Lymph heart in chick - somitic origin, development and embryonic oedema

The lymph heart is a sac-like structure on either side of avian tail. In some adult birds, it empties the lymph from the copulatory organ; however, during embryonic development, it is thought to circulate extra-embryonic lymph. Very little is known about the origin, innervation and the cellular changes it undergoes during development. Using immunohistochemistry and gene expression profiling we show that the musculature of the lymph heart is initially composed solely of striated skeletal muscle but later develops an additional layer composed of smooth myofibroblasts. Chick-quail fate-mapping demonstrates that the lymph heart originates from the hypaxial compartments of somites 34-41. The embryonic lymph heart is transiently innervated by somatic motoneurons with no autonomic input. In comparison to body muscles, the lymph heart has different sensitivity to neuromuscular junction blockers (sensitive only to decamethonium). Furthermore, its abundant bungarotoxin-positive acetylcholinesterase receptors are unique as they completely lack specific acetylcholinesterase activity. Several lines of evidence suggest that the lymph heart may possess an intrinsic pacing mechanism. Finally, we assessed the function of the lymph heart during embryogenesis and demonstrate that it is responsible for preventing embryonic oedema in birds, a role previously thought to be played by body skeletal muscle contractions.

Development and illustrative outputs of the Community Integrated Assessment System (CIAS), a multi-institutional modular integrated assessment approach for modelling climate change.

This paper describes the development and first results of the “Community Integrated Assessment System” (CIAS), a unique multi-institutional modular and flexible integrated assessment system for modelling climate change. Key to this development is the supporting software infrastructure, SoftIAM. Through it, CIAS is distributed between the community of institutions which has each contributed modules to the CIAS system. At the heart of SoftIAM is the Bespoke Framework Generator (BFG) which enables flexibility in the assembly and composition of individual modules from a pool to form coupled models within CIAS, and flexibility in their deployment onto the available software and hardware resources. Such flexibility greatly enhances modellers’ ability to re-configure the CIAS coupled models to answer different questions, thus tracking evolving policy needs. It also allows rigorous testing of the robustness of IA modelling results to the use of different component modules representing the same processes (for example, the economy). Such processes are often modelled in very different ways, using different paradigms, at the participating institutions. An illustrative application to the study of the relationship between the economy and the earth’s climate system is provided.

Uptake of a free coronary heart disease risk screening programme in community pharmacy: the Healthy Heart Assessment service

Introduction Health promotion (HP) aims to enhance good health while preventing ill-health at three levels of activity; primary (preventative), secondary (diagnostic) and tertiary (management).1 It can range from simple provision of health education to ongoing support, but the effectiveness of HP is ultimately dependent on its ability to influence change. HP as part of the Community Pharmacy Contract (CPC) aims to increase public knowledge and target ‘hard-to-reach’ individuals by focusing mainly on primary and tertiary HP. The CPC does not include screening programmes (secondary HP) as a service. Coronary heart disease (CHD) is a significant cause of morbidity and mortality in the UK. While there is evidence to support the effectiveness of some community pharmacy HP strategies in CHD, there is paucity of research in relation to screening services.2 Against this background, Alliance Pharmacy introduced a free CHD risk screening programme to provide tailored HP advice as part of a participant–pharmacist consultation. The aim of this study is to report on the CHD risk levels of participants and to provide a qualitative indication of consultation outcomes. Methods Case records for 12 733 people who accessed a free CHD risk screening service between August 2004 and April 2006 offered at 217 community pharmacies were obtained. The service involved initial self-completion of the Healthy Heart Assessment (HHA) form and measurement of height, weight, body mass index, blood pressure, total cholesterol and highdensity lipoprotein levels by pharmacists to calculate CHD risk.3 Action taken by pharmacists (lifestyle advice, statin recommendation or general practitioner (GP) referral) and qualitative statements of advice were recorded, and a copy provided to the participants. The service did not include follow-up of participants. All participants consented to taking part in evaluations of the service. Ethical committee scrutiny was not required for this service development evaluation. Results Case records for 10 035 participants (3658 male) were evaluable; 5730 (57%) were at low CHD risk (<15%); 3636 (36%) at moderate-to-high CHD risk (≥15%); and 669 (7%) had existing heart disease. A significantly higher proportion of male (48% versus 30% female) participants were at moderate- to-high risk of CHD (chi-square test; P < 0.005). A range of outcomes resulted from consultations. Lifestyle advice was provided irrespective of participants’ CHD risk or existing disease. In the moderate-to-high-risk group, of which 52% received prescribed medication, lifestyle advice was recorded for 62%, 16% were referred and 34% were advised to have a re-assessment. Statin recommendations were made in 1% of all cases. There was evidence of supportive and motivational statements in the advice recorded. Discussion Pharmacists were able to identify individuals’ level of CHD risk and provide them with bespoke advice. Identification of at-risk participants did not automatically result in referrals or statin recommendation. One-third of those accessing the screening service had moderate-to-high risk of CHD, a significantly higher proportion of whom were men. It is not known whether these individuals had been previously exposed to HP but presumably by accessing this service they may have contemplated change. As effectiveness of HP advice will depend among other factors on ability to influence change, future consultations may need to explore patients’ attitude towards change in relation to the Trans Theoretical Model4 to better tailor HP advice. The high uptake of the service by those at moderate-to-high CHD risk indicates a need for this type of screening programme in community pharmacy, perhaps specifically to reach men who access medical services less.

Characterisation and regulation of E2F-6 and E2F-6b in the rat heart: a potential target for myocardial regeneration?

The E2F transcription factors are instrumental in regulating cell cycle progression and growth, including that in cardiomyocytes, which exit the cell cycle shortly after birth. E2F-6 has been demonstrated to act as a transcriptional repressor; however, its potential role in normal cardiomyocyte proliferation and hypertrophy has not previously been investigated. Here we report the isolation and characterisation of E2F-6 and E2F-6b in rat cardiomyocytes and consider its potential as a target for myocardial regeneration following injury. At the mRNA level, both rat E2F-6 and the alternatively spliced variant, E2F-6b, were expressed in E18 myocytes and levels were maintained throughout development into adulthood. Interestingly, E2F-6 protein expression was down-regulated during myocyte development suggesting that it is regulated post-transcriptionally in these cells. During myocyte hypertrophy, the mRNA expressions of E2F-6 and E2F-6b were not regulated whereas E2F-6 protein was up-regulated significantly. Indeed, E2F-6 protein expression levels closely parallel the developmental withdrawal of myocytes from the cell cycle and the subsequent reactivation of their cell cycle machinery during hypertrophic growth. Furthermore, depletion of E2F-6, using anti-sense technology, results in death of cultured neonatal myocytes. Taken together, abrogation of E2F-6 expression in neonatal cardiomyocytes leads to a significant decrease in their viability, consistent with the notion that E2F-6 might be required for maintaining normal myocyte growth.

Inhibition of E2F abrogates the development of cardiac myocyte hypertrophy

Growth of the post- natal mammalian heart occurs primarily by cardiac myocyte hypertrophy. Previously, we and others have shown that a partial re- activation of the cell cycle machinery occurs in myocytes undergoing hypertrophy such that cells progress through the G(1)/ S transition. In this study, we have examined the regulation of the E2F family of transcription factors that are crucial for the G(1)/ S phase transition during normal cardiac development and the development of myocyte hypertrophy in the rat. Thus, mRNA and protein levels of E2F- 1, 3, and 4 and DP- 1 and DP- 2 were down- regulated during development to undetectable levels in adult myocytes. Interestingly, E2F- 5 protein levels were substantially up- regulated during development. In contrast, an induction of E2F- 1, 3, and 4 and the DP- 1 protein was observed during the development of myocyte hypertrophy in neonatal myocytes treated with serum or phenylephrine, whereas the protein levels of E2F- 5 were decreased with serum stimulation. E2F activity, as measured by a cyclin E promoter luciferase assay and E2F- DNA binding activity, increased significantly during the development of hypertrophy with serum and phenylephrine compared with non- stimulated cells. Inhibiting E2F activity with a specific peptide that blocks E2F- DP heterodimerization prevented the induction of hypertrophic markers ( atrial natriuretic factor and brain natriuretic peptide) in response to serum and phenylephrine, reduced the increase in myocyte size, and inhibited protein synthesis in stimulated cells. Thus, we have shown that the inhibition of E2F function prevents the development of hypertrophy. Targeting E2F function might be a useful approach for treating diseases that cause pathophysiological hypertrophic growth.

Cell cycle profiles and expression of p21CIP1 and P27KIP1 during myocyte development

The ability of the cardiac myocyte to divide ceases shortly after birth. Thus, following severe injury, e.g., during myocardial infarction, the mature heart is unable to regenerate new tissue to replace the dead or damaged tissue. The identification of the molecules controlling the cessation of myocyte cell division may lead to therapeutic strategies which aim to re-populate the damaged myocardial area. Hence, we have determined the cell cycle profile, expressions and activities of the cyclin-dependent kinase inhibitors (CDKIs), p21CIP1 and p27KIP1, during rat ventricular myocyte development. Fluorescent activated cell sorting (FACS) analyses showed the percentage of S phase myocytes to be decreased significantly throughout development, concomitant with a significant increase in the percentage of G0/G1 and G2/M phase cells. The expression of p21CIP1 and p27KIP1 increased significantly throughout cardiac development and complexed differentially with a number of cyclins and CDKs. Furthermore, an adult myocyte extract reduced neonatal myocyte CDK2 kinase activity significantly (>30%, p<0.05) whereas immunodepletion of p21CIP1 from adult lysates restored CDK2 kinase activity. Thus, p21CIP1 and p27KIP1 may be important for the withdrawal of cardiac myocytes from the cell cycle and for maintaining the G0/G1 and G2/M phase blockades.

Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart

Objective: Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts. Methods: Poly A+ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42-day cardiac myocytes or from whole ventricular tissue at 2, 5–11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80K/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of γ32P-ATP transferred to a specific peptide substrate. Results: RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6–8-day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day-old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood. Conclusions: Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6–8-day developmental period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.

Lipoma preferred partner is a mechanosensitive protein regulated by nitric oxide in the heart

Adaptor proteins play an important role in signaling pathways by providing a platform on which many other proteins can interact. Malfunction or mislocalization of these proteins may play a role in the development of disease. Lipoma preferred partner (LPP) is a nucleocytoplasmic shuttling adaptor protein. Previous work shows that LPP plays a role in the function of smooth muscle cells and in atherosclerosis. In this study we wanted to determine whether LPP has a role in the myocardium. LPP expression increased by 56% in hearts from pressure overload aortic-banded rats (p < 0.05 n = 4), but not after myocardial infarction, suggesting hemodynamic load regulates its expression. In vitro, LPP expression was 87% higher in cardiac fibroblasts than myocytes (p < 0.05 n = 3). LPP expression was downregulated in the absence of the actin cytoskeleton but not when microtubules were disassembled. We mechanically stretched cardiac fibroblasts using the Flexcell 4000 for 48 h (1 Hz, 5% maximum strain), which decreased total LPP total expression and membrane localization in subcellular fractions (p < 0.05, n = 5). However, L-NAME, an inhibitor of nitric oxide synthase (NOS), significantly upregulated LPP expression. These findings suggest that LPP is regulated by a complex interplay between NO and mechanical cues and may play a role in heart failure induced by increased hemodynamic load.

Conditional transgenic expression of fibroblast growth factor 9 in the adult mouse heart reduces heart failure mortality after myocardial infarction

BACKGROUND: Fibroblast growth factor 9 (FGF9) is secreted from bone marrow cells, which have been shown to improve systolic function after myocardial infarction (MI) in a clinical trial. FGF9 promotes cardiac vascularization during embryonic development but is only weakly expressed in the adult heart. METHODS AND RESULTS: We used a tetracycline-responsive binary transgene system based on the α-myosin heavy chain promoter to test whether conditional expression of FGF9 in the adult myocardium supports adaptation after MI. In sham-operated mice, transgenic FGF9 stimulated left ventricular hypertrophy with microvessel expansion and preserved systolic and diastolic function. After coronary artery ligation, transgenic FGF9 enhanced hypertrophy of the noninfarcted left ventricular myocardium with increased microvessel density, reduced interstitial fibrosis, attenuated fetal gene expression, and improved systolic function. Heart failure mortality after MI was markedly reduced by transgenic FGF9, whereas rupture rates were not affected. Adenoviral FGF9 gene transfer after MI similarly promoted left ventricular hypertrophy with improved systolic function and reduced heart failure mortality. Mechanistically, FGF9 stimulated proliferation and network formation of endothelial cells but induced no direct hypertrophic effects in neonatal or adult rat cardiomyocytes in vitro. FGF9-stimulated endothelial cell supernatants, however, induced cardiomyocyte hypertrophy via paracrine release of bone morphogenetic protein 6. In accord with this observation, expression of bone morphogenetic protein 6 and phosphorylation of its downstream targets SMAD1/5 were increased in the myocardium of FGF9 transgenic mice. CONCLUSIONS: Conditional expression of FGF9 promotes myocardial vascularization and hypertrophy with enhanced systolic function and reduced heart failure mortality after MI. These observations suggest a previously unrecognized therapeutic potential for FGF9 after MI.

Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart.

We investigated the ability of phenylephrine (PE), an alpha-adrenergic agonist and promoter of hypertrophic growth in the ventricular myocyte, to activate the three best-characterized mitogen-activated protein kinase (MAPK) subfamilies, namely p38-MAPKs, SAPKs/JNKs (i.e. stress-activated protein kinases/c-Jun N-terminal kinases) and ERKs (extracellularly responsive kinases), in perfused contracting rat hearts. Perfusion of hearts with 100 microM PE caused a rapid (maximal at 10 min) 12-fold activation of two p38-MAPK isoforms, as measured by subsequent phosphorylation of a p38-MAPK substrate, recombinant MAPK-activated protein kinase 2 (MAPKAPK2). This activation coincided with phosphorylation of p38-MAPK. Endogenous MAPKAPK2 was activated 4-5-fold in these perfusions and this was inhibited completely by the p38-MAPK inhibitor, SB203580 (10 microM). Activation of p38-MAPK and MAPKAPK2 was also detected in non-contracting hearts perfused with PE, indicating that the effects were not dependent on the positive inotropic/chronotropic properties of the agonist. Although SAPKs/JNKs were also rapidly activated, the activation (2-3-fold) was less than that of p38-MAPK. The ERKs were activated by perfusion with PE and the activation was at least 50% of that seen with 1 microM PMA, the most powerful activator of the ERKs yet identified in cardiac myocytes. These results indicate that, in addition to the ERKs, two MAPK subfamilies, whose activation is more usually associated with cellular stresses, are activated by the Gq/11-protein-coupled receptor (Gq/11PCR) agonist, PE, in whole hearts. These data indicate that Gq/11PCR agonists activate multiple MAPK signalling pathways in the heart, all of which may contribute to the overall response (e.g. the development of the hypertrophic phenotype).

Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential.

Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress-induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H(2)O(2) induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H(2)O(2) stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from approximately 30 minutes). The levels of the mitochondrial membrane marker cytochrome oxidase remained unchanged. H(2)O(2) also induced translocation of cytochrome c from the mitochondria to the cytosol within 15 to 30 minutes, which was indicative of mitochondrial dysfunction. Myocytes exposed to H(2)O(2) showed an early loss of mitochondrial membrane potential (assessed by fluorescence-activated cell sorter analysis) from 15 to 30 minutes, which was partially restored by approximately 1 hour. However, a subsequent irreversible loss of mitochondrial membrane potential occurred that correlated with cell death. These data suggest that the regulation of Bcl-2 and mitochondrial function are important factors in oxidative stress-induced cardiac myocyte apoptosis.

Activation of protein kinase cascades in the heart by hypertrophic G protein-coupled receptor agonists.

Cardiac myocyte hypertrophy involves changes in cell structure and alterations in protein expression regulated at both the transcriptional and translational levels. Hypertrophic G protein-coupled receptor (GPCR) agonists such as endothelin-(ET-1) and phenylephrine stimulate a number of protein kinase cascades in the heart. Mitogen-activated protein kinase (MAPK) cascades stimulated include the extracellularly regulated kinase cascade, the stress-activated protein kinase/c-Jun N-terminal kinase cascade, and the p38 MAPK cascade. All 3 pathways have been implicated in hypertrophy, but recent ex vivo evidence also suggests that there may be additional effects on cell survival. ET-1 and phenylephrine also stimulate the protein kinase B pathway, and this may be involved in the regulation of protein synthesis by these agonists. Thus, protein kinase-mediated signaling may be important in the regulation of the development of myocyte hypertrophy.

Targeting myocardial remodelling to develop novel therapies for heart failure: a position paper from the Working Group on Myocardial Function of the European Society of Cardiology

The failing heart is characterized by complex tissue remodelling involving increased cardiomyocyte death, and impairment of sarcomere function, metabolic activity, endothelial and vascular function, together with increased inflammation and interstitial fibrosis. For years, therapeutic approaches for heart failure (HF) relied on vasodilators and diuretics which relieve cardiac workload and HF symptoms. The introduction in the clinic of drugs interfering with beta-adrenergic and angiotensin signalling have ameliorated survival by interfering with the intimate mechanism of cardiac compensation. Current therapy, though, still has a limited capacity to restore muscle function fully, and the development of novel therapeutic targets is still an important medical need. Recent progress in understanding the molecular basis of myocardial dysfunction in HF is paving the way for development of new treatments capable of restoring muscle function and targeting specific pathological subsets of LV dysfunction. These include potentiating cardiomyocyte contractility, increasing cardiomyocyte survival and adaptive hypertrophy, increasing oxygen and nutrition supply by sustaining vessel formation, and reducing ventricular stiffness by favourable extracellular matrix remodelling. Here, we consider drugs such as omecamtiv mecarbil, nitroxyl donors, cyclosporin A, SERCA2a (sarcoplasmic/endoplasmic Ca(2 +) ATPase 2a), neuregulin, and bromocriptine, all of which are currently in clinical trials as potential HF therapies, and discuss novel molecular targets with potential therapeutic impact that are in the pre-clinical phases of investigation. Finally, we consider conceptual changes in basic science approaches to improve their translation into successful clinical applications.