Abstract-This study tested the hypothesis that atrial natriuretic peptide has direct antihypertrophic actions on the heart by modulating expression of genes involved in cardiac hypertrophy and extracellular matrix production. Hearts of male, atrial natriuretic peptide-null and control wild-type mice that had been subjected to pressure overload after transverse aortic constriction and control unoperated hearts were weighed and subjected to microarray, Northern blot, and immunohistochemical analyses. Microarray and Northern blot analyses were used to identify genes that are regulated differentially in response to stress in the presence and absence of atrial natriuretic peptide. Immunohistochemical analysis was used to identify and localize expression of the protein products of these genes. Atrial natriuretic peptide-null mice demonstrated cardiac hypertrophy at baseline and an exaggerated hypertrophic response to transverse aortic constriction associated with increased expression of the extracellular matrix molecules periostin, osteopontin, collagen I and III, and thrombospondin, as well as the extracellular matrix regulatory proteins, matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-3, and the novel growth factor pleiotrophin compared with wild-type controls. These results support the hypothesis that atrial natriuretic peptide protects against pressure overload-induced cardiac hypertrophy and remodeling by negative modulation of genes involved in extracellular matrix deposition. Key Words: atrial natriuretic factor Ⅲ constriction Ⅲ aorta Ⅲ pressure overload Ⅲ hypertrophy, cardiac Ⅲ extracellular matrix Ⅲ growth substances A trial natriuretic peptide (ANP) inhibits cell growth and proliferation and induces apoptosis in a variety of cell types, including vascular smooth muscle cells (VSMCs) and cardiomyocytes. 1-3 Intracardiac ANP might also play an important autocrine/paracrine role in modulating cardiac remodeling under stress conditions and might protect against the development of pathologic cardiac hypertrophy. 4 -7 Synthesis and release of ANP in the heart are increased under stressful conditions such as pressure and volume overloadinduced pathologic cardiac hypertrophy, exercise-induced physiologic cardiac hypertrophy, heart failure, and hypoxic pulmonary hypertension. 1,2 Expression of ANP is inversely related to cardiac growth/hypertrophy. 5,8 -11 Transgenic mice overexpressing ANP have smaller hearts than do wild-type mice, and ANP gene delivery attenuates cardiac hypertrophy in spontaneously hypertensive rats. 5,7 Conversely, transgenic mice with homozygous disruption of the pro-ANP gene (Nppa -/-mice) or the natriuretic peptide receptor-A (NPR-A) gene (Npr1 -/-mice) exhibit cardiac hypertrophy at baseline that is out of proportion to the modest elevations in blood pressure (BP) observed in these models. 8 -11 Furthermore, in Npr1 -/-mice, pressure overload induced by transverse aortic constriction (TAC) results in a greater (55%) increase in left ventricular (LV) weight than in Np...

ANP-deficient mice exhibited an exaggerated increase in heart and LV weight in response to volume overload, which was not prevented by normalization of blood pressure. The findings suggest that ANP is an important physiologic modulator of the cardiac hypertrophy induced by volume overload.

Abstract-We hypothesized that a single copy of the proatrial natriuretic peptide gene (Nppa ϩ/Ϫ ) would not be adequate to protect heterozygous mice against exaggerated cardiac hypertrophy and remodeling after pressure-overload stress. Nppa ϩ/ϩ , Nppa ϩ/Ϫ , and Nppa Ϫ/Ϫ mice were subjected to sham surgery or transverse aortic constriction and fed a basal salt diet. Heart weight varied inversely with Nppa gene load by 1 week after either surgery. Fractional shortening did not differ among genotypes at baseline and fell in Nppa Ϫ/Ϫ mice only after transverse aortic constriction. There was a graded response in collagen deposition related to atrial natriuretic peptide (ANP) expression after either surgery. A robust interstitial and perivascular fibrosis was noted in Nppa Ϫ/Ϫ and Nppa ϩ/Ϫ but not in Nppa ϩ/ϩ mice after transverse aortic constriction. Our findings are consistent with a growing body of evidence that ANP is an important modulator of cardiac hypertrophy and remodeling in response to hemodynamic stress. The observation that partial ANP deficiency results in exaggerated hypertrophy and remodeling after pressure overload suggests that genetic or environmental variation in ANP levels may play a role in the development of cardiac hypertrophy, remodeling, and failure in humans. Key Words: atrial natriuretic factor Ⅲ natriuretic peptides Ⅲ receptors, atrial natriuretic factor Ⅲ hypertrophy, cardiac Ⅲ remodeling Ⅲ extracellular matrix Ⅲ collagen R ecent studies suggest that atrial natriuretic factor (ANP) is an autocrine/paracrine modulator of cardiac hypertrophy and remodeling in response to pathologic stimuli. [1][2][3][4][5][6][7][8][9][10] Mice with homozygous deletion of the pro-ANP gene (Nppa Ϫ/Ϫ ) or the natriuretic peptide receptor-A gene (Npr1 Ϫ/Ϫ ) exhibit cardiac hypertrophy under resting conditions 3,5,11-13 and develop exaggerated hypertrophy after volume or pressure overload. 6,12,13 Furthermore, these studies raise the question as to whether variation in ANP response to hemodynamic stress is an important mediator of cardiac hypertrophy and remodeling in human hypertension and heart failure. Although studies in Nppa Ϫ/Ϫ mice have clearly demonstrated the adverse effect of ANP deletion on cardiac hypertrophy and remodeling, the effect of a modest ANP deficiency on the development of cardiac hypertrophy, remodeling, and failure remains unknown.ANP-heterozygous mice (Nppa) have normal blood pressure on either a normal-(0.5% NaCl) or an intermediate-salt (2% NaCl) diet, 6,14 in contrast to the hypertension observed in Nppa Ϫ/Ϫ mice under these conditions. 11 On a very-high-salt (8% NaCl) diet, however, Nppa ϩ/Ϫ mice develop hypertension. 6 The cardiac phenotype of the heterozygous ANP-knockout has not been rigorously studied under either basal or stress conditions. However, in their original report of this model, John et al 6 did not find a significant difference in cardiac weight between Nppa ϩ/ϩ and Nppa ϩ/Ϫ mice. The relatively normal cardiac phenotype in the heterozygous ANP-knockout might indicate...

Summary 1. Homozygous deletion of the pro‐atrial natriuretic peptide (Nppa) gene (ANP–/–) has been associated with both cardiac hypertrophy and salt‐sensitive hypertension in mice, suggesting that cardiac hypertrophy in ANP–/– mice may be related, at least in part, to increased afterload. 2. To test the hypothesis that cardiac hypertrophy in ANP–/– mice is independent of blood pressure, male ANP–/– and wild‐type ANP+/+ mice were fed a low (0.05%) or basal (0.55%) NaCl diet. Five weeks later, mean arterial pressure (MAP) was measured in conscious mice; the whole heart, atria, left and right ventricles (LV and RV, respectively), brain, lung, kidney, liver and spleen were weighed and fixed for histological analysis. Separate groups of mice were subjected to echocardiographic examination under tribromoethanol anaesthesia. 3. Mean arterial pressure and atrial, LV and RV mass were greater in ANP–/– mice than in ANP+/+ mice fed the basal salt diet. Salt depletion equalized MAP in the two genotypes, but did not alter the relative cardiac hypertrophy in ANP–/– mice. The ANP–/– mice had significant LV cardiomyocyte hypertrophy when fed either basal or low‐salt diets. 4. Left ventricle chamber dimensions did not differ between genotypes, but were significantly reduced in mice fed the low‐salt diet; LV posterior wall and septal thickness were greater in ANP–/– than ANP+/+ mice and were not altered by diet, indicating a concentric pattern of LV hypertrophy in ANP–/– mice. Left ventricle function (cardiac output, stroke volume, ejection fraction, circumferential wall stress and velocity of circumferential wall shortening) did not differ between strains on either diet; circumferential wall stress was reduced in the low‐salt groups; other functional parameters were not altered by diet. 5. These findings indicate that ANP deletion results in cardiomyocyte hypertrophy and biventricular hypertrophy independent of blood pressure, supporting the concept that ANP has direct antihypertrophic effects in the heart.

Regulation of blood pressure by angiotensin II (ANG II) is a process that involves the reactive oxygen species (ROS) and calcium. We have shown that ANG-II type 1 receptor (AT1R) and prostaglandin E2 (PGE2) type 1 receptors (EP1R) are required in the subfornical organ (SFO) for ROS-mediated hypertension induced by slow-pressor ANG-II infusion. However, the signaling pathway associated with this process remains unclear. We sought to determine mechanisms underlying the ANG II-induced ROS and calcium influx in mouse SFO cells. Ultrastructural studies showed that cyclooxygenase 1 (COX-1) codistributes with AT1R in the SFO, indicating spatial proximity. Functional studies using SFO cells revealed that ANG II potentiated PGE2 release, an effect dependent on AT1R, phospholipase A2 (PLA2) and COX-1. Furthermore, both ANG II and PGE2 increased ROS formation. While the increase in ROS initiated by ANG II, but not PGE2, required the activation of the AT1R/PLA2/COX-1 pathway, both ANG II and PGE2 were dependent on EP1R and Nox2 as downstream effectors. Finally, ANG II potentiated voltage-gated L-type Ca(2+) currents in SFO neurons via the same signaling pathway required for PGE2 production. Blockade of EP1R and Nox2-derived ROS inhibited ANG II and PGE2-mediated Ca(2+) currents. We propose a mechanism whereby ANG II increases COX-1-derived PGE2 through the AT1R/PLA2 pathway, which promotes ROS production by EP1R/Nox2 signaling in the SFO. ANG II-induced ROS are coupled with Ca(2+) influx in SFO neurons, which may influence SFO-mediated sympathoexcitation. Our findings provide the first evidence of a spatial and functional framework that underlies ANG-II signaling in the SFO and reveal novel targets for antihypertensive therapies.

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1. Atrial natriuretic peptide (ANP)-null mice (Nppa(-/-)) exhibit cardiac hypertrophy at baseline and adverse cardiac remodelling in response to transverse aortic constriction (TAC)-induced pressure overload stress. Previous studies have suggested that natriuretic peptides could potentially oppose mineralocorticoid signalling at several levels, including suppression of adrenal aldosterone production, inhibition of mineralocorticoid receptor (MR) activation or suppression of MR-mediated production of pro-inflammatory factors. Thus, we hypothesized that the MR blocker eplerenone would prevent the exaggerated left ventricular (LV) remodelling/fibrosis and dysfunction after TAC in Nppa(-/-). 2. In the present study, Nppa(-/-) and wild-type Nppa(+/+) mice fed eplerenone- or vehicle (oatmeal)-supplemented chow since weaning were subjected to TAC or sham operation. The daily dose of eplerenone administered was approximately 200 mg/kg. At 1 week after TAC, LV size and function were evaluated by echocardiogram and LV cross-sections were stained with picrosirius red for collagen volume measurement. Total RNA was extracted from the LV for real-time polymerase chain reaction analysis of osteopontin. 3. Eplerenone had no effect on baseline hypertrophy observed in sham-operated Nppa(-/-) compared with Nppa(+/+) mice. Eplerenone attenuated the TAC-induced increase in LV weight in both genotypes and completely prevented LV dilation, systolic dysfunction and interstitial collagen deposition seen in Nppa(-/-) mice after TAC. However, serum aldosterone levels were lower in Nppa(-/-) compared with Nppa(+/+) wild types. No interaction between eplerenone and genotype in osteopontin mRNA levels was observed. 4. Eplerenone prevents adverse cardiac remodelling related to pressure overload in ANP-deficient mice, mainly due to an antifibrotic effect. The mechanism whereby ANP deficiency leads to excess hypertrophy, fibrosis and early failure following TAC is increased profibrotic signals resulting from excess or unopposed MR activation, rather than increased levels of aldosterone.

Cardiac microvascular injury often occurs in patients with type 2 diabetes mellitus (T2DM) who develop hyperglycemia and hyperlipidemia. However, besides reported contradictory roles in cardiac diseases, the function of TRPV1 (transient receptor potential vanilloid 1) in cardiac microvessels is not well defined. This study was performed to determine the detailed role of TRPV1 in cardiac microvascular endothelial cells (CMECs) in T2DM. T2DM mice were established by multiple injections of low-dose streptozotocin and high-fat feeding. CMECs were cultured separately in mediums of normal glucose, high glucose (HG), high fatty acid (HF), and HG plus HF (HG-HF). HG-HF inhibited TRPV1 expression in CMECs, reducing cellular Ca content ([Ca]). T2DM impaired cardiac function, disturbed glucose uptake, and damaged microvascular barrier, which were further aggravated by TRPV1 Exposure to HG-HF, particularly in TRPV1 CMECs, led to a higher level of apoptosis and a lower level of nitric oxide production in viable CMECs. HG-HF markedly enhanced generation of reactive oxygen species and nitrotyrosine, especially in the absence of TRPV1. HO administration reduced TRPV1 expression in CMECs. HG-HF significantly depressed expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) and OPA1 (optic atrophy 1) by reducing [Ca], whereas OPA1 supplementation partly reversed those detrimental effects induced by TRPV1 Furthermore, capsaicin treatment not only attenuated CMECs injury induced by HG-HF but also mitigated cardiac microvascular injury induced by T2DM. Collectively, T2DM leads to cardiac microvascular injury by exacerbating the vicious circle of TRPV1 blockage and reactive oxygen species overload. Long-term capsaicin can protect cardiac microvessels against T2DM via suppressing oxidative/nitrative stress mediated by TRPV1/Ca/PGC-1α/OPA1 pathway in CMECs.