Pathological matrix stiffness promotes cardiac fibroblast differentiation through the POU2F1 signaling pathway.

Cardiac fibroblast (CF) differentiation into myofibroblasts is a crucial cause of cardiac fibrosis, which increases in the extracellular matrix (ECM) stiffness. The increased stiffness further promotes CF differentiation and fibrosis. However, the molecular mechanism is still unclear. We used bioinformatics analysis to find new candidates that regulate the genes involved in stiffness-induced CF differentiation, and found that there were binding sites for the POU-domain transcription factor, POU2F1 (also known as Oct-1), in the promoters of 50 differentially expressed genes (DEGs) in CFs on the stiffer substrate. Immunofluorescent staining and Western blotting revealed that pathological stiffness upregulated POU2F1 expression and increased CF differentiation on polyacrylamide hydrogel substrates and in mouse myocardial infarction tissue. A chromatin immunoprecipitation assay showed that POU2F1 bound to the promoters of fibrosis repressors IL1R2, CD69, and TGIF2. The expression of these fibrosis repressors was inhibited on pathological substrate stiffness. Knockdown of POU2F1 upregulated these repressors and attenuated CF differentiation on pathological substrate stiffness (35 kPa). Whereas, overexpression of POU2F1 downregulated these repressors and enhanced CF differentiation. In conclusion, pathological stiffness upregulates the transcription factor POU2F1 to promote CF differentiation by inhibiting fibrosis repressors. Our work elucidated the crosstalk between CF differentiation and the ECM and provided a potential target for cardiac fibrosis treatment.

Angiotensin II induces RAW264.7 macrophage polarization to the M1­type through the connexin 43/NF­κB pathway.

Angiotensin II (AngII) serves an important inflammatory role in cardiovascular disease; it can induce macrophages to differentiate into the M1­type, produce inflammatory cytokines and resist pathogen invasion, and can cause a certain degree of damage to the body. Previous studies have reported that connexin 43 (Cx43) and NF­κB (p65) are involved in the AngII­induced inflammatory pathways of macrophages; however, the mechanisms underlying the effects of Cx43 and NF­κB (p65) on AngII­induced macrophage polarization have not been determined. Thus, the present study aimed to investigate the effects of Cx43 and NF­κB (p65) on the polarization process of AngII­induced macrophages. The macrophage polarization­related proteins and mRNAs were examined by flow cytometry, western blotting, immunofluorescence, ELISA and reverse transcription­quantitative PCR analyses. RAW264.7 macrophages were treated with AngII to simulate chronic inflammation and it was subsequently found that AngII promoted RAW 264.7 macrophage polarization towards the M1­type by such effects as the release of inducible nitric oxide synthase (iNOS), tumour necrosis factor (TNF)­α, IL­1ß, the secretion of IL­6, and the expression of M1­type indicators, such as CD86. Simultaneously, compared with the control group, the protein expression levels of Cx43 and phosphorylated (p)­p65 were significantly increased following AngII treatment. The M1­related phenotypic indicators, iNOS, TNF­α, IL­1ß, IL­6 and CD86, were inhibited by the NF­κB (p65) signalling pathway inhibitor BAY117082. Similarly, the Cx43 inhibitors, Gap26 and Gap19, also inhibited the expression of M1­related factors, and the protein expression levels of p­p65 in the Gap26/Gap19 groups were significantly decreased compared with the AngII group. Altogether, these findings suggested that AngII may induce the polarization of RAW264.7 macrophages to the M1­type through the Cx43/NF­κB (p65) signalling pathway.

α1-AR overactivation induces cardiac inflammation through NLRP3 inflammasome activation.

Acute sympathetic stress causes excessive secretion of catecholamines and induces cardiac injuries, which are mainly mediated by ß-adrenergic receptors (ß-ARs). However, α1-adrenergic receptors (α1-ARs) are also expressed in the heart and are activated upon acute sympathetic stress. In the present study, we investigated whether α1-AR activation induced cardiac inflammation and the underlying mechanisms. Male C57BL/6 mice were injected with a single dose of α1-AR agonist phenylephrine (PE, 5 or 10 mg/kg, s.c.) with or without pretreatment with α-AR antagonist prazosin (5 mg/kg, s.c.). PE injection caused cardiac dysfunction and cardiac inflammation, evidenced by the increased expression of inflammatory cytokine IL-6 and chemokines MCP-1 and MCP-5, as well as macrophage infiltration in myocardium. These effects were blocked by prazosin pretreatment. Furthermore, PE injection significantly increased the expression of NOD-like receptor protein 3 (NLRP3) and the cleavage of caspase-1 (p20) and interleukin-18 in the heart; similar results were observed in both Langendorff-perfused hearts and cultured cardiomyocytes following the treatment with PE (10 µM). Moreover, PE-induced NLRP3 inflammasome activation and cardiac inflammation was blocked in Nlrp3-/- mice compared with wild-type mice. In conclusion, α1-AR overactivation induces cardiac inflammation by activating NLRP3 inflammasomes.

ß3-adrenergic receptor activation induces TGFß1 expression in cardiomyocytes via the PKG/JNK/c-Jun pathway.

In heart failure, the expression of cardiac ß3-adrenergic receptors (ß3-ARs) increases. However, the precise role of ß3-AR signaling within cardiomyocytes remains unclear. Transforming growth factor ß1 (TGFß1) is a crucial cytokine mediating the cardiac remodeling that plays a causal role in the progression of heart failure. Here, we set out to determine the effect of ß3-AR activation on TGFß1 expression in rat cardiomyocytes and examine the underlying mechanism. The selective ß3-AR agonist BRL37344 induced an increase in TGFß1 expression and the phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun in ß3-AR-overexpressing cardiomyocytes. Those effects of BRL37344 were suppressed by a ß3-AR antagonist. Moreover, the inhibition of JNK and c-Jun activity by a JNK inhibitor and c-Jun siRNA blocked the increase in TGFß1 expression upon ß3-AR activation. A protein kinase G (PKG) inhibitor also attenuated ß3-AR-agonist-induced TGFß1 expression and the phosphorylation of JNK and c-Jun. In conclusion, the ß3-AR activation in cardiomyocytes increases the expression of TGFß1 via the PKG/JNK/c-Jun pathway. These results help us further understand the role of ß3-AR signaling in heart failure.