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1.
Cardiovasc Res ; 114(6): 880-893, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29360952

ABSTRACT

Aims: Mutations in the KCNK3 gene, which encodes for an outward-rectifier K+ channel, have been identified in patients suffering from pulmonary arterial hypertension (PAH), and constitute the first described channelopathy in PAH. In human PAH and experimental pulmonary hypertension (PH), we demonstrated that KCNK3 expression and function are severely reduced in pulmonary vascular cells, promoting PH-like phenotype at the morphologic and haemodynamic levels. Since KCNK3 channel is also expressed in both the human and rodent heart, we aimed to elucidate the pathophysiological role of KCNK3 channel in right ventricular (RV) hypertrophy (RVH) related to PH. Methods and results: Using whole-cell Patch-clamp technique, we demonstrated that KCNK3 is predominantly expressed in adult rat RV cardiomyocytes compared to the left ventricle cardiomyocytes and participates in the repolarizing phase of the RV action potential. We revealed a reduction in KCNK3 function prior to development of RVH and the rise of pulmonary vascular resistance. KCNK3 function is severely reduced in RV cardiomyocytes during the development of RVH in several rat models of PH (exposure to monocrotaline, chronic hypoxia, and Sugen/hypoxia) and chronic RV pressure overload (pulmonary artery banding). In experimental PH, we revealed a reduction in KCNK3 function before any rise in pulmonary vascular resistance and the development of RVH. KCNK3 mRNA level is also reduced in human RV tissues from PAH patients compared to non-PAH patients. In line with these findings, chronic inhibition of KCNK3 in rats with the specific inhibitor (A293) induces RV hypertrophy which is associated with the re-expression of foetal genes, RV fibrosis, RV inflammation, and subsequent loss of RV performance as assessed by echocardiography. Conclusion: Our data indicate that loss of KCNK3 function and expression is a hallmark of the RV hypertrophy/dysfunction associated with PH.


Subject(s)
Hypertension, Pulmonary/metabolism , Hypertrophy, Right Ventricular/metabolism , Myocytes, Cardiac/metabolism , Nerve Tissue Proteins/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Ventricular Dysfunction, Right/metabolism , Ventricular Function, Right , Ventricular Remodeling , Action Potentials , Adolescent , Adult , Animals , Case-Control Studies , Disease Models, Animal , Down-Regulation , Female , Humans , Hypertension, Pulmonary/complications , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/physiopathology , Hypertrophy, Right Ventricular/etiology , Hypertrophy, Right Ventricular/genetics , Hypertrophy, Right Ventricular/physiopathology , Male , Middle Aged , Myocytes, Cardiac/drug effects , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Potassium Channel Blockers/pharmacology , Potassium Channels, Tandem Pore Domain/antagonists & inhibitors , Potassium Channels, Tandem Pore Domain/genetics , Rats , Signal Transduction , Sulfonamides/pharmacology , Time Factors , Ventricular Dysfunction, Right/etiology , Ventricular Dysfunction, Right/genetics , Ventricular Dysfunction, Right/physiopathology , Ventricular Function, Right/drug effects , Ventricular Remodeling/drug effects , ortho-Aminobenzoates/pharmacology
2.
Circulation ; 133(14): 1371-85, 2016 Apr 05.
Article in English | MEDLINE | ID: mdl-26912814

ABSTRACT

BACKGROUND: Mutations in the KCNK3 gene have been identified in some patients suffering from heritable pulmonary arterial hypertension (PAH). KCNK3 encodes an outward rectifier K(+) channel, and each identified mutation leads to a loss of function. However, the pathophysiological role of potassium channel subfamily K member 3 (KCNK3) in PAH is unclear. We hypothesized that loss of function of KCNK3 is a hallmark of idiopathic and heritable PAH and contributes to dysfunction of pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, leading to pulmonary artery remodeling: consequently, restoring KCNK3 function could alleviate experimental pulmonary hypertension (PH). METHODS AND RESULTS: We demonstrated that KCNK3 expression and function were reduced in human PAH and in monocrotaline-induced PH in rats. Using a patch-clamp technique in freshly isolated (not cultured) pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, we found that KCNK3 current decreased progressively during the development of monocrotaline-induced PH and correlated with plasma-membrane depolarization. We demonstrated that KCNK3 modulated pulmonary arterial tone. Long-term inhibition of KCNK3 in rats induced distal neomuscularization and early hemodynamic signs of PH, which were related to exaggerated proliferation of pulmonary artery endothelial cells, pulmonary artery smooth muscle cell, adventitial fibroblasts, and pulmonary and systemic inflammation. Lastly, in vivo pharmacological activation of KCNK3 significantly reversed monocrotaline-induced PH in rats. CONCLUSIONS: In PAH and experimental PH, KCNK3 expression and activity are strongly reduced in pulmonary artery smooth muscle cells and endothelial cells. KCNK3 inhibition promoted increased proliferation, vasoconstriction, and inflammation. In vivo pharmacological activation of KCNK3 alleviated monocrotaline-induced PH, thus demonstrating that loss of KCNK3 is a key event in PAH pathogenesis and thus could be therapeutically targeted.


Subject(s)
Hypertension, Pulmonary/physiopathology , Nerve Tissue Proteins/physiology , Potassium Channels, Tandem Pore Domain/physiology , Adventitia/pathology , Animals , Bone Morphogenetic Protein Receptors, Type II/genetics , Cell Division , Endothelium, Vascular/pathology , Fibroblasts/pathology , Genetic Predisposition to Disease , Hemodynamics , Humans , Hypertension, Pulmonary/chemically induced , Hypertension, Pulmonary/complications , Hypertension, Pulmonary/genetics , Hypertrophy, Right Ventricular/etiology , Inflammation , Male , Membrane Potentials , Monocrotaline/toxicity , Mutation , Myocytes, Smooth Muscle/pathology , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Patch-Clamp Techniques , Potassium Channels, Tandem Pore Domain/antagonists & inhibitors , Potassium Channels, Tandem Pore Domain/biosynthesis , Potassium Channels, Tandem Pore Domain/genetics , Rats , Rats, Sprague-Dawley , Rats, Wistar , Sulfonamides/pharmacology , Vascular Resistance , ortho-Aminobenzoates/pharmacology
3.
Stem Cells ; 34(1): 34-43, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26352327

ABSTRACT

Heart failure is still a major cause of hospitalization and mortality in developed countries. Many clinical trials have tested the use of multipotent stem cells as a cardiac regenerative medicine. The benefit for the patients of this therapeutic intervention has remained limited. Herein, we review the pluripotent stem cells as a cell source for cardiac regeneration. We more specifically address the various challenges of this cell therapy approach. We question the cell delivery systems, the immune tolerance of allogenic cells, the potential proarrhythmic effects, various drug mediated interventions to facilitate cell grafting and, finally, we describe the pathological conditions that may benefit from such an innovative approach. As members of a transatlantic consortium of excellence of basic science researchers and clinicians, we propose some guidelines to be applied to cell types and modes of delivery in order to translate pluripotent stem cell cardiac derivatives into safe and effective clinical trials.


Subject(s)
Heart Failure/therapy , Myocardium/cytology , Pluripotent Stem Cells/cytology , Stem Cell Transplantation , Animals , Cell Differentiation , Disease Models, Animal , Heart Failure/physiopathology , Humans
4.
J Am Coll Cardiol ; 65(7): 668-80, 2015 Feb 24.
Article in English | MEDLINE | ID: mdl-25677428

ABSTRACT

BACKGROUND: Endothelial cell (EC) dysfunction plays a central role in the pathogenesis of pulmonary arterial hypertension (PAH), promoting vasoconstriction, smooth muscle proliferation, and inflammation. OBJECTIVES: This study sought to test the hypothesis that nebivolol, a ß1-antagonist and ß2,3-agonist, may improve PAH and reverse the PAH-related phenotype of pulmonary ECs (P-EC). METHODS: We compared the effects of nebivolol with metoprolol, a first-generation ß1-selective ß-blocker, on human cultured PAH and control P-EC proliferation, vasoactive and proinflammatory factor production, and crosstalk with PA smooth muscle cells. We assessed the effects of both ß-blockers in precontracted PA rings. We also compared the effects of both ß-blockers in experimental PAH. RESULTS: PAH P-ECs overexpressed the proinflammatory mediators interleukin-6 and monocyte chemoattractant protein-1, fibroblast growth factor-2, and the potent vasoconstrictive agent endothelin-1 as compared with control cells. This pathological phenotype was corrected by nebivolol but not metoprolol in a dose-dependent fashion. We confirmed that PAH P-EC proliferate more than control cells and stimulate more PA smooth muscle cell mitosis, a growth abnormality that was normalized by nebivolol but not by metoprolol. Nebivolol but not metoprolol induced endothelium-dependent and nitric oxide-dependent relaxation of PA. Nebivolol was more potent than metoprolol in improving cardiac function, pulmonary vascular remodeling, and inflammation of rats with monocrotaline-induced pulmonary hypertension. CONCLUSIONS: Nebivolol could be a promising option for the management of PAH, improving endothelial dysfunction, pulmonary vascular remodeling, and right heart function. Until clinical studies are undertaken, however, routine use of ß-blockers in PAH cannot be recommended.


Subject(s)
Adrenergic beta-1 Receptor Antagonists/therapeutic use , Benzopyrans/therapeutic use , Endothelium, Vascular/drug effects , Ethanolamines/therapeutic use , Hypertension, Pulmonary/drug therapy , Vascular Remodeling/drug effects , Adrenergic beta-1 Receptor Antagonists/pharmacology , Animals , Benzopyrans/pharmacology , Cell Communication/drug effects , Cell Culture Techniques , Cell Proliferation , Disease Models, Animal , Endothelial Cells/drug effects , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Ethanolamines/pharmacology , Humans , Hypertension, Pulmonary/pathology , Hypertension, Pulmonary/physiopathology , Male , Metoprolol/pharmacology , Metoprolol/therapeutic use , Monocrotaline , Myocytes, Smooth Muscle , Nebivolol , Pulmonary Artery/drug effects , Pulmonary Artery/pathology , Pulmonary Artery/physiopathology , Rats , Rats, Wistar
5.
PLoS One ; 6(11): e26952, 2011.
Article in English | MEDLINE | ID: mdl-22096509

ABSTRACT

Duchenne Muscular Dystrophy (DMD) is a complex process involving multiple pathways downstream of the primary genetic insult leading to fatal muscle degeneration. Aging muscle is a multifactorial neuromuscular process characterized by impaired muscle regeneration leading to progressive atrophy. We hypothesized that these chronic atrophying situations may share specific myogenic adaptative responses at transcriptional level according to tissue remodeling. Muscle biopsies from four young DMD and four AGED subjects were referred to a group of seven muscle biopsies from young subjects without any neuromuscular disorder and explored through a dedicated expression microarray. We identified 528 differentially expressed genes (out of 2,745 analyzed), of which 328 could be validated by an exhaustive meta-analysis of public microarray datasets referring to DMD and Aging in skeletal muscle. Among the 328 validated co-expressed genes, 50% had the same expression profile in both groups and corresponded to immune/fibrosis responses and mitochondrial metabolism. Generalizing these observed meta-signatures with large compendia of public datasets reinforced our results as they could be also identified in other pathological processes and in diverse physiological conditions. Focusing on the common gene signatures in these two atrophying conditions, we observed enrichment in motifs for candidate transcription factors that may coordinate either the immune/fibrosis responses (ETS1, IRF1, NF1) or the mitochondrial metabolism (ESRRA). Deregulation in their expression could be responsible, at least in part, for the same transcriptome changes initiating the chronic muscle atrophy. This study suggests that distinct pathophysiological processes may share common gene responses and pathways related to specific transcription factors.


Subject(s)
Adaptive Immunity/physiology , Mitochondria/metabolism , Muscle, Skeletal/immunology , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/immunology , Muscular Dystrophy, Duchenne/metabolism , Adaptive Immunity/genetics , Adolescent , Aging/genetics , Child , Cluster Analysis , Humans , In Vitro Techniques , Interferon Regulatory Factor-1/genetics , Male , Muscular Dystrophy, Duchenne/genetics , Neurofibromin 1/genetics , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Protein c-ets-1/genetics , Receptors, Estrogen/genetics , Reverse Transcriptase Polymerase Chain Reaction , ERRalpha Estrogen-Related Receptor
6.
BMC Genomics ; 12: 113, 2011 Feb 16.
Article in English | MEDLINE | ID: mdl-21324190

ABSTRACT

BACKGROUND: DNA microarray technology has had a great impact on muscle research and microarray gene expression data has been widely used to identify gene signatures characteristic of the studied conditions. With the rapid accumulation of muscle microarray data, it is of great interest to understand how to compare and combine data across multiple studies. Meta-analysis of transcriptome data is a valuable method to achieve it. It enables to highlight conserved gene signatures between multiple independent studies. However, using it is made difficult by the diversity of the available data: different microarray platforms, different gene nomenclature, different species studied, etc. DESCRIPTION: We have developed a system tool dedicated to muscle transcriptome data. This system comprises a collection of microarray data as well as a query tool. This latter allows the user to extract similar clusters of co-expressed genes from the database, using an input gene list. Common and relevant gene signatures can thus be searched more easily. The dedicated database consists in a large compendium of public data (more than 500 data sets) related to muscle (skeletal and heart). These studies included seven different animal species from invertebrates (Drosophila melanogaster, Caenorhabditis elegans) and vertebrates (Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Gallus gallus). After a renormalization step, clusters of co-expressed genes were identified in each dataset. The lists of co-expressed genes were annotated using a unified re-annotation procedure. These gene lists were compared to find significant overlaps between studies. CONCLUSIONS: Applied to this large compendium of data sets, meta-analyses demonstrated that conserved patterns between species could be identified. Focusing on a specific pathology (Duchenne Muscular Dystrophy) we validated results across independent studies and revealed robust biomarkers and new pathways of interest. The meta-analyses performed with MADMuscle show the usefulness of this approach. Our method can be applied to all public transcriptome data.


Subject(s)
Computational Biology/methods , Databases, Genetic , Gene Expression Profiling , Muscles/metabolism , Animals , Cluster Analysis , Genomics , Humans , Molecular Sequence Annotation , Muscular Dystrophy, Duchenne/genetics , Oligonucleotide Array Sequence Analysis , Software
7.
In Vitro Cell Dev Biol Anim ; 43(1): 21-4, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17570030

ABSTRACT

Osteoblasts are cells specialized in extracellular matrix production and mineralization. In collaboration with osteoclasts which are bone-resorbing cells, osteoblasts regulate bone homeostasis. The study of osteoblast differentiation from the earliest states of the differentiation can be performed using embryonic stem cells. Embryonic stem cells are pluripotent cells which have the capacity to give rise to all kinds of cells of the body. The main protocol to differentiate embryonic stem cells into osteoblast uses the generation of embryoid body which is a three-dimensional structure mimicking the developing embryo. Recently, it has been shown that human embryonic stem cells have the capacity to differentiate spontaneously into osteoblasts. In this manuscript, we showed that mouse embryonic stem cells have the capacity to differentiate spontaneously into osteoblasts, which can be visualized by the appearance of mineralization nodules and osteogenic markers.


Subject(s)
Embryonic Stem Cells/cytology , Osteoblasts/cytology , Animals , Calcification, Physiologic , Cell Culture Techniques , Cell Differentiation , Mice , Osteoblasts/physiology , Osteogenesis
8.
Stem Cells ; 25(3): 544-52, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17095705

ABSTRACT

Bone remodeling involves synthesis of organic matrix by osteoblasts and bone resorption by osteoclasts. A tight collaboration between these two cell types is essential to maintain a physiological bone homeostasis. Thus, osteoblasts control bone-resorbing activities and are also involved in osteoclast differentiation. Any disturbance between these effectors leads to the development of skeletal abnormalities and/or bone diseases. In this context, the determination of key genes involved in bone cell differentiation is a new challenge to treat any skeletal disorders. Different models are used to study the differentiation process of these cells, but all of them use pre-engaged progenitor cells, allowing us to study only the latest stages of the differentiation. Embryonic stem (ES) cells come from the inner mass of the blastocyst prior its implantation to the uterine wall. Because of their capacity to differentiate into all germ layers, and so into all tissues of the body, ES cells represent the best model by which to study earliest stages of bone cell differentiation. Osteoblasts are generated by two methods, one including the generation of embryoid body, the other not. Mineralizing cells are obtained after 2 weeks of culture and express all the specific osteoblastic markers (alkaline phosphatase, type I collagen, osteocalcin, and others). Osteoclasts are generated from a single-cell suspension of ES cells seeded on a feeder monolayer, and bone-resorbing cells expressing osteoclastic markers such as tartrate-resistant alkaline phosphatase or receptor activator of nuclear factor kappaB are obtained within 11 days. The aim of this review is to present recent discoveries and advances in the differentiation of both osteoblasts and osteoclasts from ES cells.


Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Osteoblasts/cytology , Bone Diseases/etiology , Bone and Bones/abnormalities , Cell Differentiation , Embryonic Stem Cells/pathology , Humans
9.
Circ Res ; 99(10): 1076-83, 2006 Nov 10.
Article in English | MEDLINE | ID: mdl-17053194

ABSTRACT

N-terminal mutations in the KCNQ1 channel are frequently linked to fatal arrhythmias in newborn children and adolescents but the cellular mechanisms involved in this dramatic issue remain, however, to be discovered. Here, we analyzed the trafficking of a series of N-terminal truncation mutants and identified a critical trafficking motif of KCNQ1. This determinant is located in the juxtamembranous region preceding the first transmembrane domain of the protein. Three mutations (Y111C, L114P and P117L) implicated in inherited Romano-Ward LQT1 syndrome, are embedded within this domain. Reexpression studies in both COS-7 cells and cardiomyocytes showed that the mutant proteins fail to exit the endoplasmic reticulum. KCNQ1 subunits harboring Y111C or L114P exert a dominant negative effect on the wild-type KCNQ1 subunit by preventing plasma membrane trafficking of heteromultimeric channels. The P117L mutation had a less pronounced effect on the trafficking of heteromultimeric channels but altered the kinetics of the current. Furthermore, we showed that the trafficking determinant in KCNQ1 is structurally and functionally conserved in other KCNQ channels and constitutes a critical trafficking determinant of the KCNQ channel family. Computed structural predictions correlated the potential structural changes introduced by the mutations with impaired protein trafficking. In conclusion, our studies unveiled a new role of the N-terminus of KCNQ channels in their trafficking and its implication in severe forms of LQT1 syndrome.


Subject(s)
KCNQ1 Potassium Channel/genetics , KCNQ1 Potassium Channel/metabolism , Long QT Syndrome/genetics , Long QT Syndrome/metabolism , Adult , Amino Acid Sequence , Animals , CHO Cells , COS Cells , Cell Membrane/metabolism , Child , Chlorocebus aethiops , Cricetinae , Endoplasmic Reticulum/metabolism , Female , Hemagglutinins/genetics , Hemagglutinins/metabolism , Humans , KCNQ1 Potassium Channel/biosynthesis , Mice , Molecular Sequence Data , Mutagenesis , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/physiology , Protein Isoforms , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship , Transfection
10.
Br J Pharmacol ; 137(2): 153-61, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12208771

ABSTRACT

1. We have previously demonstrated that beta(3)-adrenoceptor (beta(3)-AR) stimulation induces endothelium-dependent vasorelaxation in rat aorta through the activation of an endothelial NO synthase associated with an increase in intracellular cGMP. The aim of the present study was to localise beta(3)-AR to confirm our functional study and to complete the signalling pathway of beta(3)-AR in rat aorta. 2. By RT-PCR, we have detected beta(3)-AR transcripts both in aorta and in freshly isolated endothelial cells. The absence of markers for adipsin or hormone-sensitive lipase in endothelial cells excluded the presence of beta(3)-AR from adipocytes. The localization of beta(3)-AR in aortic endothelial cells was confirmed by immunohistochemistry using a rat beta(3)-AR antibody. 3. To identify the G protein linked to beta(3)-AR, experiments were performed in rat pre-treated with PTX (10 microg kg(-1)), a G(i/0) protein inhibitor. The blockage of G(i/0) protein by PTX was confirmed by the reduction of vasorelaxation induced by UK 14304, a selective alpha(2)-AR agonist. The cumulative concentration-response curve for SR 58611A, a beta(3)-AR agonist, was not significantly modified on aorta rings from PTX pre-treated rats. 4. At the same level of contraction, the relaxations induced by 10 microM SR 58611A were significantly reduced in 30 mM-KCl pre-constricted rings (E(max)=16.7+/-8.4%, n=5), in comparison to phenylephrine (0.3 microM) pre-constricted rings (E(max)=49.11+/-11.0%, n=5, P<0.05). In addition, iberotoxin (0.1 microM), glibenclamide (1 microM) and 4-aminopyridine (1 mM), selective potassium channels blockers of K(Ca), K(ATP), and K(v) respectively, decreased the SR 58611A-mediated relaxation. 5. We conclude that beta(3)-AR is preferentially expressed in rat aortic endothelial cells. Beta(3)-AR-mediated aortic relaxation is independent of G(i/0) proteins stimulation, but results from the activation of several potassium channels, K(Ca), K(ATP), and K(v).


Subject(s)
Aorta, Thoracic/physiology , Receptors, Adrenergic, beta-3/physiology , Animals , Brimonidine Tartrate , Endothelium, Vascular/physiology , GTP-Binding Proteins/physiology , Immunohistochemistry , Male , Pertussis Toxin/pharmacology , Potassium Channels/physiology , Quinoxalines/pharmacology , RNA, Messenger/analysis , Rats , Rats, Wistar , Receptors, Adrenergic, beta-3/genetics , Tetrahydronaphthalenes/pharmacology , Vasodilation
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