Atrial Natriuretic Peptide Release Research Articles

Abstract 15078: Atrial and Brain Natriuretic Peptides Expression and Release in Pulmonary Hypertension

Introduction: Precapillary pulmonary hypertension (PH) is characterized by progressive overload for the right ventricle and right heart failure (RHF). N-terminal Brain Natriuretic Peptides (NTproBNP) and Atrial Natriuretic Peptides (ANP) are secreted from cardiomyocytes upon stretch. NTproBNP is used as biomarker for RHF. Aim: To study the association between ANP and NTproBNP and pressure overload in blood, right heart tissue and induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from PH-patients. Methods: We analyzed NTproBNP in 344 PH-patients, 9 controls, and in 9 PH-patients before and 6-months after pulmonary endarterectomy (PEA). We measured NPPA and NPPB gene expression on end-stage human right ventricular (RV) and right atrial (RA) samples from 8 PH-patients and 5 controls. In addition, we performed immunofluoresence staining for ANP and BNP on RA tissue (N=4) and RV tissue (N=10) in PH and controls (N=5). iPSC-CMs from 1 male and 1 female PH-patients were stretched for 24h at 1Hz and 10% on the Flexcell FX-6000 system, and NTproBNP release was measured on static and stretched iPSC-CMs. NTproBNP levels were measured through ECLIA. Results: NTproBNP serum levels were elevated in PH-patients versus controls (918 vs 25 pg/mL, Fig1A). Pressure unloading in CTEPH patients did not result in a decrease in NTproBNP (Fig1B). In RV tissue of PH-patients NPPA expression was upregulated, but NPPB was not (Fig1C). ANP and BNP content was higher in RA and RV tissue of PH-patients (Fig1D-E). Finally, ~5x higher NTproBNP was released from male vs female iPSC-CMs under static conditions and after mechanical stretch (Fig1F). Conclusions: ANP and BNP showed important differences at gene and protein levels in relation to pressure overload. To confirm current results, further analyses on atrial and ventricular iPSC-CMs will be performed, in addition to mid-range proANP measurements in patients’ blood and iPSC-CMs conditioned medium.

Abstract P2045: Regulation Of Atrial Natriuretic Peptide Secretion By Palmitoylation And Modulation Of The Rab3a GTPase Cycle In Cardiac Myocytes

Despite the clinical relevance to cardiovascular disease, molecular regulation of atrial natriuretic peptide (ANP) release by cardiomyocytes remains ill-defined. We uncovered novel functions for Rab3gap1 and modulation of Rab3a nucleotide cycling in membrane fusion and atrial natriuretic peptide (ANP) release in cardiomyocytes. We generated transgenic mice overexpressing the Golgi-localized S-acyltransferase, zDHHC9, and identified Rab3gap1 as a novel cardiac substrate of zDHHC9 by S-acyl proteomics. Rab3gap1 palmitoylation and retention at the cardiomyocyte Golgi, Rab3a-GTP levels, and formation of Rab3a-positive post-Golgi vesicles were all enhanced in zDHHC9 overexpressing hearts as were Rab3gap1, Rab3a, and ANP atrial protein levels but circulating ANP levels were reduced, suggesting a defect in ANP secretion. Notably, treatment of cardiomyocytes with the secretagogue phenylephrine (PE) promoted substantial ANP release as expected, but also enhanced Rab3gap1 palmitoylation and Rab3a GTP-loading and peripheral vesicle formation, whereas knockdown of zDHHC9 repressed these PE-induced effects on Rab3gap1 and Rab3a but further enhanced PE-stimulated ANP secretion, suggesting zDHHC9-mediated palmitoylation of Rab3gap1 serves as an intrinsic mechanism to limit ANP release. Consistent with a role for Rab3a nucleotide exchange in modulation of ANP release, overexpression of Rab3gap1 in cardiomyocytes promoted a profound increase in ANP release whereas a GAP-deficient mutant of Rab3gap1 (Rab3gap1 R728A ) was resistant to this effect. Collectively, these data suggest disruption of Rab3a nucleotide cycling perturbs secretory vesicle membrane fusion and ANP release in cardiac myocytes and establish critical roles for Rab3a activity and its modulation by zDHHC9-mediated palmitoylation of Rab3gap1 in governing cardiac homeostasis and endocrine signaling.

ZDHHC9 Regulates Cardiomyocyte Rab3a Activity and Atrial Natriuretic Peptide Secretion Through Palmitoylation of Rab3gap1

Production and release of natriuretic peptides by the stressed heart reduce cardiac workload by promoting vasodilation, natriuresis, and diuresis, which has been leveraged in the recent development of novel heart-failure pharmacotherapies, yet the mechanisms regulating cardiomyocyte exocytosis and natriuretic peptide release remain ill defined. We found that the Golgi S-acyltransferase zDHHC9 palmitoylates Rab3gap1 resulting in its spatial segregation from Rab3a, elevation of Rab3a-GTP levels, formation of Rab3a-positive peripheral vesicles, and impairment of exocytosis that limits atrial natriuretic peptide release. This novel pathway potentially can be exploited for targeting natriuretic peptide signaling in the treatment of heart failure.

Regulation of atrial natriuretic peptide secretion in cardiomyocytes by Rab3a and zDHHC9-mediated palmitoylation of Rab3gap1.

Natriuretic peptides are secreted by the heart in response to cardiovascular stress to reduce blood pressure and volume and thus there is interest in bolstering natriuretic peptide levels to improve outcomes in heart failure. We uncovered novel functions for the Golgi S-acyltransferase, zDHHC9, in regulating atrial natriuretic peptide (ANP) secretion in cardiomyocytes. zDHHC9 palmitoylates Rab3 GTPase activating protein 1 (Rab3gap1) in cardiomyocytes both in vitro and in vivo, resulting in enhanced Rab3gap1 retention at the Golgi, elevation of Rab3a-GTP levels, and a profound expansion of Rab3a-positive vesicles at the cardiomyocyte periphery in contrast to predominant Golgi localization in control cardiomyocytes. However, these zDHHC9-induced effects were associated with impairment of ANP release, suggesting zDHHC9-mediated disruption of Rab3a nucleotide cycling interferes with exocytosis of ANP. Consistent with this, overexpression of wildtype Rab3gap1 that promotes Rab3a GTP:GDP turnover greatly accelerated ANP secretion whereas overexpression of a Rab3gap1 GAP-defective mutant lacked this effect. Phenylephrine (PE) treatment induced Rab3gap1 palmitoylation, Rab3a-GTP levels, and Rab3a-containing post-Golgi vesicles, but this was associated with a substantial and well-established increase in ANP biosynthesis and secretion. Knockdown of zDHHC9 blunted PE-induced Rab3a GTP-loading and expansion of Rab3a-associated vesicles but evoked even greater PE-stimulated ANP release, suggesting Rab3gap1 palmitoylation provides an intrinsic regulatory mechanism to modulate cardiomyocyte exocytosis and ANP secretion. Importantly, Rab3a localized to atrial granules in vivo and transgenic mice with cardiomyocyte-specific overexpression of zDHHC9 exhibited dysregulated atrial granule morphology, accumulation of ANP in atrial cardiomyocytes, and reduced serum ANP levels, suggesting a defect in ANP release in zDHHC9-overexpressing hearts. Collectively, these data implicate zDHHC9-mediated palmitoylation of Rab3gap1 in disruption of Rab3a GTP:GDP cycling in cardiomyocytes required for proper ANP release, thereby highlighting novel roles for zDHHC9, Rab3gap1, and Rab3a in modulating ANP secretion.

MORPHOLOGICAL CHARACTERISTICS OF THE ENDOCRINE FUNCTION OF THE HEART IN COMORBID PATHOLOGY.

Aim: To study the ultrastructural remodeling of atrial myoendocrine cells (AMC) of the atrial myocardium in streptozotocin-induced diabetes (SID) under chronic immobilization stress (CIS). Materials and methods: 40 sexually mature white male rats (body weight 150-180 g) were included in the study. Four groups were formed: group 1 - animals with comorbid pathology (SID and CIS), group 2 - animals with SID, group 3 - animals with CIS, group 4 - intact animals. Results: On the 14th day of the development of SID and CIS, an increase in the functional activity of AMC is noted, which is confirmed by hyperplasia and hypertrophy of the protein-synthesizing apparatus, an increase in the volume density of secretory granules (SG), especially diffusing ones, and indicates enhanced release of atrial natriuretic peptide (ANP) from cells during the experiment. On the 56th day of the experiment, in groups 1 and 2 of , destructive changes in AMC were noted, such as vacuolar and balloon dystrophy, colliquative and partial necrosis. At the same time, the functional activity of AMC of different regions of the myocardium significantly. In animals with CIS, the volume density of young and diffusing SG in AMC is decreased. Conclusions: Therefore, in the early stages of the development of SID and CIS, an increase in the morpho-functional activity of AMC is noted. The long course of SID and its combination with CIS lead to destructive changes in AMC and to decrease in their secretory activity.

Relationship between oxytocin receptor gene polymorphism and hypertension in Turkish population

Introduction and objectivesKnown to play a key role in uterine contraction and milk ejection, the neuropeptide, oxytocin, has cardiovascular effects. To date, the known cardiovascular effects of oxytocin are blood pressure lowering (caused by natriuresis and atrial natriuretic peptide release), negative inotropic and chronotropic effect caused by parasympathetic neuromodulation, anti-stress effect and vasodilation mediated by activation of the nitric oxide pathway. The clinical significance of the rs2268498 polymorphism in oxytocin receptors in these effects is controversial. Based on the known genetic inheritance of hypertension, our research aimed to determine whether the presence of the rs2268498 oxytocin receptor (OXTR) allele C affects hypertension in our region. MethodsThis article is a case-control study conducted in the Turkish population. About 140 normotensive and 140 isolated hypertensive volunteers included in the research and genotyped with real-time PCR hybridization method via melt curve analysis. ResultsOxytocin receptor rs2268498 polymorphism was assessed in terms of the risk of hypertension and hypertensive individuals were compared to the control group. OXTR rs2268498 polymorphism was not found to be a significant risk factor for dominant, recessive and additive modeled hypertension (ORdominant: 0.966, 95% CI: 0.57-1.61, p: 0.9; ORrecessive: 1, 95% CI: 0.58-1.71, p: 1.0 and ORoverall: 0.98, chi2=0.01). ConclusionWe concluded that rs2268498 single nucleotide polymorphism is not a risk factor for hypertension in our region.

Thrombin receptor protease-activated receptor 1 (PAR1) related cytotoxic CD8+ T cell activity is associated with atrial myopathy and pro-inflammatory immune response in early atrial fibrillation

Abstract Background and aims Cytotoxic CD8+ T cells mediate myocardial damage in patients with virus induced myocarditis, plaque erosion during acute coronary syndrome, and promote adverse post-ischemic cardiac remodeling. There is emerging evidence to support a link between inflammation and atrial fibrillation (AF). Protease-activated receptor 1 (PAR1) is the thrombin receptor expressed on platelets, but it is also expressed in endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, and CD8+ T cells. The role of CD8+ T cells and PAR1 in atrial myopathy and early AF have not been studied. Methods In 80 non-anticoagulated patients with a first documented episode of AF, and 20 control subjects without AF but with a comparable cardiovascular risk profile (e.g. SCORE2, CHA2DS2-VASc, p>0.05), we studied PAR1-related cytotoxic T cell activity in the first hours (up to 24 h) of AF clinical manifestation. The Apolipoprotein E–knock out (ApoEko) mouse is a commonly used model of metabolic syndrome that develops atrial myopathy (structural correlate of AF). ApoEko male mice (Apoetm1Unc with a C57BL/6 genetic background) were fed for six weeks with a standard chew, western-type diet (high fat and high sucrose = WD) ± the specific PAR1 inhibitor vorapaxar. Results High levels of circulating, activated CD8+ T cells (CD8+CD57+ T cells with pro-inflammatory and high cytotoxic properties) were present in patients with early AF (flow cytometry). Stimulation of cytotoxic T cells, that expressed the thrombin receptor PAR1, that were isolated from patients with new onset of AF, released pro-inflammatory cytokines (e.g. IFN-γ, TNF-α). Enhanced cytotoxic activity of CD8+ T cells was suggested by elevated plasma levels of effector molecules (e.g. granulysin and granzyme). In ApoEko mice fed a WD, atrial myopathy as the structural correlate for AF, was paralleled by increased expression of effector molecules that were associated with CD8+ T cell activity (like perforin, granzyme A, granzyme B, IFN-γ). Cytotoxic T cell activity disrupted the release of the potective atrial natriuretic peptide, ANP, and increased signaling pathways associated with atrial fibrosis (e.g. TGF-β, cTGF, TGF-β receptor). The specific PAR1 inhibitor, vorapaxar, reduced inflammatory T cell activity, transcription of fibrotic mediators, and reconstituted ANP secretion in atria of ApoEko mice. Conclusions Targeting the FXa/FIIa-PAR1-CD8+ axis might be a promising approach to reduce atrial fibrosis and inflammation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): BIH Charité Clinician Scientist Program

Antagonistic Atrial Natriuretic Peptide with the Renin-Angiotensin-Aldosterone System and Effects on Systemic Blood Pressure Regulation

The atrial natriuretic peptide (ANP) presents, from the point of view of systemic blood pressure regulation, a relevant antagonistic association when compared to the renin-angiotensin-aldosterone system (RAAS). Through a careful review, the aim of the study was to evidence the process and the link between systems and hormones, from prohormone secretion, conversion, interaction with receptors, ANP action, correlating its antagonistic effects to RAAS, and the association between the mechanisms of action and SBP. The method adopted was a systematic review through electronic scientific articles in the database of the Virtual Health Library, PubMed and Cochrane. The process of searching and selecting the articles followed the rule of systematic review – PRISMA. The study demonstrates that the effects of ANP release due to cardiac atrial expansion are effectively counterregulators to the effects of RAAS malfunction, acting in a way to preserve the back, cardiac and vascular issues from blood pressure control. This mechanism acts via hydroelectrolytic regulation, especially through processes of resorption and excretion of sodium and water by the removal tubules, where the RAAS acts to increase blood volume and ANP acting to potentiate diuretic mechanisms. It is concluded that the degrading effects of the malfunction of RAAS, to some extent, be counter-regulated by the effects of ANP release, acting in the control of systemic blood pressure, alone or concomitantly with pharmacological treatment.

Abstract P1081: Palmitoylation-dependent Regulation Of Rab3gap1, Cardiomyocyte Exocytosis, And Atrial Natriuretic Peptide Secretion

Palmitoylation is the reversible post-translational modification of proteins with fatty acids catalyzed by zDHHC S-acyltransferases that functions as a potent regulatory mechanism controlling protein trafficking and signal transduction. To investigate functions of palmitoylation in cardiac homeostasis and pathophysiology, we generated transgenic mice with cardiomyocyte-specific overexpression of the Golgi enzyme, zDHHC9, which was previously reported as the Ras S-acyltransferase and is associated with X-linked intellectual disability. Although we did not observe altered Ras palmitoylation, zDHHC9 transgenic mice develop age-dependent cardiac hypertrophy that progresses to functional decompensation and cardiomyopathy. We found palmitoylation of Rab3 GTPase activating protein 1 (Rab3gap1) was significantly elevated in zDHHC9 overexpressing hearts prior to disease onset concomitant with enhanced GTP-loading of Rab3a, suggesting zDHHC9-mediated palmitoylation impairs Rab3gap1-dependent nucleotide cycling on Rab3a. Consistent with this, zDHHC9 overexpression resulted in enhanced Rab3gap1 retention at the Golgi and expansion of Rab3a-positive vesicles at the cardiomyocyte periphery. Co-immunoprecipitation analysis in HEK293AD cells confirmed that zDHHC9 elevation diminishes interaction between active Rab3a and Rab3gap1. To interrogate roles of zDHHC9 in cardiomyocyte exocytosis, we assessed secretion of atrial natriuretic peptide (ANP) and surprisingly found that while zDHHC9 overexpression increased intracellular retention and reduced release of ANP, knockdown of zDHHC9 promoted phenylephrine-induced ANP secretion. Taken together, these data suggest zDHHC9-mediated palmitoylation of Rab3gap1 represses ANP secretion by disrupting GTP:GDP cycling on Rab3a necessary for release of ANP from secretory granules. These studies reveal unexpected functions for zDHHC9-mediated palmitoylation in modulation of secretory pathway flux and regulated hormone secretion via downstream impacts on Rab3a-mediated vesicle fusion and exocytosis and suggest novel roles for zDHHC9, Rab3gap1, and Rab3a in natriuretic peptide secretion.

Exogenous ketosis suppresses diuresis and atrial natriuretic peptide during exercise.

We have previously demonstrated that exogenous ketosis reduces urine production during exercise. However, the underlying physiological mechanism of this antidiuretic effect remained unclear. Therefore, we investigated whether acute exogenous ketosis by oral ingestion of ketone ester (KE) during a simulated cycling race (RACE) affects the hormonal pathways implicated in fluid balance regulation during exercise. In a double-blind crossover design, 11 well-trained male cyclists participated in RACE consisting of a 3-h submaximal intermittent cycling (IMT180') bout followed by a 15-min time trial (TT15') in an environmental chamber set at 28°C and 60% relative humidity. Fluid intake was adjusted to maintain euhydration. Before and during RACE, the subjects received either a control drink (CON) or the ketone ester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KE), which elevated blood β-hydroxybutyrate to ∼2-4 mM. Urine output during IMT180' was ∼20% lower in KE (1,172 ± 557 mL) than in CON (1,431 ± 548 mL, P < 0.05). Compared with CON, N-terminal proatrial natriuretic peptide (NT-pro ANP) concentration during RACE was ∼20% lower in KE (P < 0.05). KE also raised plasma noradrenaline concentrations during RACE. Performance in TT15' was similar between CON and KE. In conclusion, exogenous ketosis suppresses diuresis and downregulates NT-pro ANP secretion during exercise.NEW & NOTEWORTHY We previously demonstrated that exogenous ketosis reduces urine production during exercise, however, the underlying physiological mechanism remained unclear. Here, we, for the first time demonstrate that exogenous ketosis suppresses the exercise-induced release of atrial natriuretic peptide (ANP). However, given the limited effects of ANP on renal haemodynamics during exercise, the underlying physiological mechanism remains unknown. But downregulation of ANP might explain a new physiological mechanism by which exogenous ketosis lowers blood-free fatty acid levels.

Heart-Microcirculation Connection: Effects of ANP (Atrial Natriuretic Peptide) on Pericytes Participate in the Acute and Chronic Regulation of Arterial Blood Pressure.

Cardiac ANP (atrial natriuretic peptide) moderates arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve inhibition of the renin-angiotensin-aldosterone system, increased natriuresis, endothelial permeability, and vasodilatation. The contribution of the direct vasodilating effects of ANP to blood pressure homeostasis is controversial because variable levels of the ANP receptor, GC-A (guanylyl cyclase-A), are expressed among vascular beds. Here, we show that ANP stimulates GC-A/cyclic GMP signaling in cultured microvascular pericytes and thereby the phosphorylation of the regulatory subunit of myosin phosphatase 1 by cGMP-dependent protein kinase I. Moreover, ANP prevents the calcium and contractile responses of pericytes to endothelin-1 as well as microvascular constrictions. In mice with conditional inactivation (knock-out) of GC-A in microcirculatory pericytes, such vasodilating effects of ANP on precapillary arterioles and capillaries were fully abolished. Concordantly, these mice have increased blood pressure despite preserved renal excretory function. Furthermore, acute intravascular volume expansion, which caused release of cardiac ANP, did not affect blood pressure of control mice but provoked hypertensive reactions in pericyte GC-A knock-out littermates. We conclude that GC-A/cGMP-dependent modulation of pericytes and microcirculatory tone contributes to the acute and chronic moderation of arterial blood pressure by ANP. Graphic Abstract A graphic abstract is available for this article.

The Role of Oxytocin in Cardiovascular Protection.

The beneficial effects of oxytocin on infarct size and functional recovery of the ischemic reperfused heart are well documented. The mechanisms for this cardioprotection are not well defined. Evidence indicates that oxytocin treatment improves cardiac work, reduces apoptosis and inflammation, and increases scar vascularization. Oxytocin-mediated cytoprotection involves the production of cGMP stimulated by local release of atrial natriuretic peptide and synthesis of nitric oxide. Treatment with oxytocin reduces the expression of proinflammatory cytokines and reduces immune cell infiltration. Oxytocin also stimulates differentiation stem cells to cardiomyocyte lineages as well as generation of endothelial and smooth muscle cells, promoting angiogenesis. The beneficial actions of oxytocin may include the increase in glucose uptake by cardiomyocytes, reduction in cardiomyocyte hypertrophy, decrease in oxidative stress, and mitochondrial protection of several cell types. In cardiac and cellular models of ischemia and reperfusion, acute administration of oxytocin at the onset of reperfusion enhances cardiomyocyte viability and function by activating Pi3K and Akt phosphorylation and downstream cellular signaling. Reperfusion injury salvage kinase and signal transducer and activator of transcription proteins cardioprotective pathways are involved. Oxytocin is cardioprotective by reducing the inflammatory response and improving cardiovascular and metabolic function. Because of its pleiotropic nature, this peptide demonstrates a clear potential for the treatment of cardiovascular pathologies. In this review, we discuss the possible cellular mechanisms of action of oxytocin involved in cardioprotection.

Effect of insulin on natriuretic peptide gene expression in porcine heart

Gut hormones affect cardiac function and contractility. In this study, we examined whether insulin affects the cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression and release of proANP-derived peptides in pigs. Anaesthetized pigs were included in an experimental study comparing the effect of hyperinsulinemia in 15 pigs submitted to two different protocols versus 11 control pigs receiving saline infusion. Phosphorylation of Akt on Thr308 was determined by western blotting with a pAkt-Thr308 antibody. The mRNA contents of ANP and BNP were determined with real-time PCR; plasma and cardiac tissue proANP was measured with an immunoluminometric assay targeted against the mid-region of the propeptide and a processing-independent assay. Insulin stimulation increased phosphorylation of Akt Thr308 in both left atrium and left ventricle of porcine hearts (p < 0.005). No change was observed in ANP and BNP mRNA contents in the right or left atrium. BNP mRNA contents in the left ventricle, however, decreased 3-fold (p = 0.02) compared to control animals, whereas the BNP mRNA content in the right ventricle as well as ANP mRNA content in the right and left ventricle did not change following hyperinsulinemia. Moreover, the peptide contents did not change in the four cardiac chambers. Finally, proANP concentrations in plasma did not change during the insulin infusion compared to the control animals. These results suggest that insulin does not have direct effect on atrial natriuretic peptide expression but may have a role in the left ventricle.

Chamber-specific regulation of atrial natriuretic peptide secretion in cardiac hypertrophy: atrial wall dynamics in the ANP secretion

The heart is involved in the regulation of blood pressure and body fluid homeostasis. As a blood volume sensor and effector for the regulation of the volume and pressure homeostasis, the atria are the central regulator to secrete humoral messenger cardiac natriuretic hormones into the circulation. The primary action of the atria in response to the volume change in the body is to control the secretion of atrial natriuretic peptide (ANP), a member of the family of cardiac natriuretic hormones. Although all cardiac chambers are able to secrete ANP, the major source of the cardiac hormone is the atria until reactivation of the synthesis of the ventricles. In heart disease including hypertension and cardiac hypertrophy, ventricular ANP synthesis and plasma levels of ANP are increased. However, the roles of the atria for the ANP secretion are not well defined in hypertension or heart failure. Under the high concentration of plasma levels of ANP by compensatory and/or pathophysiological reactivation of the ventricular synthesis and release of ANP, with activation of the renin-angiotensin system and changes in the atrial distensibility, the roles of the atria should be reevaluated in the heart disease. The purpose of the present review is to address modulation of the atrial role in the regulation of ANP secretion and its significance in the pathological changes in hypertension and cardiac disease and to strengthen the importance of the role of the interstitial fluid dynamics of the atrial wall in the regulation of ANP secretion.

Intradermal Administration of Atrial Natriuretic Peptide Attenuates Cutaneous Vasodilation but Not Sweating in Young Men during Exercise in the Heat

Introduction: Prolonged exercise in the heat stimulates plasma release of atrial natriuretic peptide (ANP) in association with dehydration-induced reductions in blood volume. Elevated plasma ANP levels under these conditions may indirectly attenuate cutaneous blood flow and sweating responses due to the effects of this hormone on central blood volume and plasma osmolality and the resulting stimulation of nonthermal reflexes. However, it remains unclear whether cutaneous blood flow and sweating are directly modulated by ANP at the level of the cutaneous end organs (cutaneous microvessels and eccrine sweat glands) during prolonged exercise in the heat. Objective: Therefore, we evaluated the effects of local ANP administration on forearm cutaneous vascular conductance (CVC) and local sweat rate (LSR) during rest and exercise in the heat. Methods: In 9 habitually active young men (26 ± 6 years) CVC and LSR were evaluated at 3 intradermal microdialysis sites continuously perfused with lactated Ringer solution (control) or ANP (0.1 or 1.0 μM). Participants rested in a non-heat-stress condition (25°C) for approximately 60 min followed by 70 min in the heat (35°C). They then performed 50 min of moderate-intensity cycling (approx. 55% VO_{2 peak}), with a 30-min recovery. Thereafter, 50 mM sodium nitroprusside was administered at all sites to elicit maximum CVC, which was subsequently used to normalize all values (CVC_%max). Results: No effects of ANP on CVC_%max were observed in the non-heat-stress resting condition compared to the untreated control site (both p > 0.05). Conversely during rest in the heat there was an 11% (5–17%) reduction in CVC_%max at the 1.0 μM ANP site relative to the untreated control site (p < 0.05). At the end of exercise CVC_%max was attenuated by 12% (1–23%) at the 0.1 μM ANP site and by 21% (7–35%) at the 1.0 μM ANP site relative to the untreated control site (all p < 0.05). Conversely, neither concentration of ANP influenced sweating at any time point (all p > 0.05). Conclusion: Intradermal ANP administration directly attenuated cutaneous blood flow, but not sweating, in habitually active young men during rest and exercise in the heat.

Inhibiting SLC26A4 reverses cardiac hypertrophy in H9C2 cells and in rats.

BackgroundIt has been confirmed that mutations in solute carrier family 26 member 4 (SLC26A4) contribute to pendred syndrome. However, the role of SLC26A4 in cardiac hypertrophy and the signaling pathways remain unclear.MethodsCardiomyocytes were treated by 200 µM phenylephrine (PE) to induce cardiac hypertrophy. Also, the expression of SLC26A4, GSK3, cardiac hypertrophy markers including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was detected through real-time quantitative polymerase chain reaction (RT-qPCR). Flow cytometry assay was used to test the apoptosis of PE-induced cardiomyocytes transfected by small interfere RNA (siRNA)-SLC26A4. Furthermore, we detected the expression of autophagy-related markers including light chain 3 (LC3) and P62. Finally, we established a rat model of abdominal aortic constriction (AAC)-induced cardiac hypertrophy in vivo.ResultsRT-qPCR results showed that the mRNA expression of SLC26A4 was significantly up-regulated in PE-induced cardiac hypertrophy. After inhibiting SLC26A4, the release of ANP and BNP was significantly decreased and GSK3β was elevated in vivo and in vitro. Furthermore, inhibiting SLC26A4 promoted apoptosis of cardiac hypertrophy cells. In addition, LC3 was down-regulated and P62 was enhanced after transfection of siRNA-SLC26A4.ConclusionOur findings revealed that SLC26A4 increases cardiac hypertrophy, and inhibiting SLC26A4 could decrease the release of ANP/BNP and promote the expression of GSK-3β in vitro and in vivo. Moreover, SLC26A4 silencing inhibits autophagy of cardiomyocytes and induces apoptosis of cardiomyocytes. Therefore, SLC26A4 possesses potential value to be a therapeutic target of cardiac hypertrophy, and our study provides new insights into the mechanisms of cardiac hypertrophy.

High glucose induced calcium overload via impairment of SERCA/PLN pathway and mitochondrial dysfunction leads to oxidative stress in H9c2 cells and amelioration with ferulic acid.

Oxidative stress and associated complications are the major pathological concerns of diabetic cardiomyopathy (DC). We aim to elucidate the mechanisms by which high glucose (HG) induced alteration in calcium homeostasis and evaluation of the beneficial effect of two concentrations (10 and 25 μm) of ferulic acid (FA). HG was induced in H9c2 cardiomyoblast by treating with glucose (33 mm) for 48 h, and FA was co-treated. Intracellular calcium ([Ca2+ ]i) overload was found increased significantly with HG. For elucidation of mechanism, the SERCA pathway and mitochondrial integrity (transmembrane potential and permeability transition pore) were explored. Then, we assessed oxidative stress, and cell injury with brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), and lactate dehydrogenase (LDH) release. HG caused significant [Ca2+ ]i overload through downregulation of SERCA2/1, pPLN, and pPKA C-α; and upregulation of PLN and PKA C-α and alteration in the integrity of mitochondria with HG. The [Ca2+ ]i overload in turn caused oxidative stress via generation of reactive oxygen species, lipid peroxidation, and protein carbonylation. This resulted in cell injury which was evident with significant release of BNP, ANP, and LDH. FA co-treatment was effective to mitigate all pathological changes caused by HG. From the overall results, we conclude that [Ca2+ ]i overload via SERCA pathway and altered mitochondrial integrity is the main cause for oxidative stress during HG. Based on our result, we report that FA could be an attractive nutraceutical for DC.

Nitric oxide acutely modulates hypothalamic and neurohypophyseal carbon monoxide and hydrogen sulphide production to control vasopressin, oxytocin and atrial natriuretic peptide release in rats.

Nitric oxide (NO) negatively modulates the secretion of vasopressin (AVP), oxytocin (OT) and atrial natriuretic peptide (ANP) induced by the increase in extracellular osmolality, whereas carbon monoxide (CO) and hydrogen sulphide (H2 S) act to potentiate it; however, little information is available for the osmotic challenge model about whether and how such gaseous systems modulate each other. Therefore, using an acute ex vivo model of hypothalamic and neurohypophyseal explants (obtained from male 6/7-week-old Wistar rats) under conditions of extracellular iso- and hypertonicity, we determined the effects of NO (600μmolL-1 sodium nitroprusside), CO (100μmolL-1 tricarbonylchloro[glycinato]ruthenium [II]) and H2 S (10mmolL-1 sodium sulphide) donors and nitric oxide synthase (NOS) (300μmolL-1 Nω -methyl-l-arginine [LNMMA]), haeme oxygenase (HO) (200μmolL-1 Zn(II) deuteroporphyrin IX 2,4-bis-ethylene glycol [ZnDPBG]) and cystathionine β-synthase (CBS) (100μmolL-1 aminooxyacetate [AOA]) inhibitors on the release of hypothalamic ANP and hypothalamic and neurohypophyseal AVP and OT, as well as on the activities of NOS, HO and CBS. LNMMA reversed hyperosmolality-induced NOS activity, and enhanced hormonal release by the hypothalamus and neurohypophysis, in addition to increasing CBS and hypothalamic HO activity. AOA decreased hypothalamic and neurohypophyseal CBS activity and hormonal release, whereas ZnDPBG inhibited HO activity and hypothalamic hormone release; however, in both cases, AOA did not modulate NOS and HO activity and ZnDPBG did not affect NOS and CBS activity. Thus, our data indicate that, although endogenous CO and H2 S positively modulate AVP, OT and ANP release, only NO plays a concomitant role of modulator of hormonal release and CBS activity in the hypothalamus and neurohypophysis and that of HO activity in the hypothalamus during an acute osmotic stimulus, which suggests that NO is a key gaseous controller of the neuroendocrine system.

Glucotoxicity results in apoptosis in H9c2 cells via alteration in redox homeostasis linked mitochondrial dynamics and polyol pathway and possible reversal with cinnamic acid

Several mechanisms have been proposed for the heart dysfunction during hyperglycemia. The aim of the present in vitro study is to elucidate the role of alterations in redox homeostasis in the induction of apoptosis during hyperglycemia in H9c2 cells via dysfunction in mitochondria and polyol pathway and evaluation of the beneficial effect of cinnamic acid against the same. The H9c2 cells were incubated with 33 mM glucose for 48 h to simulate the diabetic condition. Cell injury was confirmed with a significant increase of atrial natriuretic peptide and lactate dehydrogenase release. Alterations in the innate antioxidant system, polyol pathway, mitochondrial integrity, dynamics and apoptosis were investigated. Hyperglycemic insult has significantly affected redox homeostasis via depletion of superoxide dismutase, glutathione and enhanced reactive oxygen species generation. It also caused dysregulation in mitochondrial dynamics (fusion, fission proteins), dissipation of mitochondrial transmembrane potential and increased sorbitol accumulation. Finally, apoptosis was observed with upregulation of Bax, activation of caspase-3 and downregulation of Bcl-2. Cinnamic acid cotreatment increased the innate antioxidant status, improved mitochondrial function and prevented apoptosis in H9c2 cardiomyoblasts. Moreover, this in vitro model is found to be ideal for the elucidation of mechanisms at the cellular and molecular level of any physiological, pharmacological and toxicological incidents in H9c2 cells.

Effect of obstructive sleep apnea on the response to hypertension therapy

ABSTRACTObstructive sleep apnea (OSA) often precedes cardiovascular disease, partly due to treatment resistant hypertension. The nocturnal apneas of OSA trigger increased sympathetic nervous discharge during both sleep and wakefulness. Apneas also trigger cardiac release of the endogenous diuretic atrial natriuretic peptide. We hypothesized that treatment of the excess sympathetic nervous activity of OSA with a β1 blocker would lower 24 h blood pressure (BP) more than diuretic therapy. Subjects with OSA associated hypertension received 2 weeks of placebo followed by the β1 blocker nebivolol or hydrochlorothiazide (HCTZ) for 6 weeks in a blinded crossover study. BP, baroreflex sensitivity (BRS), heart rate variability (HRV), arterial reactivity, and stiffness were measured after placebo and each treatment. The β1 blocker lowered clinic BP by −11/−8 mmHg, more than the −3/−1 effect of HCTZ (P < 0.01). The β1 blocker lowered 24 h diastolic blood pressure (DBP) more than HCTZ. Although given at bedtime, neither drug increased BP dipping. Nebivolol increased HRV in the high-frequency band. Nebivolol did not alter BRS while HCTZ significantly diminished BRS compared to nebivolol (P < 0.01). Nebivolol increased flow-mediated brachial artery dilation when compared to HCTZ and slowed pulse wave velocity, indicating a decrease in arterial stiffness.Diuretic therapy failed to lower BP in OSA subjects and this might account for the frequent association of OSA with treatment resistant hypertension. However, blockade of the excess sympathetic nervous activity of OSA with a β1 blocker lowered both clinic and 24 h DBP.