[Show abstract][Hide abstract] ABSTRACT: Cardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the ?-adrenoceptor (?AR) agonist phenylephrine (PE) compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal ?1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease.
[Show abstract][Hide abstract] ABSTRACT: Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet. We studied PI3K-FOXO1A pathways during differentiation of hESC towards endothelial lineage and on proliferation, maturation and cell death of hESC-derived endothelial cells (hESC-EC). During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodelling-related genes. PI3K inhibitor LY294002 activated FOXO1A and induced formation of CD31+ hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by siRNA showed lower mRNA levels of CD31 and angiopoietin2. LY294002 decreased proliferative activity of purified hESC-EC, whilst FOXO1A siRNA increased their proliferation. LY294002 inhibits migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. After in vivo conditioning of cells in athymic nude rats, cells retain their low FOXO1A expression levels. PI3K/FOXO1A pathway is important for function and survival of hESC-EC as well as in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs.
[Show abstract][Hide abstract] ABSTRACT: Endothelial cells form a highly specialised lining of all blood vessels where they provide an anti-thrombotic surface on the luminal side and protect the underlying vascular smooth muscle on the abluminal side. Specialised functions of endothelial cells include their unique ability to release vasoactive hormones and to morphologically adapt to complex shear stress. Stem cell derived-endothelial cells have a growing number of applications and will be critical in any organ regeneration programme. Generally endothelial cells are identified in stem cell studies by well-recognised markers such as CD31. However, the ability of stem cell-derived endothelial cells to release vasoactive hormones and align with shear stress has not been studied extensively. With this in mind, we have compared directly the ability of endothelial cells derived from a range of stem cell sources, including embryonic stem cells (hESC-EC) and adult progenitors in blood (blood out growth endothelial cells, BOEC) with those cultured from mature vessels, to release the vasoconstrictor peptide endothelin (ET)-1, the cardioprotective hormone prostacyclin, and to respond morphologically to conditions of complex shear stress. All endothelial cell types, except hESC-EC, released high and comparable levels of ET-1 and prostacyclin. Under static culture conditions all endothelial cell types, except for hESC-EC, had the typical cobblestone morphology whilst hESC-EC had an elongated phenotype. When cells were grown under shear stress endothelial cells from vessels (human aorta) or BOEC elongated and aligned in the direction of shear. By contrast hESC-EC did not align in the direction of shear stress. These observations show key differences in endothelial cells derived from embryonic stem cells versus those from blood progenitor cells, and that BOEC are more similar than hESC-EC to endothelial cells from vessels. This may be advantageous in some settings particularly where an in vitro test bed is required. However, for other applications, because of low ET-1 release hESC-EC may prove to be protected from vascular inflammation.
Biochemical and Biophysical Research Communications 11/2014; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During cardiac hypertrophy, cardiomyocytes (CMs) increase in the size and expression of cytoskeletal proteins while reactivating a foetal gene programme. The process is proposed to be dependent on increased nuclear export and, since nuclear pore trafficking has limited capacity, a linked decrease in import. Our objective was to investigate the role of nuclear import and export in control of hypertrophy in rat and human heart failure (HF).
[Show abstract][Hide abstract] ABSTRACT: In Takotsubo cardiomyopathy, the left ventricle shows apical ballooning combined with basal hypercontractility. Both clinical observations in humans and recent experimental work on isolated rat ventricular myocytes suggest the dominant mechanisms of this syndrome are related to acute catecholamine overload. However, relating observed differences in single cells to the capacity of such alterations to result in the extreme changes in ventricular shape seen in Takotsubo syndrome is difficult.
By using a computational model of the rat left ventricle, we investigate which mechanisms can give rise to the typical shape of the ventricle observed in this syndrome.
Three potential dominant mechanisms related to effects of beta-adrenergic stimulation were considered: apical-basal variation of calcium transients due to differences in L-type and SERCA activation, apical-basal variation of calcium sensitivity due to differences in troponin I phosphorylation, and apical-basal variation in maximal active tension due to e.g. the negative inotropic effects of p38 MAPK. Furthermore, we investigated the interaction of these spatial variations in the presence of a failing Frank-Starling mechanism.
We conclude that a large portion of the apex needs to be affected by severe changes in calcium regulation or contractile function to result in apical ballooning, and smooth linear variation from apex to base are unlikely to result in the typical ventricular shape observed in this syndrome. A failing Frank-Starling mechanism significantly increases apical ballooning at end-systole, and may be an important additional factor underpinning Takotsubo syndrome.
American journal of physiology. Heart and circulatory physiology. 09/2014;
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.
PLoS ONE 01/2014; 9(4):e91119. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to investigate whether caveolin-3 (Cav3) regulates localization of β2-adrenergic receptor (β2AR) and its cAMP signaling in healthy or failing cardiomyocytes. We co-expressed wildtype Cav3 or its dominant-negative mutant (Cav3DN) together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps in adult rat ventricular myocytes (ARVMs). FRET and scanning ion conductance microscopy were used to locally stimulate β2AR and to measure cytosolic cAMP. Cav3 overexpression increased the number of caveolae and decreased the magnitude of β2AR-cAMP signal. Conversely, Cav3DN expression resulted in an increased β2AR-cAMP response without altering the whole-cell L-type calcium current. Following local stimulation of Cav3DN-expressing ARVMs, β2AR response could only be generated in T-tubules. However, the normally compartmentalized β2AR-cAMP signal became diffuse, similar to the situation observed in heart failure. Finally, overexpression of Cav3 in failing myocytes led to partial β2AR redistribution back into the T-tubules. In conclusion, Cav3 plays a crucial role for the localization of β2AR and compartmentation of β2AR-cAMP signaling to the T-tubules of healthy ARVMs, and overexpression of Cav3 in failing myocytes can partially restore the disrupted localization of these receptors.
Journal of Molecular and Cellular Cardiology 12/2013; · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiac cell replacement therapy by using human embryonic stem cell derivatives remains a potential approach to regenerate myocardium. The major hurdles to clinical application of this technology are immunogenicity and post-transplantation cell death. Here we examined the effects of calcineurin-targeting immunosuppressants cyclosporine A (CsA), and FK506, as well as rapamycin (RAP) and a selective inhibitor of calcineurin-binding downstream NFAT transcription factor VIVIT on the proliferative activity, function and survival of hESC-derived cardiomyocytes (hESC-CM) and endothelial cells (hESC-EC) in culture. As shown by automated microscopy, treatments with CsA, FK506 and RAP all decreased proliferation, reducing the percentage of hESC-CM and hESC-EC cells with the mitotic marker Ki67+ by as much as 60% and 74%, respectively. Administration of the cell permeable analogue 11R-VIVIT protein did not modulate their proliferative activity. All immunosuppressants reversed the pro-apoptotic effect of chelerythrine in hESC-CM demonstrating an inhibitory role of calcineurin/NFAT and mTOR pathways in hESC-CM survival (using apoptotic marker caspase-3) whereas the protection was less obvious in hESC-EC exposed to H2O2. Immunosuppressants did not affect cell viability in hESC-EC. Our results show that immunosuppressants reduce proliferation while offsetting cell loss to a smaller extent by reduction in apoptosis of hESC-CM. Immunosuppressants therapy would be compatible with stem cell transplantation but the resulting reduction in graft expansion capabilities would potentially necessitate implantation of increased cell numbers when immunosuppressants are given. The effects of NFAT-binding immunosuppressant molecules, which do not affect hESC-CM proliferation, may point the way forward for new classes of compounds better suited to cell implantation.
Stem cells and development 11/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Therapeutic options which directly enhance cardiomyocyte contractility in chronic heart failure (HF) therapy are currently limited and do not improve prognosis. In fact, most positive inotropic agents, such as β-adrenoreceptor agonists and phosphodiesterase inhibitors, which have been assessed in HF patients cause increased mortality as a result of arrhythmia and sudden cardiac death. Cardiac sarcoplasmic reticulum Ca(2+) ATPase2a (SERCA2a) is a key protein involved in sequestration of Ca(2+) into the sarcoplasmic reticulum (SR) during diastole. There is a reduction of SERCA2a protein level and function in HF, which has been successfully targeted via viral transfection of the SERCA2a gene into cardiac tissue in-vivo. This has enhanced cardiac contractility and reduced mortality in several preclinical models of HF. Theoretical concerns have been raised regarding the possibility of arrhythmogenic adverse effects of SERCA2a gene therapy due to enhanced SR Ca(2+) load and induction of SR Ca(2+) leak as a result. Contrary to these concerns SERCA2a gene therapy in a wide variety of pre-clinical models, including acute ischaemia/reperfusion, chronic pressure overload, and chronic myocardial infarction, has resulted in a reduction in ventricular arrhythmias. The potential mechanisms for this unexpected beneficial effect, as well as mechanisms of enhancement of cardiac contractile function, are reviewed in this article.
British Journal of Pharmacology 10/2013; · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Professor Sian Harding talks to Caroline Telfer, Assistant Commissioning Editor. Professor Sian Harding obtained her PhD in Pharmacology from King's College, London (UK) in 1981. She became Professor of Cardiac Pharmacology at the National Heart and Lung Institute, a division of the Imperial College Faculty of Medicine, in 2002. Her work has been funded by the British Heart Foundation, the Wellcome Trust, the Medical Research Council, the Biochemical and Biophysical Research Council, the The National Centre for the Replacement, Refinement and Reduction of Animals in Research, Pfizer, GlaxoSmithKline and SmithKline Beecham. Harding is former president of the European Section of the International Society for Heart Research and has organized international cardiovascular science meetings for this society, as well as for the European Society of Cardiology. She is the principal investigator for the first UK gene therapy trial aimed at improving cardiac contractility, organized jointly at Harefield and Papworth Hospitals. Harding is a member of the Nuffield Council on Bioethics and the Medical Research Coucil Regenerative Medicine Research Committee, and Director of a recently awarded British Heart Foundation Cardiovascular Regenerative Medicine Centre. She has been elected Fellow of the American Heart Association, European Society of Cardiology, International Society for Heart Research, Society of Biology and British Society of Pharmacology.
[Show abstract][Hide abstract] ABSTRACT: The potential of stem cell-based disease modelling is enhanced by the realisation that cardiomyocytes from human embryonic stem cells (hESC-CM) and induced pluripotent stem cells (hiPSC-CM) can be obtained with disease-specificity. Hypertrophy is a high priority target because of its central role in the transition to heart failure. Strikingly, here we found that hiPSC-CM are relatively unresponsive to major hypertrophic signals compared to hESC-CM. We show that the normal alpha-adrenergic receptor 1A subtype (ADRA1A) is not expressed robustly in either cell type. ADRA1A is reversibly silenced during differentiation, accompanied by up-regulation of ADRA1B, resulting in a distinct gene profile from that in adult human cardiomyocytes. Loss of ADRA1A is more pronounced in hiPSC-CM, due to greater epigenetic silencing and more marked up-regulation of HIF-1α, but ultimately both cell types differ from adult in their reliance on active ADRA1B rather than ADRA1A. ADRA1B up-regulation is sufficient in hESC-CM for hypertrophic changes such as cell size, cell volume and ANF. However, in hiPSC-CM, additional decreased G-protein signalling and tonically inhibitory pathway networks suppress the effect of alpha-adrenoceptor stimulation on growth. Superficial similarities between hESC-CM, hiPSC-CM and adult cardiomyocytes may mask complex differences in signalling. These data raise serious questions regarding the hiPSC-CM as a valid model system for certain aspects of cardiac disease.
Human Gene Therapy 05/2013; 24(5):A9. · 4.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The beta1-adrenoceptors (β(1)AR) and beta-2 (β(2)AR) adrenoceptors represent the predominant pathway for sympathetic control of myocardial function. Diverse mechanisms have evolved to translate signalling via these two molecules into differential effects on physiology. In this review we discuss how the functions of the βAR are o rganized from the level of secondary messengers to the whole heart to achieve this. Using novel microscopy and bio-imaging methods researchers have uncovered subtle organization of the control of cAMP, the predominant positively inotropic pathway for the βAR. The β(2)AR in particular is demonstrated to give rise to highly compartmentalized, spatially confined cAMP signals. Organization of β(2)AR within the t-tubule and caveolae of cardiomyocytes concentrates this receptor with molecules which buffer and shape its cAMP signal to give fine control. This situation is undermined in various forms of heart failure. Human and animal models of heart failure demonstrate disruption of cellular micro-architecture which contributes to the change in response to cardiac βARs. Loss of cellular structure has proven key to the observed loss of confined β(2)AR signaling. Some pharmacological and genetic treatments have been successful in returning failing cells to a more structured phenotype. Within these cells it has been possible to observe the partial restoration of normal β(2)AR signalling. At the level of the organ, expression of the two βAR sub-types varies between regions with the β(2)AR forming a greater proportion of the βAR population at the apex. This distribution may contribute to regional wall motion abnormalities in Takotsubo cardiomyopathy, a syndrome of high sympathetic activity, where the phosphorylated β(2)AR can signal via G(i) protein to produce negatively inotropic effects.
Cardiovascular Research 01/2013; · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AIM: Ca(2+) waves are thought to be important in the aetiology of ventricular tachyarrhythmias. There have been conflicting results regarding whether flecainide reduces Ca(2+) waves in isolated cardiomyocytes. We sought to confirm whether flecainide inhibits waves in the intact cardiomyocyte and to elucidate the mechanism.Methods and ResultsWe imaged spontaneous sarcoplasmic reticulum (SR) Ca(2+) release events in healthy adult rat cardiomyocytes. Variation in stimulation frequency was used to produce Ca(2+) sparks or waves. Spark frequency, wave frequency and wave velocity were reduced by flecainide in the absence of a reduction of SR Ca(2+) content. Inhibition of I(Na) via alternative pharmacological agents (tetrodotoxin, propafenone or lidocaine) produced similar changes. To assess the contribution of I(Na) to spark and wave production voltage clamping was used to activate contraction from holding potentials of -80 mV or -40 mV. This confirmed that reducing Na(+) influx during myocyte stimulation is sufficient to reduce waves and that flecainide only causes Ca(2+) wave reduction when I(Na) is active. It was found that Na(+)/Ca(2+)-exchanger (NCX)-mediated Ca(2+) efflux was significantly enhanced by flecainide and that the effects of flecainide on wave frequency could be reversed by reducing [Na(+)](o), suggesting an important downstream role for NCX function. CONCLUSIONS: Flecainide reduces spark and wave frequency in the intact rat cardiomyocyte at therapeutically relevant concentrations but the mechanism involves I(Na) reduction rather than direct ryanodine receptor (RyR2) inhibition. Reduced I(Na) results in increased Ca(2+) efflux via NCX across the sarcolemma, reducing Ca(2+) concentration in the vicinity of the RyR2.
Cardiovascular Research 01/2013; · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have been widely proposed as in vitro models of myocardial physiology and disease. A significant obstacle, however, is their immature phenotype. We hypothesised that Ca(2+) cycling of iPSC-CM is influenced by culture conditions and can be manipulated to obtain a more mature cellular behaviour. To test this hypothesis we seeded iPSC-CM onto fibronectin coated microgrooved polydimethylsiloxane (PDMS) scaffolds fabricated using photolithography, or onto unstructured PDMS membrane. After two weeks in culture, the structure and function of iPSC-CM were studied. PDMS microgrooved culture substrates brought about cellular alignment (p < 0.0001) and more organised sarcomere. The Ca(2+) cycling properties of iPSC-CM cultured on these substrates were significantly altered with a shorter time to peak amplitude (p = 0.0002 at 1 Hz), and more organised sarcoplasmic reticulum (SR) Ca(2+) release in response to caffeine (p < 0.0001), suggesting improved SR Ca(2+) cycling. These changes were not associated with modifications in gene expression. Whilst structured tissue culture may make iPSC-CM more representative of adult myocardium, further construct development and characterisation is required to optimise iPSC-CM as a model of adult myocardium.
[Show abstract][Hide abstract] ABSTRACT: Imperial College London (UK) was the showcase for the second in the 'Frontiers in Cardiovascular Biology' series, a biennial meeting of the European Society of Cardiology Council on basic cardiovascular sciences, held from 30 March to 1 April 2012. The aim of this series is to bring researchers together to learn the very latest findings in cardiac and vascular sciences, and to see state-of-the-art and developing technologies that could impact cardiovascular research. Five keynote lectures, 25 scientific symposia and two translational lunchtime symposia were grouped around the central themes of bioimaging, degeneration and regeneration, and inflammation.
[Show abstract][Hide abstract] ABSTRACT: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) are being investigated as a new source of cardiac cells for drug safety assessment. We developed a novel scalable high content microscopy-based method for the detection of cell death in hPSC-CM that can serve for future predictive in vitro cardio-toxicological screens. Using rat neonatal ventricular cardiomyocytes (RVNC) or hPSC-CM, assays for nuclear remodelling, mitochondrial status, apoptosis and necrosis were designed using a combination of fluorescent dyes and antibodies on an automated microscopy platform. This allowed the observation of a chelerythrine-induced concentration-dependent apoptosis to necrosis switch and time-dependent progression of early apoptotic cells towards a necrotic-like phenotype. Susceptibility of hPSC-CM to chelerythrine-stimulated apoptosis varied with time after differentiation, but at most time points, hPSC-CM were more resistant than RVNC. This simple and scalable humanized high-content assay generates accurate cardiotoxicity profiles that can serve as a base for further assessment of cardioprotective strategies and drug safety.
Journal of Cardiovascular Translational Research 08/2012; 5(5):593-604. · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic β(2)-adrenergic receptor (β(2)AR) from canonical stimulatory G-protein-activated cardiostimulant to inhibitory G-protein-activated cardiodepressant pathways.
We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via G(i) inactivation by pertussis toxin pretreatment. β(2)AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a β(2)AR-Gi-dependent manner. Preventing epinephrine-G(i) effects increased mortality in the Takotsubo model, whereas β-blockers that activate β(2)AR-G(i) exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality.
We suggest that biased agonism of epinephrine for β(2)AR-G(s) at low concentrations and for G(i) at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in β(2)ARs explaining the differential regional responses. We suggest this epinephrine-specific β(2)AR-G(i) signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.