Response to Mechanical Stress Is Mediated by the TRPA Channel Painless in the Drosophila Heart

Mechanotransduction modulates cellular functions as diverse as migration, proliferation, differentiation, and apoptosis. It is crucial for organ development and homeostasis and leads to pathologies when defective. However, despite considerable efforts made in the past, the molecular basis of mechanotransduction remains poorly understood. Here, we have investigated the genetic basis of mechanotransduction in Drosophila. We show that the fly heart senses and responds to mechanical forces by regulating cardiac activity. In particular, pauses in heart activity are observed under acute mechanical constraints in vivo. We further confirm by a variety of in situ tests that these cardiac arrests constitute the biological force-induced response. In order to identify molecular components of the mechanotransduction pathway, we carried out a genetic screen based on the dependence of cardiac activity upon mechanical constraints and identified Painless, a TRPA channel. We observe a clear absence of in vivo cardiac arrest following inactivation of painless and further demonstrate that painless is autonomously required in the heart to mediate the response to mechanical stress. Furthermore, direct activation of Painless is sufficient to produce pauses in heartbeat, mimicking the pressure-induced response. Painless thus constitutes part of a mechanosensitive pathway that adjusts cardiac muscle activity to mechanical constraints. This constitutes the first in vivo demonstration that a TRPA channel can mediate cardiac mechanotransduction. Furthermore, by establishing a high-throughput system to identify the molecular players involved in mechanotransduction in the cardiovascular system, our study paves the way for understanding the mechanisms underlying a mechanotransduction pathway

Contribution to the analysis of the regulation of cardiac activity in the Drosophila melanogaster heart

L’objectif général de ma thèse concerne l’étude de la régulation de l’activité cardiaque chez Drosophila melanogaster. Différentes questions ont été abordées : i) la régulation par le pH de l’activité cardiaque à travers l’étude d’un Transporteur du Bicarbonate dépendant du Na+, NDAE1 ; ii) l’implication du canal TRP Painless dans la mécanosensibilité du cœur ; iii) l’élaboration de tests quantitatifs permettant de mesurer le vieillissement cardiaque chez la mouche adulte. Le Na+-Driven Anion Exchanger (NDAE1) constitue l’unique transporteur chez la Drosophile capable de transporter le Bicarbonate dépendant du Na+, alors que l’on trouve dans le génome des mammifères 7 de ces transporteurs appartenant à la famille SLC4. NDAE1 permet l’échange de protons et de Cl- avec le Na+ et HCO3- et agit de manière réversible. Etant donné l’importance potentielle et reconnue de ce type d’échangeur durant certaines pathologies cardiaques intervenant par exemple lors d’épisodes d’ischémie-reperfusion, j’ai analysé sa fonction dans l’activité cardiaque. De manière surprenante, l’inactivation du gène spécifiquement dans le tube cardiaque par interférence à l’ARN n’a aucun effet sur les paramètres mesurables de l’activité cardiaque dans des conditions basales d’élevage, ni sur la viabilité. En revanche, la fonction de NDAE1 peut être révélée dans des conditions de stress où l’on déséquilibre l’homéostasie des ions transportées dans l’échange dépendant de nDAE1. Ainsi, une acidose provoquée dans les individus privés de la fonction de NDAE1 génère de très fortes arythmies, qui sont moins présentes dans les animaux de type sauvage, et conduisent à des arrêts cardiaques définitifs. En outre, arythmies et arrêts cardiaques sont irréversibles quand le pH physiologique est restoré, contrairement aux contrôles qui retrouvent complètement leur activité cardiaque normale. De même l’activité de NDAE1 est requise pour mieux résister aux stress provoqués par l’absence de Na+, de Cl- et de HCO3- dans le milieu extracellulaire et d’adapter à un choc osmotique. En outre, j’ai mis en évidence une forte interaction génétique de ndae1 avec ncx, qui code pour l’échangeur Sodium-Calcium, et dont la fonction est de réguler l’homéostasie calcique et sodique. Cette étude constitue la première démonstration in vivo de la fonction cardiaque des Transporteurs du Bicarbonate dépendant du Na+. J’ai d’autre part contribué à l’étude de la réponse des cardiomyocytes aux stress mécaniques et participé à la démonstration que Painless, un canal TRPA de la Drosophile, était requis pour cette réponse. Finalement, dans le cadre du programme « Identification of genetic markers of cardiac aging in Drosophila », j’ai cherché à proposer des tests capables de mesurer le déclin des performances cardiaques avec l’âge. Parmi ceux-ci, le plus prometteur consiste en une quantification des arythmies mesurée par l’analyse in vivo détaillée des battements cardiaquesMechanotransduction modulates cellular functions as diverse as migration, proliferation, differentiation and apoptosis. It is crucial for organ development and homeostasis and leads to pathologies when defective. However, despite considerable efforts made in the past, the molecular basis of mechanotransduction remains poorly understood.Here, we have investigated the genetic basis of mechanotransduction in Drosophila. We show that the fly heart senses and responds to mechanical forces by regulating cardiac activity. In particular, pauses in heart activity are observed under acute mechanical constraints in vivo. We further confirm by a variety of in situ tests that these cardiac arrests constitute the biological force-induced response.In order to identify molecular components of the mechanotransduction pathway, we carried out a genetic screen, based on the dependence of cardiac activity upon mechanical constraints and identified Painless, a TRPA channel. We observe a clear absence of in vivo cardiac arrest following inactivation of painless and further demonstrate that painless is autonomously required in the heart to mediate the response to mechanical stress. Furthermore, direct activation of Painless is sufficient to produce pauses in heartbeat, mimicking the pressure-induced response. Painless thus constitutes part of a mechanosensitive pathway that adjusts cardiac muscle activity to mechanical constraints.This constitutes the first in vivo demonstration that a TRPA channel can mediate cardiac mechanotransduction. Furthermore, by establishing a high-throughput system to identify the molecular players involved in mechanotransduction in the cardiovascular system our study paves the way for understanding the mechanisms underlying a mechanotransduction pathway

Contribution to the analysis of the regulation of cardiac activity in the Drosophila melanogaster heart

L objectif général de ma thèse concerne l étude de la régulation de l activité cardiaque chez Drosophila melanogaster. Différentes questions ont été abordées : i) la régulation par le pH de l activité cardiaque à travers l étude d un Transporteur du Bicarbonate dépendant du Na+, NDAE1 ; ii) l implication du canal TRP Painless dans la mécanosensibilité du cœur ; iii) l élaboration de tests quantitatifs permettant de mesurer le vieillissement cardiaque chez la mouche adulte. Le Na+-Driven Anion Exchanger (NDAE1) constitue l unique transporteur chez la Drosophile capable de transporter le Bicarbonate dépendant du Na+, alors que l on trouve dans le génome des mammifères 7 de ces transporteurs appartenant à la famille SLC4. NDAE1 permet l échange de protons et de Cl- avec le Na+ et HCO3- et agit de manière réversible. Etant donné l importance potentielle et reconnue de ce type d échangeur durant certaines pathologies cardiaques intervenant par exemple lors d épisodes d ischémie-reperfusion, j ai analysé sa fonction dans l activité cardiaque. De manière surprenante, l inactivation du gène spécifiquement dans le tube cardiaque par interférence à l ARN n a aucun effet sur les paramètres mesurables de l activité cardiaque dans des conditions basales d élevage, ni sur la viabilité. En revanche, la fonction de NDAE1 peut être révélée dans des conditions de stress où l on déséquilibre l homéostasie des ions transportées dans l échange dépendant de nDAE1. Ainsi, une acidose provoquée dans les individus privés de la fonction de NDAE1 génère de très fortes arythmies, qui sont moins présentes dans les animaux de type sauvage, et conduisent à des arrêts cardiaques définitifs. En outre, arythmies et arrêts cardiaques sont irréversibles quand le pH physiologique est restoré, contrairement aux contrôles qui retrouvent complètement leur activité cardiaque normale. De même l activité de NDAE1 est requise pour mieux résister aux stress provoqués par l absence de Na+, de Cl- et de HCO3- dans le milieu extracellulaire et d adapter à un choc osmotique. En outre, j ai mis en évidence une forte interaction génétique de ndae1 avec ncx, qui code pour l échangeur Sodium-Calcium, et dont la fonction est de réguler l homéostasie calcique et sodique. Cette étude constitue la première démonstration in vivo de la fonction cardiaque des Transporteurs du Bicarbonate dépendant du Na+. J ai d autre part contribué à l étude de la réponse des cardiomyocytes aux stress mécaniques et participé à la démonstration que Painless, un canal TRPA de la Drosophile, était requis pour cette réponse. Finalement, dans le cadre du programme Identification of genetic markers of cardiac aging in Drosophila , j ai cherché à proposer des tests capables de mesurer le déclin des performances cardiaques avec l âge. Parmi ceux-ci, le plus prometteur consiste en une quantification des arythmies mesurée par l analyse in vivo détaillée des battements cardiaquesMechanotransduction modulates cellular functions as diverse as migration, proliferation, differentiation and apoptosis. It is crucial for organ development and homeostasis and leads to pathologies when defective. However, despite considerable efforts made in the past, the molecular basis of mechanotransduction remains poorly understood.Here, we have investigated the genetic basis of mechanotransduction in Drosophila. We show that the fly heart senses and responds to mechanical forces by regulating cardiac activity. In particular, pauses in heart activity are observed under acute mechanical constraints in vivo. We further confirm by a variety of in situ tests that these cardiac arrests constitute the biological force-induced response.In order to identify molecular components of the mechanotransduction pathway, we carried out a genetic screen, based on the dependence of cardiac activity upon mechanical constraints and identified Painless, a TRPA channel. We observe a clear absence of in vivo cardiac arrest following inactivation of painless and further demonstrate that painless is autonomously required in the heart to mediate the response to mechanical stress. Furthermore, direct activation of Painless is sufficient to produce pauses in heartbeat, mimicking the pressure-induced response. Painless thus constitutes part of a mechanosensitive pathway that adjusts cardiac muscle activity to mechanical constraints.This constitutes the first in vivo demonstration that a TRPA channel can mediate cardiac mechanotransduction. Furthermore, by establishing a high-throughput system to identify the molecular players involved in mechanotransduction in the cardiovascular system our study paves the way for understanding the mechanisms underlying a mechanotransduction pathway.AIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest

<div><p>Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G₀ myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G₀ C2C12 myoblasts to isogenic G₀ fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G₀ is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.</p></div

Chromatin-IP analysis of Myf5 and MyoG promoters in different cellular states.

<p>Antibodies against β-cat or HBP were used to assess the association of these Wnt-regulated transcription factors with chromatin in different cellular states as described in Materials and Methods. Control pulldowns used IgG and all values shown represent fold enrichment of the specific transcription factor after normalization against control IgG values. (<b>A</b>)<b>.</b> The Wnt target transcription factor β-cat associates with the known Wnt-responsive site in Myf5 enhancer preferentially in G₀ (G₀) but shows low enrichment in either proliferating (MB) or differentiating muscle cells (MT) (Blue bars-MB; pink bars-G₀; green bars-MT). This observation is consistent with the hypothesis that Wnt signaling is active in quiescent myoblasts. Comparison of β-cat association on another myogenic promoter (Myogenin promoter) shows greater enrichment in MT. Taken together, these observations suggest that Wnt/β-cat regulates different genes in different cellular states. (<b>B</b>)<b>.</b> ChIP analysis shows that HBP1 (a Wnt-induced repressor) co-associates with the Myf5 enhancer only in G₀ (Blue bars-Mb; pink bars-G₀; green bars-MT) and does not associate with this element in either proliferating or differentiated muscle cells. This observation suggests that fine-tuning of Myf5 expression by both activating and repressive mechanisms may occur in quiescent cells by association of two types of Wnt-responsive transcription factors. Taken together, this observation would account for the absence of induction of Myf5 mRNA in quiescent myoblasts despite the association of the transcriptional activator β-cat.</p

Selected classes of genes up-regulated in quiescence.

<p>Selected classes of genes up-regulated in quiescence.</p

Wnt pathway genes are enriched in quiescent myoblasts.

<p>(A) Microarray analysis identifies a Wnt signature in quiescence. Cluster analysis of the 4 sample pairs analysed in Fig. 2 reveals induction of Wnt pathway genes in G₀ MB-many of these genes are also induced in G₀ FB but not in MT, indicating greater transcriptomal relatedness between the quiescent state of different cell types (G₀ FB vs G₀ MB) than between reversibly and irreversibly arrested cells of the same cell type (G₀ MB vs MT). (B) Schematic of Wnt signaling depicting genes induced in G₀ Mb. Genes identified as enriched in G₀ by microarray analysis are shown in black, Wnt2 and R-spondin whose expression was directly tested are in grey. Dvl, an important Wnt signaling node is depicted for clarity (was not recovered in array). Note that components of both canonical and planar cell polarity (PCP) pathways were induced as well as some components that cross-talk with Rho/Jnk pathways. (<b>C</b>) Northern blot analysis of selected Wnt-related genes on RNA isolated from proliferating (MB), quiescent (G₀ MB) and differentiated cells (MT). Numbers to the left of blots represent log ratios derived from comparison of G₀ MB to asynchronous MB. Gene symbols and mRNA sizes are shown on the right. All genes tested in this independent assay show expression patterns that support their recovery in the microarray experiment. (<b>D</b>) Q-RT-PCR analysis of two putative Wnt pathway genes Rgs2 (top graph) and Dkk3 (bottom graph) in proliferating (A), quiescent (G₀) and differentiated (MT) cells. Values represent normalized fold differences between GAPDH (control) mRNA and Rgs2/Dkk3 mRNAs in each sample calculated from cycle thresholds [2^{−(−ΔΔCt)}] (n = 3, p<0.05). (<b>E</b>) Wnt pathway genes are rapidly induced in G₀, rapidly suppressed during G1. Northern analysis of selected Wnt pathway genes during a time course of entry and exit from quiescence. Asynchronous MB (MB), cells in suspension culture for 6, 12, 24 and 48 hrs (S6, S12, S24, S48) or reactivated into the cell cycle for 20 min to 24 hrs (R20’, R1–R24). The suspended population is completely arrested by 48 hours (S48); thus ‘S48’ time point corresponds to ‘G₀ MB’ depicted in all other figures. S-phase specific (replication-dependent) histone H2B expression is suppressed as cells arrest in suspension culture, reactivated at 24 hrs after replating when cells re-enter DNA synthesis. L7 and 28S are loading controls. Wnt pathway regulatory genes- LEF1, Groucho, Dab2 as well as putative Wnt pathway genes Dkk3, Rgs2 are more tightly quiescence-dependent (expression lost by 2–6 hrs after reactivation) than Wnt target gene Ecm1 (expression down-regulated but still detected at 24 hrs after reactivation). Arrow shows a smaller LEF1 transcript seen only in G₀.</p

Enhancement of Wnt signaling subverts the quiescence program.

<p>(A) Exposure of adherent MB to rWnt3a (50 ng/ml) leads to β-cat nuclear localization, TOPflash activation and suppression of MyoD protein as compared to control cells. (B) rWnt 3a (50 ng/ml) does not enhance proliferation (BrdU incorporated in a 30′ pulse) in muscle cells: Asynchronous MB, G₀ MB, MB reactivated after synchronization in (R18) or differentiated myotubes (MT) [Note: all BrdU+ nuclei in myotube cultures were in residual mono-nucleated myobalsts]. Values represent the mean±SEM from three independent experiments. (C) Exogenous Wnt3a alters the quiescence program: Q-RTPCR analysis of control (blue bars) and Wnt-treated (pink bars) cells held in suspension for 48 hrs shows repression of MyoD and MyoG but induction of Myf5, indicating differential response of MRFs; repression of p21 and induction of CyclinD1 collectively suggesting a shift to a proliferative gene expression program; and finally, repression of quiescence-induced genes Rgs2 and Dkk3, consistent with this shift. Values represent the mean±SEM from three independent experiments. (D) Context-dependent response to Wnt enhancement. Cells in three different states (MB, G₀ or MT) were treated for 48 hours with 50ng/ml of rWnt3a. Of the MRFs, Myf5 mRNA is only induced by Wnt3a if the target cells are in G₀. Values represent the mean±SEM from three independent experiments.</p

Expression profile of Rgs2 and Dkk3 in different cell states.

<p>(<b>A</b>) Quiescence-induced genes Rgs2 and Dkk3 are not expressed throughout myogenic differentiation, confirming the distinct induction pattern in reversible arrest and not in irreversible arrest. Northern analysis of growing MB (Mb) shifted to DM for 12–96 hrs. G₀ Mb are shown for comparison. (<b>B</b>) Quiescence-induced expression of Rgs2 and Dkk3 is cell-type specific – these putative Wnt pathway genes are differentially expressed in MB and FB. Northern analysis of growing C3H10T1/2 FB (Fb), suspension-arrested FB (S48) and G₀ FB reactivated for 20 minutes to 24 hrs. Myoblast samples are shown for comparison. Rgs2 transcript is mildly induced when C3H10T1/2 FB exit from proliferation into G₀ [compare with strong G₀ induction in C2C12], but Dkk3 mRNA is not detected in FB, suggesting muscle-specific activation restricted to the quiescent state. (C,D) Induction of Rgs2 and Dkk3 protein expression in quiescent myoblasts. Western blot analysis of total protein isolated from growing (MB), quiescent (G₀) and differentiated (MT) C2C12 muscle cells and probed with antibodies against Rgs2 (C) and Dkk3 (D). In both cases the induction of protein levels is modest compared to the strong induction of the respective transcripts, potentially reflecting the strong translational repression typical of G₀. Data depicted are representative of 3 independent experiments.</p