The Role Of Heat Shock Transcription Factor1on H₂O₂-induced Cardiomyocyte Necrosis Through Suppressing High Mobility Group Box1and Potential Mechanism

Oxidative stress has been implicated in plenty of pathophsiological conditions, including inflammation, ischemia-reperfusion (I/R) injury and stroke. Intracellular reactive oxygen species (ROS), which is excessively accumulated in the oxidative stress, induces cell damage or tissue injuries.High mobility group box1(HMGB1), a30-KD nuclear protein, is secreted by active inflammatory cells or impaired tissue cells under stress conditions. Once released, extracellular HMGB1participates in various biological responses in diverse cells or tissues. On the one hand, the combination of extracellular HMGB1and endogenous stimuli, which was released from injured cells, may directly cause cell or tissue damage [1]; on the other hand, extracelullar HMGB1may amplify inflammatory responses to indirectly aggravate impairment through activating immune cells and increasing the expression of cytokines. Thus, as a marker for "necrosis", HMGB1can positively regulate the inflammation.Heat shock transcription factor1(HSF1), which is activated under various stresses and subsequently induces up-regulation of heat shock proteins (HSPs). possesses plenty of physiological functions, including directly interfering with cell death pathways, resisting inflammatory effects, approving I/R injury and inhibiting cardiac hypertrophy and myocardial remodeling [2-8]. Tang D [9] has demonstrated that HSP72inhibited the release of HMGB1by H2O2in macrophages and monocytes. However, it is still not clear whether HSF1, an upstream regulator of HSPs, can exert the inhibitory effects on HMGB1in tissue cells (e.g., cardiomyocytes).c-Jun N-terminal kinase (JNK), the member of mitogen-activated protein kinases (MAPKs) family, is activated by extensive stress stimulations including UV, cytokines, etc. As the precious report[10], JNK participates into the secretion of HMGB1induced by H2O2in the immune cells.We used H2O2to stimulate cardiomyocytes to establish the model of oxidative stress after transfecting HSF1adenovirus. And our study aimes to investigate the effects of HSF1-regulated HMGB1on H2O2-induced cardiomyocyte necrosis, and further explore the potential mechanism. Part One Effects of heat shock transcription factor1overexpression on H2O2-induced cardiomyocyte necrosisObjectives The aims of this study were to build oxidative stress model by H2O2after transfecting heat shock transcription factor1(HSF1) adenovirus, and explore the effect of HSF1overexpression on H2O2-induced cardiomyocyte necrosis.Methods Primary cultures of rat ventricular myocytes. which were obtained from one or two-day-old Sprague-Dawley rat pups, respectively transfected control adenovirus and HSF1adenovirus. After48hours, the cardiomyocytes were stimulated by0.5mmol/L H2O2for6hours and24hours. RT-PCR performed to determine the expression of HSF1in mRNA level and protein level to justify the effect of transfecting HSF1adenovirus. Moreover, the effects of HSF1overexpression on H2O2-induced cardiomyocyte necrosis were evaluated by CCK-8staining, supernatant levels of LDH.Results The transfection of HSF1adenovirus significantly elevated mRNA and protein levels of HSF1compared with control adenovirus (P<0.05). H2O2significantly reduced cardiomyocyte viability in control adenovirus group and HSF1adenovirus group (P<0.05). The cell viability exposure to H2O2for24hours in two groups was significantly lower than that for6hours, respectively, which was shown a time-dependent manner (P<0.05). Moreover, at6hours and24hours after H2O2, the cell viability in HSF1adenovirus group was markedly increased, as compared with control adenovirus group (P<0.05). In addition, we also found that H2O2significantly raised the levels of supernatant LDH in control adenovirus group and HSF1adenovirus group (P<0.05). The supernatant levels of LDH exposure to H2O2for24hours in two groups were respectively higher than that for6hours with a time-dependent manner (P<0.05). Furthermore, at6hours or24hours after H2O2, the levels of LDH in HSF1adenovirus group was attenuated, which was markedly raised in control adenovirus group, respectively (P<0.05).Conclusions We transfected HSF1adenovirus and managed to significantly elevate the HSF1expression in mRNA and protein level. Moreover, we use0.5mmol/L H2O2to successfully build the model of oxidative stress. We found H2O2markedly attenuated the cardiomyocyte viability, increased the supernatant levels of LDH in two groups with a time-dependent manner. Furthermore, under the same condition, HSF1adenovirus group perserved higher cell viability, lower levels of LDH compared with control adenovirus group. Part Two Effects of heat shock transcription factor1overexpression on H2O2-induced high mobility group box1Objectives The aim of the part is to investigate whether heat shock transcription factor1(HSF1) can negatively regulate high mobility group box1(HMGB1) to resist the effects of H2O2Methods Primary cultures of rat cardiomyocytes were cultured and respectively transfected control adenovirus and HSF1adenovirus. After48hours, the cardiomyocytes were stimulated by H2O2Western Blot performed to determine HMGB1expression from supernatant and whole cell. Besides, we also detected the location of HMGB1in cardiomyocytes by immunofluorescence stainning.Results To begin with, we determined the supernatant levels of HMGB1and found that the levels of HMGB1in control adenovirus group exposure H2O2for3hours,6hours and12hours were significantly higher than that without H2O2.(P<0.05). And the levels of HMGB1in control adenovirus group exposure H2O2for12hours and24hours were markedly reduced compared with for6hours (P<0.05). However, the levels of HMGB1in HSF1adenovirus group with H2O2for3hours and6hours were significantly higher than that without H2O2. And as compared with for3hours, the levels of HMGB1in HSF1adenovirus group with H2O2for6hours,12hours and24hours were significantly decreased (P<0.05). Besides, at the6hours,12hours and24hours after stimulations, the levels of HMGB1in HSF1adenovirus group were attenuated, which was markedly raised in control adenovirus group (P<0.05). Secondly, we measured the expression of HMGB1in the whole cells. At6h after H2O2stimulation, the expression of HMGB1in two groups was significantly lower than that without H2O2(P<0.05). Furthermore, we detected the distribution of HMGB1in cardiomyocytes. H2O2markedly increased the intensity of location of HMGB1in the cytoplasma. And the intensity of HSF1adenovirus group was significantly weaker than that of control adenovirus group.Conclusions HSF1suppresses the translocation into cytoplasma and reduces the secretion of HMGB1. which can partly account for the beneficial effects that HSF1overexpression attenuated H2O2-induced cardiomyocyte necrosis. Part Three Signaling pathways involved in the effects of heat shock transcription1on high mobility group box1proteinObjectives The aim of the part is to explore the signaling pathways involved in the effects that heat shock transcription factor1(HSF1) negatively regulated on high mobility group box1(HMGB1).Methods Primary cultures of rat cardiomyocytes were cultured and respectively transfected control adenovirus and HSF1adenovirus. After48hours, the cardiomyocytes were stimulated by H2O2Western Blot performed to determine the expression of heat shock protein27(HSP27), heat shock protein90(HSP90), phosphorylation-c-Jun N-terminal kinase (p-JNK) and c-Jun N-terminal kinase (JNK).Results The expression of HSP27and HSP90in the control adenovirus group and HSF1adenovirus group was significantly increased from6h to24h after H2O2. which was in a time-dependent manner (P<0.05). Moreover, their levels in HSF1adenovirus group were markedly higher than that in control adenovirus group during this period, respectively (P<0.05). Simultaneously, we also detected the activation of JNK. The ratio of p-JNK to JNK was significantly raised in two groups at10minutes after H2O2(P<0.05). And the ratio of HSF1adenovirus group was markedly attenuated, which was significantly increased (P<0.05).Conclusions HSF1up-regulates the expression of HSP27and HSP90, and inhibits the activation of JNK, which contributes to negatively regulating HMGB1.