Claudine Menard

Spontaneous Cardiomyocyte Differentiation From Adipose Tissue Stroma Cells

Abstract— 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.

Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: a preclinical study

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.

Transient down-regulation of L-type Ca2+ channel and dystrophin expression after balloon injury in rat aortic cells

OBJECTIVE Migration and proliferation of arterial smooth muscle cells are critical responses during restenosis after balloon angioplasty. We investigated the changes in the expression of Ca(2+) channels and dystrophin, two determinants of contraction, after balloon injury of rat aortas. METHODS Proliferation and migration of aortic myocytes were triggered in vivo by the passage of an inflated balloon catheter in the aortas of 12-week-old male Wistar rats. We used the whole-cell patch clamp technique to investigate Ba(2+) currents (I(Ba)) through Ca(2+) channels in single cells freshly isolated from media and neointima at various times after injury (days 2, 7, 15, 30 and 45). RESULTS No T-type Ca(2+) channel current was recorded in any cell at any time. In contrast, a dihydropyridine (DHP)-sensitive L-type I(Ba)was recorded consistently in the media of intact aorta. After aortic injury, I(Ba) decreased dramatically (at days 2 and 7) but recovered over time to reach normal amplitude on days 30 and 45. In the neointima, I(Ba) was absent on day 15 but also increased gradually over time as observed at days 30 and 45. The use of a specific antibody directed against the L-type Ca(2+) channel alpha(1C) subunit showed, both by immunostaining and by Western blotting, no expression of the Ca(2+) channel protein on day 15. Parallel immunodetection of dystrophin showed that this marker of the contractile phenotype of SMCs was also not detectable at this stage in neointimal cells. Both proteins were re-expressed at days 45 and 63. Balloon injury induces a transient down-regulation of I(Ba) in arterial cells. CONCLUSIONS Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca(2+) channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca(2+) homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.

Interplay between the retinoblastoma protein and LEK1 specifies stem cells toward the cardiac lineage.

The molecular mechanisms governing early cardiogenesis are still largely unknown. Interestingly, the retinoblastoma protein (Rb), a regulator of cell cycle, has recently emerged as a new candidate regulating cell differentiation. Rb−/− mice die at midgestation and mice lacking E2f1/E2f3, downstream components of the Rb‐dependent transcriptional pathway, die of heart failure. To gain insight into the function of Rb pathway in early cardiogenesis, we used Rb−/− embryonic stem (ES) cells differentiating into cardiomyocytes. Rb−/− cells displayed a dramatic delay in expression of cardiac‐specific transcription factors and in turn in the whole process of cardiac differentiation. The phenotype of Rb−/− ES cell‐derived cardiomyocytes was rescued by reintroducing Rb in cardiac progenitors, by stimulating the BMP‐dependent cardiogenic pathway or by overexpression of Nkx2.5. ES cells deficient in the recently identified factor LEK1, a murine homolog of the cardiomyogenic factor 1, or specific disruption of Rb–LEK1 interaction into the nucleus of differentiating ES cells recapitulated the delay in cardiac differentiation of Rb−/− ES cells. Thus, we provide evidence for a novel Rb/LEK1‐dependent and BMP‐independent transcriptional program, which plays a pivotal role in priming ES cells toward a cardiac fate.

Use of arginine–glycine–aspartic acid adhesion peptides coupled with a new collagen scaffold to engineer a myocardium-like tissue graft

Background Cardiac tissue engineering might be useful in treatment of diseased myocardium or cardiac malformations. The creation of functional, biocompatible contractile tissues, however, remains challenging. We hypothesized that coupling of arginine–glycine–aspartic acid–serine (RGD+) adhesion peptides would improve cardiomyocyte viability and differentiation and contractile performance of collagen-cell scaffolds.Methods Clinically approved collagen scaffolds were functionalized with RGD+ cells and seeded with cardiomyocytes. Contractile performance, cardiomyocyte viability and differentiation were analyzed at days 1 and 8 and/or after culture for 1 month.Results The method used for the RGD+ cell–collagen scaffold coupling enabled the following features: high coupling yields and complete washout of excess reagent and by-products with no need for chromatography; spectroscopic quantification of RGD+ coupling; a spacer arm of 36Å, a length reported as optimal for RGD+-peptide presentation and favorable for integrin-receptor clustering and subsequent activation. Isotonic and isometric mechanical parameters, either spontaneous or electrostimulated, exhibited good performance in RGD+ constructs. Cell number and viability was increased in RGD+ scaffolds, and we saw good organization of cell contractile apparatus with occurrence of cross-striation.Conclusions We report a novel method of engineering a highly effective collagen-cell scaffold based on RGD+ peptides cross-linked to a clinically approved collagen matrix. The main advantages were cell contractile performance, cardiomyocyte viability and differentiation.

Commitment of embryonic stem cells toward a cardiac lineage: molecular mechanisms and evidence for a promising therapeutic approach for heart failure

ANNABELLE MÉRY, EVANGELIA PAPADIMOU, DANA ZEINEDDINE, CLAUDINE MÉNARD, ATTA BEHFAR, LEONID V. ZINGMAN, DENICE M. HODGSON, JEAN-MICHEL RAUZIER, GARVAN C. KANE, CARMEN PEREZ-TERZIC, ANDRÉ TERZIC and MICHEL PUCÉAT* Centre de Recherches de Biochimie Macromoléculaire, CNRS FRE 2593, Montpellier, France; Division of Cardiovascular Diseases, Department of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA; INSERM U 390, Montpellier, France; Physical Medicine and Rehabilitation, Mayo Clinic, Rochester, MN, USA

Collagen scaffolds with or without the addition of RGD peptides support cardiomyogenesis after aggregation of mouse embryonic stem cells.

Embryonic stem (ES) cell-based cardiac muscle repair using tissue-engineered scaffolds is an attractive prospective treatment option for patients suffering from heart disease. In this study, our aim was to characterize mouse ES cell-derived cardiomyocytes growing on collagen I/III scaffolds, modified with the adhesion peptides arginine-glycine-aspartic acid (RGD). Mouse ES-derived embryoid bodies (EBs) differentiated efficiently into beating cardiomyocytes on the collagen scaffolds. QPCR analysis and immunofluorescent staining showed that cardiomyocytes expressed cardiac muscle-related transcripts and proteins. Analysis of cardiomyocytes by electron microscopy identified muscle fiber bundles and Z bands, typical of ES-derived cardiomyocytes. No differences were detected between the collagen + RGD and collagen control scaffolds. ES cells that were not differentiated as EBs prior to seeding on the scaffold, did not differentiate into cardiomyocytes. These results indicate that a collagen I/III scaffold supports cardiac muscle development and function after EB formation, and that this scaffold appears suitable for future in vivo testing. The addition of the RGD domain to the collagen scaffold did not improve cardiomyocyte development or viability, indicating that RGD signaling to integrins was not a rate-limiting event for cardiomyogenesis from EBs seeded on a collagen scaffold.

Cardiac specification of embryonic stem cells

Over the past decade, cell transplantation has been recognized as a mean of repairing infarcted myocardium. Both adult stem cells and differentiated cells have yielded encouraging results with regard to engraftment into postinfarction scars. However, these cells now feature serious restrictions. As an alternative, embryonic stem (ES) cells are particularly attractive, because of their plasticity and the subsequent possibility to drive them towards a cardiomyogenic phenotype after exposure to appropriate growth factors. An additional theoretical advantage of ES cells is their expected immune privilege. In this article, we summarize the findings obtained in cell therapy using ES cells and discuss the molecular mechanisms of cardiac specification of the cells.

Cardiac Commitment of Embryonic Stem Cells for Myocardial Repair

Embryonic stem (ES) cells represent a source for cell-based regenerative therapies of heart failure. The pluripotency and the plasticity of ES cells allow them to be committed to a cardiac lineage following treatment with growth factors of the transforming growth factor (TGF)-beta superfamily. We describe a protocol designed to turn on expression of cardiac-specific genes in undifferentiated murine ES cells stimulated with BMP2 and/or TGF-beta. Cell commitment results in a significant improvement in spontaneous cardiac differentiation of ES cells both in vitro and in vivo.

RGK Small GTPases and Regulation of CaV2 Channels

About 10 years ago, a yeast two-hybrid screen highlighted the unexpected interaction between the regulatory subunit of the voltage–gated Ca2+ channels, CaVβ, and Kir/Gem, a member of the recently identified Ras-related GTP-binding protein family RGK (Rad-Gem-Kir). It soon appeared that all the members of this family, Gem, Rad, Rem and Rem2, were able to inhibit high-voltage activated Ca2+ channels, thus opening new fields of research to understand the molecular mechanisms leading to channel inhibition and to analyze their potential physiological signification. While much of these works were first concentrated on L-type CaV1.2 channels, it is clear now that presynaptic CaV2.1 and CaV2.2 channels are also sensitive to RGK inhibition. Recent data suggest that multiple routes are used by the RGK proteins to inhibit Ca2+ channels, including modifications of channel targeting and recycling, gating-charge mobility and/or open-channel probability. A direct RGK-CaVβ interaction appears to be absolutely necessary, but additional interactions with the channel protein itself have been highlighted and suggest a finely tuned specificity at the channel level. Whether these interactions also play a role in other channel CaVα or CaVβ functions, such as synaptic transmission or transcriptional regulation, still needs to be investigated.