Diuretic effect of Lagopsis supina fraction in saline-loaded rats is mediated through inhibition of aquaporin and renin-angiotensin-aldosterone systems and up-regulation of atriopeptin.

Lagopsis supina (Steph. ex Willd.) lk. -Gal. ex Knorr. has been used as a diuretic agent in China for centuries with limited scientific evidence. This study investigated the diuretic efficacy and underlying mechanism of a macroporous adsorption resin with 30% ethanol elution fraction from L. supina (LSC) in saline-loaded rats and to identify its phytochemicals by ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-qTOF-MS/MS). As a result, 18 phenylpropanoids, 14 flavonoids and 15 others were identified in LSC, among which stachysoside A and acteoside could be the main bio-active constituents responsible for the diuretic effect. In parallel, the daily administration of LSC (16, 32 and 64 mg/kg) markedly promoted urinary excretion after 2 h of treatment. Moreover, LSC had no effect on urinary Na+ and K+ concentrations, as well as on serum Na+-K+-ATPase activity. Meanwhile, LSC significantly decreased the serum levels of angiotensin II (Ang II), anti-diuretic hormone (ADH), aldosterone (ALD), aquaporin (AQP) 1, AQP2 and AQP3, suppressed renal AQP1, AQP2, and AQP3 mRNA expressions, down-regulated AQP1, AQP2 and AQP3 protein levels, and up-regulated serum atriopeptin (ANP) level in a dose-dependent manner. These findings suggest that LSC has acute and prolonged diuretic effects by inhibiting the AQPs, RAAS, and upregulation of atriopeptin in saline-loaded rats, and this finding support LSC as a novel diuretic agent.

Natriuretic peptide system expression in murine and human submandibular salivary glands: a study of the spatial localisation of ANB, BNP, CNP and their receptors.

The natriuretic peptide (NP) system comprises of three ligands, the Atrial Natriuretic Peptide (ANP), Brain Natriuretic peptide (BNP) and C-type Natriuretic peptide (CNP), and three natriuretic peptide receptors, NPRA, NPRB and NPRC. Here we present a comprehensive study of the natriuretic peptide system in healthy murine and human submandibular salivary glands (SMGs). We show CNP is the dominant NP in mouse and human SMG and is expressed together with NP receptors in ducts, autonomic nerves and the microvasculature of the gland, suggesting CNP autocrine signalling may take place in some of these glandular structures. These data suggest the NP system may control salivary gland function during homeostasis through the regulation of electrolyte re-absorption, neural stimulation and/or blood vessel wall contraction/relaxation. We also show abnormal expression of NPRA in the stroma of a subset of human SMGs resected from patients diagnosed with oral squamous cell carcinoma (OSCC) of non-salivary gland origin. This finding warrants further research to investigate a possible correlation between early OSCC invasion and NPRA overexpression.

Recombinant production, purification and characterization of vessel dilator in E. coli.

Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E. coli BL21 (DE3), was recorded with VDII that carried the shortest fusion partner. Subsequent to the initial capture of the fusion protein by a Ni affinity column, the vessel dilator monomers were cleaved by trypsin treatment, and further purified to at least 90% homogeneity by anion exchange chromatography. De-novo sequencing and in vivo anticancer activity tests were used to verify the peptide sequence and its biological activity, respectively. The final yield was estimated to be approximately 15 mg of the purified vessel dilator per gram wet weight of the bacterial cells.

Cucurbitacin-I induces hypertrophy in H9c2 cardiomyoblasts through activation of autophagy via MEK/ERK1/2 signaling pathway.

Cucurbitacin-I, a natural triterpenoids initially identified in medicinal plants, shows a potent anticancer effect on a variety of cancer cell types. Nevertheless, the cardiotoxicity of cucurbitacin-I has not heretofore been reported. In this study, the mechanisms of cucurbitacin-I-induced cardiotoxicity were examined by investigating the role of MAPK-autophagy-dependent pathways. After being treated with 0.1-0.3µM cucurbitacin-I for 48h, H9c2 cells showed a gradual decrease in the cell viabilities, a gradual increase in cell size, and mRNA expression of ANP and BNP (cardiac hypertrophic markers). Cucurbitacin-I concentration-dependent apoptosis of H9c2 cells was also observed. The increased apoptosis of H9c2 cells was paralleling with the gradually strong autophagy levels. Furthermore, an autophagy inhibitor, 3-MA, was used to block the cucurbitacin-I-stirred autophagy, and then the hypertrophy and apoptosis induced by 0.3µM cucurbitacin-I were significantly attenuated. In addition, cucurbitacin-I exposure also activated the MAPK signaling pathways, including ERK1/2, JNK, and p38 kinases. Interestingly, only the ERK inhibitor U0126, but not the JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580, weakened the induction of 0.3µM cucurbitacin-I in hypertrophy, autophagy and apoptosis. Our findings suggest that cucurbitacin-I can increase the autophagy levels of H9c2 cells, most likely, through the activation of an ERK-autophagy dependent pathway, which results in the hypertrophy and apoptosis of cardiomyocytes.

Rapid effects of aldosterone in primary cultures of cardiomyocytes - do they suggest the existence of a membrane-bound receptor?

Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone + spironolactone + BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca(2+). Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure.

Endothelial Actions of ANP Enhance Myocardial Inflammatory Infiltration in the Early Phase After Acute Infarction.

RATIONALE: In patients after acute myocardial infarction (AMI), the initial extent of necrosis and inflammation determine clinical outcome. One early event in AMI is the increased cardiac expression of atrial natriuretic peptide (NP) and B-type NP, with their plasma levels correlating with severity of ischemia. It was shown that NPs, via their cGMP-forming guanylyl cyclase-A (GC-A) receptor and cGMP-dependent kinase I (cGKI), strengthen systemic endothelial barrier properties in acute inflammation. OBJECTIVE: We studied whether endothelial actions of local NPs modulate myocardial injury and early inflammation after AMI. METHODS AND RESULTS: Necrosis and inflammation after experimental AMI were compared between control mice and littermates with endothelial-restricted inactivation of GC-A (knockout mice with endothelial GC-A deletion) or cGKI (knockout mice with endothelial cGKI deletion). Unexpectedly, myocardial infarct size and neutrophil infiltration/activity 2 days after AMI were attenuated in knockout mice with endothelial GC-A deletion and unaltered in knockout mice with endothelial cGKI deletion. Molecular studies revealed that hypoxia and tumor necrosis factor-α, conditions accompanying AMI, reduce the endothelial expression of cGKI and enhance cGMP-stimulated phosphodiesterase 2A (PDE2A) levels. Real-time cAMP measurements in endothelial microdomains using a novel fluorescence resonance energy transfer biosensor revealed that PDE2 mediates NP/cGMP-driven decreases of submembrane cAMP levels. Finally, intravital microscopy studies of the mouse cremaster microcirculation showed that tumor necrosis factor-α-induced endothelial NP/GC-A/cGMP/PDE2 signaling impairs endothelial barrier functions. CONCLUSIONS: Hypoxia and cytokines, such as tumor necrosis factor-α, modify the endothelial postreceptor signaling pathways of NPs, with downregulation of cGKI, induction of PDE2A, and altered cGMP/cAMP cross talk. Increased expression of PDE2 can mediate hyperpermeability effects of paracrine endothelial NP/GC-A/cGMP signaling and facilitate neutrophil extravasation during the early phase after MI.

Acute Metabolic Influences on the Natriuretic Peptide System in Humans.

BACKGROUND: The cardiac natriuretic peptides (NPs), atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), have central roles in sodium and blood pressure regulation. Extracardiac factors (e.g., obesity and diabetes) influence NP production, potentially altering cardiovascular responses to volume and pressure stress. OBJECTIVES: This study examined the effects of acute carbohydrate intake on the NP system in humans, and investigated underlying mechanisms. METHODS: Normotensive subjects (N = 33) were given a high-carbohydrate shake. Venous blood was sampled to measure N-terminal (NT)-proANP and NT-proBNP levels. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and HepG2 cells were treated with glucose, and expression levels of NPs and micro ribonucleic acid 425 (miR-425), a negative regulator of ANP, were examined. The role of nuclear factor kappa B (NF-κB) in the glucose-mediated effects was investigated using a NF-κB inhibitor and expression plasmids encoding NF-κB subunits. RESULTS: We observed a 27% reduction in the levels of circulating NT-proANP (p < 0.001, maximal at 6 h) after carbohydrate challenge, with no effect on NT-proBNP levels in our human subjects. Glucose treatment of hESC-CMs for 6 h and 24 h increased levels of the primary transcript of miR-425 (pri-miR-425) and mature miR-425. A corresponding decrease in NPPA messenger RNA levels was also observed at both time points. Overexpression of NF-κB subunits in H9c2 cardiomyocytes increased miR-425 levels, whereas inhibition of NF-κB abrogated the glucose-mediated increase in pri-miR-425 levels in HepG2 cells. CONCLUSIONS: Acute carbohydrate challenge is associated with a reduction in ANP production. The mechanism appears to involve a glucose-induced increase in the expression of miR-425, mediated by NF-κB signaling.

[Prokaryotic expression, purification and identification of recombinant human atrial natriuretic peptide].

In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP3) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His6-ANP3. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.

Transcriptional and post-transcriptional regulation of CCN genes in failing heart.

BACKGROUND: CCN family of proteins has been implicated in various processes in cardiovascular physiology and pathology, including angiogenesis, regeneration and fibrosis. In this study we assessed long term changes of CCN1 and CCN2 gene products abundance in the failing ventricular myocardium. METHODS: Male, 12-14-weeks-old C57BL6/J and C57BL6/J (IL-6-/-) mice were used. To assess short term changes, a transient reversible ischemia model was utilized. Heart failure was caused by ligation of anterior descending coronary artery. The presence of systolic dysfunction was confirmed by echocardiography and left ventricular ANP RNA expression. Molecular analysis was performed on left ventricular samples from animals sacrificed 12-14 weeks after infarction. Western blotting and QT-PCR were used to investigate abundance of CCN proteins and RNAs, respectively. RESULTS: Short ischemia resulted in marked increase of CCN1 transcript. However, three months after myocardial infarction (MI), remote myocardium showed a markedly increased expression of CCN1 protein, but not RNA. In the case of CCN2, the RNA was distinctly up-regulated, whereas the protein presented only modest, non-significant increase in failing myocardium. Expression of CCN2 RNA closely correlated with expression of ANP. Long-term telmisartan administration after infarction decreased the expression of CCN1 protein. Interleukin 6 (IL-6) deficiency caused increased CCN2 protein abundance in control animals, but the difference was absent after MI. Infarction did not increase CCN1 protein in the hearts of IL-6 deficient mice. CONCLUSION: CCN genes are activated in heart failure. Their regulation is multidimensional both transcriptional and posttranscriptional. The involved pathways include angiotensin II and IL-6.

Buthionine sulfoximine, an inhibitor of glutathione biosynthesis, induces expression of soluble epoxide hydrolase and markers of cellular hypertrophy in a rat cardiomyoblast cell line: roles of the NF-κB and MAPK signaling pathways.

Evidence suggests that upregulation of soluble epoxide hydrolase (sEH) is associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac hypertrophy, and heart failure. However, the upregulation mechanism is still unknown. In this study, we treated H9C2 cells with buthionine sulfoximine (BSO) to explore whether oxidative stress upregulates sEH gene expression and to identify the molecular and cellular mechanisms behind this upregulatory response. Real-time PCR and Western blot analyses were used to measure mRNA and protein expression, respectively. We demonstrated that BSO significantly upregulated sEH at mRNA levels in a concentration- and time-dependent manner, leading to a significant increase in the cellular hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Furthermore, BSO significantly increased the cytosolic phosphorylated IκB-α and translocation of NF-κB p50 subunits, as measured by Western blot analysis. This level of translocation was paralleled by an increase in the DNA-binding activity of NF-κB P50 subunits. Moreover, our results demonstrated that pretreatment with the NF-κB inhibitor PDTC significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression in a dose-dependent manner. Additionally, mitogen-activated protein kinases (MAPKs) were transiently phosphorylated by BSO treatment. To understand further the role of MAPKs pathway in BSO-mediated induction of sEH mRNA, we examined the role of extracellular signal-regulated kinase (ERK), c-JunN-terminal kinase (JNK), and p38 MAPK. Indeed, treatment with the MEK/ERK signal transduction inhibitor, PD98059, partially blocked the activation of IκB-α and translocation of NF-κB p50 subunits induced by BSO. Moreover, pretreatment with MEK/ERK signal transduction inhibitors, PD98059 and U0126, significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression. These results clearly demonstrated that the NF-κB signaling pathway is involved in BSO-mediated induction of sEH gene expression, and appears to be associated with the activation of the MAPK pathway. Furthermore, our findings provide a strong link between sEH-induced cardiac dysfunction and involvement of NF-κB in the development of cellular hypertrophy.

Stimulation of ANP by angiotensin-(1-9) via the angiotensin type 2 receptor.

AIMS: Angiotensin-(1-9) [Ang-(1-9)] and Ang-(1-7) are cleaved by Ang converting enzyme 2 forming Ang I and Ang II, respectively, and the truncated Angs play a role in regulating atrial natriuretic peptide (ANP) secretion. Previously, we found that Ang-(1-7) stimulates ANP secretion via the Mas receptor. However, the effect of Ang-(1-9) on ANP secretion is still unknown. The aim of the present study is to determine whether Ang-(1-9) stimulates ANP secretion and to characterize the signaling pathway involved in stimulating secretion. MAIN METHODS: We examined the effects of Ang-(1-9) on ANP secretion and atrial contractility with and without inhibitors in isolated perfused atria. KEY FINDINGS: Ang-(1-9) stimulated ANP secretion and concentration without change in atrial contractility. Ang-(1-9)-induced-ANP secretion was increased from 5% to 60% by 3 µM Ang-(1-9) during the low-stretch state of the atrium. This stimulatory effect of Ang-(1-9) on ANP secretion was attenuated by pretreatment with an Ang II type 2 receptor (AT2R) antagonist but not by AT1R or Mas receptor antagonist. In addition, pretreatment with inhibitors of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) blocked Ang-(1-9)-induced ANP secretion. In the high-stretch atrial state, Ang-(1-9)-induced ANP secretion was increased more than in the low-stretch state following addition of 1 µM Ang-(1-9) (from 108% to 170%). In an in vivo experiment, acute infusion of Ang-(1-9) increased plasma ANP level without altering arterial blood pressure. This effect was attenuated by pretreatment with AT2R antagonist but not by Mas receptor antagonist. SIGNIFICANCE: These results suggest that Ang-(1-9) stimulates ANP secretion via the AT2R-PI3K-Akt-NO-cGMP pathway.

Differential expression of Nad(P)H oxidase isoforms and the effects of atorvastatin on cardiac remodeling in two-kidney two-clip hypertensive rats.

The NADPH oxidases (Noxes) are a family of ROS (reactive oxygen species)-generating enzymes which play a critical role in the development of cardiac remodeling associated with heart failure. The Noxes of their catalytic isoforms include multiple homologues in cardiovascular cells with wide range tissue distribution. It is still unclear which Noxes represent the major enzymatic source of ROS in the heart and play a predominant role in cardiac hypertrophy. In this study we investigated the differential expression changes of NAD(P)H oxidase P47phox isoform and Nox homologues in left ventricle and the effects of atorvastatin on cardiac remodeling in two-kidney two-clip(2K2C) hypertensive rats. The mRNA and protein expression of Nox2, Nox4 and P47phox showed a sustained increase at 4, 8, 12 weeks after surgery in 2K2C rats. Administration of atorvastatin attenuated cardiac dysfunction, hypertrophy and fibrosis of 2K2C rats. However, atorvastatin treatment had no effects on BP regulation. Further studies revealed that atorvastatin inhibited the increased expression of Nox2, Nox4, P47phox as well as 02"- production in 2K2C hypertensive rats. These findings indicate that Nox2, Nox4 and P47phox play a crucial role in the development of cardiac remodeling in the 2K2C hypertensive rats. Atorvastatin, independent of BP control, exerts anti-remodeling effects partially by inhibition of NAD(P)H oxidase-mediated cardiac oxidative stress.

The effects of dan-shen root on cardiomyogenic differentiation of human placenta-derived mesenchymal stem cells.

The aim of this study was to search for a good inducer agent using for cardiomyogenic differentiation of stem cells. Human placenta-derived mesenchymal stem cells (hPDMSCs) were isolated and incubated in enriched medium. Fourth passaged cells were treated with 10mg/L dan-shen root for 20 days. Morphologic characteristics were analyzed by confocal and electron microscopy. Expression of α-sarcomeric actin was analyzed by immunohistochemistry. Expression of cardiac troponin-I (TnI) was analyzed by immunohistofluorescence. Atrial natriuretic factor (ANF) and beta-myocin heavy chain (ß-MHC) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). hPDMSCs treated with dan-shen root gradually formed a stick-like morphology and connected with adjoining cells. On the 20th day, most of the induced cells stained positive with α-sarcomeric actin and TnI antibody. ANF and ß-MHC were also detected in the induced cells. Approximately 80% of the cells were successfully transdifferentiated into cardiomyocytes. In conclusion, dan-shen root is a good inducer agent used for cardiomyogenic differentiation of hPDMSCs.

CAMTA1 is a novel tumour suppressor regulated by miR-9/9* in glioblastoma stem cells.

Cancer stem cells or cancer initiating cells are believed to contribute to cancer recurrence after therapy. MicroRNAs (miRNAs) are short RNA molecules with fundamental roles in gene regulation. The role of miRNAs in cancer stem cells is only poorly understood. Here, we report miRNA expression profiles of glioblastoma stem cell-containing CD133(+) cell populations. We find that miR-9, miR-9(*) (referred to as miR-9/9(*)), miR-17 and miR-106b are highly abundant in CD133(+) cells. Furthermore, inhibition of miR-9/9(*) or miR-17 leads to reduced neurosphere formation and stimulates cell differentiation. Calmodulin-binding transcription activator 1 (CAMTA1) is a putative transcription factor, which induces the expression of the anti-proliferative cardiac hormone natriuretic peptide A (NPPA). We identify CAMTA1 as an miR-9/9(*) and miR-17 target. CAMTA1 expression leads to reduced neurosphere formation and tumour growth in nude mice, suggesting that CAMTA1 can function as tumour suppressor. Consistently, CAMTA1 and NPPA expression correlate with patient survival. Our findings could provide a basis for novel strategies of glioblastoma therapy.

Presence of dendroaspis natriuretic peptide and its binding to NPR-A receptor in rabbit kidney.

Natriuretic peptides help to maintain sodium and fluid volume homeostasis in a healthy cardio-renal environment. Since the identification of Dendroaspis natriuretic peptide (DNP) as a new member of the natriuretic peptide family, DNP has been considered as an important regulator of natriuresis and dieresis. The present study was undertaken to investigate the presence of immunoreactive Dendroaspis natriuretic peptide (DNP) and its specific receptor in rabbit. DNP was detected in heart, kidney, liver, brain, and plasma by radioimmunoassay (RIA). DNP contents of cardiac atrium and ventricle, renal cortex and medulla, liver, and brain were 1.42 ± 0.15, 1.0 6 ± 0.08, 2.55 ± 0.21, 1.81 ± 0.16, 1.36 ± 0.22, and 0.69 ± 0.15 pg/mg of wet weight, respectively. The concentration of DNP in plasma was 235.44 ± 15.44 pg/ml. By quantitative in vitro receptor autoradiography, specific ¹²5I-DNP binding sites were revealed in glomeruli, interlobular artery, acuate artery, vasa recta bundle, and inner medulla of the kidney with an apparent dissociation constant (K(d)) of 0.29 ± 0.05, 0.36 ± 0.03, 0.84 ± 0.19, 1.18 ± 0.23, and 10.91 ± 1.59 nM, respectively. Basal rate of 3', 5'-cyclic guanosine monophosphate (cGMP) production by particulate guanylyl cyclase (GC) activation of glomerular membranes was basally 13.40 ± 1.70 pmol/mg protein/min. DNP caused an increment of cGMP production in similar magnitude to that caused by ANP, BNP, and urodilatin, while the production of cGMP by CNP was significantly lower than that by DNP. Our results show that plasma levels of DNP were higher when compared to other tissues. DNP produces cGMP via the NPR-A receptor subtype in the kidney, similarly to ANP and BNP, suggesting that plasma DNP could have similar functions as ANP and BNP.

Decreased renal corin expression contributes to sodium retention in proteinuric kidney diseases.

Patients with proteinuric kidney diseases often have symptoms of salt and water retention. It has been hypothesized that dysregulated sodium absorption is due to increased proteolytic cleavage of epithelial sodium channels (ENaCs) and increased Na,K-ATPase expression. Microarray analysis identified a reduction in kidney corin mRNA expression in rat models of puromycin aminonucleoside-induced nephrotic syndrome and acute anti-Thy1 glomerulonephritis (GN). As atrial natriuretic peptide (ANP) resistance is a mechanism accounting for volume retention, we analyzed the renal expression and function of corin; a type II transmembrane serine protease that converts pro-ANP to active ANP. Immunohistochemical analysis found that corin colocalized with ANP. The nephrotic and glomerulonephritic models exhibited concomitant increased pro-ANP and decreased ANP protein levels in the kidney consistent with low amounts of corin. Importantly, kidneys from corin knockout mice had increased amounts of renal ß-ENaC and its activators, phosphodiesterase (PDE) 5 and protein kinase G II, when compared to wild-type mice. A similar expression profile was also found in cell culture suggesting the increase in PDE5 and kinase G II could account for the increase in ß-ENaC seen in nephrotic syndrome and GN. Thus, we suggest that corin might be involved in the salt retention seen in glomerular diseases.

Exacerbation of acute viral myocarditis by tobacco smoke is associated with increased viral load and cardiac apoptosis.

Exposure to tobacco smoke is known to have deleterious cardiovascular effects. In this study, we tested whether exposure to tobacco smoke exacerbates the severity of viral myocarditis in mice. Viral myocarditis was generated in 4-week-old male BALB/c mice by injection of Encephalomyocarditis virus (EMCV). Four groups were studied: (1) control (C, no smoke and no virus); (2) smoke only (S, exposure to cigarette smoke for 90 min/day for 15 days); (3) virus only (V); and (4) exposure to smoke for 5 days before plus 10 days following virus injection (S+V). We found that viral inoculation preceded by smoke exposure increased mortality more than twofold compared with virus inoculation alone. In addition, the mRNA level of atrial natriuretic factor was significantly higher in S+V than among any of the other 3 groups. Virus injection significantly decreased cardiac function compared with controls, with further deterioration observed in the S+V group. We also observed a significantly increased rate of apoptosis, with an increased activation of apoptosis-inducing factor in hearts exposed to S+V compared with those exposed to V alone. Our results suggest that preexposure to smoke significantly exacerbates the severity of viral myocarditis, likely through increased viral load and increased cardiomyocyte cell death.

Folbp1 promotes embryonic myocardial cell proliferation and apoptosis through the WNT signal transduction pathway.

The aim of this study was to evaluate the relationship between Folate-binding protein one (Folbp1) and embryonic cardiac proliferation, apoptosis, and differentiation. Folbp1 gene expression and short interference RNA expression vectors were constructed. Morphology of P19 cells during differentiation was observed by inverted microscope. Cell proliferation was tested using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazoliumbromide (MTT) method. Cell apoptosis was evaluated by DNA ladder and flow cytometry methods, and marker gene expression during differentiation, such as atrial natriuretic peptide (ANP) and cardiac troponin I (cTnI), and marker gene expression during apoptosis (Bax/Bcl-2) was measured by RT-PCR. Additionally, the critical genes (Wnt, GSK3beta, and/or beta-catenin) expressed in the Wnt signaling pathway were confirmed by RT-PCR. The Folbp1 expressing vector and the silencing vector were constructed. From day 5 of differentiation, the absorbance of cells overexpressing Folbp1 was notably higher than the controls, whereas the controls were notably higher than Folbp1 gene silenced P19 cells. P19 cell apoptosis with Folbp1 gene silencing was lower than the controls; however, more cells were driven into S phase. No significant morphological difference was observed in any of the groups. RT-PCR results show that ANP, cTnI, Wnt, Bax/Bcl-2, and beta-catenin were elevated whereas GSK3beta depressed in cells which overexpressed Folbp1 and was contradictory in Folbp1 gene silenced P19 cells. Folbp1 may be an important candidate mediator of folic acid deficiency-induced congenital cardiac anomalies which are induced by the dysfunction of proliferation and apoptosis of the myocardial cell, and possibly caused by the dysfunction of the Wnt signaling pathway.

Estrogen exerts concentration-dependent pro-and anti-hypertrophic effects on adult cultured ventricular myocytes. Role of NHE-1 in estrogen-induced hypertrophy.

Estrogen has been shown to protect the heart and attenuate myocardial hypertrophy and left ventricular remodelling through as yet to be defined mechanisms. In the present study we examined concentration-dependent effects of estrogen on hypertrophy of adult rat cardiomyocytes, potential underlying mechanisms related to intracellular pH (pHi) and possible sex-dependent responses. Cardiomyocytes were isolated from adult male and female Sprague-Dawley rats and used immediately for pHi determinations or cultured and subsequently treated for 24 h with 17beta-estradiol to assess hypertrophic responses. Fluorometric measurements with the pHi-sensitive dye BCECF demonstrated that at 1 pM 17beta-estradiol increased pHi (+0.05 pH units in females and +0.12 pH units in males, P<0.05) by a rapid non-genomic mechanism that was blocked by the sodium-hydrogen exchange isoform 1 (NHE-1) specific inhibitor AVE-4890 (AVE, 5 microM). Treatment with 1 pM 17beta-estradiol for 24 h increased cell size (females: 20%, P<0.05; males: 29%, P<0.05) and ANP expression (females: 414%, P<0.05; males: 497%, P<0.05) in a NHE-1-, and ERK1/2 MAPK-dependent manner. At 1 nM, 17beta-estradiol decreased pHi (females: -0.24 pH units, P<0.05; males: -0.07 pH units, P<0.05) which was also prevented by AVE, although at this concentration the hormone had no direct hypertrophic effect but instead prevented hypertrophy induced by phenylephrine. Our results show that low levels of estrogen produce cardiomyocyte hypertrophy through ERK/NHE-1 activation and intracellular alkalinization whereas an antihypertrophic effect is seen at high concentrations. These effects may further our understanding of the role of estrogen in heart disease particularly associated with hypertrophy.

cAMP-mediated beta-adrenergic signaling negatively regulates Gq-coupled receptor-mediated fetal gene response in cardiomyocytes.

The treatment with beta-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated beta-adrenergic signaling antagonizes alpha(1)-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the beta-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an alpha(1)-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of beta-ARs with isoproterenol, a beta-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to alpha(1)-ARs, cardiomyocytes were treated with prostaglandin F(2)alpha, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of beta-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using beta1- and beta2-AR double knockout mice, in which the predominant cardiac beta-AR subtypes are lacking, or by administering isoproterenol (ISO), a beta-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in beta(1)- and beta(2)-AR gene deficient mice. Conversely, we found that ANP, beta-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated beta-adrenergic signaling negatively regulates Gq cascade activation-induced fetal gene expression in cultured cardiomyocytes and that this inhibitory regulation is already operative in the mouse heart under physiological conditions.