Expression of constitutively active guanylate cyclase in cardiomyocytes inhibits the hypertrophic effects of isoproterenol and aortic constriction on mouse hearts

Evidence from several rodent models has suggested that a reduction of either atrial natriuretic peptide or its receptor in the heart affects cardiac remodeling by promoting the onset of cardiac hypertrophy. The atrial natriuretic peptide receptor mediates signaling at least in part via the generation of intracellular cyclic GMP. To directly test whether accumulation of intracellular cyclic GMP conveys protection against cardiac hypertrophy, we engineered transgenic mice that overexpress a catalytic fragment of constitutively active guanylate cyclase domain of the atrial natriuretic peptide receptor in a cardiomyocyte-specific manner. Expression of the transgene increased the intracellular concentration of cyclic GMP specifically within cardiomyocytes and had no detectable effect on cardiac performance under basal conditions. However, expression of the transgene attenuated the effects of the pharmacologic hypertrophic agent isoproterenol on cardiac wall thickness and prevented the onset of the fetal gene expression program normally associated with cardiac hypertrophy. Likewise, expression of the transgene inhibited the hypertrophic effects of abdominal aortic constriction, since it abolished its effects on ventricular wall thickness and greatly attenuated its effects on cardiomyocyte size. Altogether, our results suggest that cyclic GMP is a cardioprotective agent against hypertrophy that acts via a direct local effect on cardiomyocytes.     

Upregulation of adrenomedullin and its receptor components during cardiomyocyte hypertrophy induced by chronic inhibition of nitric oxide synthesis in rats

Adrenomedullin may provide a compensatory mechanism to attenuate left ventricular hypertrophy (LVH). Nitric oxide synthase inhibition, induced by chronic administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats, induces cardiac hypertrophy in some, but not all cases; there are few reports of direct assessment of cardiomyocyte parameters. The objective was to characterize hypertrophic parameters in left (LV) and right ventricular (RV) cardiomyocytes after administration of L-NAME to rats for 8 wk and to determine whether adrenomedullin and its receptor components were upregulated. After treatment with L-NAME (20 and 50 mg x kg(-1) x day(-1)), compared with nontreated animals, 1) systolic blood pressure increased (by 34.2 and 104.9 mmHg), 2) heart weight-to-body wt ratio increased 24.1% at the higher dose (P < 0.05), 3) cardiomyocyte protein mass increased (P = NS), 4) cardiomyocyte protein synthesis ([14C]phenylalanine incorporation) increased (P < 0.05), 5) expression of skeletal alpha-actin, atrial natriuretic peptide, brain natriuretic peptide, and ET-1 mRNAs was enhanced (P < 0.05) in LV but not RV cardiomyocytes at 20 and 50 mg x kg(-1) x day(-1), respectively, and 6) expression of adrenomedullin, receptor activity-modifying protein 3 (RAMP3), and RAMP2 (but not calcitonin receptor-like receptor and RAMP1) mRNAs was increased by L-NAME (20 mg x kg(-1) x day(-1)) in LV. In conclusion, L-NAME enhanced protein synthesis in both LV and RV cardiomyocytes but elicited a hypertrophic phenotype accompanied by altered expression of the counterregulatory peptide adrenomedullin and receptor components (RAMP2, RAMP3) in LV only, indicating that the former is due to impaired nitric oxide synthesis, whereas the phenotypic changes are due to pressure overload.     

Urotensin II and urocortin trigger the expression of myostatin, a negative regulator of cardiac growth, in cardiomyocytes

Urotensin II (UII) and urocortin (UCN) are potent contributors to the physiopathology of heart failure. Our study investigated the effects of UII and UCN on the expression of myostatin (Mstn) in primary culture of adult cardiomyocytes. Adult rat cardiomyocytes were stimulated for 48 h with UII and UCN. Cell size and protein content were determined. Mstn gene expression was determined by real time quantitative polymerase chain reaction. Treatment with UII and UCN stimulates hypertrophy of adult cardiomyocytes. This effect was associated with a twofold increase of Mstn gene expression. We have established for the first time that the two hypertrophic peptides UII and UCN stimulate the expression of Mstn.     

Natriuretic peptide system: a link between fat mass and cardiac hypertrophy and hypertension in fat-fed female rats

The present study was designed to develop an animal model of hypertension and cardiac hypertrophy associated with obesity in female rats. Furthermore, we studied the involvement of the natriuretic peptide system in the mechanisms of these conditions. Obesity was induced in Wistar rats by a high fat diet and ovariectomy. The rats were divided into four groups: ovariectomized or sham-operated with high-fat diet and ovariectomized or sham-operated with control diet. After 24 weeks of diet, rats were killed, and their tissues were removed. Cardiac atrial natriuretic peptide (ANP), clearance receptor (NPr-C) gene expression was determined by PCR. ANP concentrations were measured in plasma. Ovariectomized fat-fed rats (OF) showed increased body weight, visceral fat depot and blood pressure and decreased sodium excretion compared to other groups. Also, these rats showed higher heart-to-body weight and cell diameters of ventricular cardiomyocytes and lower cardiac ANP mRNA and plasma ANP than the control group. The adipocyte and renal NPr-C mRNA of OF rats were higher than the control group. These data showed that combined ovariectomy and high fat diet elicited obesity, hypertension and cardiac hypertrophy. These results suggest that the impairment of the natriuretic peptide system may be one of the mechanisms involved not only in development of hypertension but also in cardiac hypertrophy associated with obesity in ovariectomized rats.     

Salvianolic acid B protects cardiomyocytes from angiotensin II-induced hypertrophy via inhibition of PARP-1

Salvianolic acid B (SalB), one of the major bioactive components in Salviamiltiorrhiza, has plenty of cardioprotective effects. The present study was designed to investigate the effect of SalB on angiotensin II (AngII)-induced hypertrophy in neonatal rat cardiomyocytes, and to find out whether or not this effect is attributed to inhibition of poly (ADP-ribose) polymerase-1 (PARP-1), which plays a key role in cardiac hypertrophy. Our results showed that SalB prevented the cardiomyocytes from AngII-induced hypertrophy, associated with attenuation of the mRNA expressions of atrial natriuretic factor and brain natriuretic peptide, and reduction in the cell surface area. SalB inhibited the activity of PARP-1. The inhibitory effect was comparable to that of the PARP-1 inhibitor 3-Aminobenzamide (3-AB). In addition, SalB reversed the depletion of cellular NAD⁺ induced by AngII. Moreover, overexpression of PARP-1 attenuated the anti-hypertrophic effect of SalB. These observations suggested that SalB prevented the cardiomyocytes from AngII-induced hypertrophy, at least partially through inhibition of PARP-1. Moreover, SalB attenuated the generation of oxidative stress via suppression of NADPH oxidase 2 and 4, which might probably contribute to the inhibition of PARP-1. These present findings may shed new light on the understanding of the cardioprotective effect of SalB.     

Cardiovascular role of urotensin II: effect of chronic infusion in the rat

Urotensin II (UII) is a potent vaso-active peptide thought to have multiple roles in the regulation of cardiovascular physiology and pathophysiology. The actions of UII are complex and difficult to interpret given its systemic hemodynamic effects and variable action on different vascular beds and isolated vessels. Direct effects of UII on the myocardium, include myocyte hypertrophy, extracellular matrix deposition and contractility. These observations, together with elevated plasma levels found in disease, are common traits reported in other pathophysiologically implicated neurohormonal systems. In this review, we include original data obtained from chronic infusion of UII in rats. We report a reduction in first derivative of left ventricular pressure (+dP/dt), as well as an increase in the ratio of left ventricular collagen I:III, that may contribute to the reduced myocardial contractility observed in these animals.     

Hydrogen sulphide inhibits cardiomyocyte hypertrophy by up-regulating miR-133a

Hydrogen sulphide (H₂S) has been shown to play a crucial role in cardiovascular physiology and disease. However, there is no information about the possible role of H₂S in cardiomyocyte hypertrophy (CH). Our results showed that pretreatment with NaHS, an H₂S donor, significantly reduced [³H]-leucine incorporation, cell surface area, mRNA expression of brain natriuretic peptide (BNP), intracellular reactive oxygen species (ROS), miR-21 and increased atrial natriuretic peptide (ANP) and miR-133a expression in hypertrophic cardiomyocytes. Anti-miR133a inhibitor transfection partly reduced the anti-hypertrophic effect of NaHS. In conclusion, H₂S is a direct inhibitor of CH; it acts by increasing miR-133a and inhibiting the increase in intracellular ROS.     

Cardiac-specific attenuation of natriuretic peptide A receptor activity accentuates adverse cardiac remodeling and mortality in response to pressure overload

Atrial (ANP) and brain (BNP) natriuretic peptides are hormones of myocardial cell origin. These hormones bind to the natriuretic peptide A receptor (NPRA) throughout the body, stimulating cGMP production and playing a key role in blood pressure control. Because NPRA receptors are present on cardiomyocytes, we hypothesized that natriuretic peptides may have direct autocrine or paracrine effects on cardiomyocytes or adjacent cardiac cells. Because both natriuretic peptides and NPRA gene expression are upregulated in states of pressure overload, we speculated that the effects of the natriuretic peptides on cardiac structure and function would be most apparent after pressure overload. To attenuate cardiomyocyte NPRA activity, transgenic mice with cardiac specific expression of a dominant-negative (DN-NPRA) mutation (HCAT D 893A) in the NPRA receptor were created. Cardiac structure and function were assessed (avertin anesthesia) in the absence and presence of pressure overload produced by suprarenal aortic banding. In the absence of pressure overload, basal and BNP-stimulated guanylyl cyclase activity assessed in cardiac membrane fractions was reduced. However, systolic blood pressure, myocardial cGMP, log plasma ANP levels, and ventricular structure and function were similar in wild-type (WT-NPRA) and DN-NPRA mice. In the presence of pressure overload, myocardial cGMP levels were reduced, and ventricular hypertrophy, fibrosis, filling pressures, and mortality were increased in DN-NPRA compared with WT-NPRA mice. In addition to their hormonal effects, endogenous natriuretic peptides exert physiologically relevant autocrine and paracrine effects via cardiomyocyte NPRA receptors to modulate cardiac hypertrophy and fibrosis in response to pressure overload.     

Direct, autocrine and paracrine effects of cyclic stretch on growth of myocytes and fibroblasts isolated from neonatal rat ventricles

Several studies have demonstrated that static stretch of cardiomyocytes induces cardiomyocyte hypertrophy. We investigated the effects of cyclic stretch, a more physiological stimulus, on protein synthesis and DNA synthesis of rat ventricular cardiomyocytes and cardiofibroblasts. Further-more, we investigated whether these effects are caused by autocrine mechanisms. In addition, we studied the paracrine influences of stretched cardiofibroblasts on cardiomyocyte growth. Short-term cyclic stretch (0-24 h) of cardiomyocytes induced a growth response indicative of cardiomyocyte hypertrophy, given the fact that increased rates of protein synthesis and DNA synthesis were accompanied by an elevated release of atrial natriuretic peptide into the culture medium. In cardiofibroblasts, short-term cyclic stretch also induced a growth response as indicated by an increased rate of protein synthesis and DNA synthesis. Furthermore, incubation of stationary cardiofibroblasts with conditioned medium derived from stretched cardiofibroblasts revealed an autocrine effect of stretch as illustrated by an increased rate of protein synthesis in stationary cardiofibroblasts. In analogy, there was an autocrine effect of stretch on stationary cardiomyocytes incubated with conditioned medium derived from stretched cardiomyocytes. Moreover, we observed a paracrine effect of the conditioned medium derived from stretched cardiofibroblasts on stationary cardiomyocytes. Thus, short-term cyclic stretch of cardiomyocytes and cardiofibroblasts induces growth responses that are the result of direct, autocrine, and paracrine effects. These autocrine/paracrine effects of stretch are most probably due to release of factors from stretched cells.     

Circulating urotensin II levels in moderate to severe congestive heart failure: its relations with myocardial function and well established neurohormonal markers

Urotensin II (UII) is a potent vasoactive cyclic peptide thought to play a role in myocardial hypertrophy and remodelling. We therefore determined UII plasma levels in congestive heart failure (CHF) patients and its relationship with the severity of the disease and well-established markers of left ventricular function. UII was significantly higher in CHF patients (n = 57) than in controls (n = 48) [geometric mean (pg/ml), 95% PI: 1.32 (0.67-2.59) versus 0.84 (0.31-1.61), p < 0.0001], was related to the functional class of the disease and correlated negatively with left ventricular ejection fraction (r = -0.316, P = 0.016). Furthermore, UII correlated significantly with Big-ET1 (r = 0.32, p = 0.03), BNP (r = 0.42, p = 0.005) but poorly with Nt-proANP (r = 0.28, p = 0.07). Our results suggest that UII could play a role in worsening the course of congestive heart failure and is associated with established markers of cardiovascular dysfunction.     

Cytosolic CARP Promotes Angiotensin II- or Pressure Overload-Induced Cardiomyocyte Hypertrophy through Calcineurin Accumulation

The gene ankyrin repeat domain 1 (Ankrd1) is an enigmatic gene and may exert pleiotropic function dependent on its expression level, subcellular localization and even types of pathological stress, but it remains unclear how these factors influence the fate of cardiomyocytes. Here we attempted to investigate the role of CARP on cardiomyocyte hypertrophy. In neonatal rat ventricular cardiomyocytes (NRVCs), angiotensin II (Ang II) increased the expression of both calpain 1 and CARP, and also induced cytosolic translocation of CARP, which was abrogated by a calpain inhibitor. In the presence of Ang-II in NRVCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced myocyte hypertrophy, the upregulation of atrial natriuretic peptide and β-myosin heavy chain genes and calcineurin proteins as well as nuclear translocation of nuclear factor of activated T cells. Cyclosporin A attenuated Ad-Ankrd1-enhanced cardiomyocyte hypertrophy. Intra-myocardial injection of Ad-Ankrd1 in mice with transverse aortic constriction (TAC) markedly increased the cytosolic CARP level, the heart weight/body weight ratio, while short hairpin RNA targeting Ankrd1 inhibited TAC-induced hypertrophy. The expression of calcineurin was also significantly increased in Ad-Ankrd1-infected TAC mice. Olmesartan (an Ang II receptor antagonist) prevented the upregulation of CARP in both Ang II-stimulated NRVCs and hearts with pressure overload. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac remodeling through the enhancement of cytosolic translocation of CARP and upregulation of calcineurin.     

The role of angiotensin II,endothelin-1 and transforming growth factor-β as autocrine/paracrine mediators of stretch-induced cardiomyocyte hypertrophy

Cardiac hypertrophy is a compensatory response of myocardial tissue upon increased mechanical load. Of the mechanical factors, stretch is rapidly followed by hypertrophic responses. We tried to elucidate the role of angiotensin II (AII), endothelin-1 (ET-1) and transforming growth factor- (TGF-) as autocrine/paracrine mediators of stretch-induced cardiomyocyte hypertrophy. We collected conditioned medium (CM) from stretched cardiomyocytes and from other stretched cardiac cells, such as cardiac fibroblasts, endothelial cells and vascular smooth muscle cells (VSMCs). These CMs were administered to stationary cardiomyocytes with or without an AII type 1 (AT₁) receptor antagonist (losartan), an ET-1 type A (ET_A) receptor antagonist (BQ610), or anti-TGF- antibodies. By measuring the mRNA levels of the proto-oncogene c-fos and the hypertrophy marker gene atrial natriuretic peptide (ANP), the molecular phenotype of the CM-treated stationary cardiomyocytes was characterized.Our results showed that c-fos and ANP expression in stationary cardiomyocytes was increased by AII release from cardiomyocytes that had been stretched for 60 min. Stretched cardiomyocytes, cardiac fibroblasts and endothelial cells released ET-1 which led to increased c-fos and ANP expression in stationary cardiomyocytes. ET-1 released by stretched VSMCs, and TGF- released by stretched cardiac fibroblasts and endothelial cells, appeared to be paracrine mediators of ANP expression in stationary cardiomyocytes.These results indicate that AII, ET-1 and TGF- (released by cardiac and vascular cell types) act as autocrine/paracrine mediators of stretch-induced cardiomyocyte hypertrophy. Therefore, it is likely that in stretched myocardium the cardiomyocytes, cardiac fibroblasts, endothelial cells and VSMCs take part in intercellular interactions contributing to cardiomyocyte hypertrophy.     

The role of iNOS-derived NO in the antihypertrophic actions of B-type natriuretic peptide in neonatal rat cardiomyocytes

In the infarcted rat heart, the increase of NO occurs in the hypertrophied myocardium of non-infarcted areas and its antihypertrophic efficacy has been well established. As another endogenous regulator and the reliable index of heart pathology, B-type natriuretic peptide also exhibits the antihypertrophic properties in many tissues by elevating intracellular cGMP. Several studies indicate that natriuretic peptides family may exert some actions in part via a nitric oxide pathway following receptor-mediated stimulation of iNOS. Therefore, it raises our great interest to ask what role NO plays in the antihypertrophic actions of B-type natriuretic peptide in cardiomyocytes. Incubation of cardiomyocytes under mild hypoxia for 12 h caused a significant increase in cellular protein content, protein synthesis and cell surface sizes. This growth stimulation was suppressed by exogenous B-type natriuretic peptide in a concentration dependent manner. Furthermore, the generation of intracellular cGMP, the upregulation of iNOS mRNA expression, the increase of iNOS activity and subsequent nitrite generation in hypertrophic cardiomyocytes was also increased by B-type natriuretic peptide. AG, a selective iNOS inhibitor, inhibited the upregulation of iNOS expression and the increase of iNOS activity by the combination of B-type natriuretic peptide/mild hypoxia or by the combination of 8-bromo-cGMP/mild hypoxia. Rp-8-br-cGMP, cGMP dependent protein kinase inhibitor, attenuated the actions of B-type natriuretic peptide and 8-bromo-cGMP which increases intracellular cGMP independent of B-type natriuretic peptide. In conclusion, our present data suggest that B-type natriuretic peptide exerted the antihypertrophic effects in cardiomyocytes, which was partially attributed to induction of iNOS-derived NO by cGMP pathway.     

Cytosolic Dynamics of Annexin A6 Trigger Feedback Regulation of Hypertrophy via Atrial Natriuretic Peptide in Cardiomyocytes

Malfunctions in regulatory pathways that control cell size are prominent in pathological cardiac hypertrophy. Here, we show annexin A6 (Anxa6) to be a crucial regulator of atrial natriuretic peptide (ANP)-mediated counterhypertrophic responses in cardiomyocytes. Adrenergic stimulation of H9c2 cardiomyocytes by phenylephrine (PE) increased the cell size with enhanced expression of biochemical markers of hypertrophy, concomitant with elevated expression and subcellular redistribution of Anxa6. Stable cell lines with controlled increase in Anxa6 levels were protected against PE-induced adverse changes, whereas Anxa6 knockdown augmented the hypertrophic responses. Strikingly, Anxa6 knockdown also abrogated PE-induced juxtanuclear accumulation of secretory granules (SG) containing ANP propeptides (pro-ANP), a signature of maladaptive hypertrophy having counteractive functions. Mechanistically, PE treatment prompted a dynamic association of Anxa6 with pro-ANP-SG, parallel to their participation in anterograde traffic, in an isoform-specific fashion. Moreover, Anxa6 mutants that failed to associate with pro-ANP hindered ANP-mediated protection against hypertrophy, which was rescued, at least partially, by WT Anxa6. Additionally, elevated intracellular calcium (Ca²⁺) stimulated Anxa6-pro-ANP colocalization and membrane association. It also rescued pro-ANP translocation in cells expressing an Anxa6 mutant (Anxa6^ΔC). Furthermore, stable overexpression of Anxa6^T356D, a mutant with superior flexibility, provided enhanced protection against PE, compared with WT, presumably due to enhanced membrane-binding capacity. Together, the present study delivers a cooperative mechanism where Anxa6 potentiates ANP-dependent counterhypertrophic responses in cardiomyocytes by facilitating regulated traffic of pro-ANP.     

Atrial natriuretic peptide inhibits cardiomyocyte hypertrophy through mitogen-activated protein kinase phosphatase-1

Cardiac hypertrophy is formed in response to hemodynamic overload. Although a variety of factors such as catecholamines, angiotensin II (AngII), and endothelin-1 (ET-1) have been reported to induce cardiac hypertrophy, little is known regarding the factors that inhibit the development of cardiac hypertrophy. Production of atrial natriuretic peptide (ANP) is increased in the hypertrophied heart and ANP has recently been reported to inhibit the growth of various cell types. We therefore examined whether ANP inhibits the development of cardiac hypertrophy. Pretreatment of cultured cardiomyocytes with ANP inhibited the AngII- or ET-1-induced increase in the cell size and the protein synthesis. ANP also inhibited the AngII- or ET-1-induced hypertrophic responses such as activation of mitogen-activated protein kinase (MAPK) and induction of immediate early response genes and fetal type genes. To determine how ANP inhibits cardiomyocyte hypertrophy, we examined the mechanism of ANP-induced suppression of the MAPK activation. ANP strongly induced expression of MAPK phosphatase-1 (MKP-1) and overexpression of MKP-1 inhibited AngII- or ET-1-induced hypertrophic responses. These growth-inhibitory actions of ANP were mimicked by a cyclic GMP analog 8-bromo-cyclic GMP. Taken together, ANP directly inhibits the growth factor-induced cardiomyocyte hypertrophy at least partly via induction of MKP-1. Our present study suggests that the formation of cardiac hypertrophy is regulated not only by positive but by negative factors in response to hemodynamic load.