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New FindingsWhat is the central question of this study? What is the potential role of endothelial NO production via overexpression of the l-arginine transporter, CAT1, as a mitigator of cardiac hypertrophy?What is the main finding and its importance? Augmentation of endothelium-specific l-arginine transport via CAT1 can attenuate pressure-overload-dependent cardiac hypertrophy and fibrosis. Our findings support the conclusion that interventions that improve endothelial l-arginine transport may provide therapeutic utility in the setting of myocardial hypertrophy. Such modifications may be introduced by exercise training or locally delivered gene therapy, but further experimental and clinical studies are required.Endothelial dysfunction has been postulated to play a central role in the development of cardiac hypertrophy, probably as a result of reduced NO bioavailability. We tested the hypothesis that increased endothelial NO production, mediated by increased l-arginine transport, could attenuate pressure-overload-induced cardiac hypertrophy. Echocardiography and blood pressure measurements were performed 15weeks after transverse aortic constriction (TAC) in wild-type (WT) mice (n=12) and in mice with endothelium-specific overexpression of the l-arginine transporter, CAT1 (CAT+; n=12). Transverse aortic constriction induced greater increases in heart weight to body weight ratio in WT (by 47%) than CAT+ mice (by 25%) compared with the respective controls (P0.05). Likewise, the increase in left ventricular wall thickness induced by TAC was significantly attenuated in CAT+ mice (P=0.05). Cardiac collagen typeI mRNA expression was greater in WT mice with TAC (by 22%; P=0.03), but not in CAT+ mice with TAC, compared with the respective controls. Transverse aortic constriction also induced lesser increases in -myosin heavy chain mRNA expression in CAT+ mice compared with WT (P0.05). Left ventricular systolic pressure after TAC was 36 and 39% greater in WT and CAT+ mice, respectively, compared with the respective controls (P0.001). Transverse aortic constriction had little effect on left ventricular end-diastolic pressure in both genotypes. Taken together, these data indicate that augmenting endothelial function by overexpression of l-arginine transport can attenuate pressure-overload-induced cardiac hypertrophy.

Huntington's disease (HD) is a heritable neurodegenerative disorder, with heart disease implicated as one major cause of death. While the responsible mechanism remains unknown, autonomic nervous system (ANS) dysfunction may play a role. We studied the cardiac phenotype in R6/1 transgenic mice at early (3 months old) and advanced (7 months old) stages of HD. While exhibiting a modest reduction in cardiomyocyte diameter, R6/1 mice had preserved baseline cardiac function. Conscious ECG telemetry revealed the absence of 24-h variation of heart rate (HR), and higher HR levels than wild-type littermates in young but not older R6/1 mice. Older R6/1 mice had increased plasma level of noradrenaline (NA), which was associated with reduced cardiac NA content. R6/1 mice also had unstable R-R intervals that were reversed following atropine treatment, suggesting parasympathetic nervous activation, and developed brady- and tachyarrhythmias, including paroxysmal atrial fibrillation and sudden death. c-Fos immunohistochemistry revealed greater numbers of active neurons in ANS-regulatory regions of R6/1 brains. Collectively, R6/1 mice exhibit profound ANS-cardiac dysfunction involving both sympathetic and parasympathetic limbs, that may be related to altered central autonomic pathways and lead to cardiac arrhythmias and sudden death.

BACKGROUND AND PURPOSE While maintaining cardiac performance, chronic β-adrenoceptor activation eventually exacerbates the progression of cardiac remodelling and failure. We examined the adverse signalling pathways mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and reactive oxygen species (ROS) after chronic β2-adrenoceptor activation. EXPERIMENTAL APPROACH Mice with transgenic β2-adrenoceptor overexpression (β2-TG) and non-transgenic littermates were either untreated or treated with an antioxidant (N-acetylcysteine, NAC) or NADPH oxidase inhibitors (apocynin, diphenyliodonium). Levels of ROS, phosphorylated p38 mitogen-activated protein kinase (MAPK), pro-inflammatory cytokines and collagen content in the left ventricle (LV) and LV function were measured and compared. KEY RESULTS β2-TG mice showed increased ROS production, phosphorylation of p38 MAPK and heat shock protein 27 (HSP27), expression of pro-inflammatory cytokines and collagen, and progressive ventricular dysfunction. β2-adrenoceptor stimulation similarly increased ROS production and phosphorylation of p38 MAPK and HSP27 in cultured cardiomyocytes. Treatment with apocynin, diphenyliodonium or NAC reduced phosphorylation of p38 MAPK and HSP27 in both cultured cardiomyocytes and the LV of β2-TG mice. NAC treatment (500 mg·kg−1·day−1) for 2 weeks eliminated the up-regulated expression of pro-inflammatory cytokines and collagen in the LV of β2-TG mice. Chronic NAC treatment to β2-TG mice from 7 to 10 months of age largely prevented progression of ventricular dilatation, preserved contractile function (fractional shortening 37 ± 5% vs. 25 ± 3%, ejection fraction 52 ± 5% vs. 32 ± 4%, both P < 0.05), reduced cardiac fibrosis and suppressed matrix metalloproteinase activity. CONCLUSION AND IMPLICATIONS β2-adrenoceptor stimulation provoked NADPH oxidase-derived ROS production in the heart. Elevated ROS activated p38 MAPK and contributed significantly to cardiac inflammation, remodelling and failure. LINKED ARTICLE This article is commented on by Di Lisa et al., pp. 1009–1011 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01130.x

BACKGROUND AND PURPOSE Darbepoetin, a long-acting erythropoietin derivative, attenuates cardiomyocyte apoptosis and improves short-term (3 days) cardiac function, but the mechanisms responsible are unknown. We investigated potential mechanisms by which darbepoetin exerts cardioprotection following myocardial infarction in mice and the significance of the erythropoietin receptor (EPOR)–common β-chain (c-β-chain) heteroreceptor. EXPERIMENTAL APPROACH Mice underwent 60 min coronary occlusion followed by treatment with vehicle or a single dose of darbepoetin. Effects on gene expression, apoptosis and neutrophil accumulation in infarcted left ventricle were assessed 24 h later. Cardiac function, effects on vascularization and fibrosis were assessed 28 days later. The significance of EPOR–c-β-chain heteroreceptor was examined 28 days after infarction using mice deficient in c-β-chain. KEY RESULTS Twenty-four hours after darbepoetin, mRNAs encoding haeme oxygenase-1 (HO-1), iNOS and brain natriuretic peptide (BNP) were markedly elevated only in infarcted regions, and the frequency of apoptotic cells attenuated. Inflammation was also attenuated with reductions in neutrophil numbers. Darbepoetin also elevated mRNAs encoding angiogenic factors: placental growth factor, monocyte chemoattractant protein-1 and interleukin-1β. Twenty-eight days after treatment, CD31+ vessels in the infarct zone doubled and fibrosis reduced. Cardiac haemodynamics were improved. Darbepoetin also improved cardiac haemodynamics in c-β-chain-deficient mice, increased HO-1 and iNOS expression and vessel numbers and attenuated fibrosis. CONCLUSIONS AND IMPLICATIONS Darbepoetin stimulates expression of haeme oxygenase, iNOS, BNP and angiogenic factors specifically in infarcted left ventricle that attenuates inflammation, apoptosis and fibrosis; elevate vessel numbers; and improve cardiac function. The EPOR–c-β-chain heteroreceptor is not essential for these effects.

As a hallmark of heart disease, cardiac fibrosis contributes to the development of heart failure and arrhythmias and forms a key therapeutic target. There is a major unmet need for selective, potent, and safe antifibrotic drugs. Earlier studies revealed a cardiac fibrosis phenotype in relaxin-1-deficient mice. Recent studies in several rodent models of cardiac fibrosis have documented reversal of fibrosis by treatment with relaxin peptide or virally mediated relaxin gene delivery. In mice with surgically induced transmural myocardial infarction, relaxin therapy inhibited scar density. In these studies, however, functional benefits achieved by relaxin therapy were limited or less explored. Collectively, there is good experimental evidence that relaxin is able to reverse cardiac fibrosis due to distinct mechanisms. Future research needs to explore functional improvement following fibrosis reversal by relaxin and the usefulness of relaxin in antiarrhythmic or stem cell-based therapy.

1. With the generation of gene knockout (KO) or transgenic overexpression (TG) mouse models targeting adrenoceptors (AR), recent studies in vivo have investigated the role of AR subtypes in pressure overload-induced left ventricular (LV) hypertrophy and remodelling. 2. Although subjecting alpha(1B)-KO mice to transverse aortic constriction (TAC) did not reveal significant phenotype differences compared with controls, mice deficient in both alpha(1A)- and alpha(1B)-AR responded to TAC with poor survival, increased cardiomyocyte apoptosis, more severe fibrosis and dysfunction, but a similar degree of LV hypertrophy, compared with wild-type littermates. Following TAC, alpha(1B)-TG mice developed more severe hypertrophy, interstitial fibrosis and LV dysfunction. In contrast, overexpression of alpha(1A)-AR preserved cardiac function and reduced death from heart failure without affecting the degree of LV hypertrophy. Thus, alpha(1A)- and alpha(1B)-adrenoceptor signalling impacts differently on myocardial adaptation to pressure overload. 3. The absence of both beta(1)- and beta(2)-AR significantly suppressed pressure overload-evoked hypertrophy, fibrosis and expression of inflammatory or fibrogenic genes. Conversely, studies on beta(2)-TG mice with TAC revealed adverse consequences, including accelerated development of heart failure, poor survival and more severe interstitial fibrosis, but a comparable degree of hypertrophy compared with wild-type littermates. 4. Collectively, these findings suggest that the effect of ARs on pressure overload-induced myocardial adaptation is subtype specific. Whereas activation of alpha(1B)-AR or beta(2)-AR contributes to maladaptation and the onset of heart failure, activation of alpha(1A)-AR or inactivation of beta(2)-AR is beneficial in the setting of chronic pressure overload.

While the approaches of regenerating cardiac muscle remain undetermined, recent evidence indicates that paracrine function of transplanted cells contributes significantly to the beneficial effects of cell therapy. Combination of such paracrine function of grafted cells with extracellular matrix remodelling by relaxin represents a promising complement to cell-based therapy for cardiac repair and muscle regeneration.

1. Ivabradine selectively reduces heart rate (HR) by inhibiting the cardiac pacemaker I(f) current, thus prolonging the duration of spontaneous depolarization in the sinus node. The activity of ivabradine under conditions of enhanced sympathoadrenergic activity has been addressed by investigating the effects of repeated oral administration in mice with sympathoadrenergic activation due to either stress, cardiac-restricted overexpression of beta(2)-adrenergic receptors (beta(2)AR), or beta-agonist administration. HR and left ventricular fractional shortening (FS) were determined by echocardiography. 2. Initial experiments showed that the conscious restrained state was associated with stress-mediated sympathetic activation, while sympathetic withdrawal occurred under anaesthetized conditions. In wild-type mice, ivabradine reduced HR under both conscious and anaesthetized states, with a similar degree in absolute reduction under both states. FS was unchanged by the treatment. 3. Ivabradine was similarly effective in reducing HR in the beta(2)AR transgenic mice. Further, ivabradine at 10 mg kg(-1) day(-1) reduced the maximal HR increase in response to the beta-agonist isoproterenol, without modifying the response of contractile parameters. 4. These data indicate that oral administration of ivabradine in mice reduces HR while ventricular performance is maintained. This specific HR-reducing action of ivabradine is well preserved under conditions that are associated with significant activation of the sympathoadrenergic system.

The ribosomal DNA transcription-specific factor, UBF, is a key target for the regulation of ribosomal RNA synthesis and hypertrophic growth of isolated neonatal cardiomyocytes. In this study, we have examined whether UBF expression is also an important determinant of cardiac growth rates in vivo. We show that rDNA transcription, rRNA synthesis and UBF expression in left ventricular myocytes isolated from mice 1–6 weeks following transverse aortic constriction were significantly increased (2.5–3.5-fold) compared to the levels in myocytes from the left ventricle of sham-operated mice.

We examined the existence of catecholamine metabolizing enzymes (catechol-O-methyltransferase, COMT, and monoamine oxidase, MAO) in CHO cells transfected with norepinephrine (NE) transporter (NET) cDNA. NET activity was studied by incubating cells with [3H]-NE (0.5 μCi ml−1, 20 min) in a Na+ containing medium. Incubation with [3H]-NE lead to [3H] accumulation at 47797±4864 d.p.m. per well. Specific inhibitors of NET abolished this uptake. During post-uptake incubation, [3H] leaked rapidly from cells and the extracellular phase comprised 89% of total radioactivity within 40 min. Both [3H] retention and [3H]‘leakage’ were largely unaffected by inhibitors for MAO. In contrast, COMT inhibitors, U-0521 and Ro 41-0960, dose-dependently increased intracellular [3H]-NE retention with a maximal increase of 4.5 fold. The EC50 for Ro 41-0960 was 139-times lower than that of U-0521. U-0521 largely inhibited [3H]‘leakage’ and doubled the apparent Vmax for [3H]-NE uptake. Addition of U-0521 during uptake incubation increased intracellular NE content by 8 fold. Normetanephrine, the COMT-dependent metabolite of NE, was formed in large quantities during post-uptake incubation. U-0521 significantly inhibited the formation of NMN with an equal preservation of intracellular NE. CHO cells expressing NET possess COMT activity, which is responsible for the metabolism of NE to form lipophilic metabolite normetanephrine. The apparent ‘properties’ of the NET function expressed in CHO cells changed, after inhibition of COMT, in such a way closer to that described in the native neuronal preparations. British Journal of Pharmacology (1999) 128, 774–780; doi:10.1038/sj.bjp.0702831

While the expression patterns of cardiac hypertrophy-related genes have been well documented and widely used as markers for hypertrophy, recent research has revealed uncoupling of hypertrophy-related gene profiles and hypertrophic growth. The role of β-adrenergic signalling in the development of hypertrophy is incompletely understood. The finding of an upregulated expression of hypertrophy-related genes but a suppressed hypertrophy following β-blockade reveals previously unrecognized sympatho-adrenergic mechanisms of hypertrophic growth.

1. alpha 1-Adrenoceptors are known to play an important role in vasoconstriction in response to adrenergic stimulation. However, the functional importance of alpha 1-adrenoceptor subtypes at the epicardial coronary artery remains unclear. We examined alpha 1-adrenoceptor subtypes by comparing functional affinities for alpha-adrenoceptor antagonists on noradrenaline (NA)-induced vasoconstriction in porcine denuded right coronary arteries. 2. Noradrenaline induced a dose-dependent vasoconstriction in incubated vessel rings. Prazosin and phentolamine were potent and competitive antagonists for NA-induced contraction (pA2 10.27 and 9.03, respectively). In contrast, the selective alpha 2-adrenoceptor antagonist yohimbine had a low affinity (pA2 6.13). Two selective alpha 1A-adrenoceptor antagonists, WB 4101 and 5-methyl urapidil, were potent and competitive antagonists of alpha 1-adrenoceptor-induced contraction (pA2 10.67 and 8.90, respectively) and the selective alpha 1D-adrenoceptor antagonist BMY 7378 had a low affinity (pA2 6.06). Noradrenaline-induced contraction was insensitive to the alkylating effects of chlorethylclonidine. These observations indicate that the vasoconstriction is predominantly mediated by the alpha 1A-adrenoceptor subtype. This was also supported by a good correlation between pA2 values from the present study and reported binding affinities (pKi) of various alpha-adrenoceptor antagonists with cloned human alpha 1A-adrenoceptors (r = 0.98), but not for alpha 1B- or alpha 1D-adrenoceptor subtypes (r = 0.77 and 0.41, respectively). 3. Our results indicate that the alpha 1A-adrenoceptor is the main functional receptor subtype in porcine denuded coronary arteries.

SUMMARY 1. Ischaemia and anoxia induce excessive noradrenaline (NA) release in the heart by a mechanism independent of both nerve activity and extracellular Ca2+. The present study was designed to examine the potential role of intracellular Ca2+ mobilization in anoxic NA release in the heart by chelating intracellular free Ca2+. 2. In normoxic hearts, preloading with an intracellular fre. Ca2+ chelator (BAPTA) reduced neuronal NA release by 65%, confirming the effectiveness of the loading protocol. Release of NA independent of nerve activity occurred in hearts subjected to a 40 min period of anoxic, substrate-free and nominal Ca2+-free perfusion. Loading hearts with BAPTA prior to anoxia failed to reduce NA overflow (1561 ± 147 vs 1496 ± 206 pmol/g over 40 min). Infusion with BAPTA (20 μmol/L) during the first 25 min of the anoxic period reduced the quantity of anoxic NA release by approximately 25% from 2013 ± 124 to 1476 ± 207 pmol/g (P < 0.05). 3. Our results confirm that anoxic NA release is predominantly. Ca2+-independent process with Ca2+ mobilization from endogenous storage playing only a minor contributing role.

SUMMARY 1. As it has been shown that oestrogen enhances the cholinergic muscarinic activity in the central nervous system, we studied sex differences in the responses to parasympathetic nervous stimulation in the rat heart using in vivo and in vitro preparations. 2. In in situ perfused, innervated hearts, stimulation of bilateral vagus nerves (15 Hz with 1 μmol/L physostigmine) inhibited sympathetic nerve stimulation (5 Hz) induced noradrenaline release to a greater extent in female than in male rats (54 ± 5 vs 72 ± 5% of control). Similarly, vagus nerve stimulation at 1–20 Hz reduced heart rate (HR) more in females than males, and this sex difference became more marked in the presence of physostigmine. The chronotropic effect of vagal stimulation was attenuated after ovariectomy but potentiated after castration when compared with sham-operated controls. In contrast, the muscarinic agonist methacholine reduced neural NA release and HR equally well in both sexes. 3. In anaesthetized rats, reduction in HR and mean arterial pressure by vagus nerve stimulation (1–20 Hz) was more pronounced in females than in males after inhibition of acetylcholinesterase with physostigmine. 4. The results indicate that activation of parasympathetic nerve leads to greater presynaptic and postsynaptic effects in female than in male rat hearts, presumably due to a higher level of acetyl-choline release following nerve activation.

Cardiac RXFP1 was upregulated upon activation of the alpha(1)-adrenergic receptor (AR) through signal pathways involving protein kinase A and C and ERK. In contrast, beta(1)-AR activation downregulated expression of cardiac RXFP1 and it further counteracted the alpha(1)-AR-mediated RXFP1 upregulation.