This study demonstrates that proBNP, constituting a substantial portion of immunoreactive BNP in heart failure plasma, possesses significantly lower biological activity than the processed 32-amino acid hormone. These results implicate a discordance in heart failure between the high circulating levels of immunoreactive BNP and hormone activity, suggesting that some patients may be in a state of natriuretic peptide deficiency.

Abstract-The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-␤ (TGF-␤) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-␤-induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-␤-induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-␤ and BNP for 24 and 48 hours. TGF-␤, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-␤ effects; 88% and 85% of all TGF-␤-regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-␤-regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (␣-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNF␣-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate-dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-␤-induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions. Key Words: B-type natriuretic peptide Ⅲ transforming growth factor-␤ Ⅲ cardiac fibroblasts Ⅲ fibrosis N atriuretic peptides comprise a family of vasoactive hormones that play important roles in the regulation of cardiovascular and renal homeostasis. [1][2][3] Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are predominantly produced in the heart and exert vasorelaxant, natriuretic, and antigrowth activities. Binding of ANP and BNP to type-A natriuretic peptide receptor (NPRA) leads to the generation of cyclic guanosine monophosphate (cGMP), which mediates most biological effects of the peptides. Mice lacking NPRA exhibit cardiac hypertrophy, fibrosis, hypertension, and increased expression of fibrotic genes, including TGF␤1, TGF␤3, and collagen 1. 4 -6 Furthermore, targeted disruption of the BNP gene in mice results in cardiac fibrosis and enhanced fibrotic response to ventricular pressure overload, suggesting that BNP is involved in cardiac remodeling. 7 Cardiac remodeling is viewed as a key determinant of the clinical outcome in heart disease. It is characterized by a structural rearrangement of the cardiac chamber wall that involves cardiomyocyte hypertrophy, fibroblast proliferation, and increased deposition of extracellular matrix (ECM) pro...

IntroductionSustained hemodynamic overload elicits a series of functional and structural changes in the ventricular myocyte that culminate in cardiac hypertrophy. This is viewed as a compensatory response that, over the short term, results in improved cardiac performance; however, protracted exposure to the hypertrophic stimulus often results in an alteration in phenotype that leads to progressive heart failure. Clinical hypertrophy has, in fact, been linked to increased mortality independent of associated cardiovascular risk factors (1).At the cellular level, hypertrophy is thought to develop in response to a combination of mechanical (i.e., load-dependent) and neurohumoral stimuli, such as angiotensin II (AII), endothelin (ET), and adrenergic agonists. In cultured neonatal rat cardiac myocytes, both mechanical (2-4) and biochemical (5-8) stimuli effect a series of changes in gene expression that closely parallel those seen in the hypertrophied heart in vivo (9). This includes the sequential activation of immediate early genes (e.g., protooncogenes like c-jun, c-fos, and c-myc), a fetal gene program (e.g., atrial natriuretic peptide [ANP], brain natriuretic peptide [BNP], skeletal α-actin, and β-myosin heavy chain) that is typically quiescent in the nonhypertrophied adult ventricular myocardium, and the structural sarcomeric genes that contribute the protein infrastructure associated with hypertrophy (e.g., cardiac α-actin and myosin light chain-2) (9).The signaling cascades underlying the hypertrophic phenotype remain only partially understood. Protein kinase C (10), calcium/calmodulin (11), calcineurin (12-13), nonreceptor protein tyrosine kinases (14, 15), the Janus kinase/STAT system (16, 17), small G proteins (e.g., Ras, Rac, Rho, Cdc42) (18-21), and the various mitogen-activated protein kinases (i.e., ERKs, JNKs, and p38 MAPKs) (22-38) have each been implicated as playing a role in signaling hypertrophy. The MAPKs in particular have been the focus of considerable attention. Hypertrophic stimuli have been shown to activate ERK (22-24), JNK (30, 32), and p38 (34, 37, 38) in either cultured ventricular myocytes or intact myocardial tissue. Whereas interference with the ERK pathway has been shown to block some aspects of hypertrophy-dependent gene expression, it does not affect the morphological changes accompanying hypertrophy (27); in some instances (29) it has been dissociated from the hypertrophic program completely. JNKs have also been linked to hypertrophy (28-32), although recent studies have raised questions about the importance of these kinases in signaling various aspects of the phenotype (37). Activation of p38 by upstream kinases (e.g., MKK6) (35) or hypertrophic agonists (38) has been associated with effects on myocyte growth. Of note, the nature of the effect appears to be isoform specific. Overexpression of activated MKK3 (MKK3bE) elicited both characteristic hypertrophic changes and increased apoptosis in ade- Application of mechanical strain to neonatal rat ventricular myocytes in culture evo...

Using a device that applies cyclical strain (1 Hz) to ventricular cardiocytes cultured on collagen-coated silicone elastomer surfaces, we have demonstrated straindependent increases in brain natriuretic peptide (BNP) secretion, BNP mRNA levels, and expression of a transiently transfected ؊1595 human BNP-luciferase reporter. When actinomycin D (10 M) was introduced concomitantly with the strain stimulus, the strain-induced increase in BNP mRNA was eliminated, and the decay of transcripts was identical in the control and strained cells, indicating the lack of independent effects on transcript stability. Strain-dependent ؊1595 human BNPluciferase activity was completely inhibited by chelerythrine, 2-aminopurine, genistein, and W-7 and only partially or not at all by KN-62, wortmannin, and H-89. The effects of these individual agents paralleled their effects on mitogen-activated protein kinase (MAPK) activity, but not c-Jun N-terminal kinase (JNK) activity, in the cells. Overexpression of wild-type MAPK and, to a lesser extent, JNK increased strain-dependent BNP promoter activity, whereas dominant-negative mutants of MAPK kinase, JNK kinase, or Ras completely blocked strain-dependent reporter activity. These findings provide the first demonstration that mechanical strain can increase myocardial gene expression through a transcriptional mechanism and suggest important roles for MAPK and JNK in mediating this effect.The natriuretic peptides comprise a family of vasoactive hormones that play an important role in the regulation of cardiovascular and renal homeostasis (1). Their natriuretic and vasodepressor properties suggest that they represent endogenous antagonists of the various systems (e.g. renin-angiotensin system and sympathetic nervous system) that support arterial blood pressure under basal conditions and, at times, contribute to the pathophysiology of cardiovascular disease.Atrial natriuretic peptide (ANP), 1 the prototype of the group, is produced primarily in the atria of the heart. ANP is expressed in the cardiac ventricle during development and early neonatal life (2, 3). Expression decays as the animal ages and remains quiescent in the adult unless the ventricle is subjected to hemodynamic overload (i.e. mechanical strain that leads to increased wall stress and subsequently to hypertrophy of the myocardium), as occurs with systemic arterial hypertension or congestive heart failure (4 -7).Brain natriuretic peptide (BNP) is also produced in the heart. Despite the nomenclature, relatively little BNP is expressed in the mammalian brain (the exception being the porcine brain, where the peptide was identified originally). Expression of BNP in the heart is lower than that of ANP under basal conditions, and the atrial/ventricular ratio of expression is considerably less than that seen with ANP (8). Ventricular expression of BNP is activated in a fashion similar to ANP in pathophysiological states associated with hemodynamic overload (9, 10). At some stages of advanced congestive heart failure, circulating BNP le...

The peroxisome proliferator activated receptors (PPARs) appear to have beneficial effects in the cardiovascular system. PPAR gamma has been shown previously to exert an inhibitory effect on cardiac myocyte hypertrophy in vivo and in vitro. Using endothelin to activate the hypertrophic program in neonatal rat cardiac myocytes, we demonstrate that PPAR alpha ligands (fenofibrate and WY14,643) suppress hypertrophy-dependent increases in protein synthesis, cell surface area, and sarcomeric organization in vitro. This was accompanied by a decrease in brain natriuretic peptide gene expression, a marker of transcriptional activation in hypertrophy. These effects were equivalent to or greater than those seen with the PPAR gamma agonist rosiglitazone. Fenofibrate and rosiglitazone suppressed endothelin stimulation of human brain natriuretic peptide gene promoter activity, and this effect was amplified by cotransfection of PPAR alpha and PPAR gamma expression vectors, respectively. The fenofibrate-dependent suppression of endothelin's stimulatory activity was dependent upon promoter sequence positioned between -904 and -40 relative to the transcription start site and did not appear to involve a number of positive and negative regulatory elements that are known to govern transcription of this gene. These findings suggest that PPAR alpha ligands could prove to be useful in the management of disorders associated with hypertrophy and remodeling of the myocardium.

The application of mechanical strain leads to activation of human brain natriuretic peptide gene promoter activity, a marker of hypertrophy, in cultured neonatal rat ventricular myocytes. We have used a combination of transient transfection analysis and reverse transcriptase-polymerase chain reaction to examine the role of locally produced factors in contributing to this activation. Conditioned media from strained, but not static, cultures led to a dose-dependent increase in human brain natriuretic peptide gene promoter activity. This increase was completely blocked by losartan or BQ-123, implying a role for angiotensin and endothelin as autocrine/paracrine mediators of the response to strain. Inclusion of the same antagonists in the cultures themselves led to only partial inhibition (ϳ60%), whereas inclusion of exogenous endothelin or angiotensin II resulted in amplification of the strain response. Angiotensin II and endothelin appear to be arrayed in series in the regulatory circuitry; the angiotensin response was blocked by BQ-123, whereas the endothelin response was unaffected by losartan. Mechanical strain was also shown to stimulate expression of the endogenous angiotensinogen, angiotensin-converting enzyme, and endothelin genes in this system. Collectively, these data indicate that locally generated angiotensin II and endothelin, acting in series, play an important autocrine/paracrine role in mediating strain-dependent activation of cardiac-specific gene expression.Application of mechanical strain, or passive stretch, to myocardial cells in culture results in a series of phenotypic changes that closely resemble those that occur with myocyte hypertrophy in vivo. This includes activation of the immediate early gene family (e.g. c-fos, c-jun, c-myc, and egr-1), increased expression of the fetal gene program (e.g. atrial natriuretic peptide, ␣ skeletal actin, and ␤-myosin heavy chain), and increased protein synthesis (1, 2).A number of recent studies have suggested that activation of autocrine/paracrine regulatory mechanisms in the myocardium may play an important, if not dominant, role in determining the myocyte response to hemodynamic load. Specific local regulators invoked as participating in this process include angiotensin II (AII) 1 (3-6), endothelin (ET) (7-9), transforming growth factor ␤ (TGF-␤) (10, 11), fibroblast growth factor (11, 12), myotrophin (13), and cardiotrophin (14, 15), among others. Sadoshima et al. (3) suggested that locally produced angiotensin, stored in and secreted from the cardiac myocyte in vitro, plays a dominant role in effecting the response to mechanical strain. Losartan, an angiotensin type 1 (AT1) receptor antagonist, blocked strain-dependent increases in c-fos, ␣ skeletal actin and atrial natriuretic factor gene expression in their system. Yamazaki et al. (4,5) confirmed that AII plays a significant role in determining the response to strain. In their hands, the induction of MAP kinase, MAP kinase kinase, Raf-1 kinase, or protein synthesis by strain was suppressed, altho...

Abstract-The application of mechanical strain to cultured cardiac myocytes in vitro leads to activation of the brain natriuretic peptide (BNP) gene promoter, a marker of cardiac hypertrophy. We have previously shown that this activation results from both a direct mechanostimulatory event and an indirect autocrine/paracrine stimulation involving the sequential production of angiotensin II and endothelin (ET). In the present study, we examined the role of p38 mitogen-activated protein kinase (MAPK) and extracellular signal regulated kinase (ERK) in signaling the increase in promoter activity trafficking through each of these pathways. ET was shown to stimulate both p38 MAPK and ERK activity in these cultures and to activate human BNP (hBNP) promoter activity. Activation of the promoter was inhibited Ϸ45% by SB-203580, a p38 MAPK inhibitor, and Ϸ70% by PD98059, an inhibitor of the ERK-activating kinase MAPK kinase. The ET-independent (ie, direct) stimulation of the hBNP promoter by mechanical strain was inhibited Ϸ70% by SB-203580 and Ϸ60% by PD98059, implying that similar signaling circuitry is used, albeit to different degrees, by the direct and indirect pathways. The p38 MAPK component of both the ET-dependent and the ET-independent responses to strain appears to operate through a series of nuclear factor-B binding, shear stress response element-like structures in the hBNP gene promoter. Collectively, these data suggest that activation of the BNP promoter by hypertrophic stimuli involves the participation of several independent signaling pathways. Such redundancy would help to guarantee generation of the full hypertrophic phenotype independently of the nature of the hypertrophic stimulus.

Brain natriuretic peptide (BNP) gene expression is a well documented marker of hypertrophy in the cardiac myocyte. Triiodothyronine (T 3 ), the bioactive form of thyroid hormone, triggers a unique form of hypertrophy in cardiac myocytes that accompanies the selective activation or suppression of specific gene targets. In this study, we show that the BNP gene is a target of T 3 action. BNP secretion was increased 6-fold, BNP mRNA levels 3-fold, and BNP promoter activity 3-5-fold following T 3 treatment. This was accompanied by an increase in myocyte size, sarcomeric organization, and protein synthesis. Of note, several of the responses to T 3 synergized with those to the conventional hypertrophic agonist endothelin. The response to the liganded thyroid hormone receptor (TR) was mediated by an unusual thyroid hormone response element located between ؊1000 and ؊987 relative to the transcription start site. Both TR homodimers and TR⅐retinoid X receptor heterodimers associated with this element in an electrophoretic mobility shift assay. Protein fragments harboring the LXXLL motifs of the coactivators GRIP1 and SRC1 or TRAP220 interacted predominantly with the TR⅐retinoid X receptor heterodimeric pair in a liganddependent fashion. Both TR homodimers and heterodimers in the unliganded state selectively associated with glutathione S-transferase-nuclear receptor corepressor fragments harboring one of three receptor interaction domains containing the sequence (I/L)XX-(I/V)I. These interactions were dissociated following the addition of T 3 . Collectively, these findings identify the BNP gene as a potential model for the investigation of TR-dependent gene regulation in the heart.