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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 α-myosin heavy chain and sarcoplasmic reticulum Ca2+-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.

Objective : Recent studies on mice with surgically induced acute myocardial infarction (AMI) have documented the frequent occurrence of ventricular rupture, an event not previously reported in other laboratory species. We have examined the natural history, histopathology and myocardial mechanical strength in mice with AMI. Methods : AMI was induced by coronary artery occlusion and animals were monitored for fatal events. Gross and histological examinations were undertaken. Results : Rupture occurred in the left ventricular free wall at 2–6 days after AMI. Incidence of rupture in male mice varied among three strains studied (3% for FVB/N, 27% for C57B/6J, and 59% for 129sv, P

In response to pressure-overload, cardiac function deteriorates and may even progress to fulminant heart failure and death. Here we questioned if genetic enhancement of left ventricular (LV) contractility protects against pressure-overload. Transgenic (TG) mice with cardiac-restricted overexpression (66-fold) of the alpha(1A)-adrenergic receptor (alpha(1A)-AR) and their non-TG (NTG) littermates, were subjected to transverse aorta constriction (TAC)-induced pressure-overload for 12 weeks. TAC-induced hypertrophy was similar in the NTG and TG mice but the TG mice were less likely to die of heart failure compared to the non-TG animals (P0.05). The hypercontractile phenotype of the TG mice was maintained over the 12-week period following TAC with LV fractional shortening being significantly greater than in the NTG mice (42+/-2 vs 29+/-1%, P0.01). In the TG animals, 11-week beta-AR-blockade with atenolol neither induced hypertrophy nor suppressed the hypercontractile phenotype. The hypertrophic response to pressure-overload was not altered by cardiac alpha(1A)-AR overexpression. Moreover, the inotropic phenotype of alpha(1A)-AR overexpression was well maintained under conditions of pressure overload. Although the functional decline in contractility with pressure overload was similar in the TG and NTG animals, given that contractility was higher before TAC in the TG mice, their LV function was better preserved and heart failure deaths were fewer after induction of pressure overload.

Recent research using genetically modified mice has revealed significant sex differences in cardiac phenotypes. In the majority of strains, females display a lower mortality, less severe hypertrophy, better preserved function and mitigated cardiac pathology compared with male counterparts. Thus, gender is an independent determinant for the development of cardiac phenotype in murine models. While there is strong evidence for estrogen as a cardiac protector, emerging evidence indicates adverse actions of testicular hormones that might be responsible in part for the sex differences. Studies using mouse models have also revealed novel information on signalling mechanisms mediating the sex difference.

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.

Sex differences in cardiomyopathic phenotype and the role of gonadal status were studied in mice with cardiac overexpression of beta(2)-adrenergic receptors (ARs) over 6-15 months (mo) of age. Survival to 15 mo was 96% in wild-type mice but was poorer in transgenic (TG) mice and lower for males than females (13% vs. 56%, P < 0.001). Echocardiography demonstrated progressive left ventricular (LV) dilatation and reduction in LV fractional shortening in male but much less marked changes in female TG mice. Incidences of atrial thrombosis, pleural effusion and lung congestion were higher and myocyte size and fibrosis in the LV were greater in TG males than females. Deprivation of testicular hormones by castration during 3-15 mo of age improved survival and significantly ameliorated LV dysfunction, remodeling, and hypertrophy compared with intact TG males. No significant effect, except for a trend of a better survival, was detected by ovariectomy in TG females. In conclusion, cardiac beta(2)-AR overexpression at a high level leads to cardiomyopathy and heart failure with aging. Female mice had less cardiac remodeling, dysfunction, and pathology and a marked survival advantage over male mice, and this was independent of prevailing levels of ovarian hormones. TG males showed benefit from orchiectomy, suggesting a contribution by testicular hormones to the progression of the cardiomyopathic phenotype.

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.

See article by Molero et al. [14] (pages 43–51) in this issue. Gender-related differences in the risk of cardiovascular diseases have been well recognized with premenopausal women exhibiting a lower risk compared to age-matched men [1]. Estrogen is implicated as the factor responsible for this gender difference due to the facts that the female advantage over male in cardiovascular morbidity disappears following menopause and that administration of 17α-estradiol (E2) is protective against cardiovascular injury. In addition to changes in lipid metabolism, E2 at physiological levels mediates direct cardiovascular actions. The mechanisms and signal pathways responsible for the cardiovascular protection by estrogen have received great attention in recent years and a wealth of literature in this area has been recently reviewed [1–3]. Nuclear and putative membrane-bound estrogen receptors (ERs) are present in cardiovascular tissues of women and men. ER α and β isoforms mainly form homodimers (ERα and ERβ) that mediate predominant effects of estrogen. Nuclear ERα and ERβ regulate gene expression in two modes. The authentic signalling mode involves binding of ERs with specific DNA estrogen-response elements (EREs) which are located within regulatory regions of targeted genes. The binding of ERs with ERE recruits some coactivators thereby modulating machinery of gene transcription [4]. The second mode of action is independent of ERE (i.e., occurs in the absence of a direct binding of ERs to DNA) and requires interactions of ERs with promoter elements that directly bind to transcription factors. These sites include AP-1 sites that bind Jun/Fos, cyclic AMP response element (CREs) that bind c-Jun/ATF-2, and SP-1 sites [4–6]. Recently, it is believed that the rapid nongenomic actions of estrogen are mediated by membrane ERs through several pathways, including tyrosine kinase, mitogen-activated protein kinase (MAPK), adenylyl cyclase/cAMP, and guanylyl cyclase/cGMP [1,7]. Estrogen is … * Tel.: +61-3-8530-1294; fax: +61-3-8532-1100

This study aimed to investigate the cardiac response to sympathetic stimulation and norepinephrine exposure following subarachnoid hemorrhage (SAH). Cardiac functional response was assessed 3 days following an injection of 300 microl of homologous blood in the cisterna magna using an in situ perfused, innervated rat heart model. Sympathetic nervous activity was indirectly assessed from measurements of arterial plasma and tissue norepinephrine concentration and cardiac beta-receptor density. In in situ perfused hearts, sympathetic nerve stimulation (2, 4 and 8 Hz, 1 min duration) induced a frequency-dependent increase in left ventricular pressure (VP), with the response being more pronounced in the SAH group of animals at the higher frequency (P

Ca(2+) signaling plays a central role in cardiac contractility and adaptation to increased hemodynamic demand. We have generated mice with a targeted deletion of the S100A1 gene coding for the major cardiac isoform of the large multigenic S100 family of EF hand Ca(2+)-binding proteins. S100A1(-/-) mice have normal cardiac function under baseline conditions but have significantly reduced contraction rate and relaxation rate responses to beta-adrenergic stimulation that are associated with a reduced Ca(2+) sensitivity. In S100A1(-/-) mice, basal left-ventricular contractility deteriorated following 3-week pressure overload by thoracic aorta constriction despite a normal adaptive hypertrophy. Surprisingly, heterozygotes also had an impaired response to acute beta-adrenergic stimulation but maintained normal contractility in response to chronic pressure overload that coincided with S100A1 upregulation to wild-type levels. In contrast to other genetic models with impaired cardiac contractility, loss of S100A1 did not lead to cardiac hypertrophy or dilation in aged mice. The data demonstrate that high S100A1 protein levels are essential for the cardiac reserve and adaptation to acute and chronic hemodynamic stress in vivo.

Differences in the autonomic system may be due to differences in afferent receptor stimulation, in central The autonomic nervous system is of importance in the reflex transmission, in the efferent nervous system and in natural history and treatment of a number of post synaptic signaling. At each of these potential sites of pathophysiological states involving the cardiovascular difference, there may be effects due to different size or system. These include hypertension and diseases of the number of neurons, variations in receptors, differences in vasculature as well as myocardial ischaemia and cardiac neurotransmitter content or metabolism as well as func- arrhythmias. Gender differences in the incidence and tional differences in the various components of the reflex clinical course of a range of cardiovascular states are also arc. well recognised. Both short and long term prognosis after myocardial infarction are worse for women than men (1-4), whereas women with non-ischaemic car- 2. Methods of assessment of autonomic nervous diomyopathy have improved survival (5,6). In addition to activity the well known difference in age of presentation of coronary heart disease, women are more likely to suffer As indicated in the Introduction there are multiple from Raynaud's phenomenon, and to experience pre- potential sites in the autonomic nervous system which may syncopal or syncopal episodes. An appreciation of gender be subject to gender related differences. Consequently the differences in the structure and function of the autonomic examination of gender differences may require use of a nervous system is therefore important to a full understand- wide range of experimental and clinical methodologies. In ing of a number of common and important clinical this section brief mention will be made of techniques presentations (7). specifically related to this question. Gender differences in the autonomic nervous system Neurotransmitter release from efferent terminals may be may be present because of developmental differences or estimated from plasma levels, particularly for the sympa- due to the effects of prevailing levels of male and / or thetic nervous system by measuring plasma noradrenaline female sex hormones. Such prevailing hormone levels may (NA) and adrenaline levels or their urinary excretion. Such also produce differences between pre- and post-menopaus- methods, which are available for both animal and human al women and amongst pre-menopausal women at different studies, do not account for clearance or regional changes phases of the menstrual cycle, which is characterized by that may however be estimated from determination of oestrogen secretion in the late follicular (pre-ovulatory) transmitter spillover (8). It is also possible to measure phase followed by a secondary phase of secretion in the extracellular concentrations regionally from microdialysis. luteal (post-ovulatory) phase. Progesterone secretion oc- Estimation of cholinergic neurotransmission by assay of curs during the luteal phase.

Nitroxyl (HNO), a redox congener of NO•, is a novel regulator of cardiovascular function combining vasodilator and positive inotropic properties. Our previous studies have demonstrated these properties occur concomitantly in the intact heart; HNO moreover also exhibits antihypertrophic and superoxide-suppressing actions. HNO donors may thus offer favorable actions in heart failure. The impact of chronic HNO donor administration has however yet to be reported in this context. We tested the hypothesis that the HNO donor 1-nitrosocyclohexyl acetate (1-NCA) limits cardiomyocyte hypertrophy and left ventricular (LV) diastolic dysfunction in a mouse model of diabetic cardiomyopathy in vivo. Male 6 week-old FVB/N mice received either streptozotocin (55 mg/kg/day i.p. for 5 days, n=17), to induce type 1 diabetes, or citrate vehicle (n=16). After 4 weeks of hyperglycemia, mice were allocated to 1-NCA therapy (83mg/kg/day i.p.) or vehicle, and followed for a further 4 weeks. As shown in the table, blood glucose was unaffected by 1-NCA. LV diastolic dysfunction was evident in diabetic mice, measured as echocardiography-derived A wave velocity, deceleration time and E:A ratio; LV systolic function was preserved. Diabetes-induced diastolic dysfunction was accompanied by increased LV cardiomyocyte size, hypertrophic and pro-fibrotic gene expression, and upregulation of LV superoxide. These characteristics of diabetic cardiomyopathy were largely prevented by 1-NCA treatment. Selectivity of 1-NCA as a donor of HNO versus NO• was demonstrated by the sensitivity of the coronary vasodilation response of 1-NCA to the HNO scavenger L-cysteine (4mM), but not to the NO• scavenger hydroxocobalamin (50μM), in the normal rat heart ex vivo (n=3-7). Collectively, our studies provide the first evidence that HNO donors may represent a promising new strategy for the treatment of diabetic cardiomyopathy, and implies their therapeutic efficacy in settings of chronic heart failure.

Myocardial infarction (MI) is the major cause of heart failure and death in the Western world. Annexin-A1 is an endogenous, glucocorticoid-regulated anti-inflammatory protein. We have previously shown that this protein plays an important protective role in preserving left ventricular (LV) viability and function in vitro, actions that are reproduced by treatment with its N-terminal-derived peptide, Ac2-26. Little is known however about its cardioprotective actions in vivo, particularly beyond its early anti-necrotic actions in the first few hours of reperfusion. We now test the hypothesis that exogenous Ac2-26 limits multiple aspects of MI injury, over both the short and longer-term in vivo. In the first study, adult C57BL/6 mice were subjected to ischemia-reperfusion (left arterial descending coronary artery ligation, with 1-7 days reperfusion), and Ac2-26 was administered at 1mg/kg i.v. every 24h commencing 5mins before reperfusion. In the second study, mice were subjected to permanent coronary artery occlusion, with Ac2-26 administered 1mg/kg/day i.p. via osmotic pump inserted at time of surgery. As shown in the Table, Ac2-26 reduced cardiac necrosis after 24h reperfusion (infarct size, plasma troponin I levels), systemic and cardiac inflammation after 48h reperfusion (neutrophil and macrophage infiltration) and cardiac fibrosis after 7 days reperfusion. These protective actions at the level of cardiac morphology were associated with preservation of LV function 4wks after permanent occlusion, as determined on fractional shortening and velocity of circumferential fiber shortening. Taken together, our data is the first evidence of Ac2-26 cardioprotection beyond the first few hours of reperfusion in vivo, and importantly, the first to report Ac2-26-induced preservation of LV function in the heart post MI in vivo.

Heart failure (HF) and atrial fibrillation (AF) share common risk factors, frequently coexist and are associated with high mortality. Treatment of HF with AF represents a major unmet need. Here we show that a small molecule, BGP-15, improves cardiac function and reduces arrhythmic episodes in two independent mouse models, which progressively develop HF and AF. In these models, BGP-15 treatment is associated with increased phosphorylation of the insulin-like growth factor 1 receptor (IGF1R), which is depressed in atrial tissue samples from patients with AF. Cardiac-specific IGF1R transgenic overexpression in mice with HF and AF recapitulates the protection observed with BGP-15. We further demonstrate that BGP-15 and IGF1R can provide protection independent of phosphoinositide 3-kinase-Akt and heat-shock protein 70; signalling mediators often defective in the aged and diseased heart. As BGP-15 is safe and well tolerated in humans, this study uncovers a potential therapeutic approach for HF and AF.

Expression of microRNA-652 (miR-652) increases in the diseased heart, decreases in a setting of cardioprotection, and is inversely correlated with heart function. The aim of this study was to assess the therapeutic potential of inhibiting miR-652 in a mouse model with established pathological hypertrophy and cardiac dysfunction due to pressure overload. Mice were subjected to a sham operation or transverse aortic constriction (TAC) for 4 wk to induce hypertrophy and cardiac dysfunction, followed by administration of a locked nucleic acid (LNA)-antimiR-652 (miR-652 inhibitor) or LNA control. Cardiac function was assessed before and 8 wk post-treatment. Expression of miR-652 increased in hearts subjected to TAC compared to sham surgery (2.9-fold), and this was suppressed by ∼95% in LNA-antimiR-652-treated TAC mice. Inhibition of miR-652 improved cardiac function in TAC mice (fractional shortening:29±1% at 4 wk post-TAC compared to 35±1% post-treatment) and attenuated cardiac hypertrophy. Improvement in heart function was associated with reduced cardiac fibrosis, less apoptosis and B-type natriuretic peptide gene expression, and preserved angiogenesis. Mechanistically, we identified Jagged1 (a Notch1 ligand) as a novel direct target of miR-652. In summary, these studies provide the first evidence that silencing of miR-652 protects the heart against pathological remodeling and improves heart function.

SummaryInterleukin-6 (IL-6) plays a paradoxical role in inflammation and metabolism. The pro-inflammatory effects of IL-6 are mediated via IL-6 “trans-signaling,” a process where the soluble form of the IL-6 receptor (sIL-6R) binds IL-6 and activates signaling in inflammatory cells that express the gp130 but not the IL-6 receptor. Here we show that trans-signaling recruits macrophages into adipose tissue (ATM). Moreover, blocking trans-signaling with soluble gp130Fc protein prevents high-fat diet (HFD)-induced ATM accumulation, but does not improve insulin action. Importantly, however, blockade of IL-6 trans-signaling, unlike complete ablation of IL-6 signaling, does not exacerbate obesity-induced weight gain, liver steatosis, or insulin resistance. Our data identify the sIL-6R as a critical chemotactic signal for ATM recruitment and suggest that selectively blocking IL-6 trans-signaling may be a more favorable treatment option for inflammatory diseases, compared with current treatments that completely block the action of IL-6 and negatively impact upon metabolic homeostasis.

Background— Nitroxyl (HNO), a redox congener of nitric oxide (NO·), is a novel regulator of cardiovascular function, combining concomitant positive inotropic, lusitropic, and vasodilator properties. Moreover, HNO exhibits myocardial antihypertrophic and superoxide-suppressing actions. Despite these favorable actions, the impact of chronic HNO administration has yet to be reported in the context of cardiomyopathy. Diabetic cardiomyopathy is characterized by early diastolic dysfunction and adverse left ventricular (LV) structural remodeling, with LV superoxide generation playing a major causal role. We tested the hypothesis that the HNO donor 1-nitrosocyclohexylacetate (1-NCA) limits cardiomyocyte hypertrophy and LV diastolic dysfunction in a mouse model of diabetes mellitus in vivo. Methods and Results— Diabetes mellitus was induced in male FVB/N mice using streptozotocin. After 4 weeks, diabetic and nondiabetic mice were allocated to 1-NCA therapy (83 mg/kg per day IP) or vehicle and followed up for a further 4 weeks. Diabetes mellitus–induced LV diastolic dysfunction was evident on echocardiography-derived E and A wave velocities, E:A ratio, deceleration, and isovolumic relaxation times; LV systolic function was preserved. Increased LV cardiomyocyte size, hypertrophic and profibrotic gene expression, and upregulation of LV superoxide were also evident. These characteristics of diabetic cardiomyopathy were largely prevented by 1-NCA treatment. Selectivity of 1-NCA as an HNO donor was demonstrated by sensitivity of acute 1-NCA to l -cysteine but not to hydroxocobalamin in the normal rat heart ex vivo. Conclusions— Our studies provide the first evidence that HNO donors may represent a promising strategy for treatment of diabetic cardiomyopathy and implies therapeutic efficacy in settings of chronic heart failure.

HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition.

The activity of the early signaling enzyme, phospholipase Cβ1b (PLCβ1b), is elevated in diseased myocardium and activity increases with disease progression. PLCβ1b and the alternative splice variant, PLCβ1a, were expressed in mouse hearts using adeno-associated viral constructs (rAAV6-FLAG-PLCβ1b, rAAV6-FLAG- PLCβ1a) delivered intravenously. Functional responses were assessed in vivo and confirmatory mechanistic studies were conducted in neonatal rat ventricular myocytes (NRVM). FLAG-PLCβ1b was expressed in all of the chambers of the mouse heart, but was highest in left ventricle, where expression was observed in >90% of the cells and was localized to the sarcolemma and T-tubules. Heightened PLCβ1b expression caused a rapid loss of contractility and down-regulation of Phospholamban expression. The loss of contractility induced by PLCβ1b was reversed by inhibition of protein kinase Cα (PKCα). PLCβ1a did not affect contractile function or phospholamban expression. Mechanistic analysis performed in neonatal rat cardiomyocytes confirmed PLCβ1b increased the membrane association of PKCα as well as downstream dephosphorylation of phospholamban and depletion of the Ca2+ stores of the sarcoplasmic reticulum, both of which were mediated by PKCα. Trans-aortic constriction (TAC) resulted in progressive hypertrophy together with reduced contractility in PLCβ1a expressing mice. In PLCβ1b-expressing mice, TAC induced a similar hypertrophic response, but did not cause further contractile depression above that due to PLCβ1b expression alone, suggesting that PLCβ1b is responsible for lowering contractility in response to pressure overload. We conclude that heightened PLCβ1b activity observed in diseased myocardium contributes to pathology by PKCα-mediated contractile dysfunction. PLCβ1b is a cardiac-specific signaling system, and thus provides an ideal therapeutic target for the development of well-tolerated inotropic agents for use in failing myocardium.

Introduction: Targeting microRNAs differentially regulated in settings of stress and protection could represent a new approach for the treatment of heart failure. miR-652 expression increased in hearts of a cardiac stress mouse model and was downregulated in a model of cardiac protection. Aim: To assess the therapeutic potential of silencing miR-652 in a mouse model with established pathological hypertrophy and cardiac dysfunction due to pressure overload. Methods: Mice were subjected to a sham operation (n=10) or transverse aortic constriction (TAC, n=14) for 4 weeks to induce hypertrophy and cardiac dysfunction. Mice were subcutaneously administered a locked nucleic acid (LNA)-antimiR-652 or LNA-control. Cardiac function was assessed by echocardiography before and 8 weeks post treatment, followed by molecular and histological analyses. Results: Expression of miR-652 increased in hearts subjected to pressure overload compared to sham operated mice (2.9 fold, n=3-5, P Conclusion: Therapeutic silencing of miR-652 protects the heart against pathological cardiac remodeling and improves heart function via mechanisms that are associated with preserved angiogenesis, decreased fibrosis and upregulation of a miR-652 target, Jagged1. These studies provide the first evidence that targeted inhibition of miR-652 could represent an attractive approach for the treatment of heart failure.

Relaxin, a new drug for heart failure therapy, exerts its cardiac actions through relaxin family peptide receptor 1 (RXFP1). Factors regulating RXFP1 expression remain unknown. We have investigated effects of activation of adrenoceptors (AR), an important modulator in the development and prognosis of heart failure, on expression of RXFP1 in rat cardiomyocytes and mouse left ventricles (LV). Expression of RXFP1 at mRNA (real-time PCR) and protein levels (immunoblotting) was measured in cardiomyocytes treated with α- and β-AR agonists or antagonists. RXFP1 expression was also determined in the LV of transgenic mouse strains with cardiac-restricted overexpression of α1A-, α1B- or β2-AR. Specific inhibitors were used to explore signal pathways involved in α1-AR mediated regulation of RXFP1 in cardiomyocytes. In cultured cardiomyocytes, α1-AR stimulation resulted in 2–3 fold increase in RXFP1 mRNA (P

Over the past few decades, research on the peptide hormone, relaxin, has significantly improved our understanding of its biological actions under physiological and diseased conditions. This has facilitated the conducting of clinical trials to explore the use of serelaxin (human recombinant relaxin). Acute heart failure (AHF) is a very difficult to treat clinical entity, with limited success so far in developing new drugs to combat it. A recent phase-III RELAX-AHF trial using serelaxin therapy given during hospitalization revealed acute (ameliorated dyspnea) and chronic (improved 180-day survival) effects. Although these findings support a substantial improvement by serelaxin therapy over currently available therapies for AHF, they also raise key questions and stimulate new hypotheses. To facilitate the development of serelaxin as a new drug for heart disease, joint efforts of clinicians, research scientists and pharmacological industries are necessary to study these questions and hypotheses. In this review, after providing a brief summary of clinical findings and the pathophysiology of AHF, we present a working hypothesis of the mechanisms responsible for the observed efficacy of serelaxin in AHF patients. The existing clinical and preclinical data supporting our hypotheses are summarized and discussed. The development of serelaxin as a drug provides an excellent example of the bilateral nature of translational research.

Therapeutic inhibition of the miR-34 family (miR-34a,-b,-c), or miR-34a alone, have emerged as promising strategies for the treatment of cardiac pathology. However, before advancing these approaches further for potential entry into the clinic, a more comprehensive assessment of the therapeutic potential of inhibiting miR-34a is required for two key reasons. First, miR-34a has ∼40% fewer predicted targets than the miR-34 family. Hence, in cardiac stress settings in which inhibition of miR-34a provides adequate protection, this approach is likely to result in less potential off-target effects. Secondly, silencing of miR-34a alone may be insufficient in settings of established cardiac pathology. We recently demonstrated that inhibition of the miR-34 family, but not miR-34a alone, provided benefit in a chronic model of myocardial infarction. Inhibition of miR-34 also attenuated cardiac remodeling and improved heart function following pressure overload, however, silencing of miR-34a alone was not examined. The aim of this study was to assess whether inhibition of miR-34a could attenuate cardiac remodeling in a mouse model with pre-existing pathological hypertrophy. Mice were subjected to pressure overload via constriction of the transverse aorta for four weeks and echocardiography was performed to confirm left ventricular hypertrophy and systolic dysfunction. After four weeks of pressure overload (before treatment), two distinct groups of animals became apparent: (1) mice with moderate pathology (fractional shortening decreased ∼20%) and (2) mice with severe pathology (fractional shortening decreased ∼37%). Mice were administered locked nucleic acid (LNA)-antimiR-34a or LNA-control with an eight week follow-up. Inhibition of miR-34a in mice with moderate cardiac pathology attenuated atrial enlargement and maintained cardiac function, but had no significant effect on fetal gene expression or cardiac fibrosis. Inhibition of miR-34a in mice with severe pathology provided no therapeutic benefit. Thus, therapies that inhibit miR-34a alone may have limited potential in settings of established cardiac pathology.

Reperfusion of globally ischemic rat hearts causes rapid generation of inositol(1,4,5) trisphosphate [Ins(1,4,5)P(3)] and the development of arrhythmias, following stimulation of alpha(1)-adrenergic receptors by norepinephrine released from the cardiac sympathetic nerves. The heightened inositol phosphate response in reperfusion depends on the activation of the Na(+)/H(+) exchanger, which might reflect a central role for increased Ca(2+)following reverse mode activation of the Na(+)/Ca(2+) exchanger (NCX). Isolated, perfused rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and the content of Ins(1,4,5)P(3) measured by mass analysis or by anion-exchange high performance liquid chromatography (HPLC) following [(3)H]inositol labeling. Reperfusion caused generation of Ins(1,4,5)P(3) (1266+/-401 to 3387+/-256 cpm/g tissue, mean+/-s.e.m., n=6, P

Post-ischemic reperfusion causes a change in inositol phosphate responses to norepinephrine from primary generation of inositol(1,4) bis phosphate (Ins(1,4)P(2)) to generation of inositol(1,4,5) tris phosphate (Ins(1,4,5)P(3)) that is required for the initiation of reperfusion arrhythmias. The current study was undertaken to investigate the role of Na(+)/H(+)exchange in facilitating this transient change in inositol phosphate response. Rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and Ins(1, 4,5)P(3)content was measured by mass analysis or by anion-exchange HPLC following [(3)H]inositol labeling. Reperfusion caused generation of [(3)H]Ins(1,4,5)P(3)(1732+/-398 to 3103+/-214, cpm/g tissue, mean+/-S.E.M., n=5, P

Cardiac hypertrophy and function were studied 6 wk after constriction of the thoracic aorta (TAC) in transgenic (TG) mice expressing constitutively active mutant α1B-adrenergic receptors (ARs) in the heart. Hearts from sham-operated TG animals and nontransgenic littermates (WT) were similar in size, but hearts from TAC/TG mice were larger than those from TAC/WT mice, and atrial natriuretic peptide mRNA expression was also higher. Lung weight was markedly increased in TAC/TG animals, and the incidence of left atrial thrombus formation was significantly higher. Ventricular contractility in anesthetized animals, although it was increased in TAC/WT hearts, was unchanged in TAC/TG hearts, implying cardiac decompensation and progression to failure in TG mice. There was no increase in α1A-AR mRNA expression in TAC/WT hearts, and expression was significantly reduced in TAC/TG hearts. These findings show that cardiac expression of constitutively actively mutant α1B-ARs is detrimental in terms of hypertrophy and cardiac function after pressure overload and that increased α1A-AR mRNA expression is not a feature of the hypertrophic response in this murine model.

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

Objective: To seek direct evidence for a cause–effect relation between sympathetic activation and arrhythmogenesis. Methods: Rats underwent open-chest surgery with either coronary artery occlusion or sham operation, and were studied 8 weeks later using in situ heart perfusion and nerve stimulation methods. Results: Infarcted rats showed cardiac functional impairment and increased heart and lung weight. The extent of these changes correlated well with infarct size (IS). In in situ perfused hearts, sympathetic nerve stimulation (2 and 4 Hz, 45 s duration) induced a frequency-dependent release of norepinephrine (NE). NE release was lower in MI than that in control groups. In hearts with large IS (≥40%, n =19) ventricular arrhythmias were rare at baseline, but nerve stimulation evoked the onset of ventricular premature beats (95%), tachycardia (37%) and fibrillation (26%). IS and stimulation frequency were key determinants for the inducibility of arrhythmias. Lower K+ concentration enhanced arrhythmia inducibility. β-blockade inhibited the frequency of arrhythmias produced by nerve stimulation. Conclusion: In infarcted rat hearts sympathetic activation is a potent trigger for the onset of ventricular tachyarrhythmias.

Amiodarone has a "reserpine-like" sympatholytic action in the heart. The aims of this study were to test whether desethylamiodarone (DEA), the in vivo bioactive metabolite of amiodarone, has this action and whether this action could be demonstrated in a neuronal preparation. Experiments were performed in intact rats, perfused hearts, or brain synaptosomes treated with DEA and amiodarone, and concentrations of norepinephrine (NE) and dihydroxyphenylglycol (DHPG), the intraneuronal metabolite of NE, were assayed in plasma, coronary effluent, and synaptosomes. In perfused hearts, DEA at 1, 3, and 10 microM increased DHPG overflow by threefold, sixfold, and ninefold, respectively (all p < 0.01 vs. control). DEA at 1 microM was more potent than amiodarone in increasing DHPG overflow. DEA at 1 and 3 microM also inhibited NE release evoked by sympathetic nerve stimulation (p < 0.05). In intact rats, intravenous DEA at 15 mg/kg elicited onefold increase in plasma DHPG level, and oral pretreatment with amiodarone did not interfere with the sympatholytic action of intravenous amiodarone. In synaptosomes, 40-min incubation with amiodarone, DEA (both 10 microM), and reserpine reduced synaptosomal NE content by 42, 45, and 60%, respectively. Thus similar to its parent drug, DEA exerts a presynaptic sympatholytic action in rat hearts in vivo and in vitro. This action of amiodarone and DEA also was observed in synaptosomes.

The recent development of techniques in gene targeted manipulation, murine cardiac physiology and microsurgery in the mouse allow scientists to address some of the most fundamental questions concerning cardiac diseases and heart failure. Surgically induced heart disease models in mice have recently been described, but detailed information about the surgical techniques and functional characteristics of these models is limited. We describe the surgical details for induction of transverse aortic stenosis and myocardial infarction, 2 models which are ideal for studies on cardiac hypertrophy and failure, as evidenced by the functional results.

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.

Reperfusion of ischemic rat hearts initiates the generation of inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] and arrhythmias, provided that either norepinephrine or thrombin is present. In the current study, effects on endothelin-1 (ET-1) responses were investigated. Reperfusion of catecholamine-depleted, [3H]inositol-labeled hearts in the presence of ET-1 caused an increase in [3H]inositol phosphates (7,073 +/- 1,004 to 17,300 +/- 206 counts.min-1.g tissue-1, means +/- SE, n = 4, P < 0.01), which was quantitatively greater than the release observed under normoxic conditions, but there was no increase in [3H]Ins(1,4,5)P3. Gentamicin (150 microM) inhibited inositol phosphate responses in the presence of either norepinephrine or thrombin but did not inhibit the response to ET-1, providing additional evidence that the inositol phosphate response to ET-1 does not involve formation of Ins(1,4,5)P3, even under reperfusion conditions. In contrast to norepinephrine and thrombin, ET-1 did not initiate reperfusion arrhythmias (4.4% ventricular fibrillation compared with 0% ventricular fibrillation in catecholamine-depleted controls). The data provide strong evidence that the effect of ischemia-reperfusion on inositol phosphate responses is specific for particular receptor types and eliminates G proteins, phospholipase C enzymes, and substrate availability as the primary factors responsible for Ins(1,4,5)P3 generation under reperfusion conditions.

Precisely regulated patterns of gene expression are dependent on the binding of transcription factors and chromatin-associated determinants referred to as co-activators and co-repressors. These regulatory components function with the core transcriptional machinery to serve in critical activities to alter chromatin modification and regulate gene expression. While we are beginning to understand that cell-type specific patterns of gene expression are necessary to achieve selective cardiovascular developmental programs, we still do not know the molecular machineries that localize these determinants in the heart. With clear implications for the epigenetic control of gene expression signatures, the ENCODE (Encyclopedia of DNA Elements) Project Consortium determined that about 90% of the human genome is transcribed while only 1-2% of transcripts encode proteins. Emerging evidence suggests that non-coding RNA (ncRNA) serves as a signal for decoding chromatin modifications and provides a potential molecular basis for cell type-specific and promoter-specific patterns of gene expression. The discovery of the histone methyltransferase enzyme EZH2 in the regulation of gene expression patterns implicated in cardiac hypertrophy suggests a novel role for chromatin-associated ncRNAs and is the focus of this article.

The Polycomb-group protein, Ezh2, is required for epigenetic gene silencing in the adult heart by unknown mechanism. We investigated the role of Ezh2 and non-coding RNAs in a mouse model of pressure overload using transverse aortic constriction (TAC) attenuated by the prototypical histone deacetylase inhibitor, trichostatin A (TSA). Chromatin immunoprecipitation of TAC and TAC+TSA hearts suggests interaction of Ezh2 and primary microRNA-208b (pri-miR-208b) in the regulation of hypertrophic gene expression. RNAi silencing of pri-miR-208b and Ezh2 validate pri-miR-208b-mediated transcriptional silencing of genes implicated in cardiac hypertrophy including the suppression of the bi-directional promoter (bdP) of the cardiac myosin heavy chain genes. In TAC mouse heart, TSA attenuated Ezh2 binding to bdP and restored antisense β-MHC and α-MHC gene expression. RNA-chromatin immunoprecipitation experiments in TAC hearts also show increased pri-miR-208b dependent-chromatin binding. These results are the first description by which primary miR interactions serve to integrate chromatin modifications and the transcriptional response to distinct signaling cues in the heart. These studies provide a framework for MHC expression and regulation of genes implicated in pathological remodeling of ventricular hypertrophy.

Myocardial infarction (MI) provokes regional inflammation which facilitates the healing, whereas excessive inflammation leads to adverse cardiac remodelling. Our aim was to determine the role of macrophage migration inhibitory factor (MIF) in inflammation and cardiac remodelling following MI. Wild type (WT) or global MIF deficient (MIFKO) mice were subjected to coronary artery occlusion. Compared to WT mice, MIFKO mice had a significantly lower incidence of post-MI cardiac rupture (27% vs. 53%) and amelioration of cardiac remodelling. These were associated with suppressed myocardial leukocyte infiltration, inflammatory mediators' expression, and reduced activity of MMP-2, MMP-9, p38 and JNK MAPK. Infarct myocardium-derived or exogenous MIF mediated macrophage chemotaxis in vitro that was suppressed by inhibition of p38 MAPK or NF-κB. To further dissect the role of MIF derived from different cellular sources in post-MI cardiac remodelling, we generated chimeric mice with MIF deficiency either in bone marrow derived-cells (WT(KO)) or in somatic-cells (KO(WT)). Compared to WT and KO(WT) mice, WT(KO) mice had reduced rupture risk and ameliorated cardiac remodelling, associated with attenuated regional leukocyte infiltration and expression of inflammatory mediators. In contrast, KO(WT) mice had delayed healing and enhanced expression of M1 macrophage markers, but diminished expression of M2 markers during the early healing phase. In conclusion, global MIF deletion protects the heart from post-infarct cardiac rupture and remodelling through suppression of leukocyte infiltration and inflammation. Leukocyte-derived MIF promotes inflammatory responses after MI, whereas cardiac-derived MIF affects early but not ultimate healing process.

Catecholamines regulate the β-adrenoceptor/cyclic AMP-regulated protein kinase A (cAMP/PKA) pathway. Deregulation of this pathway can cause apoptotic cell death and is implicated in a range of human diseases, such as neuronal loss during aging, cardiomyopathy and septic shock. The molecular mechanism of this process is, however, only poorly understood. Here we demonstrate that the β-adrenoceptor/cAMP/PKA pathway triggers apoptosis through the transcriptional induction of the pro-apoptotic BH3-only Bcl-2 family member Bim in tissues such as the thymus and the heart. In these cell types, the catecholamine-mediated apoptosis is abrogated by loss of Bim. Induction of Bim is driven by the transcriptional co-activator CBP (CREB-binding protein) together with the proto-oncogene c-Myc. Association of CBP with c-Myc leads to altered histone acetylation and methylation pattern at the Bim promoter site. Our findings have implications for understanding pathophysiology associated with a deregulated neuroendocrine system and for developing novel therapeutic strategies for these diseases.