The WNT/Ca2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-ß activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria.

WNT signaling plays an important role in cardiac development, but abnormal activity is often associated with cardiac hypertrophy, myocardial infarction, remodeling, and heart failure. The effect of WNT signaling on regulation of atrial natriuretic peptide (ANP) secretion is unclear. Therefore, the purpose of this study was to investigate the effect of Wnt agonist 1 (Wnta1) on ANP secretion and mechanical dynamics in beating rat atria. Wnta1 treatment significantly increased atrial ANP secretion and pulse pressure; these effects were blocked by U73122, an antagonist of phospholipase C. U73122 also abolished the effects of Wnta1-mediated upregulation of protein kinase C (PKC) ß and γ expression, and the PKC antagonist Go 6983 eliminated Wnta1-induced secretion of ANP. In addition, Wnta1 upregulated levels of phospho-transforming growth factor-ß activated kinase 1 (p-TAK1), TAK1 banding 1 (TAB1) and phospho-activating transcription factor 2 (p-ATF2); these effects were blocked by both U73122 and Go 6983. Wnta1-induced ATF2 was abrogated by inhibition of TAK1. Furthermore, Wnta1 upregulated the expression of T cell factor (TCF) 3, TCF4, and lymphoid enhancer factor 1 (LEF1), and these effects were blocked by U73122 and Go 6983. Tak1 inhibition abolished the Wnta1-induced expression of TCF3, TCF4, and LEF1 and Wnta1-mediated ANP secretion and changes in mechanical dynamics. These results suggest that Wnta1 increased the secretion of ANP and mechanical dynamics in beating rat atria by activation of PKC-TAK1-ATF2-TCF3/LEF1 and TCF4/LEF1 signaling mainly via the WNT/Ca2+ pathway. It is also suggested that WNT-ANP signaling is implicated in cardiac physiology and pathophysiology.

NOX4/Src regulates ANP secretion through activating ERK1/2 and Akt/GATA4 signaling in beating rat hypoxic atria.

Nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are the major enzymatic source of reactive oxygen species (ROS). NOX2 and NOX4 are expressed in the heart but its role in hypoxia-induced atrial natriuretic peptide (ANP) secretion is unclear. This study investigated the effect of NOX on ANP secretion induced by hypoxia in isolated beating rat atria. The results showed that hypoxia significantly upregulated NOX4 but not NOX2 expression, which was completely abolished by endothelin-1 (ET-1) type A and B receptor antagonists BQ123 (0.3 µM) and BQ788 (0.3 µM). ET-1-upregulated NOX4 expression was also blocked by antagonists of secreted phospholipase A2 (sPLA2; varespladib, 5.0 µM) and cytosolic PLA2 (cPLA2; CAY10650, 120.0 nM), and ET-1-induced cPLA2 expression was inhibited by varespladib under normoxia. Moreover, hypoxia-increased ANP secretion was evidently attenuated by the NOX4 antagonist GLX351322 (35.0 µM) and inhibitor of ROS N-Acetyl-D-cysteine (NAC, 15.0 mM), and hypoxia-increased production of ROS was blocked by GLX351322. In addition, hypoxia markedly upregulated Src expression, which was blocked by ET receptors, NOX4, and ROS antagonists. ET-1-increased Src expression was also inhibited by NAC under normoxia. Furthermore, hypoxiaactivated extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) were completely abolished by Src inhibitor 1 (1.0 µM), and hypoxia-increased GATA4 was inhibited by the ERK1/2 and Akt antagonists PD98059 (10.0 µM) and LY294002 (10.0 µM), respectively. However, hypoxia-induced ANP secretion was substantially inhibited by Src inhibitor. These results indicate that NOX4/Src modulated by ET-1 regulates ANP secretion by activating ERK1/2 and Akt/GATA4 signaling in isolated beating rat hypoxic atria.

Cholecystokinin Octapeptide Promotes ANP Secretion through Activation of NOX4-PGC-1α-PPARα/PPARγ Signaling in Isolated Beating Rat Atria.

Atrial natriuretic peptide (ANP), a canonical cardiac hormone, is mainly secreted from atrial myocytes and is involved in the regulation of body fluid, blood pressure homeostasis, and antioxidants. Cholecystokinin (CCK) is also found in cardiomyocytes as a novel cardiac hormone and induces multiple cardiovascular regulations. However, the direct role of CCK on the atrial mechanical dynamics and ANP secretion is unclear. The current study was to investigate the effect of CCK octapeptide (CCK-8) on the regulation of atrial dynamics and ANP secretion. Experiments were performed in isolated perfused beating rat atria. ANP was measured using radioimmunoassay. The levels of hydrogen peroxide (H2O2) and arachidonic acid (AA) were determined using ELISA Kits. The levels of relative proteins and mRNA were detected by Western blot and RT-qPCR. The results showed that sulfated CCK-8 (CCK-8s) rather than desulfated CCK-8 increased the levels of phosphorylated cytosolic phospholipase A2 and AA release through activation of CCK receptors. This led to the upregulation of NADPH oxidase 4 (NOX4) expression levels and H2O2 production and played a negative inotropic effect on atrial mechanical dynamics via activation of ATP-sensitive potassium channels and large-conductance calcium-activated potassium channels. In addition, CCK-8s-induced NOX4 subsequently upregulated peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) expression levels through activation of p38 mitogen-activated protein kinase as well as the serine/threonine kinase signaling, ultimately promoting the secretion of ANP via activation of PPARα and PPARγ. In the presence of the ANP receptor inhibitor, the CCK-8-induced increase of AA release, H2O2 production, and the upregulation of NOX4 and CAT expressions was augmented but the SOD expression induced by CCK-8s was repealed. These findings indicate that CCK-8s promotes the secretion of ANP through activation of NOX4-PGC-1α-PPARα/PPARγ signaling, in which ANP is involved in resistance for NOX4 expression and ROS production and regulation of SOD expression.

NOX4 stimulates ANF secretion via activation of the Sirt1/Nrf2/ATF3/4 axis in hypoxic beating rat atria.

Silent information regulator factor 2­related enzyme 1 (Sirt1) is involved in the regulation of cell senescence, gene transcription, energy balance and oxidative stress. However, the effect of Sirt1 on atrial natriuretic factor (ANF) secretion, especially under hypoxic conditions is unclear. The present study aimed to investigate the effect of Sirt1, regulated by NADPH oxidase 4 (NOX4), on ANF secretion in isolated beating rat atria during hypoxia. ANF secretion was analyzed using radioimmunoassays and protein expression levels were determined by western blotting and immunofluorescence staining. Intra­atrial pressure was recorded using a physiograph. Hypoxia significantly upregulated Sirt1 and nuclear factor erythroid­2­related factor 2 (Nrf2) protein expression levels, together with significantly increased ANF secretion. Hypoxia­induced protein expression of Sirt1 was significantly blocked by a NOX4 inhibitor, GLX351322, and Nrf2 protein expression levels were significantly abolished using the Sirt1 inhibitor, EX527. Hypoxia also significantly elevated the protein expression levels of phosphorylated­Akt and sequestosome 1 and significantly downregulated Kelch­like ECH­associated protein 1 protein expression levels. These effects were significantly blocked by EX527, preventing hypoxia­induced Nrf2 expression. An Nrf2 inhibitor, ML385, significantly abolished the hypoxia­induced upregulation of activating transcription factor (ATF)3, ATF4, T cell factor (TCF)3 and TCF4/lymphoid enhancer factor 1 (LEF1) protein expression levels, and significantly attenuated hypoxia­induced ANF secretion. These results indicated that Sirt1 and Nrf2, regulated by NOX4, can potentially stimulate TCF3 and TCF4/LEF1 signaling via ATF3 and ATF4 activation, thereby potentially participating in the regulation of ANF secretion in beating rat atria during hypoxia. In conclusion, intervening with the Sirt1/Nrf2/ATF signaling pathway may be an effective strategy for resisting oxidative stress damage in the heart during hypoxia.

Src-IL-18 signaling regulates the secretion of atrial natriuretic factor in hypoxic beating rat atria.

BACKGROUND: Interleukin (IL)-18 is produced mainly in the heart and can be associated with the development of cardiac hypertrophy that leads to cardiac dysfunction. However, the effects of hypoxia on IL-18 expression and atrial natriuretic factor (ANF) secretion remain largely unknown. AIM: The aim of this study was to assess the effect of hypoxia on IL-18 production and its role in ANF secretion by using an isolated perfused beating rat atrial model. METHODS: The level of ANF in the perfusates was determined by radioimmunoassay, and the protein levels of Src, IL-18 and its receptors (IL-18-Rα and IL-18-Rß), Rho guanine nucleotide exchange factor (RhoGEF) and RhoA, activating transcription factor 3 (ATF3), T cell factor (TCF) 3 and 4, and lymphoid enhancer factor (LEF) 1 in atrial tissue samples were detected by Western blotting. RESULTS: Hypoxia significantly upregulated the expression of the non-receptor tyrosine kinase Src, and this effect was blocked by endothelin-1 receptor type A (BQ123) and type B (BQ788) antagonists. Hypoxia also enhanced the expression of RhoGEF and RhoA concomitantly with the upregulation of IL-18, IL-18-Rα and IL-18-Rß. The hypoxia-induced RhoGEF and RhoA were abolished by Src inhibitor 1 (SrcI), and the protein levels of IL-18 and its two receptors were also blocked by SrcI. Moreover, the hypoxia-induced expression levels of ATF3, TCF3, TCF4 and LEF1 were repealed by IL-18 binding protein, and the hypoxia-promoted secretion of ANF was also obviously attenuated by this binding protein. CONCLUSIONS: These findings imply that Src-IL-18 signaling is involved in the release of ANF in hypoxic beating rat atria.

Endogenous ET-1 promotes ANP secretion through activation of COX2-L-PGDS-PPARγ signaling in hypoxic beating rat atria.

Endothelin-1 (ET-1) is a potent stimulus for the secretion of atrial natriuretic peptide (ANP) and hypoxia stimulates the release of ET-1, which is involved in the regulation of atrial ANP secretion. However, the precise mechanism of endogenous ET-1 in the regulation of hypoxia-induced ANP secretion is unclear. Therefore, this study aimed to investigate the mechanism of hypoxia-induced endogenous ET-1 regulation of ANP secretion in isolated perfused hypoxic beating rat atria. The results of this study showed that acute hypoxia significantly stimulated ET-1 release and upregulated the expression of its type A as well as type B receptors (ETA and ETB receptors). Endogenous ET-1 induced by hypoxia markedly upregulated the expression of cyclooxygenase 2 (COX2) through activation of its two receptors, leading to an increase in lipocalin-type prostaglandin D synthase (L-PGDS) expression and prostaglandin D2 (PGD2) production. L-PGDS-derived PGD2 activated peroxisome proliferator-activated receptor γ (PPARγ), ultimately promoting hypoxia-induced ANP secretion. Conversely, L-PGDS-derived PGD2 may in turn regulate L-PGDS expression by a nuclear factor erythroid-2-related factor 2 (NRF2)-mediated feedback mechanism. These results indicate that endogenous ET-1 induced by hypoxia promotes hypoxia-induced ANP secretion by activation of COX2-L-PGDS-PPARγ signaling in beating rat atria. In addition, the positive feedback loop between L-PGDS-derived PGD2 and L-PGDS expression induced by hypoxia is part of the mechanism of hypoxia-induced ANP secretion by endogenous ET-1.