He Chang

Cholesterol modulates function of connexin 43 gap junction channel via PKC pathway in H9c2 cells

It has been shown that cholesterol modulates activity of protein kinase C (PKC), and PKC phosphorylates connexin 43 (Cx43) to regulate its function, respectively. However, it is not known whether cholesterol modulates function of Cx43 through regulating activity of PKC. In the present study, we demonstrated that cholesterol enrichment reduced the dye transfer ability of Cx43 in cultured H9c2 cells. Western blot analysis indicated that cholesterol enrichment enhanced the phosphorylated state of Cx43. Immunofluorescent images showed that cholesterol enrichment made the Cx43 distribution from condensed to diffused manner in the interface between the cells. In cholesterol enriched cells, PKC antagonists partially restored the dye transfer ability among the cells, downregulated the phosphorylation of Cx43 and redistributed Cx43 from the diffused manner to the condensed manner in the cell interface. In addition, reduction of cholesterol level suppressed PKC activity to phosphorylate Cx43 and restored Cx43 function in PKC agonist-treated cells. Furthermore, we demonstrated that cholesterol enrichment upregulated the phosphorylated state of Cx43 at Ser368, while PKC antagonists reversed the effect. Taken together, cholesterol level in the cells plays important roles in regulating Cx43 function through activation of the PKC signaling pathway.

Hydrodynamics-based delivery of an interleukin-1 receptor II fusion gene ameliorates rat autoimmune myocarditis by inhibiting IL-1 and Th17 cell polarization

Type II interleukin-1 receptor (IL-1RII) is a non-signaling decoy receptor that blocks the activity of interleukin-1 (IL-1), a pro-inflammatory cytokine involved in experimental autoimmune myocarditis (EAM). The aim of this study was to examine the effects of hydrodynamics-based delivery of a recombinant plasmid encoding IL-1RII-Ig and to elucidate the role of IL-1RII in EAM rats. Rats were immunized on day 0 and injected with a recombinant plasmid encoding IL-1RII-Ig or pCAGGS-SP-Ig (control plasmid) on day 6. IL-1RII-Ig gene therapy effectively controlled EAM as indicated by a decreased heart weight-to-body weight ratio, reduced areas of myocarditis, reduced expression of genes encoding atrial natriuretic peptide and brain natriuretic peptide in the heart, and improved cardiac function. IL-1RII-Ig significantly inhibited the expression of IL-1-related cytokines such as IL-1β, prostaglandin E2 synthase, cyclooxygenase, and monocyte chemotactic protein-1 in EAM hearts. Furthermore, the effect of serum containing IL-1RII-Ig on the expression of immune-related genes in IL-1-stimulated splenocytes cultured from EAM rats was examined. The results showed that the expression of IL-6, transforming growth factor-β, retinoic acid-related orphan nuclear receptor (RORγt) and IL-17, was significantly decreased upon exposure to serum containing IL-1RII-Ig. In conclusion, hydrodynamics-based delivery of a recombinant plasmid encoding IL-1RII-Ig effectively prevented progression of left ventricular remodeling and myocardial damage in EAM rats. Moreover, IL-1RII may ameliorate experimental autoimmune myocarditis by blocking IL-1 and inhibiting production of the cytokines important for the polarization of T cells toward a Th17 phenotype.

The apolipoprotein A-I mimetic peptide, D-4F, alleviates ox-LDL-induced oxidative stress and promotes endothelial repair through the eNOS/HO-1 pathway

Apolipoprotein A-I (apoA-I) mimetic peptide exerts many anti-atherogenic properties. However, the underlying mechanisms related to the endothelial protective effects remain elusive. In this study, the apoA-I mimetic peptide, D-4F, was used. Proliferation assay, wound healing, and transwell migration experiments showed that D-4F improved the impaired endothelial proliferation and migration resulting from ox-LDL. Endothelial adhesion molecules expression and monocyte adhesion assay demonstrated that D-4F inhibited endothelial inflammation. Caspase-3 activation and TUNEL stain indicated that D-4F reduced endothelial cell apoptosis. A pivotal anti-oxidant enzyme, heme oxygenase-1 (HO-1) was upregulated by D-4F. The Akt/AMPK/eNOS pathways were involved in the expression of HO-1 induced by D-4F. Moreover, the anti-oxidation, pro-proliferation, and pro-migration capacities of D-4F were diminished by the inhibitors of both eNOS (L-NAME) and HO-1 (Znpp). Additionally, downregulation of ATP-binding cassette transporter A1 (ABCA1) by siRNA abolished the activation of Akt, AMPK and eNOS, and reduced the upregulation of HO-1 triggered by D-4F. Furthermore, D-4F promoted the reendothelialization of injured intima in carotid artery injury model of C57BL/6J mice in vivo. In summary, these findings suggested that D-4F might be a powerful candidate in the protection of endothelial cells and the prevention of cardiovascular disease (CVD).

Effect of PKC inhibitor on experimental autoimmune myocarditis in Lewis rats

Myocarditis is a major cause of sudden, unexpected death in young people. However, it is still one of the most challenging diseases to treat in cardiology. In the present study, we showed that both expression level and activity of PKC-α were up-regulated in the rat heart of experimental autoimmune myocarditis (EAM). Intraperitoneal administration of PKC inhibitor (Ro-32-0432) at the end of the most severe inflammation period of EAM still significantly reduced the EAM induced expression of failure biomarkers. Furthermore, Ro-32-0432 reduced the ratio of Bax/Bcl-2 and suppressed the expression of cleaved caspase-3, both of which were increased in the heart of the EAM rats, suggesting an anti-apoptotic role of Ro-32-0432. Besides, Ro-32-0432 suppressed EAM-induced cardiac fibrosis and release of pro-inflammatory cytokines IL-1β and IL-17. These results suggest that inhibition of PKC may serve as a potential therapeutic strategy for the treatment of myocarditis.Myocarditis is a major cause of sudden, unexpected death in young people. However, it is still one of the most challenging diseases to treat in cardiology. In the present study, we showed that both expression level and activity of PKC-α were up-regulated in the rat heart of experimental autoimmune myocarditis (EAM). Intraperitoneal administration of PKC inhibitor (Ro-32-0432) at the end of the most severe inflammation period of EAM still significantly reduced the EAM induced expression of failure biomarkers. Furthermore, Ro-32-0432 reduced the ratio of Bax/Bcl-2 and suppressed the expression of cleaved caspase-3, both of which were increased in the heart of the EAM rats, suggesting an anti-apoptotic role of Ro-32-0432. Besides, Ro-32-0432 suppressed EAM-induced cardiac fibrosis and release of pro-inflammatory cytokines IL-1β and IL-17. These results suggest that inhibition of PKC may serve as a potential therapeutic strategy for the treatment of myocarditis.

The apolipoprotein A‐I mimetic peptide, D‐4F, restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.

Up‐regulated Cx43 phosphorylation at Ser368 prolongs QRS duration in myocarditis

Prolongation of QRS duration in electrocardiogram is one of the risk factors for morbidity and mortality in many kinds of cardiac diseases. However, its molecular mechanism is unknown. In this study, utilizing experimental autoimmune myocarditis (EAM) as a disease model, we show that the prolongation of QRS duration is accompanied by elevated phosphorylation of connexin 43 (Cx43) at Ser368 (pS368Cx43). In cultured cells, inflammatory cytokine IL‐1β activates p38 MAPK to up‐regulate pS368Cx43 and impairs cell‐to‐cell communication. In isolated hearts of normal rats, perfusion of IL‐1β not only increases pS368Cx43 but also impairs cell‐to‐cell communication and prolongs QRS duration. Furthermore, blockade of p38 MAPK down‐regulates pS368Cx43, improves cell‐to‐cell communication and reduces QRS duration in EAM. These findings suggest that up‐regulation of pS368Cx43 by IL‐1β via p38 MAPK contributes to the prolongation of QRS duration and could be a therapeutic target for myocarditis‐induced prolongation of QRS duration.