B-type Natriuretic Peptide Expression Research Articles

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.

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.

Downregulation of lncRNA MALAT1 Inhibits Angiotensin II-induced Hypertrophic Effects of Cardiomyocytes by Regulating SIRT4 via miR-93-5p.

Cardiac hypertrophy is a leading risk for heart failure and sudden death. Long non-coding RNAs (lncRNAs) have been implicated in a variety of human diseases, including cardiac hypertrophy. We aimed to investigate the potential role and functional mechanism of lncRNA metastasis-associated in lung adenocarcinoma transcript 1 (MALAT1) in cardiac hypertrophy. C57BL/6 mice underwent transverse aortic constriction (TAC) to induce cardiac hypertrophy in vivo. The expression of MALAT1, miR-93-5p, and sirtuin 4 (SIRT4) mRNA was detected using a quantitative real-time polymerase chain reaction. The protein levels of cardiac hypertrophy-related markers, including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), and SIRT4 were measured via western blotting. The putative interaction between miR-93-5p and MALAT1 or SIRT4 was verified using a dual-luciferase reporter assay, RNA immunoprecipitation assay, or pull-down assay. Consequently, the expression of MALAT1 and SIRT4 was increased in TAC-treated mouse heart and angiotensin II (Ang-II)-induced cardiomyocytes, whereas the expression of miR-93-5p was decreased. Ang-II promoted the expression of ANP, BNP, and β-MHC and the surface area of cardiomyocytes, whereas MALAT1 downregulation impaired their expression and cell area. MiR-93-5p was a target of MALAT1, and its inhibition reversed the effects of MALAT1 downregulation. More importantly, MALAT1 modulated SIRT4 expression by degrading miR-93-5p. The expression of ANP, BNP, and β-MHC suppressed by miR-93-5p restoration was recovered by SIRT4 promotion. Overall, MALAT1 knockdown ameliorated cardiac hypertrophy partly by regulating the miR-93-5p/SIRT4 network, indicating that MALAT1 was a substantial indicator of cardiac hypertrophy.

Catalase Mediates the Inhibitory Actions of PPARδ against Angiotensin II-Triggered Hypertrophy in H9c2 Cardiomyocytes.

Hypertrophy of myocytes has been implicated in cardiac dysfunctions affecting wall stress and patterns of gene expression. However, molecular targets potentially preventing cardiac hypertrophy have not been fully elucidated. In the present study, we demonstrate that upregulation of catalase by peroxisome proliferator-activated receptor δ (PPARδ) is involved in the anti-hypertrophic activity of PPARδ in angiotensin II (Ang II)-treated H9c2 cardiomyocytes. Activation of PPARδ by a specific ligand GW501516 significantly inhibited Ang II-induced hypertrophy and the generation of reactive oxygen species (ROS) in H9c2 cardiomyocytes. These effects of GW501516 were almost completely abolished in cells stably expressing small hairpin (sh)RNA targeting PPARδ, indicating that PPARδ mediates these effects. Significant concentration and time-dependent increases in catalase at both mRNA and protein levels were observed in GW501516-treated H9c2 cardiomyocytes. In addition, GW501516-activated PPARδ significantly enhanced catalase promoter activity and protein expression, even in the presence of Ang II. GW501516-activated PPARδ also inhibited the expression of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), which are both marker proteins for hypertrophy. The effects of GW501516 on the expression of ANP and BNP were reversed by 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor. Inhibition or downregulation of catalase by 3-AT or small interfering (si)RNA, respectively, abrogated the effects of PPARδ on Ang II-induced hypertrophy and ROS generation, indicating that these effects of PPARδ are mediated through catalase induction. Furthermore, GW501516-activated PPARδ exerted catalase-dependent inhibitory effects on Ang II-induced hypertrophy by blocking p38 mitogen-activated protein kinase. Taken together, these results indicate that the anti-hypertrophic activity of PPARδ may be achieved, at least in part, by sequestering ROS through fine-tuning the expression of catalase in cardiomyocytes.

Apelin-13 regulates angiotensin ii-induced Cx43 downregulation and autophagy via the AMPK/mTOR signaling pathway in HL-1 cells.

Atrial fibrillation is associated with atrial remodeling, in which connexin 43 (Cx43) and cell hypertrophy play important roles. In this study, apelin-13, an aliphatic peptide, was used to explore the protective effects of the adenosine monophosphate-activated protein kinase (AMPK)/mTOR signaling pathway on Cx43 expression and autophagy, using murine atrial HL-1 cells. The expression of Cx43, AMPK, B-type natriuretic peptide (BNP) and pathway-related proteins was detected by Western blot analysis. Cellular fluorescence imaging was used to visualize Cx43 distribution and the cytoskeleton. Our results showed that the Cx43 expression was significantly decreased in HL-1 cells treated with angiotensin II but increased in cells additionally treated with apelin-13. Meanwhile, apelin-13 decreased BNP expression and increased AMPK expression. However, the expression of Cx43 and LC3 increased by apelin-13 was inhibited by treatment with compound C, an AMPK inhibitor. In addition, rapamycin, an mTOR inhibitor, promoted the development of autophagy, further inhibited the protective effect on Cx43 expression and increased cell hypertrophy. Thus, apelin-13 enhances Cx43 expression and autophagy via the AMPK/mTOR signaling pathway, and serving as a potential therapeutic target for atrial fibrillation.

Associations of B-Type Natriuretic Peptide and Its Coding Gene Promoter Methylation With Functional Outcome of Acute Ischemic Stroke: A Mediation Analysis.

BackgroundThe prognostic role of B‐type natriuretic peptide (BNP) in stroke has been suggested, but limited studies have shown mixed results and unknown underlying mechanisms. DNA methylation, a molecular modification that alters gene expression, may represent a candidate mechanism for this purpose. We aimed to examine the associations of BNP and methylation of its coding gene (natriuretic peptide B [NPPB]) with the functional outcome in a large sample of patients with acute ischemic stroke from CATIS (China Antihypertensive Trial in Acute Ischemic Stroke).Methods and ResultsLeveraging participants from CATIS with available specimens, serum proBNP (equimolarly produced with BNP) was measured in 3216 patients (mean age, 62 years; 64% men), and peripheral blood DNA methylation of the NPPB promoter was quantified by targeted bisulfite sequencing in 806 patients (mean age, 62 years; 54% men). The functional outcome was defined as an ordered modified Rankin Scale score assessed at 14 days or hospital discharge after stroke onset. Mediation analysis was conducted to test the potential mediating effect of proBNP on the relationship between NPPB methylation and functional outcome. The results showed that a higher level of proBNP was significantly associated with a higher risk of having a poorer functional outcome (odds ratio [OR], 1.14; P=0.006). Every 5% of hypermethylation at 2 (Chr1:11919160 [OR, 0.93; P=0.022] and Chr1:11918989 [OR, 0.92; P=0.032]) of 11 CpG loci assayed was associated with 7% and 8% lower risk, respectively, of having a poor functional outcome. In addition, proBNP was negatively correlated to hypermethylation at 1 CpG (Chr1:11918989 [β=−0.029; P=0.009]) and mediated approximately 7.69% (95% CI, 2.50%–13.82%) of the association between this CpG methylation and the functional outcome.ConclusionsHypermethylation at the NPPB promoter is associated with the functional outcome after ischemic stroke, at least partially by suppressing BNP expression or excretion.

Benefits of Tai Chi Exercise Among Adults With Chronic Heart Failure: A Systematic Review and Meta-Analysis.

Exercise-based cardiac rehabilitation is safe and effective for adults with chronic heart failure (CHF), yet services are greatly underutilized. However, tai chi is a popular and safe form of exercise among older adults with chronic health conditions. A systematic review and meta-analysis was conducted to examine the benefits of tai chi exercise among persons with CHF. An electronic literature search of 10 databases (Allied and Complementary Medicine Database, Cumulative Index to Nursing and Allied Health Literature, Embase, OpenGrey, PsycARTICLES, PsycINFO, PubMed, Scopus, SPORTDiscus, and Web of Science) was conducted from January 1, 2004, to August 1, 2019. Clinical trials that examined tai chi exercise, were published in English or German languages, and conducted among participants with CHF were included. Comprehensive Meta-Analysis version 2.0 software (Biostat, Inc) was used to calculate effect sizes (ie, Hedges g) and 95% confidence intervals using random effects models. A total of 6 studies met the inclusion criteria, enrolling 229 participants (mean age, 68 years; 28% women; mean ejection fraction = 37%). At least 3 studies reported outcomes for exercise capacity (n = 5 studies), quality of life (n = 5 studies), depression (n = 4 studies), and b-type natriuretic peptide (n = 4 studies), allowing for meta-analysis. Compared with controls, tai chi participants had significantly better exercise capacity (g = 0.353; P = .026, I = 32.72%), improved quality of life (g = 0.617; P = .000, I = 0%), with less depression (g = 0.627; P = .000, I = 0%), and decreased b-type natriuretic peptide expression (g = 0.333; P = .016, I = 0%). Tai chi can be easily integrated into existing cardiac rehabilitation programs. Further research is needed with rigorous study designs and larger samples before widespread recommendations can be made.

B-type natriuretic peptide is upregulated by c-Jun N-terminal kinase and contributes to septic hypotension.

B-type natriuretic peptide (BNP) is secreted by ventricular cardiomyocytes in response to various types of cardiac stress and has been used as a heart failure marker. In septic patients, increased BNP suggests poor prognosis; however, no causal link has been established. Among various effects, BNP decreases systemic vascular resistance and increases natriuresis that leads to lower blood pressure. We previously observed that JNK inhibition corrects cardiac dysfunction and suppresses cardiac BNP mRNA in endotoxemia. In this study, we investigated the transcriptional mechanism that regulates BNP expression and the involvement of plasma BNP in causing septic hypotension. Our in vitro and in vivo findings confirmed that activation of JNK signaling increases BNP expression in sepsis via direct binding of c-Jun in activating protein–1 (AP-1) regulatory elements of the Nppb promoter. Accordingly, genetic ablation of BNP, as well as treatment with a potentially novel neutralizing anti-BNP monoclonal antibody (19B3) or suppression of its expression via administration of JNK inhibitor SP600125 improved cardiac output, stabilized blood pressure, and improved survival in mice with polymicrobial sepsis. Therefore, inhibition of JNK signaling or BNP in sepsis appears to stabilize blood pressure and improve survival.

Abstract P229: Benefits of Tai Chi Exercise Among Adults With Chronic Heart Failure: A Systematic Review and Meta-analysis

Background: Regular exercise is beneficial for adults with chronic heart failure (CHF). Tai Chi is popular among older adults and may offer additional exercise options for those with CHF. Objectives: A systematic review and meta-analysis was conducted to examine the benefits of Tai Chi exercise among adults with CHF. Methods: An electronic literature search of ten databases (AMED, CINAHL, Embase, OpenGrey, PsycARTICLES, PsycINFO, PubMed, Scopus, SPORTDiscus, and Web of Science) was conducted from January 1, 2004 to August 1, 2019. Clinical trials that examined Tai Chi exercise, were published in English or German languages, among participants with CHF were included. Study quality was assessed independently by two reviewers, using the modified Downs and Black Quality Index checklist (low quality = score ≤14, moderate quality = score 15-23, high quality = score ≥ 24). Comprehensive Meta-Analysis version 2.0 software (Biostat, Inc.) was used to calculate the effect sizes (i.e., Hedges’ g) and the 95% confidence intervals using random effects models. Results: A total of six studies met the inclusion criteria (five RCTs and one quasi-experimental study with a comparison group), enrolling 229 participants (mean age=68 years old, 28% women, mean ejection fraction=37%). The Yang style of Tai Chi was most commonly practiced in these studies (n=5, 83%), with usual care the most common control condition. Study quality was moderate (mean score=23). At least three studies reported outcomes for exercise capacity, quality of life (QOL), depression, and b-type natriuretic peptide (BNP), allowing for meta-analysis. Compared to controls, Tai Chi participants had significantly better exercise capacity (Hedge’s g=0.353; p=0.026, I 2 =32.72%), improved QOL (Hedge’s g=0.617; p=0.000, I 2 =0%), with less depression (Hedge’s g=0.627; p=0.000, I 2 =0%), and decreased BNP expression (Hedge’s g=0.333; p=0.016, I 2 =0%). Conclusion: Among adults with CHF, Tai Chi was effective in improving exercise capacity and quality of life, with less depression and BNP levels observed, when compared to controls. Tai Chi is popular and safe form of exercise among older adults, yet few studies have been conducted during the past 15 years examining the benefits of Tai Chi among adults with CHF. Tai Chi can be easily integrated into existing cardiac rehabilitation programs. Further research is needed with more rigorous study designs and larger samples, before widespread recommendations can be made.

THE GROWTH HORMONE SECRETAGOGUE RECEPTOR, GHRELIN, AND BNP IN HUMAN HEART DISEASE WITH PRESERVED EJECTION FRACTION

Heart disease (HD) diagnosis is based on clinical features, imaging, and circulating biomarkers, notably B-type natriuretic peptide (BNP), the gold standard marker of heart failure. However, a cardiac-specific biomarker detecting contractile dysfunction at early stages is lacking. The growth hormone secretagogue receptor (GHSR) and its ligand ghrelin may be cardiac-specific biomarkers. We have characterized a fluorescent analog of ghrelin, Cy5-ghrelin (1-19) that detects changes in cardiac GHSR in situ in diabetic cardiomyopathy and heart failure. We are now using this tool to map changes in regional and cellular distribution of GHSR in HD. We obtained tissue from the left ventricle (LV) and right atrium (RA) from 10 cardiac surgery patients with preserved ejection fraction (pEF) and from 10 deceased donors with no history of HD. GHSR levels were measured by quantitative fluorescence microscopy using Cy5-ghrelin(1-19). Ghrelin and BNP were measured by immunofluorescence. High-resolution images were acquired using a Nikon A1R confocal microscope and fluorescence intensities were quantified using ImageJ FIJI's built-in algorithms (Shanbhag for GHSR or RenyiEntropy for ghrelin and BNP). Values of fluorescence intensities were compared based on individual algorithms and colocalization analysis was conducted with the NIS-Elements software. Data were analysed by t-test, one-way ANOVA, and Pearson correlation coefficient (PCC) with significance set at p < 0.05. There were significantly lower levels of GHSR in RA tissue samples from cardiac surgery patients (p=0.041) and no significant differences in ghrelin or BNP expression (Figure 1). There was strong colocalization of ghrelin and BNP in both healthy (PCC=0.935) and diseased (PCC=0.856) tissue samples. In healthy tissue, colocalization appeared as large, discretely defined punctate areas near the nucleus, whereas in the tissue from cardiac surgery patients, ghrelin and BNP signals were more diffuse and contained in smaller areas surrounding the nucleus (Figure 2, arrows). We have shown expression of GHSR, ghrelin and BNP in both healthy and diseased cardiac tissue with only GHSR significantly changing in HD. This may indicate GHSR expression is decreased in HD with pEF whereas BNP (elevated in end stage HF) and ghrelin are not changed. The strong correlation between ghrelin and BNP indicates that these markers may be stored in the same intracellular compartment near the nucleus where they are released upon cellular stress induced by HD. This is the first study showing colocalization between ghrelin and BNP in human cardiac tissue. We are currently stratifying these results based on sex, type of HD, and region of the heart.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Protective effects of berberine against exhaustion exercise induced myocardial injury in rats

Objective: To investigate the effect and possible mechanism of berberine (Ber) on myocardial injury induced by exhaustion exercise (Ee). Methods: Forty healthy male SPF Sprague-Dawley rats were randomly divided into 5 groups using the random unit group design method: control group, Ee group and Ee plus Ber group (low: 50 mg·kg(-1)·d(-1), medium: 100 mg·kg(-1)·d(-1) and high dose: 150 mg·kg(-1)·d(-1), n=8 each). Ber (1.5 ml) or equal volume saline was given per gavage for 14 days. Rats assigned to Ee groups underwent Ee swimming once daily and rats in control group remain sedentary. After 14 days, echocardiographic measurements were performed and left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), left ventricular diastolic diameter (LVIDd) and left ventricular systolic diameter (LVIDs) were obtained. The morphological structure of heart was detected by HE and Masson staining. Serum superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by enzyme-linked immunosorbent assay. Cardiomyocytes apoptosis was detected by TUNEL method. The protein expression of myocardial hypertrophy marker protein B-type natriuretic peptide (BNP) and apoptotic marker protein (Bcl-2, Bax) in rat myocardial tissue was detected by Western blot. Results: (1) Both LVFS and LVEF were significantly lower, and LVIDs and LVIDd were significantly larger in Ee group than those in control group (all P<0.01). The LVFS and LVEF in medium dose of Ber and high-dose Ber groups were significantly higher, and the LVIDs and LVIDd were significantly smaller than those in Ee group (all P<0.01). (2) The results of HE staining showed that the myocardial cells in control group were closely arranged, regular, normal in morphology, clear in structure, and uniform in staining. The myocardial cells of rats in Ee group were disarranged, cell staining was uneven, and vacuoles appeared in the cytoplasm. The disorder of myocardial cell arrangement and unequal staining in the medium dose of Ber were attenuated than in Ee group. The Masson staining results showed that the myocardial cells in control group were closely arranged, regular, normal in shape, clear in structure, and rarely blue-stained (fibrosis). Myocardial cells in rats in Ee group showed obvious fibrosis. The myocardial cell fibrosis in rats with medium dose of Ber was significantly reduced than exercise group. (3) MDA content in myocardial tissue of rats in Ee group was significantly higher than that of control group, and MDA content in myocardial tissue of rats in medium dose of Ber group was significantly lower than in Ee group (P<0.01). The SOD activity of myocardial tissue in rats was significantly lower than that of control group, while that of rats with medium dose of Ber was significantly higher than that of rats in Ee group (P<0.01). (4) TUNEL staining results showed that only a small amount of apoptosis myocardial cells were seen in control group, and a large number of apoptosis myocardial cells were seen in rats in Ee group. However, the number of apoptotic cardiomyocytes in medium dose of Ber was significantly lower than that in Ee group. The AI of rat cardiomyocytes was significantly higher than that of control group (P<0.01), and the AI of rat cardiomyocytes in median dose of Ber group was significantly lower than in Ee group (P<0.01). (5) BNP and Bax protein expression in the myocardial tissues of rats in Ee group were significantly higher than in control group (P<0.01). BNP and Bax protein expression in the myocardial tissues in median dose of Ber group were significantly lower than that of Ee group (P<0.01). The myocardial protein expression level of Bax was significantly higher, and the myocardial protein level of Bcl-2 was significantly lower in Ee group than in control group (both P<0.01), treatment with median dose of Ber could partly reverse above changes (both P<0.01). Conclusion: Ber can attenuate exhaustion exercise induced myocardial injury and remodeling in rats, and the beneficial effects of Ber might possibly be mediated by reducing free radical release and cardiomyocytes apoptosis.

Abstract 907: Understanding Cardiomyocyte Mechanosensing Utilizing CRISPR-Cas9 Based Screening Methods

Heart failure (HF) is a leading cause of premature death that affects approximately 6.5 million patients in the United States and costs over $30 billion annually. HF is a complex disease that involves genetic and environmental factors, and is characterized by gradual deterioration of cardiac pump function. A key process implicated in the development of HF is mechanotransduction—the ability of cardiomyocytes to sense and respond to mechanical forces. While mechanotransduction signaling may play an adaptive role in response to changes in mechanical load, this process can become maladaptive, and ultimately lead to pathological hypertrophy and eventual HF. Gaining insight into cardiac mechanotransduction would solidify our understanding of how cardiomyocytes sense mechanical perturbations and will provide novel targets for the prevention and management of cardiomyopathy and HF. To study perturbations in cardiomyocyte mechanosensing, we have developed a B-type natriuretic peptide (BNP) reporter assay in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), which uses bright red fluorescent protein (tdTomato) to parallel endogenous BNP expression. Our reporter has been validated as a robust, scalable, and quantitative molecular marker of sarcomere function (inside-out) and substrate stiffness (outside-in). We have also generated a custom CRISPR guide RNA (gRNA) library targeting 75 extrinsic and intrinsic components of the cardiomyocyte mechanotransduction apparatus, enabling efficient analysis of single gene knockouts in our BNP reporter and providing a novel high-throughput model for interrogating the mechanical regulation of BNP expression. These gene candidates will be further tested in an in vitro HF model to assess functional response and the potential for phenotypic correction.

Correlation of miR-21 and BNP with pregnancy-induced hypertension complicated with heart failure and the diagnostic value.

Correlation of miR-21 and B-type natriuretic peptide (BNP) with pregnancy-induced hypertension (PIH) complicated with heart failure and the diagnostic value was investigated. Sixty patients with PIH complicated with heart failure admitted to Affiliated Hospital of Chengde Medical University from July 2016 to July 2017 were enrolled as the experimental group, and 35 normal pregnant women as the control group. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method was used to determine the expression level of plasma miR-21 expression level. An automatic biochemical analyzer was used to determine plasma BNP expression level. Spearmans correlation analysis was used for the correlation analysis of miR-21 and BNP. ROC curve was used for evaluating the diagnostic values of miR-21 and BNP for PIH complicated with heart failure. miR-21 and BNP expression levels were higher in patients with PIH complicated with heart failure than those in the normal individuals, and were increased in line with the heart failure grade (P<0.001). The plasma miR-21 expression was positively correlated with BNP in patients with PIH complicated with heart failure (r=0.685, P<0.001). Both miR-21 and BNP had higher diagnostic values for PIH complicated with heart failure, in the diagnosis, the best cut-off value [odds ratio (OR)] of miR-21 was 1.113, with an area under curve (AUC) of 0.889 and a 95% confidence interval (CI) of 82.05–95.76%; the OR of BNP was 123, with an AUC of 0.747 and a 95% CI of 64.95–84.38%. Blood pressure, six-minute walk test (6MWT), left ventricular ejection fraction (LVEF) and left ventricular end diastolic diameter (LVEDD) were independent risk factors for the occurrence of PIH complicated with heart failure (P<0.05). In conclusion, miR-21 and BNP, highly expressed in patients with PIH complicated with heart failure, are expected to become important biomarkers for diagnosing PIH complicated with heart failure and judging the degree of heart failure in the patients, and worthy of clinical popularization and application.

EFFECTS OF NANOCURCUMIN IN RATS WITH DIABETES INDUCED WITH STREPTOZOTOCIN AND NICOTINAMIDE: MRNA EXPRESSION OF B-TYPE NATRIURETIC PEPTIDE

Objective: This study aimed to determine the effects of nanocurcumin on cardiomyopathy, assessed by the expression of B-type natriuretic peptide(BNP) mRNA in heart tissue.Methods: Type 2 diabetic rats were induced with streptozotocin and nicotinamide. Nanocurcumin was orally administered (100 mg/kg/day)for 30 days. BNP-45 mRNA expression in the heart tissue was measured using quantitative reverse transcription-polymerase chain reaction andcalculated using the Livak method.Results: BNP-45 levels increased significantly (p&lt;0.05) in diabetic rats compared with the normal group. Nanocurcumin treatment at a dose of100 mg/kg for 30 days significantly decreased BNP-45 expression levels (p&lt;0.05) compared with diabetic rats without treatment.Conclusion: Nanocurcumin may be beneficial in inhibiting the progression of diabetic cardiomyopathy by suppressing the expression of BNP-45.

Comparison of effects of MHBFC on cardiac hypertrophy after banding of the abdominal aorta in wild-type mice and eNOS knockout mice

17-Methoxyl-7-hydroxy-benzene-furanchalcone (MHBFC) was isolated from the Millettia pulchra (Benth.) Kurz var. Laxior (Dunn) Z. Wei (Papilionaceae) (MKL) roots. This study aimed to study the effects of MHBFC on cardiac hypertrophy after banding of the abdominal aorta in wild-type mice and endothelial nitric oxide synthase (eNOS) knockout mice in order to verify whether MHBFC depends on the eNOS gene in reversing cardiac hypertrophy. By observing the blood pressure, body weight, ventricular hypertrophy index, expression of B-type natriuretic peptide (BNP), myosin heavy chain beta (β-MHC), atrial natriuretic peptide (ANP), serum amyloid A SAA mRNA, level of endothelin-1 (ET-1) protein, histopathological changes of myocardial cells, and the myocardial ultrastructure, we found that MHBFC significantly ameliorated the changes in the structure of the heart and aorta, significantly decreased the blood pressure, myocardial cell cross-section area, level of ET-1 protein, and mRNA levels of BNP, β-MHC, ANP and SAA, prevented myocardial ultrastructure alterations, and relieved myocardial and perivascular fibrosis of wild-type mice. However, MHBFC could not induce any significant change in eNOS gene knockout mice. These results show that MHBFC is beneficial to protect against cardiac hypertrophy after banding of the abdominal aorta in wild-type mice, but has no such effect on eNOS gene knockout mice, which indicates that MHBFC is dependent on the existence of the eNOS gene in reversing cardiac hypertrophy.

B-type natriuretic peptide reference interval of newborns from healthy and pre-eclamptic women: a prospective, multicentre, cross-sectional study

ObjectiveTo define and compare the reference interval of B-type natriuretic peptide (BNP) in healthy newborns (HN) from healthy mothers and with severe pre-eclampsia.DesignProspective, multicentre, cross-sectional study.SettingFour obstetric wards of second-level...

Effect of parathyroid hormone on cardiac function in rats with cardiomyopathy.

The present study investigated the role of parathyroid hormone (PTH) in non-ischemic cardiomyopathy (CM) and its underlying mechanism. A total of 30 Sprague-Dawley male rats were randomly divided into a control group (n=6) and an experimental group (n=24). To induce CM in the rats of the experimental group, 2 mg/kg Adriamycin (ADR) was administered intraperitoneally with 5 equal injections every third day followed by 5 weekly injections resulting in a cumulative dose of 20 mg/kg. Following establishment of the model, rats in the experimental group were subdivided into a PTH-untreated CM group that received daily normal saline subcutaneous injections for 7 days and three treated CM groups that received daily subcutaneous injections of 5, 10, or 20 µg/kg of recombinant PTH for 7 days. Rats in the control group accordingly received intraperitoneal and subcutaneous injections of normal saline. Blood sample analysis revealed that B-type natriuretic peptide (BNP), troponin T, C-reactive protein (CRP), creatinine and phosphorus concentrations were increased in the PTH-untreated CM group compared with that in the control group, whereas PTH and calcium concentrations were decreased. Administration of PTH dose-dependently decreased BNP, CRP, creatinine and phosphorus levels, and increased PTH and calcium levels. Notably, there were significant differences in PTH, BNP, troponin T, CRP, creatinine, calcium, and phosphorus levels among the rats in the five groups (P<0.01). Cardiac ultrasonography results indicated that the left ventricular ejection fraction (LVEF) was significantly decreased in rats treated with ADR compared with the rats from the control group (P<0.01). However, the LVEF gradually recovered with elevated PTH treatment doses. The overall differences of LVEF and left ventricular end-systolic volume in the five experimental groups were statistically significant (P<0.01). Furthermore, there were dose-dependent increases in LV mass and left ventricular end-diastolic volume in PTH-treated rats; however, the differences between any two groups did not reach statistical significance (P>0.05). Immunohistochemical staining and western blot analysis using an anti-PTH polyclonal antibody was performed to evaluate the protein expression levels of PTH in myocardial tissues. The mRNA expression levels of PTH and BNP were measured using reverse transcription-quantitative polymerase chain reaction. The results demonstrated that the mRNA and protein expression levels of PTH in myocardial tissues were significantly decreased in ADR-treated rats compared with the levels in the control group rats. Injection of recombinant PTH significantly increased PTH expression and reduced BNP expression in dose-dependent manners (P<0.05). These findings demonstrated that PTH can improve cardiac function in rats with ADR-induced CM, suggesting a potential therapeutic application for PTH in non-ischemic CM.

Expression and Implication of Clusterin in Left Ventricular Remodeling After Myocardial Infarction

Left ventricular remodeling (LVR) after myocardial infarction is associated with an increased risk of heart failure and death. In spite of a modern therapeutic approach, LVR remains relatively frequent and difficult to predict in clinical practice. Our aim was to identify new biomarkers of LVR and understand their involvement in its development. Proteomic analysis of plasma from the REVE-2 study (Remodelage Ventriculaire)-a study dedicated to the analysis of LVR which included 246 patients after a first anterior myocardial infarction-identified increased plasma levels of CLU (clusterin) in patients with high LVR. We used a rat model of myocardial infarction to analyze CLU expression in the LV and found a significant increase that was correlated with LVR parameters. We found increased CLU expression and secretion in primary cultures of rat neonate cardiomyocytes hypertrophied by isoproterenol. Silencing of CLU in hypertrophied neonate cardiomyocytes induced a significant decrease in cell size, ANP (atrial natriuretic peptide), and BNP (B-type natriuretic peptide) expression, associated with a decreased ERK (extracellular signal-regulated kinase) 1/2 activity, suggesting a prohypertrophic role of CLU. We then confirmed a significant increase of both intracellular p-CLU (precursor form of CLU) and m-CLU (mature form of CLU) in failing human hearts. Finally, the circulating levels of CLU (secreted form) were increased in patients with chronic heart failure who died from cardiovascular cause during a 3-year follow-up (n=99) compared with survivors (n=99). Our results show for the first time that plasma CLU levels are associated with LVR post-myocardial infarction, have in part a cardiac origin, and are a predictor of early death in heart failure patients.

The Histone Demethylase JMJD2A Modulates the Induction of Hypertrophy Markers in iPSC-Derived Cardiomyocytes.

The development of cardiovascular pathologies is partly attributed to epigenetic causes, including histone methylation, which appears to be an important marker in hearts that develop cardiac hypertrophy. Previous studies showed that the histone demethylase JMJD2A can regulate the hypertrophic process in murine cardiomyocytes. However, the influence of JMJD2A on cardiac hypertrophy in a human cardiomyocyte model is still poorly understood. In the present study, cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) were used. Hypertrophy was induced by angiotensin II and endothelin-1 (ET-1), and transfections were performed to overexpress JMJD2A and for small interfering RNA (siRNA)-induced silencing of JMJD2A. Gene expression analyses were determined using RT-PCR and Western blot. The expression levels of B-type natriuretic peptide (BNP), natriuretic peptide A (ANP), and beta myosin heavy chain (β-MHC) were increased by nearly 2–10-fold with ET-1 compared with the control. However, a higher level of JMJD2A and UTX was detected, whereas the level of JMJD2C was lower. When cardiomyocytes were transiently transfected with JMJD2A, an increase close to 150% in BNP was observed, and this increase was greater after treatment with ET-1. To verify the specificity of JMJD2A activity, a knockdown was performed by means of siRNA-JMJD2A, which led to a significant reduction in BNP. The involvement of JMJD2A suggests that histone-specific modifications are associated with genes encoding proteins that are actively transcribed during the hypertrophy process. Since BNP is closely related to JMJD2A expression, we suggest that there could be a direct influence of JMJD2A on the expression of BNP. These results may be studied further to reduce cardiac hypertrophy via the regulation of epigenetic modifiers.