrane cytoskeleton of cardiomyocyte after LPS treatment in comparatively short time;(2) LPS can induce the change of distribution of the intracellular cytoskeleton in comparatively short time (1 h), which may gradually become looser with the extension of the LPS treatment time. While the LPS can increase the volume of the cytoskeleton in comparative long time (8h);(3) LPS can induce the cardiomyocyte hypertrophy;(4) The cardiomyocyte hypertrophy induced by LPS may be related to the decrease of Na+-K+-ATPase activity of cardiomyocytes induced by LPS and the increase of inflow of Ca2+, which may play an important role in the process of cardiomyocyte hypertrophy. |
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