Cardiomyocyte Gap Junction Intercellular Communication and Autophagy
Date Issued
2010
Date
2010
Author(s)
Abstract
Study background: The gap junction intercellular communication may involve in signal exchange for both cell death and cell survival. The cell destiny depends on the cell status and environmental context of the cell that receives the signals. Autophagy was once considered as a protective mechanism in cells. Through removing the diseased organelles or protein, autophagy functioned in maintaining cellular homeostasis, nutrient or energy preservation and providing cell survival. Scientists who devoted in autophagy study are trying to transform autophagy concept into therapeutic tool enthusiastically. However, the best studied intracellular regulation of autophagy was known to be complex, and in the contrast the interaction with apoptosis was well-documented. In fact, the intercellular communication of autophagy signal between cells was never investigated and the role of gap junction in autophagy was unknown.
Study purpose: We investigate the contribution of gap junction to cardiomyocyte autophagy which was produced by oxidative stress. We hypothesized that autophagy signal between individual cardiomyocyte was conducted through gap junction.
Material and methods: We use H9c2 cell line as study model, 1-Heptanol as gap junction uncoupler, and hydrogen peroxide as oxidative stresser for autophagy induction. Autophagy activity was investigated by reverse-transcription polymerase chain reaction (RT-PCR) and Western blot for light chain 3 and Beclin-1 detection. Quantity analysis of autophagy was determined by densitometry by Alpha Innotech and Gaussia luciferase report assay after transfecting pCMV-Lc3-Gluc to H9c2 cell. Our study composed of 3 main parts. The first part of the study was designed to establish the laboratory model. We test the effects of 1-Heptanol and hydrogen peroxide on H9c2 and measure the change of autophagy expression, connexin 43 expressions and survival. The second part of the study included constructing pCMV-Lc3-Gluc plasmid and transfecting the plasmid into H9c2. The whole system was taken as Gaussia luciferase report system. We determined the effect of transfection on autophagy and testify the feasibility of Gaussia luciferase report system for the role of reporting LC3. During the third part of study, we divided wild type H9c2 and Gaussia luciferase report system transfected H9c2 into 4 groups, including the Control group, the Heptanol group, the H2O2 group and the H2O2+Heptanol group. In the H2O2+Heptanol group, 1-Heptanol was administered for gap junction blockade along with hydrogen peroxide for generating oxidative stress-induced autophagy. In the Heptanol group, we use 1-Heptanol only. We also setup negative control (the Control group) using DMEM culture medium and positive control with hydrogen peroxide only (the H2O2 group).
Results: We found that 100 to 400μM hydrogen peroxide could induce autophagy in H9c2 when treatment duration is longer than 6 hours. However, higher concentration of hydrogen peroxide (>200μM) might decrease H9c2 survival measured by MTT assay. When hydrogen peroxide treatment was longer than 14 hours, H9c2 Cx43 expression might reduce. Heptanol when used at 0.5mM will not decrease Cx43 expression. Blocking gap junction by 1-heptanol (H2O2+Heptanol group), we found there is about 24.2% reduction of LC3 mRNA expression when compare to H2O2 group which use hydrogen peroxide (P>0.05, n=3). Using Gaussia luciferase report assay, the LC3 expression was attenuated by 20.6 % when compare to H9c2 cell that receive 1-Heptanol pre-treatment before hydrogen peroxide stressing (P<0.001, n=4).
Conclusion: Blocking the gap junction inter-cellular communication showed trends in decreasing oxidative stress induced autophagy activity. It’s possible that autophagy regulation required complex machinery which involved autocrine, paracrine and probable gap junction intercellular communication. Further studies are required to figure out the exact signal molecules, the true effect of autophagy flux and the existence other intercellular regulation.
Subjects
autophagy
oxidative stress
H9c2
gap junction
Heptanol
hydrogen peroxide connexin
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