Novel Therapeutic Targets in Heart Failure
Since the discovery of the cardiac hormone atrial natriuretic peptide by de Bold and colleagues in 1981, the field of natriuretic peptides has significantly advanced with translation of new knowledge to the clinical practice of heart failure. This new knowledge builds on the importance of cardiorenal mechanisms that contribute to optimal cardiovascular regulation. Recent investigations by our group and others have also established the direct myocardial actions of the natriuretic peptides, broadening their therapeutic potential beyond renal mechanisms. Indeed, a potential therapeutic target is cardiac remodeling and fibrosis based on the unique cardiorenal and humoral protective properties that natriuretic peptides possess. We review new insights into the natriuretic peptide system and specifically focus on the possible role of natriuretic peptides as a new therapeutic strategy to limit cardiac remodeling and fibrosis to delay worsening of cardiac function and the progression of heart failure.
- atrial natriuretic peptide
- brain natriuretic peptide
- particulate guanylate cyclase
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SODIUM- AND ALDOSTERONE-REGULATING ACTIONS
The natriuretic peptide system (NPS) consists of three known peptides, with each being a distinct gene product with similar structure. Atrial natriuretic peptide (ANP)1and brain natriuretic peptide (BNP)2are primarily from cardiomyocytes, and C-type natriuretic peptide (CNP) is chiefly from endothelial cells.3-5These three peptides function through the second messenger cyclic guanosine monophosphate (cGMP), where ANP and BNP bind to natriuretic peptide receptor (NPR)-A and CNP binds to NPR-B.6,7All three peptides are cleared by the clearance receptor NPR-C, which is a nonparticulate guanylyl cyclase-linked receptor.6These peptides are degraded enzymatically by widely distributed neutral endopeptidase (NEP) 24.11.7A comprehensive view of the biology of these peptides has emerged following studies in cell systems, murine models of altered natriuretic peptide production or receptor function, and integrative physiologic studies in disease models and in humans. The biologic properties of these peptides, which include natriuresis, vasodilatation, inhibition of the renin-angiotensin-aldosterone system (RAAS), positive lusitropism, and inhibition of fibrosis, have led to the unique concept of cardiorenal protection by activation of cGMP.8-14These biologic properties have supported the development of the natriuretic peptides as therapeutic agents for cardiorenal disease syndromes.15-21
Whereas CNP lacks renal actions,22BNP is the most potent among the other members of the natriuretic peptide family in augmenting sodium excretion and exerting direct myocardial actions. This natriuretic effect of BNP, owing to its unique glomerular filtration rate (GFR)-enhancing properties, contributes to unloading of the heart and improving congestion in heart failure (HF). Specifically, in a model of severe HF, we demonstrated that BNP is markedly more potent in augmenting sodium excretion compared with ANP or with the renally synthesized form of ANP, urodilatin.23The renal-enhancing action of BNP has understandable importance in developing BNP-based therapies for the treatment of HF. Increasing evidence supports the concept that the kidney plays a crucial role in HF and that the presence of reduced GFR is the most robust marker for poor survival. Thus, the possibility of improving or preserving renal function is key in the management of HF.24Recently, the concept of enhancing renal function in HF has emerged as an important therapeutic strategy to delay disease progression.25,26Further, the development of renal resistance to the natriuretic peptides may be an ominous sign of a very high risk of increased mortality.27Sackner-Bernstein and colleagues recently showed in two meta-analysis studies that the use of BNP in patients with acute congestive heart failure (CHF) increased the risk of worsening renal function and had a trend to increase mortality.28,29It should be noted, however, that these studies were not designed to assess renal function or mortality. Furthermore, baseline clinical characteristics were not assessed; thus, the authors concluded that these studies are hypothesis-generating findings rather than conclusive evidence of harm. Therefore, there is a need for new investigations on the role of BNP in the setting of acute CHF, which, based on preclinical research, may suggest that an opportunity exists for the use of the natriuretic peptides in early stages of cardiac diseases to delay disease progression. Indeed, Martin and colleagues recently reported that chronic enhancement of the NPS via oral NEP inhibition, which suppresses the enzymatic degradation of the natriuretic peptides in a progressive CHF model, resulted in a delay of renal impairment onset together with greater suppression of aldosterone in advanced HF.30This natriuretic peptide-based therapeutic strategy, which preserved renal function, was also associated with an enhanced clinical score and a maintained cardiac function assessed by echocardiography. Again, these studies underscore the importance of the NPS in renoprotection in the setting of evolving HF.
An additional mechanism by which the natriuretic peptides may enhance renal function is related to their anti-renin-aldosterone properties. Importantly, one of the first demonstrations of the biologic action of ANP was its inhibition of angiotensin II-mediated synthesis and release of aldosterone.31-34Chen and colleagues previously reported that 10-day subcutaneous BNP in experimental HF had beneficial effects to unload the heart without evidence of tolerance.35,36We extended this concept of chronic subcutaneous BNP to human studies and reported the first demonstration that a 3-day period of subcutaneous BNP increases circulating concentrations of BNP, activates plasma and urinary cGMP, and, more importantly, enhances sodium excretion and reduces activation of the RAAS.37More recently, we demonstrated that the antialdosterone properties of BNP are maintained in a model of overt HF even in the presence of a loop diuretic. Specifically, Cataliotti and colleagues reported that the coadministration of intravenous high-dose BNP (10 pmol/kg/min) and intravenous furosemide (1 mg/kg/h) enhanced the GFR.38Further, low- and high-dose BNP plus furosemide demonstrated a greater diuretic and natriuretic response than furosemide alone (1 mg/kg/h). Most importantly, there was a lack of activation of aldosterone with coadministration of BNP and furosemide in contrast to a marked activation of aldosterone with the loop diuretic alone. In addition, there was the greater unloading of the heart that occurred with coadministration of both sodium-regulating compounds. Noteworthy in this study, the authors did not precede BNP infusion with a bolus because it is indicated in the current clinical practice. The lack of bolus perhaps prevented a significant reduction in blood pressure with the combination of furosemide and BNP. Indeed, this maintained blood pressure level may be a key factor to preserve the beneficial renal actions associated with BNP. This study underscores the potential benefit of chronic BNP therapy in HF to modulate aldosterone, enhance renal sodium and water excretion, and maintain the GFR. These findings were also supported by our investigations with the natriuretic peptide-based therapeutic agent omapatrilat, which simultaneously inhibits NEP and angiotensin-converting enzyme (ACE).39Specifically, in a model of early-stage HF, we observed that omapatrilat administered with furosemide was superior in enhancing renal function, unloading the heart, and inhibiting activation of aldosterone compared with a combination of ACE inhibitors and furosemide. Therefore, improvement of the natriuretic peptide system via inhibition of NEP or by direct stimulation of the NPR-A via BNP attenuated furosemide activation of aldosterone. In addition, there were greater GFR-enhancing actions with both natriuretic peptide-based therapies. Indeed, these data support a rationale for a more physiologic approach to enhancing renal function in the early stage of HF with natriuretic peptide-based therapy with the additional benefit of suppressing aldosterone. The use of new diuretic agents that may improve the renal actions of conventional diuretics is most timely because conventional loop diuretics have been associated with adverse effects, especially with potassium-wasting properties, excessive activation of the RAAS, and worsening of renal function.40-45
There also is an emerging alternative novel strategy to activation of cGMP other than via the natriuretic peptides. Unlike conventional organic nitrates, which are associated with the development of tolerance, direct soluble guanylate cyclase (sGC) stimulation with a novel new molecule, BAY 41-2272, can be considered an effective therapy for cardiovascular diseases. BAY 41-2272 is orally available, directly stimulates the heterodimeric hemeprotein sGC while increasing its sensitivity to its natural stimulator, nitric oxide, and lacks tolerance.46We investigated whether the biologic actions of the natriuretic peptides are specific to particulate guanylyl cyclase (pGC) activation or if these actions can be mimicked by direct stimulation of sGC. Specifically, Boerrigter and colleagues used BAY 41-2272 direct sGC stimulation to demonstrate potent cardiovascular actions with unloading of the heart, particularly pulmonary capillary wedge pressure with enhancement of renal blood flow at high doses.47In contrast to BNP, however, there was no enhancement of GFR and sodium excretion or suppression of an already activated RAAS. Thus, pGC activation by BNP is unique because of its renal actions and differs from cGMP activation through direct sGC stimulation, which lacks renal effects.
Another very recent therapeutic innovation involves significant activation of pGC by the use of a newly developed oral form of human BNP.48Specifically, the recent development of alkylPEGylation (in which short, monodispersed, amphiphilic oligomers are covalently attached to specific sites on proteins) has made it possible to modify the hydrophilicity and hydrophobicity of BNP. This technology protects the protein from gastric proteolysis and renders the resultant conjugate suitable for oral delivery while retaining the activity inherent in the native peptide. Ultimately, this new conjugation process makes oral chronic BNP administration feasible and may extend the use of natriuretic peptides to other cardiovascular diseases, such as hypertension, and perhaps as a preventive strategy in evolving heart failure. Indeed, such early stages of cardiac disease in which renal function is still preserved may benefit the most from the chronic stimulation of pGC.
DIRECT MYOCARDIAL ACTIONS
Acute intravenous BNP in humans with HF enhances ventricular function.49,50The basis for BNP approval as an intravenous agent for the treatment of acute human HF was its superiority, compared with the cGMP-activating drug nitroglycerin, in reducing cardiac filling pressures over a 3-hour period in association with improved symptoms compared with placebo.21In experimental overt HF, intravenous BNP was more potent than ANP in enhancing diastolic function by reducing both end-systolic and end-diastolic volume.11Further, only BNP, and not ANP or CNP, decreased both end-diastolic volume and end-systolic volume in experimental HF, with a greater improvement in diastolic function. This increase in the rate of left ventricular relaxation may improve left ventricular filling and maintain cardiac output. Importantly, the reduction in both end-systolic pressure and end-systolic volume, as well as the more prominent improvement in end-diastolic volume with BNP compared with ANP, would be therapeutically useful because it tends to reduce congestion while maintaining cardiac output. Therefore, these data provide additional insight into the potential superior therapeutic actions of BNP in the treatment of HF compared with the other natriuretic peptides.
CARDIAC FIBROSIS AND THE NATRIURETIC PEPTIDES
A hallmark of heart failure is cardiac fibrosis. Indeed, cardiac fibrosis contributes to impaired myocardial contraction and relaxation and cellular hypoxia owing to impaired myocardial perfusion secondary to reduced diffusion of oxygen from blood vessels to cardiomyocytes. In this regard, an additional favorable action of intravenous BNP is to increase myocardial perfusion and reduce oxygen consumption in normal humans.51Cardiac fibrosis is also known to predispose patients to atrial and ventricular dysrhythmias, which, in turn, may aggravate the impaired myocardial perfusion, thus creating a vicious circle that leads to reduced myocardial function.52-55This is especially important in chronic HF, in which atrial fibrosis may serve as a participating mechanism of atrial fibrillation with further impairment of cardiac function.56As we have only limited modalities to target cardiac fibrosis clinically, the significance of the proposed use of natriuretic peptides in the treatment of HF is high.
Whereas myocytes occupy most of the space in the myocardium, cardiac fibroblasts (CFs) account for two-thirds of the total population of cells in the heart and play a key role in the regulation of myocardial structure and function.57,58CFs regulate the production of collagen and through induction of matrix metalloproteinases (MMPs) the degradation of collagen.59,60A fine balance of production and degradation of collagen maintains myocardial structure to optimally preserve cardiac function. In contrast, an excessive degradation of collagen leads to ventricular dilatation and systolic dysfunction, whereas an excessive proliferation of CFs and increased production of collagen lead to cardiac fibrosis and contribute to diastolic dysfunction.
A number of different humoral players are involved in the regulation of this fine equilibrium between synthesis and degradation of collagen. Some of these humoral factors are responsible for increased production of collagen and cardiac fibrosis, whereas others are responsible for excessive collagen degradation. Aldosterone and angiotensin II are considered among the most potent stimulators of collagen synthesis. Although this is still controversial and the ultimate mechanisms are far from being understood,61-63there is solid evidence that the use of an ACE inhibitor or aldosterone antagonist may prevent cardiac fibrosis.64,65Two recent clinical trials brought renewed focus on the potential importance of cardiac fibrosis as a target for cardiovascular drug therapies using an aldosterone antagonist. First, the Randomized Aldactone Evaluation Study (RALES) in CHF reported that a relatively low dose of the nonselective aldosterone antagonist spironolactone, in addition to the standard medical regimen, reduced mortality and hospitalization for worsening HF.66The mechanism for the beneficial effect of spironolactone remains unknown, but it was speculated that this aldosterone receptor antagonist blocked the profibrotic actions of aldosterone. This speculation was supported by the observation that a biomarker for collagen (procollagen type III peptide) decreased in the spironolactone-treated subgroup compared with placebo.67Recently, the results of the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) were reported.68Eplerenone is a selective aldosterone antagonist that acts as a competitive antagonist of the mineralocorticoid receptor. EPHESUS showed that eplerenone added to optimal medical therapy reduced morbidity and mortality in patients who had left ventricular dysfunction and HF after an acute myocardial infarction. Again, the investigators speculated that the benefit was related to a favorable impact of therapy on ventricular remodeling and fibrosis.
The report that genetic suppression of the NPS in mice results in cardiac fibrosis set the stage for considering the natriuretic peptides as antifibrotic therapies. Mouse models of disrupted NPS pathways were the first to demonstrate the direct myocardial structural actions of the natriuretic peptides independent of renal actions. Indeed, the natriuretic peptides have been shown to possess specific angiotensin- and aldosterone-inhibiting properties, thus explaining in part their antifibrotic actions. We recently investigated a possible direct role for BNP on the regulation of CF activities. Tsuruda and colleagues first investigated whether CFs were a source of BNP.69Immunohistochemical staining demonstrated that BNP was diffusely present in the cytoplasm of CFs. Furthermore, CFs secreted BNP into the medium at a rate of 11.2 ± 1.0 pg/105cells per 48 hours, and this secretion was significantly augmented in a time-dependent fashion with tumor necrosis factor (TNF)-α. BNP was analyzed by high-performance liquid chromatography, which revealed one major peak that appeared at an elution position identical to that of synthetic full-length canine BNP1-32. Our next goal was to determine BNP's action on CFs with the specific hypothesis that we could demonstrate the direct antifibrotic properties of BNP. Indeed, BNP (1 μM) significantly increased intracellular cGMP. The effect of BNP on de novo collagen synthesis was assessed by measuring [3H]proline incorporation. BNP significantly inhibited [3H]proline incorporation by 29%, clearly demonstrating antifibrotic properties in vitro in CFs. Further, to determine if BNP could also degrade collagen, we sought to define its action on several MMPs. We accomplished this by defining zymographic MMP abundance. For detection of MMP and tissue inhibitor of metalloproteinase proteins in CFs, Western blotting was performed. Furthermore, together with direct actions on the CFs, we also reported that BNP could attenuate the profibrotic actions of endothelin 1 (ET-1). BNP alone significantly increased zymographic total MMP-2 abundance at 24 hours, but TNF-α (10-7 M) alone did not. However, TNF-α in combination with BNP significantly increased BNP-induced MMP-2 abundance. ET-1 (10-7 M) significantly down-regulated the zymographic MMP-2 abundance after 3 hours of incubation. Coincubation of BNP (10-6 M) and ET-1 (10-7 M) reversed the action of ET-1, resulting in increased zymographic MMP-2 abundance. Thus, these data underscore the important role of BNP on collagen degradation and inhibition of cardiac fibrosis.
More recently, Kapoun and colleagues reported further evidence of the potent antifibrotic effects of BNP in CFs.70Recognizing the reported properties of BNP in CFs and because transforming growth factor (TGF)-β is associated with profibrotic processes, they tested whether BNP could inhibit TGF-β-induced effects on primary human CFs. Indeed, BNP inhibited TGF-β-induced cell proliferation and the production of collagen 1 and fibronectin. Complementary deoxyribonucleic acid (DNA) microarray analysis revealed that TGF-β, but not BNP treatment, resulted in a significant change in the ribonucleic acid profile. However, BNP treatment resulted in an 85% alteration of all TGF-β-regulated messenger ribonucleic acids. BNP opposed TGF-β-regulated genes related to fibrosis and inflammation, and these actions were linked to cGMP. These studies therefore demonstrated that BNP has direct effects on CFs to inhibit fibrotic responses by the extracellular signal-regulated kinase pathway via cGMP signaling, thus suggesting that BNP functions as an antifibrotic factor in the heart. Therefore, BNP may prevent cardiac fibrosis owing to these recently described actions on CFs that go beyond its well-characterized antiangiotensin and antialdosterone properties.
As previously discussed, the importance of the natriuretic peptides in controlling cardiac fibrosis is also underscored by a number of experimental studies in which the deletion of BNP leads to accelerated and enhanced cardiac fibrosis.71This report established that independent of increases in arterial pressure, genetic deletion of the BNP gene resulted in an increase in ventricular fibrosis. In this murine model, increases in afterload produced by aortic constriction also resulted in exaggerated cardiac fibrosis compared with wild-type animals. Most recently, targeted deletion of the NPR-A in the myocytes of a murine model was associated with cardiac hypertrophy and altered ventricular relaxation.72That BNP could play a key role in the heart as an antifibrotic factor has a major clinical impact because cardiac fibrosis is a hallmark of HF and has been one of the therapeutic targets of two landmark clinical trials in human HF.66,68Fujisaki and colleagues determined that the natriuretic peptides inhibited angiotensin II- or ET-1-induced increases in DNA synthesis.73Interestingly, BNP was approximately twofold more potent than ANP with respect to the inhibition of DNA synthesis. They also confirmed that intracellular cGMP formation plays an important role in the NP-mediated inhibition of DNA synthesis and pre pro endothelin 1 messenger ribonucleic acid expression in CFs. Most recently, Horio and colleagues reported that adult rat CFs express and secrete CNP and that CNP potentially regulates fibrosis in an autocrine fashion.74
Although the natriuretic peptides possess both direct antifibrotic and antialdosterone actions that are thought to be related to cGMP activation by pGC, an equally important question related to therapy for fibrosis is whether sGC activation—especially by novel orally available molecules—can be considered efficacious. Calderone and colleagues investigated the effect of ANP and the nitric oxide donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) on norepinephrine-stimulated neonatal rat myocytes and CFs.75Both SNAP and ANP significantly reduced norepinephrine-induced [3H]leucine incorporation in myocytes and [3H]thymidine in CFs. 8-Bromo-cGMP mimicked this action. These data strongly suggest that sGC can also be a target for inhibition of cardiac fibrosis. As discussed above, we recently reported the efficacious cardiovascular actions of a novel orally available sGC stimulator (BAY 41-2272) in experimental severe CHF.47The use of this molecule therefore provides the opportunity to clarify if the mechanism of cGMP activation is important in determining the antifibrotic actions of natriuretic peptides and provides an additional potential therapy.
In summary, ongoing research supports the emerging concept that the antifibrotic properties of cGMP-activating compounds such as the natriuretic peptides have unique multitissue actions that support their therapeutic properties in HF and cardiac fibrosis. First, a direct action on CF proliferation emerges as a seminal action. Second, the intrinsic natriuretic and diuretic properties owing to both vascular and renal actions that contribute to a decrease in myocardial load represent an additional favorable mechanism. Further, the known ability of the natriuretic peptides to retard activation of the RAAS and ET-1 is another humoral action that complements their cardiovascular protective properties. These properties strongly underscore the therapeutic utility of these proteins in cardiovascular disease management.
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