| Microtubules are a main component of the cytoskeleton in cardiomyocytes,and are involved in several biological processes, such as cell morphogenesis. Microtubule depolymerization caused by hypoxia is essential for cardiac dysfunction in "Shock heart" theory of severe burns.However, the role that microtubule depolymerization play in electrophysiological properties,as the basis of myocardial cell activity and function, has been a lack of systematic research and observation. In addition, the study of microtubule system has been highly dependent on the microscopy and photomicrography techniques, this traditional approach has a fatal flaw:observer bias. Therefore, how to objectively quantify microtubule morphology will bring new understanding about hypoxia-induced microtubule depolymerization.In this study, we took neonatal rat cardiomyocytes as research subjects, Microtubule depolymerization caused by fixed-time treatment of a certain concentration of colchicine simulated the destruction of microtubules in hypoxic environment. In the experimental condition, we observed the changes of electrophysiological indicators, in order to recognize the electrophysiological characterization caused by microtubule depolymerization and explore their mechanism. Meanwhile, based on fluorescence images labeled microtubules, characteristic parameters of grayscale histogram quantified microtubule depolymerization caused by drugs and hypoxia, to further clarify both corresponding relationship.Ⅰ.Materials and methods1.Neonatal rat cardiomyocytesisolatedand cultured in vitro were divided into control group and depolymerization group. Microtubules were observed after colchicine treatment. The distribution and morphological changes of microtubules were observed with laser confocal microscopy after α-tubulin immunofluorescence staining. Then the content of polymerized and dissociative α-tubulin was detected with Westernblot.2.Neonatal rat cardiomyocytes isolated and cultured in vitro were divided into control group and depolymerization group.Electrophysiological properties and oxygen consumption changes were observed after colchicine treatment. Spontaneous beating of the cultured cells were recorded under an inverted-stage microscope and camera, and calculate beating rates. Based on the whole-cell patch clamp technique, we record action potential,delayed rectifier potassium current(IK) and L-type Ca2+ current(ICa-L). Dissolved oxygen concentration of medium was determinated by oxygen microelectrode.3. H9C2 cardiomyocytes were divided into normal group, hypoxia group and colchicine group. Based on fluorescence images, microtubule quantitative analysis method was established: α-tubulin immunofluorescence staining, digital images of microtubules were obtained through confocal microscopy. Matlab software was used for image preprocessing: single cell division, de-noising, proportional partitioning and extraction of characteristic parameters.Ⅱ.Results1.In control group, microtubules of cardiac myocytes were in linear tubiform,distributed;the tubiform structure of the microtubule in depolymerization group were indistinct, crimped and with weakened immune fluorescence intensity, but the continuity was not entirely lost. In depolymerization group, the expression of polymerizedα-tubulin was obviously lower than that in control group, dissociative α-tubulin was significantly higher.2.In depolymerization group, spontaneous beating rate of neonatal rat cardiomyocytes was distinctly higher than that of control group. Correspondingly, the oxygen consumption increased after treatment with colchicine.3.Action potential morphology of neonatal rat cardiomyocytes in depolymerization group changed significantly, repolarization plateau phase was not obviou. The action potential duration reduced apparently, especially APD50, APD90. Resting potential and action potential amplitude had no significant changes.4.Compared with that of control group, microtubule depolymerization increased IK obviously under each test voltage(0-40mV) and its I-V curves moved up. However there was no alteration in ICa-L under each test voltage(-30-50mV)between control and depolymerization group, their I-V curves were nearly overlap.5.Quantitative characteristics of microtubule distribution were similar after culturing cells under hypoxic conditions or after treatment with colchicine. In addition, we provide a method for internal segmentation of the cells, which revealed that microtubular depolymerized evidently near the cell membrane following hypoxia or colchicine treatment.Ⅲ.Conclusions1.After the microtubule depolymerization, enhanced IK and changeless ICa-Lmakes AP repolarization faster, APD shortened. Then the rapid beating rate increase oxygen consumption of cardiac myocytes.This may be an important factor in myocardial damages caused by ischemia.2.Based on fluorescent images,the parameters of grayscale histogram(mean, variance, skewness, kurtosis, energy and entropy) can be used to quantitatively describe the distribution of microtubules in cells. and grayscale histograms. A conclusion confirmed by this method is that microtubule morphology quantitative characteristics of cardiomyocytes is similar after hypoxia and colchicine treatment. There is a certain corresponding relationship between them.GLH quantitative analysis can descript microtubule morphology of cardiomyocytes. It is more accurate to determine myocardial microtubule damages caused by ischemia.It is of great significance. |
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