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Right ventricular (RV) failure is a major concern in grown-up congenital heart diseases. Cell-based myocardial repair may be an innovative approach to restore overloaded RV function. A repaired Tetralogy of Fallot pig model was used. After 5 months, cell therapy was surgically administrated using human embryonic stem cell-derived Nkx2.5+ cardiac progenitor cell-seeded collagen patch. A large patch containing 107 cells was engineered to cover the whole RV free wall and applied on the epicardium of 4 pigs (treated group). A control group (n = 4) received a cell-free patch (3) or no patch (1). Myocardial function was assessed by echocardiography using standard and strain parameters before and 2 months after cell graft. The fate of progenitors was tracked using human-and cardiac specific antibodies. Indexed RV diameter and telediastolic RV area significantly decreased in treated group whereas increased in control. The free wall thickness significantly increased in treated group compared to control. In the treated group, RV free wall strain significantly improved by contrast to control (respectively from −13.4 to −15.9% vs. −18.9 to −12.9%, P Fig. 1 ). Although still immature, some cells featured TnT+ actinin+ sarcomeric units. Proliferating small Ki-67+ TnT+ labelled pig myocytes were observed. Expression of collagen genes col1a3, col3a1 monitored in real-Time QPCR reflecting myocardial fibrosis status significantly decreased in the treated group. Human cardiac progenitors migrated and differentiated, contributing together with triggered endogenous mechanism of repair to maintain the RV adaptation to overload and to improve free wall contractility. They also induced a proliferation of host cells supporting potential paracrine effects.

The origins of congenital heart diseases, the most common congenital diseases are still largely unknown. Environmental factors are now emerging as major causes of these diseases. Vitamin D deficiency has become a public health burden, notably for childbearing age, pregnant and breastfeeding women. Since maternal 25-hydroxyvitamin D (25(OH)D) determined fetal and neonatal 25(OH)D status, foetuses exposed to insufficient levels of vitamin D, may feature developmental defects.Herein, we investigated the effects of maternal vitamin D deficiency on cardiovascular defects in early and later life of offsprings in two generations as well as the molecular mechanisms underlying vitamin D effect.Eight weeks before and during pregnancy, C57BL/6JRj female mice received a sufficient or vitamin D deficient diet ((1.0 IU/g in control vs 0.0 IU/g in Vitamin D Deficient (VDD) group). E16.5 Embryos of maternal VDD diet featured hypertrophic heart revealed by a thicker left ventricular (LV) wall and septum. RNAseq analysis of LV revealed 1555 transcripts differentially expressed in the VDD group and among them cardiac transcription factors and constitutive cardiac genes (tbx5, gata4, myl2). Anti-Vitamin D receptor (VDR) Chip-seq from chromatin of E16.5 LV uncovered different targeting of tbx5 and tbx3 loci by VDR in the VDD vs control embryos. Anti-CTCF ChIP-loop experiments focusing on the Tbx3 and Tbx5 loci uncovered a change in the Topology Associated Domains associated with these loci.Echocardiography of 2-months-old VDD offspring revealed a significantly thicker left ventricle and increased fractional shortening while 6-months-old mice featured cardiac decompensation and in turn failing LV.Maternal vitamin D deficiency severely affects heart formation following a change in chromatin conformation on cardiac gene loci and impacts function of adult hearts in two generations. These defects are likely to be at the origin of cardiovascular diseases in the adulthood.

International audience; In utero environment is crucial to ensure normal development of the fetus and to program metabolic health throughout the life. Beside macronutrients, the role of micronutrients, including vitamin D, begins to be explore. The aim of this study was to decipher the impact of maternal vitamin D deficiency (VDD), in normal and high‐fat (HF) diet context, on adipose tissue metabolism and energy homeostasis in offspring, considering sex‐specific responses. Body weight, energy expenditure, and spontaneous activity was differential impacted in juvenile male and female offspring born from VDD mice. In adulthood, a HF diet combined with maternal VDD disrupted glucose homeostasis and adiposity in male offspring but not in females. Such phenotypes were associated to different transcriptomic profiles in adipose tissue, which could be related to differential modulation of plasma 17β‐estradiol concentrations. Thus, maternal VDD sex‐dependently modulated metabolic fate of the offspring, especially when associated with HF diet in adulthood.

Epicardium, the mesothelium covering the heart, is composed of multipotent cells and is reactivated following myocardial injury in adults. Herein, we provide evidence for activation of atrial epicardium in aged patients with diseased atria and in murine models of atrial remodeling. Epicardial activation contributed to fibro-fatty infiltration of sub-epicardium that contained a number of cells co-expressing markers of epicardial progenitors and fibroblasts. Indeed, using genetic lineage tracing of adult epicardium, we demonstrate the epicardial origin of fibroblasts within fibro-fatty infiltrates. A subpopulation of adult epicardial-derived cells (aEPDCs) expressing PDGFRα, niched in the sub-epicardium, were isolated and differentiated into myofibroblast in the presence of angiotensin-II. Furthermore, single cell RNA-seq analysis identified several clusters of aEPDCs and revealed transition from adipogenic to fibrogenic cells. In conclusion, a subset of aEPDCs, pre-programmed towards a specific cell fate, contributes to fibro-fatty infiltration of sub-epicardium of diseased atria.

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International audience; Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Cardiac fibroblasts are a major cell population of the heart and are characterized by their capacity to produce extracellular matrix (ECM). In hearts subjected to pressure overload, excessive fibroblast accumulation is responsible for fibrosis of the myocardium, a major clinical issue. Hence, understanding mechanisms generating fibroblasts in this context has become a key question in the cardiovascular field. Recent studies now point to the activation of resident fibroblasts as the underlying cause of fibrosis. However, de novo generation of fibroblasts from endothelium and circulating hematopoietic cells has also been proposed to significantly contribute to fibrosis. Here, we discuss the latest findings on fibroblast origins, with a particular emphasis on the pressure overload model, and the implication of these findings for the development of anti-fibrotic therapies that are currently lacking.

Myocardial infarction is a major cause of adult mortality worldwide. The origin(s) of cardiac fibroblasts that constitute the postinfarct scar remain controversial, in particular the potential contribution of bone marrow lineages to activated fibroblasts within the scar. The aim of this study was to establish the origin(s) of infarct fibroblasts using lineage tracing and bone marrow transplants and a robust marker for cardiac fibroblasts, the Collagen1a1-green fluorescent protein reporter. Using genetic lineage tracing or bone marrow transplant, we found no evidence for collagen-producing fibroblasts derived from hematopoietic or bone marrow lineages in hearts subjected to permanent left anterior descending coronary artery ligation. In fact, fibroblasts within the infarcted area were largely of epicardial origin. Intriguingly, collagen-producing fibrocytes from hematopoietic lineages were observed attached to the epicardial surface of infarcted and sham-operated hearts in which a suture was placed around the left anterior descending coronary artery. In this controversial field, our study demonstrated that the vast majority of infarct fibroblasts were of epicardial origin and not derived from bone marrow lineages, endothelial-to-mesenchymal transition, or blood. We also noted the presence of collagen-producing fibrocytes on the epicardial surface that resulted at least in part from the surgical procedure. T.M. Moore-Morris was supported by American Heart Association postdoctoral fellowship 11POST7310066. P. Cattaneo was supported by the Marie Curie International Outgoing Fellowship within the 7th European Community Framework Program under grant agreement No 623739—The Cardiac Code. M. Cedenilla received funding from the Spanish Ministry of Education, Culture, and Sports, Predoctoral Fellowship program (FPU, AP2008-00508) and Travel Grant Program (TTFPU11-AP2008-00508). J. Bogomolovas is supported by the European Commission’s Marie Sklodowska-Curie Individual Fellowship (Titin Signals, 656636). S.M. Evans is funded by grants from the National Heart, Lung, and Blood Institute and the Leducq Foundation. M. Pucéat and T.M. Moore-Morris acknowledge the generosity of the Leducq Foundation (SHAPEHEART). Sí

Recently, a preclinical report assessed the efficacy, safety, and feasibility of using human embryonic stem cells to regenerate infarcted myocardium in a nonhuman primate model. This commentary evaluates that study by pointing out key weaknesses, offering an alternative perspective, and summarizing major unresolved issues. Our conclusion is that significant challenges remain before human embryonic stem cells are ready for use in clinical trials. Ischemic heart disease (IHD) affects millions of patients. The cardiac dysfunction that results from IHD is secondary to the death of cardiac muscle cells. Current IHD treatments are inadequate, and there is a need to develop novel therapies to improve the lives of affected patients. New cellular therapies are being developed for repairing damaged hearts via induction of cardiac myocyte regeneration. In our view, approaches that can regenerate new, functional myocardium need to be tested first in small animal models and then in appropriate large animal models. Those approaches that are shown to be safe and to improve cardiac function in preclinical studies should then be considered for clinical application. In a recent report in Nature , Chong et al1 describe experiments designed to test the potential of human embryonic stem cell–derived cardiac myocytes (hESC-CMs) to regenerate infarcted myocardium in a nonhuman primate model. This study was proposed by the authors as proof of concept for trials of these cells in patients. Our commentary looks at this report with an eye toward safety and efficacy of hESC-CMs. A major advance in the study of Chong et al1 is that an approach has been developed to produce large quantities of hESC-CMs that seem to be sufficient for future use in humans with IHD. The authors were able to amplify 1 billion hESC-CMs, a number purported sufficient to regenerate an adult human infarcted heart. This approach addresses …

International audience; The abundance of epicardial adipose tissue (EAT) is associated with atrial fibrillation (AF), the most frequent cardiac arrhythmia. However, both the origin and the factors involved in EAT expansion are unknown. Here, we found that adult human atrial epicardial cells were highly adipogenic through an epithelial-mesenchymal transition both in vitro and in vivo. In a genetic lineage tracing the WT1(CreERT2+/-)Rosa(tdT+/-)mouse model subjected to a high-fat diet, adipocytes of atrial EAT derived from a subset of epicardial progenitors. Atrial myocardium secretome induces the adipogenic differentiation of adult mesenchymal epicardium-derived cells by modulating the balance between mesenchymal Wingless-type Mouse Mammary Tumor Virus integration site family, member 10B (Wnt10b)/beta-catenin and adipogenic ERK/MAPK signaling pathways. The adipogenic property of the atrial secretome was enhanced in AF patients. The atrial natriuretic peptide secreted by atrial myocytes is a major adipogenic factor operating at a low concentration by binding to its natriuretic peptide receptor A (NPRA) receptor and, in turn, by activating a cGMP-dependent pathway. Hence, our data indicate cross-talk between EAT expansion and mechanical function of the atrial myocardium.

Background and aims The accumulation of the adipose tissue (AT) in the sup- and subepicardium of the atrial myocardium is associated with a high risk of atrial fibrillation. Here we addressed the question of the cellular origin of atrial AT. Methods Human right atrial specimen obtained during cardiac surgery were used for histological, biochemical studies and to harvest epicardial progenitors. Cells were characterized using flow cytometry, proteomic and genic expression assays and maintained in culture conditions, in some experiments they were co cultured with human atrial myocytes using a Transwell system. Results In atrial section, epicardial progenitors expressing the pre-adipocyte factor 1 (Pref-1) were detected in the epicardial and sub-epicardial layer which contained a number of progenitor cells referred as epicardic progenitorderived cells (EPDCs). Some EPDCs expressed Pref-1 suggesting that they could engaged in the adipocyte pathways. This was tested in vitro using EPDCs harvested from human right atrial samples (n=20). From 1 st to 5-6 cells passages, EPDCs underwent an epithelial-to-mesenchymal transition (EMT), acquired mesenchymal phenotypes and could differentiate into osteocyte or chondrocyte. When cultured using an adipogenic medium, around 40±6% of EPDCs cells showed lipid droplet colored with oil red and expressed mature adipocyte perilipin marker. Culture medium enriched with the secretome of human atria too induced the differentiation of EPDCs in adipocytes (n=6). A shift from pre- to mature adipocytes was indicated by the detection of Pref-1 at 7 but not 30 days of culture, it was the opposite for perilipin. Human atrial myocyte could be the source of pro-adipogenic factor. In fact, after 7 days in coculture, EPDCs are differentiated into adipocytes (n=6). Conclusion Human EPDCs from atria can differentiate into adipocyte; factors secreted by atrial myocytes regulate this differentiation process. The author hereby declares no conflict of interest

Cardiac fibroblasts produce the extracellular matrix (ECM) scaffold within which the various cellular components of the heart are organized. As well as providing structural support, it is becoming evident that the quality and quantity of ECM is a key factor for determining cardiac cell behavior during development and in pathological contexts such as heart failure involving fibrosis. Cardiac fibroblasts have long remained a poorly characterized cardiac lineage. Well characterized markers are now paving the way for a better understanding of the roles of these cells in various developmental and disease contexts. Notably, the relevance of processes including endothelial-tomesenchymal transition and the recruitment of circulating fibroblast progenitors in heart failure has been challenged. This review describes the latest findings on cardiac fibroblast markers and developmental origins, and discusses their importance in myocardial remodeling. Effective modulation of cardiac fibroblast activity would likely contribute to successful treatment of various cardiac disorders.

In the adult, the heart rate is driven by spontaneous and repetitive depolarizations of pacemaker cells to generate a firing of action potentials propagating along the conduction system and spreading into the ventricles. In the early embryo before E9.5, the pacemaker ionic channel responsible for the spontaneous depolarization of cells is not yet functional. Thus the mechanisms that initiate early heart rhythm during cardiogenesis are puzzling. In the absence of a functional pacemaker ionic channel, the oscillatory nature of inositol 1,4,5-trisphosphate (InsP3)-induced intracellular Ca2+signaling could provide an alternative pacemaking mechanism. To test this hypothesis, we have engineered pacemaker cells from embryonic stem (ES) cells, a model that faithfully recapitulates early stages of heart development. We show that InsP3-dependent shuttle of free Ca2+in and out of the endoplasmic reticulum is essential for a proper generation of pacemaker activity during early cardiogenesis and fetal life.

Mutations of genes encoding contractile proteins are responsible for familial hypertrophic cardiomyopathies. Understanding the process of differentiation of cardiomyocytes carrying a mutated protein is a crucial step towards potential treatments of inherited cardiac disorders. Embryonic Stem (ES) cells which faithfully recapitulate in vitro the process of cardiac cell differentiation can be genetically modified to incorporate a mutation mimicking a cardiomyopathy. ES cell lines engineered to express a wild-type (MLC2vGFP) or a mutated form (R58QMLC2vGFP) of ventricular myosin light chain 2 (MLC2v) fused to GFP were differentiated into cardiomyocytes within embryoid bodies (EBs). Visualization of GFP combined with sarcomeric actinin immunofluorescence of EBs revealed that mutated MLC2v dramatically prevented myofibrillogenesis. Cardiomyocytes expressing wild-type MLC2v featured spontaneous Ca(2+) spiking, but not those harboring the mutation. Expression of cardiac transcription factors Mef2c, GATAs, myocardin and Nkx2.5 was not affected by cell expression of mutated MLC2v. A dramatic decrease in expression of mRNAs encoding alpha-actin, MLC2a and MLC2v was observed in R58QMLC2vGFP EBs. This event was attributed to a failure of Mef2c to translocate into the nucleus, a Ca(2+)-dependent process. Expression in mutated cells of a constitutively active Ca(2+)- and calmodulin-dependent kinase II or treating EBs with ionomycin fully restored translocation of Mef2c into the nucleus and expression of mRNAs encoding sarcomeric proteins partially rescued contractile activity of EBs. Alteration of Ca(2+) homeostasis in mutated cardioblasts affects the transcriptional program of cardiac cell differentiation leading to a defect in myofibrillogenesis, and, in turn, in contractility. Genetically modified ES cells provide a unique cell model to determine abnormalities in Ca(2+) homeostasis underlying progression of human cardiomyopathies.

The present study was designed to investigate whether and how the purinergic stimulation of rat ventricular myocytes modulates the cAMP-dependent pathway. Stimulation of cardiomyocytes with ATPgammaS in the presence of the phosphodiesterase inhibitor IBMX increases by 3-fold intracellular cAMP content. In contrast to beta-adrenergic stimulation, the purinergic stimulation of adenylyl cyclase was not inhibited by activation or enhanced by inhibition of a Gi protein. Forskolin did not potentiate the effect of extracellular ATPgammaS on intracellular cAMP content but the effect of isoproterenol did. Like isoproterenol, the purinergic agonist decreased subsequent ADP-ribosylation of a 45 kDa G(alpha s) by cholera toxin. ATPgammaS also increased cAMP content in neonatal rat cardiomyocytes, a cell type that expresses a long form of Gs protein (alpha(s), 52 kDa) in contrast to adult rat cardiomyocytes that express mostly a short form of Gs protein (alpha(s), 45 kDa). Both purinergic and beta-adrenergic agonists increased cAMP in HEK 293 cells expressing type V adenylyl cyclase while cAMP was only increased by beta-adrenergic stimulation of HEK expressing type IV or VI adenylyl cyclases. Thus, we propose that the purinergic and beta-adrenergic stimulations differentially activate adenylyl cyclase isoforms in rat cardiomyocytes and that adenylyl cyclase V is the specific target of the purinergic stimulation.

International audience; The functional and architectural benefits of embryonic stem cells (ESC) and myoblasts (Mb) transplantations into infarcted myocardium have been investigated extensively. Whereas ESC repopulated fibrotic areas and contributed to myocardial regeneration, Mb exerted their effects through paracrine secretions and scar remodeling. This therapeutic perspective, however, has been less explored in the setting of nonischemic dilated cardiomyopathies (DCMs). Our aim was to compare the integration and functional efficacy of ESC committed to cardiac fate by bone morphogenic protein 2 (BMP-2) pretreatment and Mb used as gold standard following their transplantation into the myocardium of a mouse model of laminopathy exhibiting a progressive and lethal DCM. After 4 and 8 weeks of transplantation, stabilization was observed in Mb-transplanted mice (P = 0.008) but not in groups of ESC-transplanted or medium-injected animals, where the left ventricular fractional shortening (LVFS) decreased by 32 ± 8% and 41 ± 8% respectively. Engrafted differentiated cells were consistently detected in myocardia of mice receiving Mb, whereas few or no cells were detected in the hearts of mice receiving ESC, except in two cases where teratomas were formed. These data suggest that committed ESC fail to integrate in DCM where scar tissue is absent to provide the appropriate niche, whereas the functional benefits of Mb transplantation might extend to nonischemic cardiomyopathy.

The effects of beta-adrenergic agonists on pHi were studied on single ventricular myocytes isolated from adult rat heart and loaded with the acetoxymethyl ester (AM) form of the pH indicator SNARF-1. In modified Krebs' solution containing 20 mmol/L HEPES and 4.4 mmol/L HCO3-, isoproterenol (1 mumol/L) caused a significant decrease of steady-state pHi from 7.20 +/- 0.02 to 7.13 +/- 0.02 (mean +/- SEM) within 2 minutes. This acidification, which was also observed in myocytes that were preloaded with the Ca2+ chelator BAPTA and superfused with nominally Ca(2+)-free solution, was blocked by propranolol as well as by the specific beta 1-antagonist CGP 20712 A but not by the beta 2-antagonist ICI 118,551. Forskolin (10 mumol/L) induced a similar reversible decrease of pHi (average decrease, 0.11 +/- 0.02 pH unit). Furthermore, adenosine (100 mumol/L) substantially attenuated the isoproterenol-induced decrease of pHi. The effect of isoproterenol was not prevented by inhibitors of the Na(+)-H+ antiport, amiloride (1 mmol/L) and 2-N,N-hexamethylene amiloride (20 mumol/L). On the other hand, blockers of Cl- transport mechanisms, DIDS (200 mumol/L) and probenecid (100 mumol/L), inhibited this acidification, Isoproterenol also failed to induce a decrease of steady-state pHi in myocytes incubated in Cl(-)-free medium. Rather, the initial rate of rise of pHi observed on removal of external Cl- ions was significantly increased in the presence of isoproterenol or dibutyryl cAMP. Because the alkalinization induced by removal of Cl- ions is mainly due to reversal of the Cl(-)-HCO3- exchanger, the augmentation of this initial rate of pHi rise directly points to a beta-adrenergic stimulation of the exchanger. Furthermore, the pHi recovery following NH4Cl exposure was accelerated by isoproterenol in the presence of probenecid, indicating that the Na(+)-HCO3- cotransport and/or the Na(+)-H+ antiport also could be activated.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the effects of an alpha 1-adrenoceptor (phenylephrine) and a purinoceptor agonist (ATP), both of which accelerate the phosphoinositide turnover, on the Na-H antiport activity of rat single cardiac cells using the pH-sensitive fluorescent indicator seminaphthorhodafluor-1 (SNARF-1). Both phenylephrine, in the presence of a beta-adrenoceptor blocker, and ATP enhanced the ability of the cell to regulate its intracellular pH (pHi) after an imposed acid load. This effect was observed in HCO3-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and prevented by Na-H antiport inhibitors ethylisopropylamiloride (EIPA) or amiloride. Similar results were obtained when cells were bathed in an acidic extracellular medium. Hence, the alpha 1-adrenoceptor and purinoceptor agonists activate the Na-H antiport even when it is partially inhibited by extracellular protons. To further evaluate the effects of the two neurohormones, the rate of proton efflux was estimated as a function of the magnitude of the imposed acid load. The results indicate that the agonist-induced modulation of the Na-H antiport is caused by an acceleration of its exchange activity and by a shift of its dependence on pHi toward more alkaline pH values. The agonist-mediated stimulation of the antiport was also observed in partially depolarized cells and was not dependent on intracellular Ca. Phorbol 12-myristate 13-acetate was not able to reproduce the effects of the agonists on the Na-H antiport. Conversely, the inhibitors of protein kinase C did not prevent the activation of the antiport by the neurohormones. Thus our data suggest that neither a Ca-calmodulin-dependent kinase nor protein kinase C is responsible for the alpha 1-adrenoceptor- and purinoceptor-mediated stimulation of the antiport.

Background Despite the prevalence of right ventricular (RV) failure in congenital heart diseases, cell therapy applied to RV is poorly studied. Our aim is to evaluate in a large animal model of overloaded RV dysfunction such therapy using cardiac progenitors (CP) issued from human embryonic stem cells. Methods A combined overloaded RV dysfunction was created in pigs using a surgical procedure mimicking repaired tetralogy of Fallot. At 4 months, cell therapy was surgically administrated using either multiple transepicardial injections of HUES-24 derived CP into RV myocardium or CP seeded collagen patches sewn on RV free wall. SHAM animals received either multiple transepicardial injections of medium or acellular patches. Myocardial function was determined 3 months later by conductance catheter technique with maximal elastance (Emax) slope. Ventricular arrhythmia risks were tested by programmed ventricular stimulation. A histological study analyzed the structural remodelling. CP fate was studied using anti- Ki67, CD31, CD34, GFP, Islet1 and Connexin 43 antibodies. All pigs were immunosuppressed by Tacrolimus. Results All pigs survived. Neither complication nor ventricular arrhythmia occurred. In injected animals (SHAM: n=6, HUES-24: n=6) the Emax slope value evoluated similarly in both groups. Whereas total fibrosis increased significantly with time in the SHAM group, it returned to baseline in HUES-24 group. However, CP could not be found. In contrast, in patched animals, CP were found in the patch zone and close to the myocardium. These CP were able to proliferate, migrate, express cardiac markers and establish connexions. Conclusion Cell therapy using transepicardial injections of human CP seems to have a beneficial effect on overloaded RV tissue remodelling, but this administration mode did not improve myocardial contractility. Seeded patches seem to be more conservative for engrafted cells; their impact on overloaded RV function requires further experiments.

Background— The safety and efficacy of myocardial regeneration using embryonic stem cells are limited by the risk of teratoma and the high rate of cell death. Methods and Results— To address these issues, we developed a composite construct made of a sheet of adipose tissue–derived stroma cells and embryonic stem cell–derived cardiac progenitors. Ten Rhesus monkeys underwent a transient coronary artery occlusion followed, 2 weeks later, by the open-chest delivery of the composite cell sheet over the infarcted area or a sham operation. The sheet was made of adipose tissue–derived stroma cells grown from a biopsy of autologous adipose tissue and cultured onto temperature-responsive dishes. Allogeneic Rhesus embryonic stem cells were committed to a cardiac lineage and immunomagnetically sorted to yield SSEA-1 + cardiac progenitors, which were then deposited onto the cell sheet. Cyclosporine was given for 2 months until the animals were euthanized. Preimplantation studies showed that the SSEA-1 + progenitors expressed cardiac markers and had lost pluripotency. After 2 months, there was no teratoma in any of the 5 cell-treated monkeys. Analysis of >1500 histological sections showed that the SSEA-1 + cardiac progenitors had differentiated into cardiomyocytes, as evidenced by immunofluorescence and real-time polymerase chain reaction. There were also a robust engraftment of autologous adipose tissue–derived stroma cells and increased angiogenesis compared with the sham animals. Conclusions— These data collected in a clinically relevant nonhuman primate model show that developmentally restricted SSEA-1 + cardiac progenitors appear to be safe and highlight the benefit of the epicardial delivery of a construct harboring cells with a cardiomyogenic differentiation potential and cells providing them the necessary trophic support.

P2-purinergic stimulation of the L-type Ca current induced by the external application of 100 microM ATP gamma S was investigated in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The purinergic-induced increase in ICa was slow and monophasic and reached a steady state within 3 min. In contrast to beta-adrenergic stimulation, after a brief agonist application the current did not continue to increase on washout; recovery started immediately after agonist removal. The P2-purinergic increase in ICa was significantly less in the presence of GDP beta S, but it occurred much faster and was twice as large when a low dose of GTP gamma S (100 microM) was added to a GTP-containing internal medium. This suggests that the ICa increase was mediated by a G protein. Based on electrophoretic mobility and susceptibility to cholera toxin and anti-G alpha s serum, it is proposed that the G protein involved during purinergic-induced ICa stimulation is an isoform of Gs not coupled to the adenylyl cyclase, since the cyclic AMP level was unaffected. High intracellular GTP gamma S (1 mM) maximally activated ICa so that neither beta-adrenergic nor P2-purinergic agonists further increased ICa. In the absence of GTP and an ATP-regenerating system, GTP gamma S was much more potent in increasing basal ICa and supporting purinergic stimulation. This indicates that a nucleoside diphosphate kinase activity might replenish endogenous GTP; GTP exchange with GTP gamma S on the G protein was promoted by the P2-purinergic stimulation and led to a reversible and reproducible increase in ICa. In the presence of 3 mM internal ATP gamma S, the P2-purinergic stimulation was also reversible and reproducible. Moreover, under these conditions (ATP gamma S or GTP gamma S) the increase in ICa was not maintained during prolonged agonist application. Such an inhibition occurred slowly and irreversibly; it might be related to the threefold increase in cyclic GMP. In conclusion, we propose that extracellular ATP induces both a stimulatory and an inhibitory effect on ICa, probably mediated by subtypes of P2-purinergic receptors. An isoform of the Gs protein is likely to mediate the stimulation.

Before its degradation to adenosine, extracellular ATP released by nerves, or by cardiac cells during ischemia, induces positive inotropy related to stimulation of calcium conductance by P2-purinoceptors. Transiently ATP may also depolarize the cells and trigger arrhythmias by activating a new P3-purinoceptor that requires Mg ions.

1755-3245 (Electronic) Journal Article Research Support, Non-U.S. Gov't; AIMS: Cells derived from the stroma vascular fraction (SVF) of mouse adipose tissue can spontaneously give rise to rare, functional, cardiac-like cells in vitro. This study aimed to improve the production of adipose-derived cardiomyogenic cells (AD-CMG), to characterize them and to assess their cardiac fate and functional outcomes after their administration in a mouse model of acute myocardial infarction. METHODS AND RESULTS: The culture process optimized to improve in vitro cardiac specification consisted of a primary culture of murine SVF cells in semi-solid methylcellulose medium, a selection of AD-CMG cell clusters, and a secondary culture and expansion in BHK21 medium. AD-CMG cells were CD29(+), CD31(-), CD34(-), CD44(+), CD45(-), CD81(+), CD90(-), CD117(-), and Flk-1(-) and expressed several cardiac contractile proteins. After 1, 2, and 4 weeks of their injection in mice having acute myocardial infarction, a strong presence of green fluorescent protein-positive cells was identified by immunohistochemistry as well as quantitative polymerase chain reaction. Echocardiography showed a significant reduction of remodelling and stability of left ventricle ejection fraction in the AD-CMG cell-treated group vs. controls. Vascular density analysis revealed that AD-CMG administration was also associated with stimulation of angiogenesis in peri-infarct areas. CONCLUSION: Cardiomyogenic cells can be selected and expanded in large amounts from mouse adipose tissue. They can survive and differentiate in an acute myocardial infarction model, avoiding remodelling and impairment of cardiac function, and can promote neo-vascularization in the ischaemic heart.

To investigate the changes in the properties of cardiac contractile proteins due to neurohormonal stimulation, different agonists were applied to single cells isolated from rat ventricle. Cells were then rapidly skinned by Triton X-100, and force was recorded after gluing the cells to a strain gauge. The skinned cells had mechanical properties very similar to those described for thin trabeculas. Tension-pCa relations were highly reproducible from one cell to another, with sarcomere length fixed at 2.1 microns. The application of alpha 1-adrenergic and muscarinic agonists, which increase the turnover of phosphatidylinositol, for 5 minutes before skinning the cells increased the sensitivity of the myofilaments to calcium, as indicated by a leftward shift of the tension-pCa relation, whereas beta-adrenergic stimulation induced a rightward shift. The increase in calcium sensitivity was also evoked by protein kinase C activators such as 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate but not by protein kinase C itself or by purinergic agonists, although the latter also increased the turnover of phosphatidylinositol. Incubation of the skinned cells with phosphatase reversed the alterations in calcium sensitivity induced by previous agonist stimulation of the intact cells. In conclusion, this study demonstrates a potentially influential mechanism for the physiological regulation of cardiac muscle contractility.

Background Cell therapy using intramyocardial injections of cardiac progenitors issued from human embryonic stem cells showed benefits on overloaded right ventricular (RV) tissue remodelling and arrhythmic susceptibility but this delivery mode failed to improve RV function. Our aim was to evaluate in a porcine model of overloaded RV dysfunction a new delivery mode of such therapy. Methods A combined overloaded RV dysfunction was obtained in piglets using a surgical procedure mimicking repaired tetralogy of Fallot. After 4 months, cell therapy was surgically administrated using 2 types of human NKX2.5+ cardiac progenitor cell-seeded collagen patches applied on the epicardium: QGel® and pressured-patches. Myocardial function was measured 1 month after transplantation by conductance catheter technique and echocardiography (standard and strain). The fate of progenitors was studied using antibodies directed against Ki67, CD31, actinin and Islet1. Results All pigs survived without any complication. Pressured-patches allowed human progenitors to migrate across the complete myocardium while QGel® patches restricted the cell migration to only a third of the myocardium. In both cases, progenitors differentiated toward the cardiac lineage assessed by Islet1 and actinin expression and maintained their proliferation capacity. Concerning RV function, only pressured-patches (N=3) tended to improve the contractility (Emax slope). By contrast, this parameter decreased in QGel® patches animals (N=2). Moreover, in 2 pressured-patch animals, standard echocardiographic functional parameters (FAC, TAPSE, s’wave) were maintained while 2D strain and strain rate values increased. Conclusion Cell therapy using seeded-patches was more conservative for engrafted cells than intramyocardial injections but only pressured-patches seemed to give benefits on overloaded RV function and contractility. These first promising results need to be checked at longer term.

Despite the increasing incidence of right ventricular (RV) failure in adult patients with congenital heart disease, current therapeutic options are still limited. By contrast to left-heart diseases, cell-based myocardial regeneration applied to the right ventricle is poorly studied, even though it may be a therapeutic solution. As human embryonic stem cell-derived cardiac progenitors seem to be good candidates owing to their proliferation capacity, our aim was to assess, in a large animal model of overloaded RV dysfunction, the feasibility and effects of such a cell therapy.Human MesP1(+)/SSEA-1(+) cardiogenic mesodermal cells were administered using multiple intramyocardial injections 4 months after a surgical procedure mimicking the repaired tetralogy of Fallot, and their effects were observed 3 months later on hemodynamic, rhythmic, and histologic parameters.All pigs (sham n = 6, treated n = 6) survived without complication, and cell therapy was clinically well tolerated. Although functional, contractility, and energetics parameters evolved similarly in both groups, benefits regarding arrhythmic susceptibility were observed in the treated group, associated with a significant decrease of peri-myocyte fibrosis (5.71% ± 2.49% vs 12.12% ± 1.85%; P.01) without interstitial fibrosis change (5.18% ± 0.81% vs 5.49% ± 1.01%). Such a decrease could be related to paracrine effects, as no human cells could be detected within the myocardium.Cell therapy using intramyocardial injections of human MesP1(+)/SSEA-1(+) cardiogenic mesodermal cells seems to have benefits regarding overloaded RV tissue remodeling and arrhythmic susceptibility, but this mode of administration is not sufficient to obtain a significant improvement in RV function.

Summary Background Heart failure develops after myocardial infarction and is a major cause of morbidity and mortality. The ability to direct differentiation of embryonic stem cells (ESC) towards a cardiomyogenic phenotype makes them an attractive therapeutic option for cardiac repair, but species-specific and individual-specific immunological imprinting remains a hurdle. Our aim was to ascertain whether the purported immune privilege of ESC allows for their cross-species engraftment in a clinically relevant large-animal model. Methods We studied engraftment and differentiation of cardiac-committed mouse ESC in 18 sheep in which a myocardial infarction had been induced; nine controls received medium and nine sheep (five of which were immunosuppressed) received ESC. The gain in myocardial function was measured by echocardiography 1 month after cell transplantation. Findings Cardiac-committed murine ESC engrafted in infarcted myocardium of immunosuppressed and immunocompetent sheep, and differentiated into mature cardiomyocytes that expressed connexins. Colonisation of the scar area by ESC was accompanied by a functional benefit of the damaged myocardium. Left-ventricular ejection fraction deteriorated in the control group by a median of 9·9% (range −20 to 0·3) relative to baseline (p=0·011) whereas in the treated group it improved by 6·6% (−5·7 to 50·8; comparison between groups p=0·002). Interpretation These findings obtained in a clinically relevant large-animal model of heart failure strengthen the potential therapeutic use of ESC to regenerate the severely dysfunctional myocardium and bring additional evidence for an immune privilege of these cells.

The mitotic capacity of heart muscle is too limited to fully substitute for cells lost following myocardial infarction. Emerging stem cell-based strategies have been proposed to overcome the self-renewal shortfall of native cardiomyocytes, yet there is limited evidence for their capability to achieve safe de novo cardiogenesis and repair. We present our recent experience in treating long-term, infarcted hearts with embryonic stem cells, a prototype source for allogenic cell therapy. The cardiogenic potential of the engrafted murine embryonic stem cell colony was pre-tested by in vitro differentiation, with derived cells positive for nuclear cardiac transcription factors, sarcomeric proteins and functional excitation-contraction coupling. Eight weeks after infarct, rats were randomized into sham- or embryonic stem cell-treated groups. Acellular sham controls or embryonic stem cells, engineered to express enhanced cyan fluorescent protein (ECFP) under control of the cardiac actin promoter, were injected through a 28-gauge needle at three sites into the peri-infarct zone for serial assessment of functional and structural impact. In contrast to results with sham-treated animals, stem cell therapy yielded, over the 5-month follow-up period, new ECFP-positive cardiomyocytes that integrated with the infarcted myocardium. The stem cell-treated group showed a stable contractile performance benefit with normalization of myocardial architecture post infarction. Transition of embryonic stem cells into cardiomyocytes required host signaling to support cardiac-specific differentiation and could result in tumorigenesis if the stem cell dose exceeded the heart's cardioinductive capacity. Supported by the host environment, proper dosing and administration of embryonic stem cells is thus here shown useful in the chronic management of cardiac injury promoting sustained repair.

Conventional therapies for myocardial infarction attenuate disease progression without contributing significantly to repair. Because of the capacity for de novo cardiogenesis, embryonic stem cells are considered a potential source for myocardial regeneration, yet limited information is available on their ultimate therapeutic value. We treated infarcted rat hearts with CGR8 embryonic stem cells preexamined for cardiogenicity, serially probed left ventricular function, and determined final pathological outcome. Stem cell delivery generated new cardiomyocytes of embryonic stem cell origin that integrated with host myocardium within infarct regions. This resulted in a functional benefit within 3 wk that remained sustained over 12 wk of continuous follow-up and included a vigorous inotropic response to beta-adrenergic challenge. Integration of stem cell-derived cardiomyocytes was associated with normalized ventricular architecture, little scar, and a decrease in signs of myocardial necrosis. In contrast, sham-treated infarcted hearts exhibited ventricular cavity dilation and aneurysm formation, poor ventricular function, and a lack of response to beta-adrenergic stimulation. No evidence of graft rejection, ectopy, sudden cardiac death, or tumor formation was observed after therapy. These findings indicate that embryonic stem cells, through differentiation within the host myocardium, can contribute to a stable beneficial outcome on contractile function and ventricular remodeling in the infarcted heart.

Cardiomyocyte regeneration is limited in adult life. Thus, the identification of a putative source of cardiomyocyte progenitors is of great interest to provide a usable model in vitro and new perspective in regenerative therapy. As adipose tissues were recently demonstrated to contain pluripotent stem cells, the emergence of cardiomyocyte phenotype from adipose-derived cells was investigated. We demonstrated that rare beating cells with cardiomyocyte features could be identified after culture of adipose stroma cells without addition of 5-azacytidine. The cardiomyocyte phenotype was first identified by morphological observation, confirmed with expression of specific cardiac markers, immunocytochemistry staining, and ultrastructural analysis, revealing the presence of ventricle- and atrial-like cells. Electrophysiological studies performed on early culture revealed a pacemaker activity of the cells. Finally, functional studies showed that adrenergic agonist stimulated the beating rate whereas cholinergic agonist decreased it. Taken together, this study demonstrated that functional cardiomyocyte- like cells could be directly obtained from adipose tissue. According to the large amount of this tissue in adult mammal, it could represent a useful source of cardiomyocyte progenitors. Garcia Verdugo, Jose Manuel, J.Manuel.Garcia@uv.es

Before its degradation to adenosine, extracellular ATP released by nerves, or by cardiac cells during ischemia, induces a positive inotropy related to stimulation of the calcium conductance by P2-purinoceptors. This stimulation is additive with the β-adrenergic one and seems to occur via the activation of an isoform of a Gs-protein sensitive to cholera toxin, although with no increase in cAMP. No Phosphorylation of the CA Channel is required since the P2-purinergic stimulation is rapidly reversible even in the presence of the poorly hydrolysable compounds ATPγS or GTPγS intracellularly. Transiently, ATP may also depolarize the cells and trigger arrhythmias by binding to a new P3-purinoceptor type which requires Mg ions. Such a receptor induced a tyrosine phosphorylation of CI/HCO3 Exchanger, a band 3-like protein which under such activation induces a large, transient acidosis. © 1993 Wiley-Liss, Inc.

This editorial refers to ‘Dissociation of calmodulin from cardiac ryanodine receptor causes aberrant Ca2+ release in heart failure’, by M. Ono et al. , pp. 609–617, this issue. The ryanodine receptor (RyR) releases intracellular Ca2+ from the sarcoplasmic reticulum (SR) of beating cardiomyocytes following Ca2+ influx via the voltage-dependent sarcolemmal Ca2+ channel. This crucial cellular event, named ‘Ca2+-induced Ca2+ release' and discovered three decades ago by Fabiato and Fabiato,1 is mandatory for the rhythmic contraction as well as for maintaining the force–frequency relationship of the myocardium. RyR type 1 (RyR1), cardiac RyR2, and RyR3 have been the subject of intensive investigation for many years. More recently, Ikemoto and Yamamoto provided evidence of structure–function relationships in the RyRs. They reported that the N-terminal and central domains of RyRs form an interacting domain pair. Unzipping and zipping actions of such a domain pair determines the opening and closing probabilities of the RyR Ca2+ channel, respectively.2 A dysregulation of this domain pair leads to a defect in heart function. Unzipping the N-terminal from the central domain leads to RyR2 hyperactivation and Ca2+ leak from the SR, … *Corresponding author. Tel: +33 160 878 923; fax: +33 160 878 999, Email: michel.puceat{at}inserm.fr

Mineralocorticoid hormones regulate many physiological functions in the cardiovascular system. Although high affinity binding sites for aldosterone have been found in myocardium, aldosterone effects on pHi regulatory systems in cardiac cells have not been described. We have addressed this issue by using microspectrofluorimetric monitoring of intracellular pH in developing neonatal rat cardiomyocytes cultured for 2 weeks. Developmental changes in cell morphology were controlled by anti-myosin light chain antibody staining of the sarcomeric units using confocal laser scanning microscopy. The data obtained demonstrate that from early stages of the development, pHi in neonatal cardiac cells is regulated by three ion transporting mechanisms, namely, Na/H antiport, Na- and HCO3-dependent transporter and Cl/HCO3 exchanger. A 24-h treatment of the cells with aldosterone increases the activity of the Cl/HCO3 exchanger at day 6 of cell culture while the Na/H antiport activity is enhanced in the cells treated with the hormone at days 9 and 13 of culture. Thus, by affecting the activity of ionic transporters, aldosterone modulates acid-base balance in cardiac cells.

To expel the excess protons generated during a cellular acidification and to fully recover basal intracellular pH (pHi), cardiac cells rely on the amiloride-sensitive Na/H antiport. We report that rat single ventricular cardiomyocytes, loaded with the fluorescent pH indicator Snarf-1 and treated with inhibitors of the Na/H antiport, amiloride or its analogues, partially restored their pHi through a bicarbonate-dependent mechanism following an acidosis (imposed by the ammonia-pulse technique). In the presence of ethylisopropylamiloride (10 microM) or amiloride (1 mM) and 25 mM bicarbonate in the extracellular solution, the average time that cells needed to recover half of their pHi, following the removal of 20 mM NH4Cl, was 3.4 min, while the rate of proton efflux was calculated to be 2.0 mM/min. The stilbene derivative, 4-4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS 200 microM), a known blocker of anion transporters, inhibited this recovery. Both phenylephrine (100 microM, 3 microM propranolol present), an alpha 1-adrenoceptor agonist, and ATP (10 microM), a purinergic agonist, significantly enhanced the rate of proton efflux that was due to this HCO3-dependent alkalinizing mechanism. Phenylephrine and ATP also shortened by three-fold the time that a myocyte needed to recover half of its initial pHi. This bicarbonate-dependent alkalinizing mechanism could provide an additional means by which cardiac cells recover their pHi from acidosis, especially under conditions in which the Na/H antiport is inhibited. Furthermore, catecholamines and ATP, which are released under various pathophysiological conditions often associated with intracellular acidosis, could play an important role in the modulation of pHi under these conditions.

1. The cellular effects of alpha 1-adrenoceptor stimulation by phenylephrine were studied in the presence of propranolol in single cells isolated from the ventricles of rat hearts. 2. Phenylephrine (10-100 microM) induced a biphasic pattern of inotropism in these cells: a transient negative followed by a sustained positive inotropic effect as usually observed in cardiac tissues. 3. In Snarf-1-loaded cells, phenylephrine induced an alkalinization. This effect was reversible on wash-out and inhibited by prazosin, an alpha 1-adrenoceptor antagonist. 4. The alpha 1-adrenoceptor-mediated increase in intracellular pH (pHi) was 0.1 pH unit in HEPES buffer containing 4.4 mM-NaHCO3 and in Krebs buffer containing 25 mM-NaHCO3. 5. The alkalinization was blocked by the Na(+)-H+ antiport blocker, ethylisopropylamiloride (EIPA). 6. The recovery from an acidosis induced by a NH4Cl pre-pulse was accelerated by phenylephrine. The phenylephrine-induced alkalinization was attributed to activation of the Na(+)-H+ antiport. 7. Despite its ability to increase pHi, phenylephrine did not alter Ca2+ current amplitude and kinetics. 8. Ca2+ transients recorded in Indo-1-loaded cells were not augmented by phenylephrine. Diastolic calcium level was decreased. 9. In single skinned cells, the Ca2+ sensitivity of the contractile proteins was increased by a pre-treatment with phenylephrine even when the alpha 1-adrenoceptor-mediated alkalinizing effect had been prevented by EIPA. 10. These results lead us to propose that the alpha 1-adrenergic-induced positive inotropic response of heart muscle could result from an increased sensitivity of the myofilaments to Ca2+ ions. This alpha 1-adrenoceptor-mediated Ca2+ sensitization could result both from an intracellular alkalinization and from a direct effect on contractile proteins.

Human Embryonic or pluripotent stem cells hold many promises in regenerative medicine. They also provide the scientific community with powerful models of early human development including cardiogenesis under normal or pathological (congenital and genetic diseases) situations. Furthermore their cardiac derivatives turn out to be very useful to study human cardiac electrophysiology, pharmacology or cardiac toxicology. The current overview provides the basic knowledge on developmental biology of the heart which can be applied to stem cell research to study early cardiogenesis. We summarize both the cardiogenic transcriptional network and the role of morphogens involved in early cardiogenesis. We review protocols of cardiac differentiation of pluripotent stem cells so far available. We finally discuss the translation of basic stem cell research into clinical applications.

We have immunologically identified the isoforms of protein kinase C (PKC) present in neonatal and adult rat cardiomyocytes and examined their regulation by hormones and phorbol ester. Both cell types express the Ca(2+)-dependent alpha-PKC and the Ca(2+)-independent epsilon- and delta-PKC isoforms. The atypical zeta-PKC isoform is also expressed in neonatal, but only weakly in adult cells. Stimulation of the alpha 1-adrenergic or purinergic receptor with phenylephrine or ATP, respectively, increases membrane-associated immunoreactivity of both epsilon- and delta-PKC in neonatal and adult cells; endothelin and carbachol are also effective in adult cells. In contrast, none of the agonists leads to increases in membrane-associated alpha-PKC in cardiomyocytes. PKC zeta is also unaffected by receptor stimulation. The phorbol ester phorbol 12-myristate 13-acetate causes redistribution and subsequently down-regulation of alpha-, epsilon-, and delta- but not zeta-PKC. The three isoforms are down-regulated at distinctively different rates, with alpha-PKC being the most rapid and epsilon-PKC the slowest. We used selective down-regulation of alpha-, epsilon-, and delta-PKC to investigate the role of these isoforms in PKC phosphorylation-dependent events in neonatal myocytes. Our findings suggest that epsilon-PKC is responsible for the phenylephrine-induced phosphorylation of MARCKS, an endogenous PKC-specific substrate. In contrast, agonist-induced c-fos expression is unlikely to be mediated by epsilon-PKC since the response is rapidly down-regulated and apparently Ca(2+)-dependent. Our finding that the PKC isoforms are differentially responsive to neurohormones suggests that they play distinct and specific roles in cardiac function.