Prevention of concentric hypertrophy and diastolic impairment in aortic-banded rats treated with resveratrol

This study was designed to examine the effects of the antioxidant resveratrol on cardiac structure and function in pressure overload (PO)-induced cardiac hypertrophy. Male Sprague-Dawley rats were subjected to sham operation and the aortic banding procedure. A subgroup of sham control and aortic-banded rats were treated with resveratrol for 2 wk after surgery. Echocardiographic analysis of cardiac structure and function along with Western blot analysis of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and redox factor-1 (ref-1) were performed in all groups after 4 wk of surgery. Banded rats showed significantly increased left ventricle-to-body weight ratio. Echocardiographic analysis showed that the interventricular septal wall thickness and left ventricular posterior wall thickness at systole and diastole were significantly increased in banded rats. Also, a significant increase in isovolumic relaxation time was observed in banded rats. Measured eNOS, iNOS, and ref-1 protein levels were significantly reduced in banded rats. Resveratrol treatment prevented the above changes in cardiac structure, function, and protein expression in banded rats. Aortic banding after 4 wk resulted in concentric remodeling and impaired contractile function due to PO on the heart. The 2-wk treatment with resveratrol was found to abolish PO-induced cardiac hypertrophy. Resveratrol may therefore be beneficial against PO-induced cardiac hypertrophy found in clinical settings of hypertension and aortic valve stenosis.     

Attenuation of cardiac hypertrophy by inhibiting both mTOR and NFkappaB activation in vivo

A role for the PI3K/Akt/mTOR pathway in cardiac hypertrophy has been well documented. We reported that NFκB activation is needed for cardiac hypertrophy in vivo. To investigate whether both NFκB activation and PI3K/Akt/mTOR signaling participate in the development of cardiac hypertrophy, two models of cardiac hypertrophy, namely, induction in caAkt-transgenic mice and by aortic banding in mice, were employed. Rapamycin (2 mg/kg/daily), an inhibitor of the mammalian target of rapamycin, and the antioxidant pyrrolidine dithiocarbamate (PDTC; 120 mg/kg/daily), which can inhibit NFκB activation, were administered to caAkt mice at 8 weeks of age for 2 weeks. Both rapamycin and PDTC were also administered to the mice immediately after aortic banding for 2 weeks. Administration of either rapamycin or PDTC separately or together to caAkt mice reduced the ratio of heart weight/body weight by 21.54, 32.68, and 42.07% compared with untreated caAkt mice. PDTC administration significantly reduced cardiac NFκB activation by 46.67% and rapamycin significantly decreased the levels of p70S6K by 34.20% compared with untreated caAkt mice. Similar results were observed in aortic-banding-induced cardiac hypertrophy in mice. Our results suggest that both NFκB activation and the PI3K/Akt signaling pathway participate in the development of cardiac hypertrophy in vivo.     

Alteration of gene expression during progression of hypertension-induced cardiac dysfunction in rats

Hypertension induced by high-salt diet in Dahl salt-sensitive rats leads to compensatory cardiac hypertrophy by approximately 11 wk, cardiac dysfunction at approximately 17 wk, and death from cardiac dysfunction at approximately 21 wk. It is unclear what molecular hallmarks distinguish the compensatory hypertrophy from the decompensated cardiac dysfunction phase. Here we compared the gene expression in rat cardiac tissue from the compensatory hypertrophic phase (11 wk, n = 6) with the cardiac dysfunction phase (17 wk, n = 6) and with age-matched normotensive controls. Messenger RNA levels of 93 genes, selected based on predicted association with cardiac dysfunction, were measured by quantitative real-time PCR. In the hypertrophic phase, the expression of three genes, atrial natriuretic peptide (ANP; P = 0.0089), brain natriuretic peptide (P = 0.0012), and endothelin-1 precursor (P = 0.028), significantly increased, whereas there was decreased expression of 24 other genes including SOD2 (P = 0.0148), sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (P = 0.0002), and ryanodine receptor 2 (P = 0.0319). In the subsequent heart cardiac dysfunction phase, the expression of an additional 20 genes including inducible nitric oxide synthase (NOS; P = 0.0135), angiotensin I-converting enzyme (P = 0.0082), and IL-1beta (P < 0.0001) increased, whereas the expression of seven genes decreased compared with those of age-matched controls. Furthermore, the expression of 22 genes, including prepro-endothelin-1, ANP, angiotensin I-converting enzyme, beta(1)-adrenergic receptor, SOD2, and endothelial NOS, significantly changed in the cardiac dysfunction phase compared with the compensatory hypertrophic phase. Finally, principal component analysis successfully segregated animals with decompensatory cardiac dysfunction from controls, as well as from animals at the compensated hypertrophy phase, suggesting that we have identified molecular markers for each stage of the disease.     

Blockade of MyD88 attenuates cardiac hypertrophy and decreases cardiac myocyte apoptosis in pressure overload-induced cardiac hypertrophy in vivo

In this study, we evaluated whether blocking myeloid differentiation factor-88 (MyD88) could decrease cardiac myocyte apoptosis following pressure overload. Adenovirus expressing dominant negative MyD88 (Ad5-dnMyD88) or Ad5-green fluorescent protein (GFP) (Ad5-GFP) was transfected into rat hearts (n = 8/group) immediately followed by aortic banding for 3 wk. One group of rats (n = 8) was subjected to aortic banding for 3 wk without transfection. Sham surgical operation (n = 8) served as control. The ratios of heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) were calculated. Cardiomyocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. Cardiac myocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and myocardial interstitial fibrosis was examined by Masson's Trichrome staining. Aortic banding significantly increased the HW/BW by 41.0% (0.44 +/- 0.013 vs. 0.31 +/- 0.008), HW/TL by 47.2% (42.7 +/- 1.30 vs. 29.0 +/- 0.69), cardiac myocyte size by 49.6%, and cardiac myocyte apoptosis by 11.5%, and myocardial fibrosis and decreased cardiac function compared with sham controls. Transfection of Ad5-dnMyD88 significantly reduced the HW/BW by 18.2% (0.36 +/- 0.006 vs. 0.44 +/- 0.013) and HW/TL by 22.3% (33.2 +/- 0.95 vs. 42.7 +/- 1.30) and decreased cardiomyocyte size by 56.8%, cardiac myocyte apoptosis by 76.2%, as well as fibrosis, and improved cardiac function compared with aortic-banded group. Our results suggest that MyD88 is an important component in the Toll-like receptor-4-mediated nuclear factor-kappaB activation pathway that contributes to the development of cardiac hypertrophy. Blockade of MyD88 significantly reduced cardiac hypertrophy, cardiac myocyte apoptosis, and improved cardiac function in vivo.     

Pyrrolidine dithiocarbamate restores endothelial cell membrane integrity and attenuates monocrotaline-induced pulmonary artery hypertension

Monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) in rats is preceded by an inflammatory response, progressive endothelial cell membrane disruption, reduction in the expression of caveolin-1, and reciprocal activation of STAT3 (PY-STAT3). Superoxide and NF-kappaB have been implicated in PAH. To evaluate the role of caveolin-1, PY-STAT3 activation, and superoxide in PAH, MCT-injected rats were treated daily with pyrrolidine dithiocarbamate (PDTC; starting on days 1, 3, and 14 x 2 wk), an inhibitor of inflammation and NF-kappaB activation. Hemodynamic data, the expression of inhibitory (I)-kappaBalpha, caveolin-1, and Tie2 (a membrane protein), activation of PY-STAT3 and NF-kappaB, and superoxide chemiluminescence were examined. Rats developed progressive PAH at 2 wk post-MCT. There was progressive reduction in the expression of caveolin-1, Tie2, and activation of PY-STAT3 in the lungs. Reduction in I-kappaBalpha expression was present at 2 and 4 wk post-MCT. Superoxide chemiluminescence and NF-kappaB activation were observed only at 2 wk post-MCT and both decreased by 4 wk post-MCT despite progressive PAH. PDTC (starting on days 1 and 3) rescued caveolin-1 and Tie2, reversed MCT-induced PY-STAT3 activation, and attenuated PAH. In addition, PDTC restored I-kappaBalpha expression and reduced superoxide chemiluminescence at 2 wk but did not inhibit NF-kappaB activation despite attenuation of PAH. PDTC had no effect on established PAH. Increased superoxide chemiluminescence and NF-kappaB activation appear to be a transient phenomenon in the MCT model. Thus the disruption of endothelial cell membrane integrity resulting in caveolin-1 loss and reciprocal activation of PY-STAT3 plays a key role in the MCT-induced PAH.     

GH-releasing peptides improve cardiac dysfunction and cachexia and suppress stress-related hormones and cardiomyocyte apoptosis in rats with heart failure

Growth hormone (GH)-releasing peptides (GHRP), a class of synthetic peptidyl GH secretagogues, have been reported to exert a cardioprotective effect on cardiac ischemia. However, whether GHRP have a beneficial effect on chronic heart failure (CHF) is unclear, and the present work aims to clarify this issue. At 9 wk after pressure-overload CHF was created by abdominal aortic banding in rats, one of four variants of GHRP (GHRP-1, -2, and -6 and hexarelin, 100 mug/kg) or saline was injected subcutaneously twice a day for 3 wk. Echocardiography and cardiac catheterization were performed to monitor cardiac function and obtain blood samples for hormone assay. GHRP treatment significantly improved left ventricular (LV) function and remodeling in CHF rats, as indicated by increased LV ejection fraction, LV end-systolic pressure, and diastolic posterior wall thickness and decreased LV end-diastolic pressure and LV end-diastolic dimension. GHRP also significantly alleviated development of cardiac cachexia, as shown by increases in body weight and tibial length in CHF rats. Plasma CA, renin, ANG II, aldosterone, endothelin-1, and atrial natriuretic peptide were significantly elevated in CHF rats but were significantly decreased in GHRP-treated CHF rats. GHRP suppressed cardiomyocyte apoptosis and increased cardiac GH secretagogue receptor mRNA expression in CHF rats. GHRP also decreased myocardial creatine kinase release in hypophysectomized rats subjected to acute myocardial ischemia. We conclude that chronic administration of GHRP alleviates LV dysfunction, pathological remodeling, and cardiac cachexia in CHF rats, at least in part by suppressing stress-induced neurohormonal activations and cardiomyocyte apoptosis.     

Increased natriuretic peptide receptor A and C gene expression in rats with pressure-overload cardiac hypertrophy

Both atrial (ANP) and brain (BNP) natriuretic peptide affect development of cardiac hypertrophy and fibrosis via binding to natriuretic peptide receptor (NPR)-A in the heart. A putative clearance receptor, NPR-C, is believed to regulate cardiac levels of ANP and BNP. The renin-angiotensin system also affects cardiac hypertrophy and fibrosis. In this study we examined the expression of genes for the NPRs in rats with pressure-overload cardiac hypertrophy. The ANG II type 1 receptor was blocked with losartan (10 mg.kg(-1).day(-1)) to investigate a possible role of the renin-angiotensin system in regulation of natriuretic peptide and NPR gene expression. The ascending aorta was banded in 84 rats during Hypnorm/Dormicum-isoflurane anesthesia; after 4 wk the rats were randomized to treatment with losartan or placebo. The left ventricle of the heart was removed 1, 2, or 4 wk later. Aortic banding increased left ventricular expression of NPR-A and NPR-C mRNA by 110% (P < 0.001) and 520% (P < 0.01), respectively, after 8 wk; as expected, it also increased the expression of ANP and BNP mRNAs. Losartan induced a slight reduction of left ventricular weight but did not affect the expression of mRNAs for the natriuretic peptides or their receptors. Although increased gene expression does not necessarily convey a higher concentration of the protein, the data suggest that pressure overload is accompanied by upregulation of not only ANP and BNP but also their receptors NPR-A and NPR-C in the left ventricle.     

Cardiomyocyte-restricted inhibition of G protein-coupled receptor kinase-3 attenuates cardiac dysfunction after chronic pressure overload

Transgenic mice with cardiac-specific expression of a peptide inhibitor of G protein-coupled receptor kinase (GRK)3 [transgenic COOH-terminal GRK3 (GRK3ct) mice] display myocardial hypercontractility without hypertrophy and enhanced α(1)-adrenergic receptor signaling. A role for GRK3 in the pathogenesis of heart failure (HF) has not been investigated, but inhibition of its isozyme, GRK2, has been beneficial in several HF models. Here, we tested whether inhibition of GRK3 modulated evolving cardiac hypertrophy and dysfunction after pressure overload. Weight-matched male GRK3ct transgenic and nontransgenic littermate control (NLC) mice subjected to chronic pressure overload by abdominal aortic banding (AB) were compared with sham-operated (SH) mice. At 6 wk after AB, a significant increase of cardiac mass consistent with induction of hypertrophy was found, but no differences between GRK3ct-AB and NLC-AB mice were discerned. Simultaneous left ventricular (LV) pressure-volume analysis of electrically paced, ex vivo perfused working hearts revealed substantially reduced systolic and diastolic function in NLC-AB mice (n = 7), which was completely preserved in GRK3ct-AB mice (n = 7). An additional cohort was subjected to in vivo cardiac catheterization and LV pressure-volume analysis at 12 wk after AB. NLC-AB mice (n = 11) displayed elevated end-diastolic pressure (8.5 ± 3.1 vs. 2.9 ± 1.2 mmHg, P < 0.05), reduced cardiac output (3,448 ± 323 vs. 4,488 ± 342 μl/min, P < 0.05), and reduced dP/dt(max) and dP/dt(min) (both P < 0.05) compared with GRK3ct-AB mice (n = 16), corroborating the preserved cardiac structure and function observed in GRK3ct-AB hearts assessed ex vivo. Increased cardiac mass and myocardial mRNA expression of β-myosin heavy chain confirmed the similar induction of cardiac hypertrophy in both AB groups, but only NLC-AB hearts displayed significantly elevated mRNA levels of brain natriuretic peptide and myocardial collagen contents as well as reduced β(1)-adrenergic receptor responsiveness to isoproterenol, indicating increased LV wall stress and the transition to HF. Inhibition of cardiac GRK3 in mice does not alter the hypertrophic response but attenuates cardiac dysfunction and HF after chronic pressure overload.     

PYK2 expression and phosphorylation increases in pressure overload-induced left ventricular hypertrophy

Proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor protein tyrosine kinases. PYK2 has been implicated in linking G protein-coupled receptors to activation of mitogen-activated protein kinase cascades and cellular growth in a variety of cell types. To determine whether PYK2 expression and phosphorylation is altered in left ventricular (LV) myocardium undergoing LV hypertrophy (LVH) and heart failure in vivo, suprarenal abdominal aortic coarctation was performed in 160-g male Sprague-Dawley rats. Immunohistochemistry and Western blotting were performed on LV tissue 1, 8, and 24 wk after aortic banding. Aortic banding produced sustained hypertension and gradually developing LVH. PYK2 levels were increased 1.8 +/- 0.2-, 2.7 +/- 0.6-, and 2.0 +/- 0.2-fold in 1-, 8-, and 24-wk banded animals compared with their respective sham-operated controls. The increase in PYK2 expression was paralleled by an increase in PYK2 phosphorylation, both of which preceded the development of LVH. Immunohistochemistry revealed that enhanced PYK2 expression occurred predominantly in the cardiomyocyte population. Furthermore, there was a high degree of correlation (R = 0.75; P < 0.001) between the level of PYK2 and the degree of LVH in 24-wk sham and banded animals. In contrast, FAK levels and FAK phosphorylation were not increased before the development of LVH. However, there was a high degree of correlation (R = 0.68; P < 0.001) between the level of FAK and the degree of LVH in 24-wk sham and banded rats. There was also a significant increase in the ratio of phosphospecific anti-FAK to FAK at this time point. These data are consistent with a role for PYK2 in the induction of pressure overload-induced cardiomyocyte hypertrophy, and suggest that PYK2 and FAK have distinctly different roles in LVH progression.     

Urotensin II induces hypertrophic responses in cultured cardiomyocytes from neonatal rats.

Urotensin II (UII), a cyclic neuropeptide, functions not only in the central nervous system but also in non-neural systems including cardiovascular systems. In the present study we examined whether UII regulates hypertrophy in cardiomyocytes. The exposure of cultured cardiomyocytes from neonatal rats to UII dose-dependently activated extracellular signal-regulated kinases (ERKs), important molecules in the development of cardiac hypertrophy. ERK activation by UII at 100 nM peaked at 8 min after stimulation. UII markedly induced expression of specific genes encoding atrial natriuretic peptide and brain natriuretic peptide, and significantly increased amino acid incorporation into proteins. Incubation of cardiomyocytes with UII increased cell size and myofibril organisation. UII, then, might participate in cardiomyocyte hypertrophy.     

Chronic Akt blockade aggravates pathological hypertrophy and inhibits physiological hypertrophy

The attenuation of adverse myocardial remodeling and pathological left ventricular (LV) hypertrophy is one of the hallmarks for improving the prognosis after myocardial infarction (MI). The protein kinase Akt plays a central role in regulating cardiac hypertrophy, but the in vivo effects of chronic pharmacological inhibition of Akt are unknown. We investigated the effect of chronic Akt blockade with deguelin on the development of pathological [MI and aortic banding (AB)] and physiological (controlled treadmill running) hypertrophy. Primary cardiomyocyte cultures were incubated with 10 μmol deguelin for 48 h, and Wistar rats were treated orally with deguelin (4.0 mg·kg(-1)·day(-1)) for 4 wk starting 1 day after the induction of MI or AB. Exercise-trained animals received deguelin for 4 wk during the training period. In vitro, we observed reduced phosphorylation of Akt and glycogen synthase kinase (GSK)-3β after an incubation with deguelin, whereas MAPK signaling was not significantly affected. In vivo, treatment with deguelin led to attenuated phosphorylation of Akt and GSK-3β 4 wk after MI. These animals showed significantly increased heart weights and impaired LV function with increased end-diastolic diameters (12.0 ± 0.3 vs. 11.1 ± 0.3 mm, P < 0.05), end-diastolic volumes (439 ± 8 vs. 388 ± 18 μl, P < 0.05), and cardiomyocyte sizes (+20%, P < 0.05) compared with MI animals receiving vehicle treatment. Furthermore, activation of Ca(2+)/calmodulin-dependent kinase II in deguelin-treated MI animals was increased compared with the vehicle-treated group. Four wk after AB, we observed an augmentation of pathological hypertrophy in the deguelin-treated group with a significant increase in heart weights and cardiomyocyte sizes (>20%, P < 0.05). In contrast, the development of physiological hypertrophy was inhibited by deguelin treatment in exercise-trained animals. In conclusion, chronic Akt blockade with deguelin aggravates adverse myocardial remodeling and antagonizes physiological hypertrophy.     

Effects of sex differences on constitutive nitric oxide synthase expression and activity in response to pressure overload in rats

Clinical studies have documented sex differences in left ventricular (LV) hypertrophy patterns, but the mechanisms are so far poorly defined. This study aimed to determine whether 1) severe pressure overload altered expression and/or activity of cardiac constitutive nitric oxide synthase (NOS1 and NOS3) and 2) these changes were modulated according to sex. Analyses were performed 0.4-20 wk after thoracic aortic constriction (TAC) in male and female Wistar rats. Male rats with TAC exhibited early signs of cardiac dysfunction, as shown by echocardiographic and LV end-diastolic pressure measurements, whereas females with TAC exhibited higher LV hypertrophy (+96% vs. males at 20 wk; P < 0.05). After TAC, cardiac NOS1 expression was rapidly induced (0.4 wk) and stable afterward in males (P < 0.05 vs. sham groups), whereas it was delayed in females. Accordingly, specific NOS1 activity was increased by 2 wk in male rats with TAC (+122%; P < 0.001 vs. sham groups) and only by 20 wk in females (+220%; P < 0.001 vs. sham groups). NOS1 activity was correlated with NOS1 level. Regarding cardiac NOS3, expression was unaffected by TAC, and the decrease in activity observed at early and late times in male and female rats with TAC, respectively, is shown to be related to NOS3 allosteric regulator caveolin-1 level. The data demonstrated a unique sex-dependent regulation of the constitutive NOSs in response to TAC in rats; such a difference might play a role in the sex-dependent adaptability of the heart in response to pressure overload.     

Regression of pressure overload-induced left ventricular hypertrophy in mice

As a prelude to investigating the mechanism of regression of pressure overload-induced left ventricular (LV) hypertrophy (LVH), we studied the time course for the development and subsequent regression of LVH as well as accompanying alterations in cardiac function, histology, and gene expression. Mice were subjected to aortic banding for 4 or 8 wk to establish LVH, and regression was initiated by release of aortic banding for 6 wk. Progressive increase in LV mass and gradual chamber dilatation and dysfunction occurred after aortic banding. LVH was also associated with myocyte enlargement, interstitial fibrosis, and enhanced expression of atrial natriuretic peptide, collagen I, collagen III, and matrix metalloproteinase-2 but suppressed expression of alpha-myosin heavy chain and sarcoplasmic reticulum Ca(2+)-ATPase. Aortic debanding completely or partially reversed LVH, chamber dilatation and dysfunction, myocyte size, interstitial fibrosis, and gene expression pattern, each with a distinct time course. The extent of LVH regression was dependent on the duration of pressure overload, evidenced by the fact that restoration of LV structure and function was complete in animals subjected to 4 wk of aortic banding but incomplete in animals subjected to 8 wk of aortic banding. In conclusion, LVH regression comprises a variety of morphological, functional, and genetic components that show distinct time courses. A longer period of pressure overload is associated with a slower rate of LVH regression.     

Myocardial remodeling and arrhythmogenesis in moderate cardiac hypertrophy in rats

In 47 male adult Wistar rats with 4-wk aortic coarctation (AC) and 39 age-matched sham-operated rats (SO) chronically instrumented for telemetry electrocardiogram recording, we investigated the mechanisms of arrhythmogenesis in moderate cardiac hypertrophy, with an approach from "in vivo" toward the cellular level, analyzing 1) stress-induced cardiac arrhythmias in all rats and 2) myocardial fibrosis in 35 animals and action potential duration and density of hyperpolarization-activated current in 19 others at the ventricular level. Aortic banding increased arterial blood pressure, cardiac weight, and ventricular myocyte volume by 11, 25, and 14%, respectively (P < 0.001-0.05). Ventricular arrhythmias occurred at similar rates in AC and SO rats throughout the stress procedure. Action potential duration and hyperpolarization-activated current were about twice as great and myocardial fibrosis about four times greater in AC animals (P < 0.005-0.05). Electrocardiogram data also revealed more supraventricular arrhythmias in AC rats during the baseline period and after stress and fewer atrioventricular block episodes after stress (P < 0.05). Thus stress-induced supraventricular and atrioventricular nodal, but not ventricular, arrhythmias were affected in moderate cardiac hypertrophy when ventricular morphofunctional alterations were evident.     

Differential change in expression of pulmonary ET-1 and eNOS in rats after chronic left ventricular pressure overload

Pressure overload in the left ventricle of the heart follows a chronic and progressive course, resulting in eventual left heart failure and pulmonary hypertension (PH). The purpose of this research was to determine whether a differential pulmonary gene change of endothelin (ET)-1 and endothelial nitric oxide synthase (eNOS) occurred in adult rats with left ventricular overload. Eight groups of eight rats each were used (four rats with banding and four rats with sham operations). The rats underwent ascending aortic banding for 1 day, 2 weeks, 4 weeks, and 12 weeks before sacrifice. Significant medial hypertrophy of the pulmonary arterioles developed in two groups (4 and 12 weeks). Increased pulmonary arterial pressures were noted in three groups (1 day, 4 weeks, and 12 weeks). The aortic banding led to significant increases in pulmonary preproET-1 messenger RNA (mRNA) at 1 day and 12 weeks, and in pulmonary eNOS mRNA at 1 day and 12 weeks. In addition, there was increased pulmonary eNOS content at 1 day and 12 weeks in the banded rats, and increased lung cGMP levels were observed at 1 day. Increased lung ET-1 levels were also noted at 1 day (banded, 310 +/- 12 ng/g protein; sham, 201 +/- 12 ng/g protein; P < 0.01), 4 weeks (banded, 232 +/- 12 ng/g protein; sham, 201 +/- 12 ng/g protein; P < 0.01) and 12 weeks (banded, 242 +/- 12 ng/g protein; sham, 202 +/- 12 ng/g protein; P < 0.01). This indicates that the upregulated expression of ET-1 developed at least 4 weeks before eNOS expression in the course of PH, and, thus, medication against ET-1 could play a crucial role in treating PH with cardiac dysfunction secondary to aortic banding.     

Pressure overload-induced hypertrophy in transgenic mice selectively overexpressing AT2 receptors in ventricular myocytes

The role of the angiotensin II type 2 (AT2) receptor in cardiac hypertrophy remains controversial. We studied the effects of AT2 receptors on chronic pressure overload-induced cardiac hypertrophy in transgenic mice selectively overexpressing AT2 receptors in ventricular myocytes. Left ventricular (LV) hypertrophy was induced by ascending aorta banding (AS). Transgenic mice overexpressing AT2 (AT2TG-AS) and nontransgenic mice (NTG-AS) were studied after 70 days of aortic banding. Nonbanded NTG mice were used as controls. LV function was determined by catheterization via LV puncture and cardiac magnetic resonance imaging. LV myocyte diameter and interstitial collagen were determined by confocal microscopy. Atrial natriuretic polypeptide (ANP) and brain natriuretic peptide (BNP) were analyzed by Northern blot. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2, inducible nitric oxide synthase (iNOS), endothelial NOS, ERK1/2, p70S6K, Src-homology 2 domain-containing protein tyrosine phosphatase-1, and protein serine/threonine phosphatase 2A were analyzed by Western blot. LV myocyte diameter and collagen were significantly reduced in AT2TG-AS compared with NTG-AS mice. LV anterior and posterior wall thickness were not different between AT2TG-AS and NTG-AS mice. LV systolic and diastolic dimensions were significantly higher in AT2TG-AS than in NTG-AS mice. LV systolic pressure and end-diastolic pressure were lower in AT2TG-AS than in NTG-AS mice. ANP, BNP, and SERCA2 were not different between AT2TG-AS and NTG-AS mice. Phospholamban (PLB) and the PLB-to-SERCA2 ratio were significantly higher in AT2TG-AS than in NTG-AS mice. iNOS was higher in AT2TG-AS than in NTG-AS mice but not significantly different. Our results indicate that AT2 receptor overexpression modified the pathological hypertrophic response to aortic banding in transgenic mice.     

Reduced cGMP signaling activates NF-kappaB in hypertrophied hearts of mice lacking natriuretic peptide receptor-A

Mice lacking natriuretic peptide receptor-A (NPRA) develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for cardiac hypertrophic growth in the absence of NPRA signaling are not yet known. We sought to determine the activation of nuclear factor-kappaB (NF-kappaB) in Npr1 (coding for NPRA) gene-knockout (Npr1-/-) mice exhibiting cardiac hypertrophy and fibrosis. NF-kappaB binding activity was 4-fold greater in the nuclear extract of Npr1-/- mutant mice hearts as compared with wild-type (Npr1+/+) mice hearts. In parallel, inhibitory kappaB kinase-beta activity and IkappaB-alpha protein phosphorylation were also increased 3- and 4-fold, respectively, in hypertrophied hearts of mutant mice. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates NF-kappaB binding activity associated with hypertrophic growth of mutant mice hearts.     

Adapter molecule DOC-2 is differentially expressed in pressure and volume overload hypertrophy and inhibits collagen synthesis in cardiac fibroblasts.

DOC-2 (differentially expressed in ovarian carcinoma) is involved in Ras-, beta-integrin-, PKC-, and transforming growth factor-beta-mediated cell signaling. These pathways are implicated in the accumulation of extracellular matrix proteins during progression of hypertrophy to heart failure; however, the role of DOC-2 in cardiac pathophysiology has never been examined. This study was undertaken to 1) analyze DOC-2 expression in primary cultures of cardiac fibroblasts and cardiac myocytes and in the heart following different types of hemodynamic overloads and 2) examine its role in growth factor-mediated ERK activation and collagen production. Pressure overload and volume overload were induced for 10 wk in Sprague-Dawley rats by aortic constriction and by aortocaval shunt, respectively. ANG II (0.3 mg.kg(-1).day(-1)) was infused for 2 wk. Results showed that, compared with myocytes, DOC-2 was found abundantly expressed in cardiac fibroblasts. Treatment of cardiac fibroblasts with ANG II and TPA resulted in increased expression of DOC-2. Overexpression of DOC-2 in cardiac fibroblasts led to inhibition of hypertrophy agonist-stimulated ERK activation and collagen expression. An inverse correlation between collagen and DOC-2 was observed in in vivo models of cardiac hypertrophy; in pressure overload and after ANG II infusion, increased collagen mRNA correlated with reduced DOC-2 levels, whereas in volume overload increased DOC-2 levels were accompanied by unchanged collagen mRNA. These data for the first time describe expression of DOC-2 in the heart and demonstrate its modulation by growth-promoting agents in cultured cardiac fibroblasts and in in vivo models of heart hypertrophy. Results suggest a role of DOC-2 in cardiac remodeling involving collagen expression during chronic hemodynamic overload.