Dataset Information

The potential roles of stress-induced phosphoprotein 1 and connexin 43 in rats with reperfusion arrhythmia.

ABSTRACT:

Objective

Connexin 43 (Cx43) is a critical gene for maintaining myocardial homeostasis. Interestingly, Cx43 and stress-induced phosphoprotein 1 (STIP1) were recorded to be lowly expressed in ischemia/reperfusion (I/R). However, their impacts on reperfusion arrhythmia (RA) remain to be explored. Our study aimed to find out the related underlying mechanisms.

Methods

After the establishment of an isolated heart model through Langendorff perfusion, the heart rate, conduction activation time, conduction velocity, and conduction direction of the left ventricle were evaluated, along with the apoptotic rate detection in the collected myocardial tissues. After the construction of a hypoxia/reoxygenation (H/R)-induced cellular model, cell apoptosis, intercellular communication, cell viability, and the content of reactive oxygen species, superoxide dismutase, malondialdehyde, and lactic dehydrogenase were measured. The expression of Cx43 and STIP1 was determined in both rat heart and cell models. The bindings of STIP3 and Cx43 to heat shock protein 90 (HSP90) and heat shock protein 70 (HSP70) were verified.

Results

Relative to the corresponding controls, Cx43 and STIP1 were decreased in myocardial tissues of RA rats and H/R-stimulated H9C2 cells, where Cx43-binding HSP70 and HSP90 were respectively increased and decreased, and ubiquitination level of Cx43 was enhanced. STIP1 overexpression promoted protein expression of Cx43, intercellular communication, and cell viability, and reduced cell apoptosis and oxidative stress in H/R-stimulated H9C2 cells.

Conclusion

STIP1 promoted Cx43 expression to improve intercellular communication and reduce oxidative stress in H/R-stimulated H9C2 cells.

SUBMITTER: An L

PROVIDER: S-EPMC10546868 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Publications

The potential roles of stress-induced phosphoprotein 1 and connexin 43 in rats with reperfusion arrhythmia.

An Li L Gao Hong H Gao Hong H Zhong Yi Y Liu Yanqiu Y Cao Ying Y Yi Jing J Huang Xiang X Wen Chunlei C Tong Rui R Pan Zhijun Z Yan Xu X Liu Meiyan M Wang Shengzhao S Wu Hao H Hu Tingju T

Immunity, inflammation and disease 20231001 10

<h4>Objective</h4>Connexin 43 (Cx43) is a critical gene for maintaining myocardial homeostasis. Interestingly, Cx43 and stress-induced phosphoprotein 1 (STIP1) were recorded to be lowly expressed in ischemia/reperfusion (I/R). However, their impacts on reperfusion arrhythmia (RA) remain to be explored. Our study aimed to find out the related underlying mechanisms.<h4>Methods</h4>After the establishment of an isolated heart model through Langendorff perfusion, the heart rate, conduction activatio ...[more]

PMID: 37904692

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Project description:Background Connexin 43 (Cx43), the predominant gap junction protein in hearts, is modified by specific (de)phosphorylation events under physiological and pathological states to affect myocardium function and structure. Previously we found that deficiency in Cx43 S282 phosphorylation could impair intercellular communication and contribute to cardiomyocyte apoptosis by activating p38 mitogen-activated protein kinase (p38 MAPK)/factor-associated suicide (Fas)/Fas-associating protein with a novel death domain (FADD) pathway, which is involved in myocardium injury in ischemia/reperfusion (I/R) heart. In addition, mutant at Cx43 S282 substituted with alanine heterozygous mice (S282A+/−) exhibited different degrees of ventricular arrhythmias and only some underwent myocardium apoptosis. In this study, we aimed to investigate the role of Cx43 pS282 in different cardiac pathological phenotypes. Methods We examined cardiac function, structure, and relevant protein expression in S282A+/− mice (aged 2, 10 and 30 weeks) by electrocardiograph, echocardiography, histological staining, and co-immunoprecipitation followed by Western blot. Intraperitoneal isoprenaline injection and I/R surgery were applied in S282A+/− mice as external stimulus. 2,3,5-triphenyltetrazolium chloride staining was used for myocardium infarction evaluation. Results Adult S282A+/− mice (aged 10 and 30 weeks) still exhibited spontaneous arrhythmia. Unlike neonatal stage (aged around 2 weeks), no apoptosis-related manifestations and the activation of p38 MAPK-Fas-FADD apoptotic pathway were observed in adult S282A+/− hearts. S282A+/− neonatal mice with cardiomyocytes apoptosis exhibited more than 60% dephosphorylation at Cx43 S282 than WT mice, while less than 40% S282 dephosphorylation were found in adult S282A+/− mice. In addition, although S282A+/− mice displayed normal cardiac function, they were highly susceptible to isoproterenol-induced ECG alternans and prone to cardiac injury and deaths upon I/R attack. Conclusions These results reinforce that Cx43 S282 dephosphorylation acts as a susceptibility factor in regulating cardiomyocyte survival and cardiac electrical homeostasis in basal conditions and contributes to myocardium injury in the setting of I/R. Cx43 S282 phosphorylation was competent to induce spontaneous arrhythmias, cardiomyocyte apoptosis and deaths based on the degree of S282 dephosphorylation.

Project description:AimArgirein (rhein-arginine) is a derivative of rhein isolated from Chinese rhubarb (Rheum Officinale Baill.) that exhibits antioxidant and anti-inflammatory activities. In the present study we investigated the effects of argirein on stress-induced (hypergonadotrophic) and diabetic (hypogonadotrophic) hypogonadism in male rats.MethodsStress-induced and diabetic hypogonadism was induced in male rats via injection of isoproterenol (ISO) or streptozotocin (STZ). ISO-injected rats were treated with argirein (30 mg·kg(-1)·d(-1), po) or testosterone replacement (0.5 mg·kg(-1)·d(-1), sc) for 5 days, and STZ-injected rats were treated with argirein (40-120 mg·kg(-1)·d(-1), po) or aminoguanidine (100 mg·kg(-1)·d(-1), po) for 4 weeks. After the rats were euthanized, blood samples and testes were collected. Serum hormone levels were measured, and the expression of endothelin receptor A (ETA), connexin 43 (Cx43) and other proteins in testes was detected. For in vitro experiments, testis homogenate was prepared from normal male rats, and incubated with ISO (1 μmol/L) or high glucose (27 mmol/L).ResultsISO injection induced hyper-gonadotrophic hypogonadism characterized by low testosterone and high FSH and LH levels in the serum, whereas STZ injection induced hypogonadotrophic hypogonadism as evidenced by low testosterone and low FSH and LH levels in the serum. In the testes of ISO- and STZ-injected rats, the expression of ETA, MMP-9, NADPH oxidase and pPKCε was significantly increased, and the expression of Cx43 was decreased. Administration of argirein attenuated both the abnormal serum hormone levels and the testis changes in ISO- and STZ-injected rats, and aminoguanidine produced similar actions in STZ-injected rats; testosterone replacement reversed the abnormal serum hormone levels, but did not affect the testis changes in ISO-injected rats. Argirein (0.3-3 μmol/L) exerted similar effects in testis homogenate incubated with ISO or high glucose in vitro.ConclusionTwo types of hypogonadism of male rats exhibit increased expression of ETA and depressed expression of Cx43 in testes, despite different patterns of serum FSH and LH. Argirein alleviates the two types of male hypogonadism via normalizing ETA and Cx43 in testes.

Project description:Oxidative stress is widely accepted as a key factor of doxorubicin (Doxo)‑induced cardiotoxicity. There is evidence to indicate that nitrosative stress is involved in this process, and that Doxo interacts by amplifying cell damage. Mitochondrial connexin 43 (mitoCx43) can confer cardioprotective effects through the reduction of mitochondrial reactive oxygen species production during Doxo‑induced cardiotoxicity. The present study aimed to evaluate the involvement of mitoCx43 in Doxo‑induced nitrosative stress. Rat H9c2 cardiomyoblasts were treated with Doxo in the absence or presence of radicicol, an inhibitor of Hsp90, the molecular chaperone involved in Cx43 translocation to the mitochondria that underlies its role in cardioprotection. FACS analysis and RT‑qPCR revealed that Doxo increased superoxide dismutase, and catalase gene and protein expression. As shown by hypodiploid nuclei and confirmed by western blot analysis, Doxo increased caspase 9 expression and reduced procaspase 3 levels, which induced cell death. Moreover, a significant increase in the activation of the NF‑κB signaling pathway was observed. It is well known that the increased expression of inducible nitric oxide synthase results in nitric oxide overproduction, which then rapidly reacts with hydrogen peroxide or superoxide generated by the mitochondria, to form highly reactive and harmful peroxynitrite, which ultimately induces nitrotyrosine formation. Herein, these interactions were confirmed and increased effects were observed in the presence of radicicol. On the whole, the data of the present study indicate that an interplay between oxidative and nitrosative stress is involved in Doxo‑induced cardiotoxicity, and that both aspects are responsible for the induction of apoptosis. Furthermore, it is demonstrated that the mechanisms that further increase mitochondrial superoxide generation (e.g., the inhibition of Cx43 translocation into the mitochondria) significantly accelerate the occurrence of cell death.

Project description:BackgroundGlia-mediated neuroinflammation is related to brain injury exacerbation after cerebral ischemia/reperfusion (I/R) injury. Astrocytic hemichannels or gap junctions, which were mainly formed by connexin-43, have been implicated in I/R damage. However, the exact roles of astrocytic hemichannels and gap junction in neuroinflammatory responses induced by I/R injury remain unknown.MethodsPrimary cultured astrocytes were subjected to OGD/R injury, an in vitro model of I/R injury. Salvianolic acid B (SalB) or carbenoxolone (CBX) were applied for those astrocytes. Besides, Cx43 mimetic peptides Gap19 or Gap26 were also applied during OGD/R injury; Cx43 protein levels were determined by western blot and cytoimmunofluorescene staining, hemichannel activities by Ethidium bromide uptake and ATP concentration detection, and gap junction intercellular communication (GJIC) permeability by parachute assay. Further, astrocyte-conditioned medium (ACM) was collected and incubated with microglia. Meanwhile, ATP or apyrase were applied to explore the role of ATP during OGD/R injury. Microglial activation, M1/M2 phenotypes, and M1/M2-related cytokines were detected. Also, microglia-conditioned medium (MEM) was collected and incubated with astrocytes to further investigate its influence on astrocytic hemichannel activity and GJIC permeability. Lastly, effects of ACM and MCM on neuronal viability were detected by flow cytometry.ResultsWe found that OGD/R induced abnormally opened hemichannels with increased ATP release and EtBr uptake but reduced GJIC permeability. WB tests showed decreased astrocytic plasma membrane's Cx43, while showing an increase in cytoplasma. Treating OGD/R-injured microglia with ATP or OGD/R-ACM induced further microglial activation and secondary pro-inflammatory cytokine release, with the M1 phenotype predominating. Conversely, astrocytes incubated with OGD/R-MCM exhibited increased hemichannel opening but reduced GJIC coupling. Both SalB and CBX inhibited abnormal astrocytic hemichannel opening and ATP release and switched the activated microglial phenotype from M1 to M2, thus providing effective neuroprotection. Application of Gap19 or Gap26 showed similar results with CBX. We also found that OGD/R injury caused both plasma membrane p-Cx43(Ser265) and p-Src(Tyr416) significantly upregulated; application of SalB may be inhibiting Src kinase and attenuating Cx43 internalization. Meanwhile, CBX treatment induced obviously downregulation of p-Cx43(Ser368) and p-PKC(Ser729) protein levels in plasma membrane.ConclusionsWe propose a vicious cycle exists between astrocytic hemichannel and microglial activation after OGD/R injury, which would aggravate neuroinflammatory responses and neuronal damage. Astrocytic Cx43, hemichannels, and GJIC play critical roles in OGD/R injury-induced neuroinflammatory responses; treatment differentially targeting astrocytic Cx43, hemichannels, and GJIC may provide novel avenues for therapeutics during cerebral I/R injury.

Dataset Information

The potential roles of stress-induced phosphoprotein 1 and connexin 43 in rats with reperfusion arrhythmia.

Objective

Methods

Results

Conclusion

Publications

The potential roles of stress-induced phosphoprotein 1 and connexin 43 in rats with reperfusion arrhythmia.

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