Natriuretic Peptide Gene Expression Research Articles

Abstract 4136605: Effect Of A Bone Morphogenetic Protein Receptor Type 2 Mutation On The Cardiomyocyte Response To Pressure Overload

Introduction: A mutation in the Bone Morphogenetic Protein Receptor Type 2 ( BMPR2 ) gene is in 70% of hereditary pulmonary arterial hypertension (hPAH) patients the causative mutation of the disease. Previous work demonstrated that right ventricular function is more impaired in hPAH patients with a BMPR2 mutation. However, the underlying mechanism remains elusive. Moreover, natriuretic peptides, such as N-terminal Brain Natriuretic Peptides (NTproBNP) and Mid Regional proAtrial Natriuretic Peptides (MRproANP), are secreted from cardiomyocytes upon stretch as adaptation to increased pressure overload. We hypothesize that the BMPR2 mutation impairs the response to increased pressure overload. For this purpose, we make use of ventricular induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of hPAH patients carrying a BMPR2 mutation and their isogenic controls in which the mutation was corrected. Aim: To study the effect of a BMPR2 mutation on the response to pressure overload in vitro . Methods: iPSCs from 2 hPAH patients and its isogenic controls were used. iPSCs were differentiated into ventricular iPSC-CMs and stretched 10% equiaxially for 24h at 1Hz on the Flexcell FX-6000 system. Natriuretic peptides gene expressions were quantified via RT-PCR. NTproBNP and MRproANP release were measured on cell supernatants throughout specific immunoassays. Normality of the data was checked, and statistics were performed accordingly. Results: No differences were observed on natriuretic peptides gene expression on static and stretched iPSC-CMs. There is a clear correlation between NTproBNP and MRproANP release (R 2 =0.83, p<0.0001, Figure 1A). Interestingly, hPAH iPSC-CMs showed higher baseline values for NTproBNP release compared to isogenic iPSC-CMs (p<0.01, Figure 1B). Furthermore, stretching resulted in increased NTproBNP and MRproANP secretion in both hPAH and isogenic iPSC-CMs (Figure 1B-C). Conclusions: Secretion of NTproBNP and MRproANP was closely correlated in iPSC-CMs. iPSC-CMs of hPAH patients with a BMPR2 mutation showed higher baseline values of NTproBNP. Increased secretion of NTproBNP and MRproANP upon mechanical stimulation was similar between hPAH and isogenic iPSC-CMs. Experiments in atrial iPSC-CMs are currently ongoing to test potential differences in natriuretic peptide secretion between ventricular and atrial cardiomyocytes as the right atrium is also under stretch in PAH.

Suppression of B-type natriuretic peptide gene expression in cardiomyocytes under anoxic conditions

Several cell biology studies have focused on the effects of hypoxic environments on cardiomyocytes. However, the effect of anoxic conditions on cardiomyocytes remains largely unexplored. In the present study, we investigated the direct effects of anoxia on B-type natriuretic peptide (BNP) gene expression in cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) were exposed to anoxia using an airtight chamber saturated with 95% N2/5% CO2. BNP mRNA levels in NRCM were substantially reduced after more than 8hours of anoxia exposure, whereas after reoxygenation, BNP gene expression levels recovered in a time-dependent manner and significantly increased after 24hours of reoxygenation. BNP mRNA levels suppressed under anoxic conditions were significantly increased by aldosterone-induced activation of sodium-proton exchanger 1 (NHE1), which was cancelled by an NHE1 inhibitor, suggesting that anoxia reduces BNP gene expression, at least in part, in an NHE1-dependent manner. In summary, we found that BNP gene expression in cardiomyocytes decreases under anoxic conditions, in contrast to previous research findings that BNP expression increases under hypoxic conditions. These findings reveal a new insight that, within a single heart tissue in various cardiovascular diseases, such as myocardial infarction, the biological responses of cardiomyocytes are fundamentally different in regions of anoxia and hypoxia.

Imatinib‑ and ponatinib‑mediated cardiotoxicity in zebrafish embryos and H9c2 cardiomyoblasts.

Tyrosine kinase inhibitors (TKIs) offer targeted therapy for cancers but can cause severe cardiotoxicities. Determining their dose‑dependent impact on cardiac function is required to optimize therapy and minimize adverse effects. The dose‑dependent cardiotoxic effects of two TKIs, imatinib and ponatinib, were assessed in vitro using H9c2 cardiomyoblasts and in vivo using zebrafish embryos. In vitro, H9c2 cardiomyocyte viability, apoptosis, size, and surface area were evaluated to assess the impact on cellular health. In vivo, zebrafish embryos were analyzed for heart rate, blood flow velocity, and morphological malformations to determine functional and structural changes. Additionally, reverse transcription‑quantitative PCR (RT‑qPCR) was employed to measure the gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), established markers of cardiac injury. This comprehensive approach, utilizing both in vitro and in vivo models alongside functional and molecular analyses, provides a robust assessment of the potential cardiotoxic effects. TKI exposure decreased viability and surface area in H9c2 cells in a dose‑dependent manner. Similarly, zebrafish embryos exposed to TKIs exhibited dose‑dependent heart malformation. Both TKIs upregulated ANP and BNP expression, indicating heart injury. The present study demonstrated dose‑dependent cardiotoxic effects of imatinib and ponatinib in H9c2 cells and zebrafish models. These findings emphasize the importance of tailoring TKI dosage to minimize cardiac risks while maintaining therapeutic efficacy. Future research should explore the underlying mechanisms and potential mitigation strategies of TKI‑induced cardiotoxicities.

Endurance Exercise Prevented Diabetic Cardiomyopathy through the Inhibition of Fibrosis and Hypertrophy in Rats.

Exercise training could be essential in preventing pathological cardiac remodeling in diabetes. Therefore, the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) singly or plus metformin on diabetes-induced cardiomyopathy were investigated in this study. Forty-nine Wistar rats (male) were recruited. Seven groups of animals were treated for six weeks as control, diabetes, MICT (15 m/min, 40 min/day), HIIT (20 m/min, 40 min/day), metformin (300 mg/kg), HIIT+metformin (Met-HIIT), and MICT+metformin (Met-MICT). The metformin was orally administered with an intragastrical needle, and the exercised rats were trained (5 days/week) with a motorized treadmill. Metabolic parameters, echocardiographic indices, histopathology evaluation, and assessment of gene expression connected with cardiac fibrosis, hypertrophy, mitochondrial performance, and intracellular calcium homeostasis were investigated. Our results demonstrated that all the interventions prevented weight loss and enhanced heart weight/body weight ratio and fasting plasma glucose in diabetic rats. Both types of exercise and their metformin combinations improved diabetic animals' echocardiography indices by enhancing heart rate, fractional shortening (FS), ejection fraction (EF) and reducing end-systolic and end-diastolic diameter of left ventricular (LVESD and LVEDD). Gene expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), transforming growth factor (TGF)- , and collagen increased in the diabetes group. In contrast, the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 ), AMP-activated protein kinase (AMPK), ryanodine receptors (RyR), and ATPase pump of the sarcoplasmic reticulum (SERCA) was reduced in diabetic animals. Exercise training alone or in combination with metformin reversed these changes. Moreover, diabetes-induced cardiac fibrosis was ameliorated in treated groups. All indicators of diabetic cardiomyopathy were improved more in the Met-HIIT group than in other groups. Exercise training, notably with metformin combination, alleviated diabetes-induced cardiac complications. The beneficial effects of exercise could be related to improving pathological cardiac remodeling and enhancing cardiac function.

Angiotensin-(1-7) Treatment Early in Life Prevents Cardiac Hypertrophy in Adult Hypertensive Rats.

Angiotensin (Ang)-(1-7) is a cardioprotective peptide of the renin-angiotensin system. Prepuberty has been considered as a later susceptible window of development, and stressful factors in this life phase can induce chronic diseases in adulthood. We aimed to investigate whether the treatment with Ang-(1-7) during the prepuberty could attenuate the development of hypertension and cardiac injury in adult spontaneously hypertensive rats (SHRs). SHRs were treated with Ang-(1-7) (24 μg/kg/h) from age 4 to 7 weeks. Systolic blood pressure was measured by tail-cuff plethysmography up to 17th week. Thereafter, echocardiography was performed, and the rats were euthanized for the collection of tissues and blood. Ang-(1-7) did not change the systolic blood pressure but reduced the septal and posterior wall thickness, and cardiomyocyte hypertrophy and fibrosis in SHR. In addition, Ang-(1-7) reduced the gene expression of atrial natriuretic peptide and brain natriuretic peptide, increased the metalloproteinase 9 expression, and reduced the extracellular signal-regulated kinases 1/2 phosphorylation. Ang-(1-7) also prevented the reduction of Mas receptor but did not change the protein expression of angiotensin-converting enzyme, angiotensin-converting enzyme 2, AT1, and AT2. The treatment with Ang-(1-7) decreased the malondialdehyde (MDA) levels and increased superoxide dismutase-1 and catalase activities and protein expression of catalase. Our findings demonstrate that the treatment of SHR with Ang-(1-7) for 3 weeks early in life promotes beneficial effects in the heart later in life, even without altering blood pressure, through mechanisms involving the reduction of oxidative stress and ERK1/2 phosphorylation. In addition, this study supports the prepuberty as an important programming window.

Comparison of Protective Effect of Tri-circulator and Coenzyme Q10 on Myocardial Injury and the Mechanism Study by Zebrafish Model.

Tri-Circulator (TC) is a product comprising coenzyme Q10 (CoQ10), Salvia miltiorrhiza, and Panax notoginseng. Individually, each of these constituents has demonstrated protective effects on myocardial injury. The purpose of this study is to evaluate the protective efficacy of TC on heart function and compare the differential effects between CoQ10 and TC. Two myocardial injury models of zebrafish, the hypoxia-reoxygenation model (H/R) and the isoproterenol (ISO, a β-receptor agonist) model, were used in this experiment. The zebrafish subjects were divided into 4 groups: control, H/R, TC, and CoQ10. Heart rate, stroke volume (SV), cardiac output (CO), ejection fraction (EF), fractional area change (FAC), and pericardial height were monitored to assess changes in heart function. The gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was studied as markers of injury/stress. TC significantly suppresses elevated heart rate induced by H/R and prevents the decrease of heart rate induced by ISO. It alleviates the pericardial infusion induced by ISO, whereas CoQ10 does not possess a similar effect. Both TC and CoQ10 significantly inhibit the decline in SV, CO, EF, and FAC induced by H/R and ISO, and suppress the expression of ANP and BNP in cardiomyocytes induced by ISO. It is noteworthy that TC demonstrates a more pronounced effect on EF, FAC, ANP, and BNP gene expression compared to CoQ10. Both TC and CoQ10 have a protective effect on myocardial injury of zebrafish. However, TC exhibits a greater efficacy compared to CoQ10 alone in mitigating myocardial injury.

Melanocortin 1 receptor deficiency protects against pathological cardiac hypertrophy

Abstract Background The melanocortin system, including adrenocorticotropic hormone, melanocyte-stimulating hormones and five melanocortin receptors (MC-R), regulates important physiological functions such as pigmentation and energy homeostasis. It has been also recently implicated in the regulation of pathological cardiac remodeling but the role of different MC-R subtypes in the heart remain unknown. Purpose Based on the abundant cardiac expression of MC1-R, we hypothesized that it is functionally active in the heart and regulates pathological cardiac remodeling. Methods Rat H9c2 cells were used to study the effects of the selective MC1-R agonist LD211 on intracellular signaling cascades, gene expression and cellular growth response by using Western blotting, quantitative RT-PCR and 3H-leucine incorporation assay, respectively. Recessive yellow mice (Mc1re/e) were used as a model of global MC1-R deficiency. Inducible cardiomyocyte-specific MC1-R knockout mouse model (Mc1r-cKO) was engineered by intercrossing MC1-R floxed mice (Mc1rfl/fl) with tamoxifen-inducible Myh6-MerCreMer transgenic mice (Myh6-MCM). Mc1re/e and Mc1r-cKO mice were subjected to transverse aortic constriction (TAC, 8 wks) or subcutaneous infusion of angiotensin II (Ang II, 4 wks) to induce pathological cardiac hypertrophy. Cardiac structure and function were measured by echocardiography and by histological examination of H&E-stained heart sections. Results Treatment of H9c2 cells with LD211 increased MAP-kinase p-38 phosphorylation (+53 %, P=0.005 vs control), upregulated the expression of B-type natriuretic peptide (Nppb, +40 %, P=0.03 vs control) and increased 3H-leucine incorporation (+27 %, P=0.006 vs control). Mc1re/e mice showed reduction in ventricular weight (-29 mg, P=0.03) compared to WT mice after TAC operation, and reduced left ventricular (LV) expression of atrial natriuretic peptide (Nppa) and fibrosis-related genes (Ctgf and Mmp2). In terms of LV systolic performance, TAC-operated Mc1re/e mice showed tendency towards improved LV ejection fraction (EF, +6 %, P=0.06) compared to WT mice. To further investigate whether the phenotype of Mc1re/e mice is driven by deficient MC1-R signaling in cardiomyocytes, we characterized Mc1r-cKO mice, which recapitulated the phenotypic features of Mc1re/e mice. Specifically, TAC-operated Mc1r-cKO mice displayed reduced ventricular weight (-38 mg, P=0.007) and downregulation of LV expression of Nppa and fibrosis-related genes compared to Myh6-MCM mice. Likewise, in a model of Ang II-induced cardiac hypertrophy, Mc1r-cKO mice showed reduced ventricular weight (-10 mg, P=0.015) compared to Myh6-MCM mice, indicating that the protection against pathological cardiac hypertrophy was model-independent. Conclusion Our studies demonstrate that MC1-R activation promotes cardiomyocyte growth in vitro, while global and cardiomyocyte-specific MC1-R deficiency protects against pathological cardiac hypertrophy induced by TAC or Ang II infusion.

Abstract 9486: miR-425/CFL2 Regulates Natriuretic Peptides Expression in Human CVD

Introduction: Previously, miR-425 have been implicated in the inhibition of the cardiac natriuretic peptide (NP) ANP gene expression with their role on BNP undefined. The mechanism by which miR-425 modulates NP expression remains to be explored. In heart failure (HF), adrenergic and neurohormone pathways are aberrantly activated. Our goal was to investigate the role of miR-425 and its downstream target Cofilin 2 (CFL2) on ANP and BNP expression in human CV disease (CVD). Hypothesis: We hypothesized miR-425, via CFL2, supresses NPs expression in vitro. MiR-425, CFL2 and NP levels were also determined in CVD patients. Methods: In vitro, human cardiomyocytes (HCM) were treated with vehicle (Veh), catecholamine isoproterenol (ISO) and cytokine endothelin (ET), miR-425 mimic. Cells were transfected with CFL2 plasmid or CFL2 siRNA. Normal (NM, n=100), Hypertension (HTN, n=30) and Acute HF (AHF, n=74) cohorts were studied. A confirmatory cohort was recruited. Plasma NT-proBNP, miR-425 were measured. In left ventricles (LV) from NM or HF (n=5 each), NP, CFL2 and miR-425 expression were assessed. Data expressed as means±SEM. * p<0.05 vs NM or Veh. Results: In vitro in HCM, treatment with ISO or ET in HCM reduced miR-425 and elevated NP mRNA levels. Furthermore, miR-425 mimic reduced Nppa (Veh: 1.0±0.03, mimic: 0.7±0.02*) and Nppb levels (Veh: 1.0±0.03, mimic: 0.5±0.01*). We showed that a top predicted target of miR-425, CFL2, was also reduced by the mimic. Further, NP mRNA was elevated by CFL2 overexpression and reduced by the knockdown of CFL2. In humans, MiR-425 was lower in HTN and AHF patients (NM: 0.15±0.03, HTN: 0.03±0.005*, HF: 0.01±0.003*) than NM. MiR-425 negatively correlates with NT-proBNP in NM and AHF cohorts. Downregulation of miR-425 was validated in a second AHF cohort. Similarly, in HF LV tissues, miR-425 (NM: 2.5±0.5, HF: 1.0±0.5*) was lower than NM group, while CFL2 and NP gene and protein expression were increased. Conclusion: In vitro, cardiac stress hormones downregulate miR-425 and increase NP gene expression, potentially through a novel target, CFL2. In CVD patients, miR-425 is lower, accompanied by an elevation of CFL2 and NP. Our findings implicate the pathway of stress hormones/miR-425/CFL2/NP production in CVD.

Supplementation with the Prebiotic High-Esterified Pectin Improves Blood Pressure and Cardiovascular Risk Biomarker Profile, Counteracting Metabolic Malprogramming.

Supplementation with the prebiotic pectin is associated with beneficial health effects. We aimed to characterize the cardioprotective actions of chronic high-esterified pectin (HEP) supplementation (10%) in a model of metabolic malprogramming in rats, prone to obesity and associated disorders: the progeny of mild calorie-restricted dams during the first half of pregnancy. Results show that pectin supplementation reverses metabolic malprogramming associated with gestational undernutrition. In this sense, HEP supplementation improved blood pressure, reduced heart lipid content, and regulated cardiac gene expression of atrial natriuretic peptide and lipid metabolism-related genes. Moreover, it caused an elevation in circulating levels of fibroblast growth factor 21 and a higher expression of its co-receptor β-klotho in the heart. Most effects are correlated with the gut levels of beneficial bacteria promoted by HEP. Therefore, chronic HEP supplementation shows cardioprotective actions, and hence, it is worth considering as a strategy to prevent programmed cardiometabolic alterations.

S1P lyase inhibition improves post-ischemic cardiac remodeling independently of infarct size via S1P receptor 1

Abstract Background Revascularization of the infarct vessel is the golden standard in acute myocardial infarction (AMI). Even in Western countries, more than ten percent of AMI patients are so-called “latecomers” and it is controversial if revascularization of the infarct vessel is beneficial in these patients. Current guidelines even discourage revascularization of the infarcted artery if symptom onset was >48 hours and the patient is asymptomatic. These patients are at high risk for cardiovascular events and heart failure with reduced ejection fraction (HFrEF). HFrEF has an enormous socio-economic impact, high morbidity, and mortality. Purpose Therapeutic options targeting post-ischemic cardiac remodeling are sparse. The bioactive sphingolipid sphingosine-1-phosphate (S1P) reduces ischemia/reperfusion injury when administered in advance. However, its impact on post-ischemic remodeling independently of its infarct size (IS)-reducing effect is yet unknown. Methods Acute myocardial infarction (AMI) in mice was induced by permanent ligation of the left anterior descending artery (LAD). C57Bl/6J were treated with the S1P lyase inhibitor 4-deoxypyridoxine (DOP) starting seven days prior to AMI to increase endogenous S1P concentrations. Cardiac function and myocardial healing were assessed by cardiovascular magnetic resonance imaging (cMRI), histomorphology and gene expression analysis. DOP effects were investigated in cardiomyocyte-specific S1P receptor 1 deficient (S1PR1 Cardio Cre+ and Cre− control) mice, and S1P concentrations measured by LC-MS/MS. Results S1P concentrations in plasma before induction of AMI were increased fourfold by DOP (Control 0.97±0.09μM [n=6] vs. DOP 3,80±0,09μM [n=6]). Scar size determined by MRI, as well as ejection fraction (EF), did not differ 24 h post AMI. In contrast, after 21 days, there was a clear difference between the two groups (scar size vehicle: 19.3±6.2% vs. DOP 13.4±5.7%; EF: Vehicle: 26.4±8.7% vs. DOP 38.2±11.8%). In addition, in the remote area 21 days post AMI in the DOP-treated animals, a reduced gene expression of brain natriuretic peptide, atrial natriuretic peptide and collagen 1a2. Finally, cardiomyocyte diameter in the remote myocardium was 21% smaller in DOP-treated (Vehicle: 21.95±1.59μm vs. DOP 17.35±0.77μm). The benefit of DOP-treatment was abolished in cardiomyocyte-specific S1PR1-deficient mice. Conclusion S1P improves cardiac function and myocardial healing post AMI independently of initial infarct size via S1PR1. Hence, in addition to its beneficial effects on I/R injury, S1PR1 may be a promising target in post-infarction myocardial remodeling as adjunctive therapy to revascularization and in patients who are not eligible for standard interventional procedures. Funding Acknowledgement Type of funding sources: None.

In Vivo Inhibition of miR-34a Modestly Limits Cardiac Enlargement and Fibrosis in a Mouse Model with Established Type 1 Diabetes-Induced Cardiomyopathy, but Does Not Improve Diastolic Function.

MicroRNA 34a (miR-34a) is elevated in the heart in a setting of cardiac stress or pathology, and we previously reported that inhibition of miR-34a in vivo provided protection in a setting of pressure overload-induced pathological cardiac hypertrophy and dilated cardiomyopathy. Prior work had also shown that circulating or cardiac miR-34a was elevated in a setting of diabetes. However, the therapeutic potential of inhibiting miR-34a in vivo in the diabetic heart had not been assessed. In the current study, type 1 diabetes was induced in adult male mice with 5 daily injections of streptozotocin (STZ). At 8 weeks post-STZ, when mice had established type 1 diabetes and diastolic dysfunction, mice were administered locked nucleic acid (LNA)-antimiR-34a or saline-control with an eight-week follow-up. Cardiac function, cardiac morphology, cardiac fibrosis, capillary density and gene expression were assessed. Diabetic mice presented with high blood glucose, elevated liver and kidney weights, diastolic dysfunction, mild cardiac enlargement, cardiac fibrosis and reduced myocardial capillary density. miR-34a was elevated in the heart of diabetic mice in comparison to non-diabetic mice. Inhibition of miR-34a had no significant effect on diastolic function or atrial enlargement, but had a mild effect on preventing an elevation in cardiac enlargement, fibrosis and ventricular gene expression of B-type natriuretic peptide (BNP) and the anti-angiogenic miRNA (miR-92a). A miR-34a target, vinculin, was inversely correlated with miR-34a expression, but other miR-34a targets were unchanged. In summary, inhibition of miR-34a provided limited protection in a mouse model with established type 1 diabetes-induced cardiomyopathy and failed to improve diastolic function. Given diabetes represents a systemic disorder with numerous miRNAs dysregulated in the diabetic heart, as well as other organs, strategies targeting multiple miRNAs and/or earlier intervention is likely to be required.

Substantial impact of 3-iodothyronamine (T1AM) on the regulations of fluorescent thermoprobe-measured cellular temperature and natriuretic peptide expression in cardiomyocytes

There is growing interest in 3-iodothyronamine (T1AM), an active thyroid hormone metabolite, that induces negative inotropic and chronotropic actions in the heart and exerts systemic hypothermic action. We explored the direct impact of T1AM on cardiomyocytes with a focus on the regulation of the intracellular temperature and natriuretic peptide (NP) expression. A thermoprobe was successfully introduced into neonatal rat cardiomyocytes, and the temperature-dependent changes in the fluorescence intensity ratio were measured using a fluorescence microscope. After one-hour incubation with T1AM, the degree of change in the fluorescence intensity ratio was significantly lower in T1AM-treated cardiomyocytes than in equivalent solvent-treated controls (P < 0.01), indicating the direct hypothermic action of T1AM on cardiomyocytes. Furthermore, T1AM treatment upregulated B-type NP (BNP) gene expression comparable to treatment with endothelin-1 or phenylephrine. Of note, ERK phosphorylation was markedly increased after T1AM treatment, and inhibition of ERK phosphorylation by an MEK inhibitor completely cancelled both T1AM-induced decrease in thermoprobe-measured temperature and the increase in BNP expression. In summary, T1AM decreases fluorescent thermoprobe-measured temperatures (estimated intracellular temperatures) and increases BNP expression in cardiomyocytes by activating the MEK/ERK pathway. The present findings provide new insight into the direct myocardial cellular actions of T1AM in patients with severe heart failure.

B-Type Natriuretic Peptide (BNP) Revisited—Is BNP Still a Biomarker for Heart Failure in the Angiotensin Receptor/Neprilysin Inhibitor Era?

Simple SummaryActive BNP-32, less active proBNP-108, and inactive N-terminal proBNP-76 all circulate in the blood. The circulating protease neprilysin has lower substrate specificity for BNP than ANP, while proBNP and N-terminal proBNP are not degraded by neprilysin. Currently available BNP immunoassays react with both mature BNP and proBNP; therefore, measured plasma BNP is mature BNP + proBNP. Because ARNI administration increases mature BNP, measured plasma BNP initially increases with ARNI administration by the amount of the increase in mature BNP. Later, ARNI administration reduces myocardial wall stress, and the resultant reduction in BNP production more than offsets the increase of mature BNP due to inhibition of degradation by neprilysin, resulting in lower plasma BNP levels. In the ARNI era, BNP remains a useful biomarker for heart failure, though mild increases early during ARNI administration should be taken into consideration.Myocardial wall stress, cytokines, hormones, and ischemia all stimulate B-type (or brain) natriuretic peptide (BNP) gene expression. Within the myocardium, ProBNP-108, a BNP precursor, undergoes glycosylation, after which a portion is cleaved by furin into mature BNP-32 and N-terminal proBNP-76, depending on the glycosylation status. As a result, active BNP, less active proBNP, and inactive N-terminal proBNP all circulate in the blood. There are three major pathways for BNP clearance: (1) cellular internalization via natriuretic peptide receptor (NPR)-A and NPR-C; (2) degradation by proteases in the blood, including neprilysin, dipeptidyl-peptidase-IV, insulin degrading enzyme, etc.; and (3) excretion in the urine. Because neprilysin has lower substrate specificity for BNP than atrial natriuretic peptide (ANP), the increase in plasma BNP after angiotensin receptor neprilysin inhibitor (ARNI) administration is much smaller than the increase in plasma ANP. Currently available BNP immunoassays react with both mature BNP and proBNP. Therefore, BNP measured with an immunoassay is mature BNP + proBNP. ARNI administration increases mature BNP but not proBNP, as the latter is not degraded by neprilysin. Consequently, measured plasma BNP initially increases with ARNI administration by the amount of the increase in mature BNP. Later, ARNI reduces myocardial wall stress, and the resultant reduction in BNP production more than offsets the increase in mature BNP mediated by inhibiting degradation by neprilysin, which lowers plasma BNP levels. These results suggest that even in the ARNI era, BNP can be used for diagnosis and assessment of the pathophysiology and prognosis of heart failure, though the mild increases early during ARNI administration should be taken into consideration.

Smooth Muscle‐Specific Deletion of Neuropilin‐1 Increases Vascular Contractility and Blood Pressure

IntroductionNeuropilin‐1 (NRP1) is a transmembrane glycoprotein that acts as a co‐receptor for the Class 3 Semaphorin (SEMA) ligand SEMA3A and mediates the inhibition of RhoA signaling. NRP1 is expressed in vascular smooth muscle cells (VSMCs), however, its role in regulating VSMC contractility and blood pressure in vivo is unknown.HypothesisWe hypothesize that SEMA3A‐NRP1 signaling is essential in mitigating RhoA activity in VSMCs and that loss of NRP1 increases vascular tone and blood pressure in vivo.MethodsTo study the role of NRP1 in SMCs, mice with inducible, smooth muscle cell‐specific deletion of NRP1 (SM22a‐CreERT2; Nrp1flox/flox) were generated. Following recombination using 4‐hydroxy tamoxifen (SM‐NRP1 KO) in male and female adult mice (8‐12 weeks), systolic blood pressure (SBP) was measured using a tail cuff and compared to age‐ and sex‐matched mice that did not receive tamoxifen (control). Vascular reactivity to various contractile agonists was measured using ex vivo tension myography.ResultsSBP was significantly increased in SM‐NRP1 KO mice following recombination compared to control mice (SBP: 136.5 ± 10.9 vs 112.9 ± 5.6 mmHg; p=0.0006). Aortas of SM‐NRP1 KO mice displayed significantly enhanced contractile response to phenylephrine, KCl, and the thromboxane agonist U44619, and reduced relaxation to the endothelium‐independent vasodilator sodium nitroprusside. In vitro, treatment of murine primary aortic VSMC with SEMA3A decreased angiotensin II‐induced Rho‐GTP activation. Gene expression of Sema3a in the aorta was downregulated in two models of hypertension, angiotensin II‐dependent and genetic model of hypertension, as identified by qPCR and transcriptomic analysis respectively, further supporting the role of SEMA3A‐NRP1 signaling in basal blood pressure regulation. Additionally, control and SM‐NRP1 KO mice (starting at 2 weeks post‐recombination) were administered angiotensin II (490 ng/kg/day) for 4 weeks. While there was no significant difference in SBP at weeks 1 and 2, SM‐NRP1 KO mice had significantly lower SBP at weeks 3 and 4 following angiotensin II infusion compared to controls (Week 4 SBP: 150 ± 1.4 vs 130.5 ± 2.5 mmHg; p=0.02), suggesting a low ejection fraction and cardiac dysfunction in these mice. In support of this observation, gene expression of atrial natriuretic peptide was increased (p=0.06) in hearts of angiotensin II‐infused SM‐NRP1 KO mice. Preliminary analyses indicated an increase in aortic medial thickness at baseline and an increase in aortic adventitial collagen following angiotensin II infusion in SM‐NRP1 KO mice compared to controls.ConclusionTogether, our data point to the role of NRP1 as a novel regulator of basal vascular tone and blood pressure, and the loss of NRP1 leads to the onset of hypertension and exacerbates pathological vascular remodeling and cardiac dysfunction.

Relationship Between Circulating MicroRNAs and Left Ventricular Hypertrophy in Hypertensive Patients.

ObjectiveLeft ventricular hypertrophy (LVH) is a common complication of hypertension and microRNAs (miRNAs) are considered to play an important role in cardiac hypertrophy development. This study evaluated the relationship between circulating miRNAs and LVH in hypertensive patients.MethodsTwo cohorts [exploratory (n = 42) and validation (n = 297)] of hypertensive patients were evaluated by clinical, laboratory and echocardiography analysis. The serum expression of 754 miRNAs in the exploratory cohort and 6 miRNAs in the validation cohort was evaluated by the TaqMan OpenArray® system and quantitative polymerase chain reaction, respectively.ResultsAmong the 754 analyzed miRNAs, ten miRNAs (miR-30a-5p, miR-let7c, miR-92a, miR-451, miR-145-5p, miR-185, miR-338, miR-296, miR-375, and miR-10) had differential expression between individuals with and without LVH in the exploratory cohort. Results of multivariable regression analysis adjusted for confounding variables showed that three miRNAs (miR-145-5p, miR-451, and miR-let7c) were independently associated with LVH and left ventricular mass index in the validation cohort. Functional enrichment analysis demonstrated that these three miRNAs can regulate various genes and pathways related to cardiac remodeling. Furthermore, in vitro experiments using cardiac myocytes demonstrated that miR-145-5p mimic transfection up-regulated the expression of brain and atrial natriuretic peptide genes, which are markers of cardiac hypertrophy, while anti-miR-145-5p transfection abrogated the expression of these genes in response to norepinephrine stimulus.ConclusionsOur data demonstrated that circulating levels of several miRNAs, in particular miR-145-5p, miR-451, and let7c, were associated with LVH in hypertensive patients, indicating that these miRNAS may be potential circulating biomarkers or involved in hypertension-induced LV remodeling.

Cardiomyopathy Associated with Worsening Intravascular Hemolysis in Transgenic Knock-in Mouse Model of Sickle Cell Disease

Cardiopulmonary complications are the leading cause of death in sickle cell disease (SCD). Cardiac complications, including left ventricular (LV) dysfunction, are prevalent in both young and adult SCD patients. Our studies in the Townes transgenic murine model indicate mice with homozygous SCD (SS) recapitulate the age-related clinical deterioration seen in humans. This is evidenced by the sudden decline in survival of SS mice during the transition from adolescence to adulthood (between the ages of three to six months); we have previously determined that the severity of intravascular hemolysis increases during this transition period in SS mice (Ghosh et al JCI Insight 2016; 1(4): e81090). Hitherto, the cardiac phenotype of this murine model of SCD, which appears the most versatile, has not been defined. In this study, we imaged the hearts of one, three, and six month old Townes SS mice and littermates with sickle cell trait (AS mice) using high resolution ultrasound (Vevo 2100, VisualSonics). Standard indices of LV systolic function, size, and mass, were derived from measurements made on short axis B-mode images of the heart. Ejection fraction and fractional shortening were similar for AS and SS mice, signifying preservation of systolic function in both groups up to 6 months of age. The LV mass, LV internal dimensions (LVID), systolic and diastolic volumes as well as stroke volume significantly increased with age in the SS but not in the AS. Major differences in the cardiac phenotype in SS compared to AS littermates were observed only at six-months. At six months, the SS mice had larger hearts ( 236.5 ± 20.1 mg, n=7, p=0.005) compared to AS littermates (180.2 ± 9.6 mg, n=6). The LVID in both systole (3.1 ± 0.2 mm versus2.1± 0.3, p=0.03) and diastole (4.7 ± 0.2 mmversus 3.9 ± 0.1 mm, p=0.002) were larger in the SS mice, accompanied by higher LV Mass Index (4.6 ± 0.2, n=7 versus 3.1 ± 0.3, n= 6 , p=0.03) indicating LV hypertrophy in the SS mice. RT-PCR analysis of hypertrophy markers in the LV revealed increased expression of Atrial Natriuretic Peptide (ANP, p=0.001) and β-Myosin Heavy Chain (p=0.03) genes in the SS mice compared to AS mice. In agreement with findings in humans, at 6 months, systolic volume (38.4± 6.6 µ L, n=7,versus 17.2±5.3 µ L, n=6, p=0.003) , diastolic volume (101.3± 8.4 µ L, n=7, versus 62.3 ± 4.6 µ L , n=6, p=0.0002), stroke Index (2.2± 0.2, n=7, versus 1.5±0.2, n=6, p=0.03)and cardiac index (1.0±0.1, n=7, versus 0.7±0.1, n=6, p=0.047)were all higher in the six-month SS mice compared to AS littermates. To evaluate vascular inflammation in the heart, P-selectin was measured by ELISA; Compared to AS littermates, SS mice hearts showed a 2-fold higher expression of P-selectin (p=0.009). The severity of anemia and intravascular hemolysis increased with aging in the SS mice. Our study shows progression of LV enlargement and dilation in the Townes SS mice concomitant with increased severity of intravascular hemolysis with aging. Future studies are aimed at delineating the specific molecules and cognate pathways downstream of intravascular hemolysis that promote cardiomyopathy in SCD. DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties.

Cardiac Gq Receptors and Calcineurin Activation Are Not Required for the Hypertrophic Response to Mechanical Left Ventricular Pressure Overload.

RationaleGq-coupled receptors are thought to play a critical role in the induction of left ventricular hypertrophy (LVH) secondary to pressure overload, although mechano-sensitive channel activation by a variety of mechanisms has also been proposed, and the relative importance of calcineurin- and calmodulin kinase II (CaMKII)-dependent hypertrophic pathways remains controversial.ObjectiveTo determine the mechanisms regulating the induction of LVH in response to mechanical pressure overload.Methods and ResultsTransgenic mice with cardiac-targeted inhibition of Gq-coupled receptors (GqI mice) and their non-transgenic littermates (NTL) were subjected to neurohumoral stimulation (continuous, subcutaneous angiotensin II (AngII) infusion for 14 days) or mechanical pressure overload (transverse aortic arch constriction (TAC) for 21 days) to induce LVH. Candidate signaling pathway activation was examined. As expected, LVH observed in NTL mice with AngII infusion was attenuated in heterozygous (GqI+/–) mice and absent in homozygous (GqI–/–) mice. In contrast, LVH due to TAC was unaltered by either heterozygous or homozygous Gq inhibition. Gene expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and α-skeletal actin (α-SA) was increased 48 h after AngII infusion or TAC in NTL mice; in GqI mice, the increases in ANP, BNP and α-SA in response to AngII were completely absent, as expected, but all three increased after TAC. Increased nuclear translocation of nuclear factor of activated T-cells c4 (NFATc4), indicating calcineurin pathway activation, occurred in NTL mice with AngII infusion but not TAC, and was prevented in GqI mice infused with AngII. Nuclear and cytoplasmic CaMKIIδ levels increased in both NTL and GqI mice after TAC but not AngII infusion, with increased cytoplasmic phospho- and total histone deacetylase 4 (HDAC4) and increased nuclear myocyte enhancer factor 2 (MEF2) levels.ConclusionCardiac Gq receptors and calcineurin activation are required for neurohumorally mediated LVH but not for LVH induced by mechanical pressure overload (TAC). Rather, TAC-induced LVH is associated with activation of the CaMKII-HDAC4-MEF2 pathway.

Role of expression of atrial natriuretic peptide gene in essential hypertension among Egyptian patients

ABSTRACT Introduction Essential Hypertension has been a great burden on public health services for a long time, with many life-threatening complications. Therefore, we decided to study Atrial Natriuretic peptide (ANP) gene expression as one of the most important blood pressure controlling genes, in order to use ANP gene as a potential diagnostic or therapeutic marker in the near future. Methods One hundred essential hypertensive patients and 100 normotensive controls were included. Study Subjects were subjected to ANP gene expression analysis, together with blood pressure measurement, Lab investigations, and BMI analysis. Results There was a statistical difference between ANP gene expression and blood pressure, with lower ANP gene expression level (median of 0.3) being present among hypertensive patients and higher ANP gene expression level (median of 1.6) among normotensive controls (p < 0.001). Discussion We proved that ANP gene expression to be low in essential hypertension patients compared with normotensive individuals.

Abstract 17325: Obesity, Blood Pressure, and Diurnal Variation in Natriuretic Peptides: A Clinical Trial

Background: Obese individuals have disturbed blood pressure (BP) rhythmicity and relative natriuretic peptide (NP) deficiency. The relationship of diurnal variation in NP levels and 24h BP rhythm is not known. Furthermore, mechanisms behind difference in circulating NPs in healthy obese and lean individuals has not been explored. We conducted a prospective clinical trial to evaluate 1) the diurnal rhythmicity of NPs and its relationship with 24-hour BP rhythm between healthy lean and obese individuals, and 2) elucidate mechanism behind NP deficiency in obese. Methods: Healthy, normotensive, lean (BMI:18.5-25 kg/m 2 ) and obese (BMI:30-45 kg/m 2 ) individuals aged 18-40 years, underwent 24-hour standardized inpatient protocol involving ambulatory BP monitoring (ABPM), controlled light intensity, and 10 blood draws, following 5-days of standardized diet. NP gene expression was evaluated in a cohort of 37 healthy donor heart tissues obtained from the MAGNet repository. Results: Among 52 participants screened, a total of 40 participants (18 lean; 22 obese) were enrolled. Diurnal variation in MRproANP levels was seen in both lean and obese individuals (p for rhythmicity=0.001). The mesor of the NP rhythm was 15% (8.5-21.6%) lower in obese. The diurnal variation in MRproANP was in antiphase with diurnal variation of systolic BP (p&lt;0.001) ( Figure ). Obese participants had lower 24h renin levels (p=0.06) and higher nocturnal sodium excretion (p=0.08). Among obese, there was lower expression of NP production genes ( NPPA, NPPB) (p&lt;0.05) , and higher expression of clearance gene ( NPR3 ) (p&lt;0.001) in heart tissue. Conclusions: In a mechanistic human trial, we elucidate key neurohormonal differences in rhythm and evidence of poor salt handling in obese. Decreased production and increased clearance may contribute to the NP deficiency in obese. Targeting the diurnal NP-BP rhythm axis may reduce the cardiovascular risk burden, specifically in obese individuals.

Natriuretic Peptides and MicroRNAs 155 And 425 in Human Heart Failure: Biomarker Roles and Pathophysiologic Regulation

Introduction MicroRNAs (miRNAs) have recently emerged as important molecular entities in heart failure (HF) pathophysiology and diagnosis. To date, miR-155 and miR-425 have been implicated in the inhibition of the cardiac natriuretic peptide (NP) ANP gene with their role on BNP undefined. In HF, adrenergic and neurohormonal pathways are dysregulated. Our goal was to advance our understanding of the regulation of these two miRNAs and their predictive value in human HF. Hypothesis We hypothesized that miR-155 and -425 levels are reduced in HF compared to normal subjects. We also investigated the molecular mechanisms of NPs expression regulated by the miR-425 and -155. Methods Normal (healthy, NM, n=100) and Acute HF (AHF, n=74) cohorts were studied. Two confirmatory cohorts were also recruited: NM (n=20) and AHF (n=28). Plasma NT-proBNP was measured. Plasma miR-155 and -425 expression were quantified with RT-PCR. In left ventricular myocardium (LV) from NM or HF (n=5 each), Nppa (ANP gene), Nppb (BNP gene), miR-155, and -425 expressions were measured. In vitro, human cardiomyocytes (HCM) were treated with vehicle (Veh), 10-4 M catecholamine isoproterenol (ISO) and 10-6 M cytokine endothelin (ET) which are known to be activated in HF, mimics of miR-155 or -425, followed by gene expression assessment. Data expressed as means±SEM. * p Results MiR-155 and miR-425 were significantly lower in AHF patients (miR-155: NM: 1.8±0.29, HF: 0.13±0.02*), (miR-425: NM: 0.15±0.03, HF: 0.01±0.003*) than NM. The area under the curve (AUC) of the two miRNAs are 0.86 and 0.87 in AHF cohorts respectively (vs NT-proBNP 0.98). MiR-425 negatively correlates with NT-proBNP in AHF cohorts (r=-0.33*). Downregulation of both miRNAs were validated in a second AHF cohort. Similarly, in HF LV tissues, HF displayed lower miR-155 and miR-425 levels than NM group, while NP gene and protein expression were increased. In vitro, treatment with ISO or ET in HCM reduced miR-155, miR-425 levels and significantly elevated Nppa and Nppb levels. Furthermore in HCM, treatment with mimic of miR-425 reduced Nppa (Veh: 1.0±0.03, miR-425 mimic: 0.7±0.02*) and Nppb levels (Veh: 1.0±0.03, miR-425 mimic: 0.5±0.01*). We also demonstrated that apoptosis signal-regulating kinase 1 (ASK1) and Cofilin 2, which have been reported to enhance Nppa and Nppb expression, was also reduced by miR-425. Conclusion MiR-155 and miR-425 are reduced in HF, which may be one of the mechanisms contributing to an increase in circulating ANP and BNP. MiR-425 also displays a negative correlation with NT-proBNP. In vitro, cardiac stress hormones known to be activated in HF downregulate these 2 miRNAs and increase NP gene expression which provides further insight into mechanisms of NP activation in HF. The findings for the first time implicate the pathway of stress hormones/miR-425/NP production in human HF simulated in vitro and further our understanding of NP activation in HF. These studies also support biomarker value for miR-155 and miR-425 and the compensatory nature of this decline in HF.