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Background Galectin-3 (Gal-3) is a clinical biomarker for risk of cardiovascular disease and a disease mediator forming a therapeutic target. However, the mechanism(s) that regulate cardiac expression of Gal-3 remains unknown. Activation of the sympatho-β-adrenergic system is a hallmark of heart disease, but the relationship of βAR activation and cardiac content of Gal-3 remains unknown. Purpose To determine the role of βAR activation in regulating cardiac Gal-3 level and the responsible mechanism focusing on the Hippo signalling pathway. Methods Wild-type and Gal-3 gene deleted (Gal3-KO) mice were used. To test the role of the Hippo pathway, we used transgenic (TG) mouse strains with cardiac overexpression of mammalian-20-like sterile kinase 1 (Mst1, mammalian orthology of Drosophila Hippo kinase) either in wild-type form (TG-Mst1) or dominative-negative kinase dead mutant form (TG-dnMst1). Effects of β-antagonist (isoprenaline, ISO) and antagonists were determined. We measured phosphorylation (Ser127) of YAP as a transcription co-regulator acting as the main signal output of the Hippo pathway. Results In wild-type mice, treatment with ISO led to a time- and dose-dependent increase in cardiac expression of Gal-3 (Fig. A) accompanied by elevated circulating Gal-3 levels (Fig. B). ISO treatment stimulated cardiac expression of Mst1 and YAP hyper-phosphorylation (i.e. inactivation, Fig. C), indicating activation of the Hippo signalling. These effects of ISO were inhibited by β-blockers (propranolol, Prop; carvedilol, Carv; Fig. D,E). Relative to non-TG controls, ISO-induced expression of Gal-3 was inhibited by 75% in TG-dnMst1 mice (inactivated Mst1), but exaggerated by 7-fold in TG-Mst1 mice (activated Mst1). Mst1-TG mice had a 45-fold increase in Gal-3 content, YAP hyper-phosphorylation and enhanced pro-fibrotic signaling. In Mst1-TG mice, whilst blood Gal-3 level was unchanged, treatment with ISO (6 mg, 2 days) evoked a marked increase in cardiac and blood Gal-3 levels. Using rat cardiomyoblasts, we showed that ISO-mediated Mst1 expression and YAP phosphorylation were PKA-dependent and that siRNA-mediated YAP knockdown led to Gal-3 upregulation. The role of Gal-3 in mediating ISO-induced cardiomyopathy was examined by treating wild-type and Gal3-KO mice with ISO (30 mg/kg, 7 days). ISO-treated wild-type mice had 8-fold increase in cardiac Gal-3, ventricular dysfunction, fibrosis, hypertrophy and activated inflammatory or fibrotic signalling. All these changes, except hypertrophy, were abolished by Gal3-KO. beta-AR regulates galectin-3 Conclusion βAR stimulation increases cardiac expression of Gal-3 through activation of the Hippo signalling pathway. This is accompanied by elevated circulating Gal-3 level. βAR antagonists inhibited βAR-Mst1 (Hippo) signalling and cardiac Gal-3 expression, actions likely contributing to the overall efficacy of β-blockers. Acknowledgement/Funding NHMRC of Australia; Nature Science Fund of China

Objective The aims of this study were, first, to determine if granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) improved left ventricular function in the setting of a reperfusion model of myocardial infarction (MI) and, second, to evaluate the effects of G-CSF/SCF on cellular repair and, in particular, the fate of bone marrow cells homing to the site of tissue injury. Methods MI was induced in mice by transient ligation of the left descending coronary artery. G-CSF/SCF were administered for 5 days after MI. Cardiac function was assessed 28 days after MI. The effect of G-CSF/SCF on the cellular composition of the infarct region was assessed by immunohistochemistry. MI was performed in mice reconstituted with bone marrow cells expressing DsRed to track the fate of bone marrow-derived cells within the infarct region. Results G-CSF/SCF-treated mice had significantly improved left ventricular (LV) function as determined by LV developed pressure, LV±d p /d t max/min, and LV end-diastolic pressure. G-CSF alone produced similar improvements in cardiac function. These improvements in LV function were associated with 70% more blood vessels and a doubling of cells expressing cardiomyocyte-specific transcription factors GATA-4, Nkx2.5 and alpha-actinin cells within the infarct zone. Cells within the infarct expressing stromal-derived factor also increased by 200%. To elucidate the origin of these cells, bone marrow chimeras, where hematopoietic cells expressed the fluorescent marker DsRed, were treated with G-CSF/SCF after MI. Bone marrow-derived, DsRed-expressing cells in the infarct region of G-CSF/SCF-treated chimeras increased by an average of 12-fold; however, the vast majority of DsRed cells expressed the hematopoietic-specific marker CD45 but not blood vessel or cardiomyocyte markers. Conclusions G-CSF/SCF therapy improved cardiac function when delivered after MI, increasing the number of blood vessels and cells of cardiomyogenic lineage. However, these cells were of myocardial rather than bone marrow origin.

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

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.

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

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.

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.

Experimental studies have provided evidence that the autonomic nervous activity is modulated by oestrogen. Such modulation at central and peripheral levels tends to suppress sympathetic but elevate parasympathetic tone to the cardiovascular system. Thus, available data support the view that cardiovascular protection by oestrogen may, at least in part, be mediated by its influence on autonomic nervous function.

Objective: The aim was to examine the time course of exocytotic and “spontaneous” noradrenaline overflow and the influence of an Uptake1, inhibitor, desipramine, in rat hearts subjected to anoxic and substrate-free perfusion. Methods: Hearts were perfused with a constant flow and exocytotic noradrenaline overflow was elicited either by electrical stimulation of the left stellate ganglion or by K+ depolarisation. Noradrenaline overflow was measured by HPLC. Results: Energy depletion for a period of 30 min resulted in an enhanced spontaneous noradrenaline overflow and a progressive decline in the nerve stimulation induced noradrenaline overflow. However, noradrenaline overflow induced by 40 mM K+ was enhanced by three- to fourfold in the energy depleted conditions. During anoxia, desipramine (0.3 μM) inhibited the spontaneous noradrenaline overflow and partly increased, in the early phase of anoxia, noradrenaline overflow by nerve stimulation, but showed no effect on K+ induced overflow. Further experiments showed that K+ at 10 mM failed to evoke noradrenaline overflow in normoxic hearts but induced a significant overflow in energy depleted hearts, either in the presence or absence of desipramine; quantities of noradrenaline overflow in response to 10-40 mM K+were substantially higher in anoxia. This difference in noradrenaline overflow caused by K+ during normoxia and anoxia was partly narrowed by desipramine which enhanced overflow in normoxia. Conclusions: “Spontaneous” and exocytotic noradrenaline release coexist within the 30 min period of anoxia but their responses to Uptake1 inhibitor differ. K+-induced noradrenaline overflow was markedly augmented by energy depletion due to a combination of failed neuronal reuptake and enhanced exocytosis. Cardiovascular Research 1993; 27 :2011-2015

See article by Guo et al. ([1], pages 930–938) in this issue. Ventricular arrhythmias occur frequently following acute myocardial infarction, during reperfusion and in heart failure. They are the likely major cause of sudden cardiac death in the majority of instances and as such represent a continuing and major cardiac health problem. The role of the sympathetic nervous system (SNS) in the genesis of arrhythmias under these conditions has been extensively studied for many years. Nevertheless, the importance of SNS activation in arrhythmogenesis remains controversial. This is again apparent in the current issue of Cardiovascular Research, where the findings from two papers with apparently contradictory interpretations are presented [1,2]. Numerous experimental approaches have been applied to manipulate the activity of the SNS, from dennervation and catecholamine depletion to cardiac sympathetic nerve stimulation or treatment with adrenergic agonists. These studies have been reviewed thoroughly [3–7]. Several lines of evidence have suggested an important or perhaps even pivotal role for SNS activation in initiating arrhythmias following myocardial infarction, reperfusion or heart failure. Thus both high levels of circulating catecholamines as well as increased cardiac noradrenaline (NA) spillover [8], increased sympathetic nerve firing rates and suppressed baroreflex sensitivity are all associated with increased incidences of arrhythmias and mortality [5]. β-blockers have been shown to be particularly beneficial in reducing cardiac mortality following myocardial infarction or heart failure, although evidence that their mechanism is by an anti-arrhythmic effect is less clear cut. There is strong evidence that ventricular fibrillation and sudden cardiac death may be triggered in some patients by psychologic stressors [9,10]. … * Corresponding author. Tel.: +61-3-9522-4399; fax: +61-3-9521-1362 xiaojun.du{at}baker.edu.au