Jean-François Renaud

A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.

Structural Localization and Expression of CXCL12 and CXCR4 in Rat Heart and Isolated Cardiac Myocytes

CXCL12 (SDF-1), which binds CXCR4, is involved in several physiological and pathophysiological processes. In heart, this axis seems to play a key role in cardiogenesis and is involved in the neovascularization of ischemic tissues. Rats have three known CXCL12 mRNA isoforms, of which only α and γ are present in the normal heart. However, little is known about CXCL12 protein expression and localization. We investigated the pattern of protein expression and the localization of both CXCR4 and CXCL12 in the heart, using isolated cardiomyocytes and a rat myocardial infarction model. Western blots showed that cardiomyocytes contained a specific 67-kDa CXCR4 isoform and a 12-kDa CXCL12 isoform. Confocal and electron microscopy clearly showed that CXCR4 was present at the plasmalemma and CXCL12 in continuity of the Z-line, in the proximal part of T-tubules. In conclusion, we provide the first description of the expression and fine localization of CXCR4 and CXCL12 proteins in normal rat heart and cardiomyocytes. These results suggest that the CXCL12/CXCR4 axis may be involved in cardiomyocyte calcium homeostasis regulation. Our work and the well-known chemoattraction properties of the CXCL12/CXCR4 axis highlight the importance of deciphering the function of this axis in both normal and pathological hearts.

T-type Ca2+ signalling regulates aldosterone-induced CREB activation and cell death through PP2A activation in neonatal cardiomyocytes

Aims We have investigated Ca2+ signalling generated by aldosterone-induced T-type current (ICaT), the effects of ICaT in neonatal cardiomyocytes, and a putative role for ICaT in cardiomyocytes during cardiac pathology induced by stenosis in an adult rat. Methods and results Neonatal rat cardiomyocytes treated with aldosterone showed an increase in ICaT density, principally due to the upregulation of the T-type channel Cav3.1 (by 80%). Aldosterone activated cAMP-response element-binding protein (CREB), and this activation was enhanced by blocking ICaT or by inhibiting protein phosphatase 2A (PP2A) activity. Aldosterone induced PP2A activity, an induction that was prevented upon ICaT blockade. ICaT exerted a negative feedback regulation on the transcription of the Cav3.1 gene, and the activation of PP2A by ICaT led to increased levels of the pro-apoptotic markers caspase 9 and Bcl-xS and decreased levels of the anti-apoptotic marker Bcl-2. These findings were corroborated by flow cytometry analysis for apoptosis and necrosis. Similarly, in a rat model of cardiac disease, ICaT re-emergence was associated with a decrease in CREB activation and was correlated with increases in caspase 9 and Bcl-xS and a decrease in Bcl-2 levels. Conclusion Our findings establish PP2A/CREB as targets of ICaT-generated Ca2+ signalling and identify an important role for ICaT in cardiomyocyte cell death.

Isoflurane alters angiotensin II-induced Ca2+ mobilization in aortic smooth muscle cells from hypertensive rats: implication of cytoskeleton.

BACKGROUND Angiotensin II (AngII) is a potent vasoconstrictor involved in the short-term control of arterial blood pressure. Isoflurane was reported to decrease vascular tone through an alteration of vascular smooth muscle cell vasomotor response to several agonists, but its effect on AngII signaling is not known. On the other hand, vascular response to AngII is altered in hypertension. In this study, the authors tested the hypothesis that (1) isoflurane alters AngII-induced intracellular Ca mobilization in aortic vascular smooth muscle cell from Wistar Kyoto and spontaneously hypertensive rats, and (2) this effect could be associated with an alteration of the organization of microtubular network, reported to be involved in AngII signaling. METHODS The effect of 0.5-3% isoflurane was studied (1) on AngII (10 m)-induced intracellular Ca mobilization, intracellular Ca release from internal stores, and Ca influx in Fura-2 loaded cultured aortic vascular smooth muscle cell isolated from 6-week-old Wistar Kyoto and spontaneously hypertensive rats, using fluorescent imaging microscopy; and (2) on the organization of cytoskeletal elements, using immunofluorescence labeling. RESULTS In both stains, isoflurane decreased in a concentration-dependent manner AngII-induced intracellular Ca mobilization, Ca release from internal stores, and Ca influx through nifedipine-insensitive Ca channels. This effect occurred at a lower concentrations of isoflurane in Wistar Kyoto rats than in spontaneously hypertensive rats. In both strains, the effect of isoflurane on AngII- Ca mobilization was abolished by impairment with nocodazole, vinblastine, or paclitaxel of microtubules polymerization. Isoflurane directly altered tubular network organization in a concentration-dependent and reversible manner. CONCLUSIONS Isoflurane decreased AngII-induced Ca mobilization at clinically relevant concentrations, suggesting that vascular response to AngII could be altered during isoflurane anesthesia. The hypertensive strain was found less sensitive than the normotensive one. In both strains, the isoflurane effect was associated with a microtubular network interaction.

Effect of Extracellular Matrix Elements on Angiotensin II–Induced Calcium Release in Vascular Smooth Muscle Cells From Normotensive and Hypertensive Rats

The interaction of the vascular smooth muscle cells (VSMCs) with the components of the matrix determines several functions of the cell, such as growth and differentiation. In contrast, an alteration in angiotensin (Ang) II-induced Ca2+ mechanisms in VSMCs was reported in genetic hypertension. In this study, we wished to assess the effect of different components of the extracellular matrix on the increase of [Ca2+]i induced by Ang II in VSMCs from spontaneously hypertensive rats (SHR) compared with those from normotensive Wistar-Kyoto rats (WKY). Results demonstrate for the first time that elements of the extracellular matrix modulate the Ang II-induced Ca2+ transport mechanisms. This modulation is different in cells from WKY compared with those from SHR. Thus, growing cells from SHR on collagen I, collagen IV, fibronectin, vitronectin, or Matrigel induced a significant decrease in Ang II-induced Ca2+ release from internal stores, whereas in cells from WKY, no effect could be observed except for those grown on collagen I, which increased Ca2+ release. Fibronectin and vitronectin, however, induced a decrease in Ang II-induced Ca2+ influx in WKY, whereas no effect could be observed in SHR. Conversely, collagen I and collagen IV induced an increase in this influx in SHR but not in WKY, whereas Matrigel increased the influx in both strains. These results suggest a modulation of the Ang II-associated signaling events by the matrix elements via the focal adhesion points. The understanding of these synergies should provide insight into issues such as development of hypertrophy of large vessels in hypertension.

Transforming growth factor-β1 modulates angiotensin II-induced calcium release in vascular smooth muscle cells from spontaneously hypertensive rats

Objectives To investigate the role of transforming growth factor-β1 (TGF-β1) on Ca2+-dependent mechanisms elicited by angiotensin II in aortic vascular smooth muscle cells (VSMC) of Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Methods Cai2+ release induced by angiotensin II (1 μmol/l) was studied in cultured VSMC isolated from the aortas of 6-week-old WKY rats and SHR. Intracellular Ca2+ (Cai2+) was assessed in Fura-2 loaded cells using fluorescent imaging microscopy. Angiotensin II receptors were analysed by binding studies. Results Pretreatment of VSMC for 24 h with TGF-β1 significantly increased angiotensin II-induced Cai2+ mobilization from internal stores in SHR, while Ca2+ influx was not altered. This effect involves tyrosine kinase and is not due to an increase in angiotensin II binding sites, or a change in the affinity of the receptors. By contrast, TGF-β1 did not modify the response of VSMC from WKY rats to angiotensin II. Conclusions These results help our understanding of the interactions between the pathways activated by TGF-β1 and the G protein-coupled receptor signalling pathway, and their role in genetic hypertension.

Differences in aortic response to vasoactive stimuli in Japanese and Lyon rats. The role of hypertension.

OBJECTIVE We have previously shown that conduit arteries of normotensive (WKY) and hypertensive (SHR) Japanese rats differ from normotensive (LN) and hypertensive (LH) Lyon rats in terms of lower aortic thickness and higher collagen III content, whereas differences in vasoactive properties are unknown. METHODS Aortic rings with (E+) and without (E-) endothelium were studied under resting and noradrenaline-stimulated conditions in the presence of N(omega)-nitro-L-arginine (L-NNA) alone or in association with indomethacin, bosentan and/or BQ123. RESULTS Under resting conditions, aortas of normotensive and hypertensive Japanese rats differed from Lyon rats by higher developed tension in the presence of L-NNA and endothelium. In the absence of endothelium, normotensives differed from hypertensives in terms of stronger developed tensions in the presence of L-NNA in the two strains. Addition of indomethacin to L-NNA induced relaxation in E+ SHR and E- WKY and contraction in E-LH. By contrast, tensions were unchanged after addition of bosentan and BQ123. Under stimulated conditions, tensions were equally increased by L-NNA in E+ and unchanged in E- both in Japanese and Lyon rats whether they were normotensive or hypertensive, and indomethacin (but not bosentan) elicited higher response in Lyon than in Japanese rats in E+ and E- aorta. CONCLUSION Under NO synthase inhibition, the vasoactive properties of Japanese and Lyon aorta differ in the presence of a cyclo-oxygenase blocker but not endothelin blockers. These results indicate that the aorta vasorelaxant tone is associated to prostanoid regulation in Lyon but not in Japanese rats. This observation appears dependent on the genetic and/or environmental background linked to the origin and not the presence of hypertension.

Inhibition of L-type but not T-type calcium channel current by a new dihydropyridine derivative, S11568.

S11568, (±){[(amino-2-ethoxy)-2-ethoxy]-methyl}-2-(dichloro-2‘,3’-phenyl)-4-ethoxycarbonyl-3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyridine HCl, is a new dihydropyridine derivative that is water soluble and relatively insensitive to light. The Ca2+ channel inhibitory activity of S11568 was tested in whole-cell patch clamp recordings from cultured embryonic chick cardiomyocytes in 40 mM Ba2+-containing medium that revealed T-type and L-type components of inward current through calcium channels. S11568 inhibited L-type Ca2+ current with an IC50 value near 1 μM but was without effect on the T-type barium current.

Alkali cation permeability and caesium blockade of cromakalim-activated current in guinea-pig ventricular myocytes

1 The sensitivity of cromakalim‐activated current (Icrom) to manipulations of extracellular cationic composition was examined in whole‐cell voltage clamp recordings from freshly‐dispersed, adult guinea‐pig ventricular myocytes. In bathing media with different concentrations of K+ (1, 2.5, 5.4 and 12 mm) the Icrom reversal potential (Erev) varied in strict correspondance with the K+ equilibrium potential and inward Icrom amplitude was proportional to the external K+ concentration. 2 Replacement of 12 mm K+ with 12 mm Rb+ induced a slight positive shift of Erev indicating that PRb+/PK+ = 1.06. K+ replacement with 12 mm Cs+ reduced or abolished inward Icrom and produced a negative shift of Erev by at least 50 mV; an upper limit of PCs+/PK+ was fixed at 0.18. 3 Addition of Rb+ (1–30 mm) to 2.5 mm K+‐containing medium produced a concentration‐dependent increase in inward Icrom and positive shift of Erev suggesting that K+ and Rb+ have similar permeabilities and conductivities and do not interfere with each other in the channel. 4 Cs+ (0.01–30 mm), added to medium containing 12 mm Rb+, induced a potent, voltage‐dependent inhibition of inwardly rectifying current (IK1; IC50 = 0.2–3 mm). Voltage‐dependent inhibition of inward Icrom was observed only at considerably higher Cs+ concentrations (IC50 = 4–30 mm). Extracellular Rb+ and Cs+ did not substantially alter the amplitude of outward Icrom. 5 The results support the contention that the ATP‐sensitive K+ channel is the primary target of cromakalim action in ventricular myocytes.

T-type Ca2 + signalling downregulates MEK1/2 phosphorylation and cross-talk with the RAAS transcriptional response in cardiac myocytes ☆

Cardiac dysfunction is often associated with an increase in the activity of the renin-angiotensin II-aldosterone system (RAAS). Here, we highlight the cross-talk between the Ca(2+) signalling generated by cardiac T-type current (I(CaT)) and RAAS signalling. Neonatal rat cardiomyocytes exposed to aldosterone, angiotensin II or aldosterone plus angiotensin II co-treatment (AA) show an increase in I(CaT) density, with no cumulative effect of the AA co-treatment. AA increases the amount of T-type channel Ca(v)3.1 mRNA in a time-dependent manner. Angiotensin II increases Ca(v)3.1 mRNA stability, whereas aldosterone increases the transcriptional activity of the Ca(v)3.1 gene promoter. However, in AA-treated cells, angiotensin II decreases aldosterone-induced promoter activity, and aldosterone decreases angiotensin II-induced mRNA stability. The mitogen-activated protein kinase kinase (MEK1/2), which is synergically phosphorylated in AA-treated cells, alters the translocation of glucocorticoid receptors (GR) into the nucleus and attenuates aldosterone-induced promoter activity. In contrast, MEK1/2 has no effect on the NFkB-induced increase in Ca(v)3.1 mRNA and MEK1/2 promoted CREB-target gene transcription. Aldosterone and AA-induced I(CaT) signalling result in a time-dependent activation of the phosphatase PP2A, which dephosphorylates MEK1/2 and CREB. Finally, angiotensin II alone also activates PP2A, which targets MEK1/2, but this activation is independent of I(CaT) calcium signalling and has no effect on CREB phosphorylation. In conclusion, our data demonstrate the cross-talk between a GR-mediated aldosterone response, angiotensin II and the I(CaT) signalling pathways and identify MEK1/2 as a point of connection. This cross-talk results in the fine control of GR- and/or CREB-target gene expression.