Mariana Casas | Universidad de Chile (original) (raw)
Papers by Mariana Casas
Exercise produces oxidants from a variety of intracellular sources, including NADPH oxidases (NOX... more Exercise produces oxidants from a variety of intracellular sources, including NADPH oxidases (NOX) and mitochondria. Exercise-derived ROS are beneficial, and the amount and location of these ROS are important to avoid muscle damage associated with oxidative stress. We discuss here some of the evidence that involves ROS production associated with skeletal muscle contraction and the potential oxidative stress associated with muscle contraction. We also discuss the potential role of H2O2 produced after NOX activation in the regulation of glucose transport in skeletal muscle. Finally, we propose a model based on evidence for the role of different populations of mitochondria in skeletal muscle in the regulation of ATP production upon exercise. The sub-sarcolemmal population of mitochondria has the enzymatic and metabolic components to establish a high mitochondrial membrane potential when fissioned at rest but lacks the capacity to produce ATP; calcium entry to the mitochondria will further increase the metabolic input. Upon exercise, sub-sarcolemmal mitochondria will fuse to intermyofibrillar mitochondria and will transfer the membrane potential to them. These mitochondria are rich in ATP synthase and will subsequentially produce the ATP needed for muscle contraction in long-term exercise. These events will optimize energy use and minimize mitochondria ROS production.
The Royal Society of Chemistry eBooks, Oct 8, 2015
Cells have developed a variety of mechanisms to keep free calcium ion concentrations at very low ... more Cells have developed a variety of mechanisms to keep free calcium ion concentrations at very low levels in the cytosol. These mechanisms allow transient increases in cell calcium concentrations to be used as signals to trigger a variety of cellular processes, gene expression being one of them. Skeletal muscle relies on nerve activity both for contraction and also for the expression of genes related to pathways that include survival and the plastic changes required for adaptation to exercise. A particular pathway that involves Cav1.1 as a voltage sensor for nerve activity, pannexin-1 channels to release ATP to the extracellular milieu, purinergic P2Y receptors to link the signal via G protein to PI3 kinase and phospholipase C, will finally give rise to slow, long-lasting calcium transients in the nuclear region that can be linked to either expression or repression of a variety of genes. This mechanism appears to be the basis of fast to slow muscle fiber transition.
The FASEB Journal, Apr 1, 2019
Diabetologia, Aug 25, 2020
Aims/hypothesis Disrupted intracellular Ca 2+ handling is known to play a role in diabetic cardio... more Aims/hypothesis Disrupted intracellular Ca 2+ handling is known to play a role in diabetic cardiomyopathy but it has also been postulated to contribute to obesity-and type 2 diabetes-associated skeletal muscle dysfunction. Still, there is so far very limited functional insight into whether, and if so to what extent, muscular Ca 2+ homeostasis is affected in this situation, so as to potentially determine or contribute to muscle weakness. In differentiated muscle, force production is under the control of the excitationcontraction coupling process: upon plasma membrane electrical activity, the Ca V 1.1 voltage sensor/Ca 2+ channel in the plasma membrane triggers opening of the ryanodine receptor Ca 2+ release channel in the sarcoplasmic reticulum (SR) membrane. Opening of the ryanodine receptor triggers the rise in cytosolic Ca 2+ , which activates contraction while Ca 2+ uptake by the SR ATPase Ca 2+pump promotes relaxation. These are the core mechanisms underlying the tight control of muscle force by neuronal electrical activity. This study aimed at characterising their inherent physiological function in a diet-induced mouse model of obesity and type 2 diabetes. Methods Intact muscle fibres were isolated from mice fed either with a standard chow diet or with a high-fat, high-sucrose diet generating obesity, insulin resistance and glucose intolerance. Properties of muscle fibres were investigated with a combination of whole-cell voltage-clamp electrophysiology and confocal fluorescence imaging. The integrity and density of the plasma membrane network (transverse tubules) that carries the membrane excitation throughout the muscle fibres was assessed with the dye Di-8-ANEPPS. Ca V 1.1 Ca 2+ channel activity was studied by measuring the changes in current across the plasma membrane elicited by voltage-clamp depolarising pulses of increasing amplitude. SR Ca 2+ release through ryanodine receptors was simultaneously detected with the Ca 2+-sensitive dye Rhod-2 in the cytosol. Ca V 1.1 voltage-sensing activity was separately characterised from the properties of intra-plasma-membrane charge movement produced by short voltage-clamp depolarising pulses. Spontaneous Ca 2+ release at rest was assessed with the Ca 2+-sensitive dye Fluo-4. The rate of SR Ca 2+ uptake was assessed from the time course of cytosolic Ca 2+ recovery after the end of voltage excitation using the Ca 2+-sensitive dye Fluo-4FF. The response to a fatigue-stimulation protocol was determined from the time course of decline of the peak Fluo-4FF Ca 2+ transients elicited by 30 trains of 5-ms-long depolarising pulses delivered at 100 Hz. Results The transverse tubule network architecture and density were well preserved in the fibres from the obese mice. The Ca V 1.1 Ca 2+ current and voltage-sensing properties were also largely unaffected with mean values for maximum conductance and maximum amount of charge of 234 ± 12 S/F and 30.7 ± 1.6 nC/μF compared with 196 ± 13 S/F and 32.9 ± 2.0 nC/μF in fibres from mice fed with the standard diet, respectively. Voltage-activated SR Ca 2+ release through ryanodine receptors also exhibited very similar properties in the two groups with mean values for maximum rate of Ca 2+ release of 76.0 ± 6.5 and 78.1 ± 4.4 μmol l-1 ms-1 , in fibres from control and obese mice, respectively. The response to a fatigue protocol was also largely unaffected in fibres from the obese mice, and so were the rate of cytosolic Ca 2+ removal and the spontaneous Ca 2+ release activity at rest.
Journal of Molecular Endocrinology, Oct 1, 2020
Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that is considered a myokine... more Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that is considered a myokine playing a role in a variety of endocrine functions, including regulation of glucose transport and lipid metabolism. Although FGF21 has been associated with glucose metabolism in skeletal muscle cells, its cellular mechanism in adult skeletal muscle fibers glucose uptake is poorly understood. In the present study, we found that FGF21 induced a dose−response effect, increasing glucose uptake in skeletal muscle fibers from the flexor digitorum brevis muscle of mice, evaluated using the fluorescent glucose analog 2-NBDG (300 µM) in single living fibers. This effect was prevented by the use of either cytochalasin B (5 µM) or indinavir (100 µM), both antagonists of GLUT4 activity. The use of PI3K inhibitors such as LY294002 (50 µM) completely prevented the FGF21-dependent glucose uptake. In fibers electroporated with the construct encoding GLUT4myc-eGFP chimera and stimulated with FGF21 (100 ng/mL), a strong sarcolemmal GLUT4 label was detected. This effect promoted by FGF21 was demonstrated to be dependent on atypical PKC-ζ, by using selective PKC inhibitors. FGF21 at low concentrations potentiated the effect of insulin on glucose uptake but at high concentrations, completely inhibited the uptake in the presence of insulin. These results suggest that FGF21 regulates glucose uptake by a mechanism mediated by GLUT4 and dependent on atypical PKC-ζ in skeletal muscle.
The Journal of General Physiology, Oct 12, 2021
One of the most important functions of skeletal muscle is to respond to nerve stimuli by contract... more One of the most important functions of skeletal muscle is to respond to nerve stimuli by contracting. This function ensures body movement but also participates in other important physiological roles, like regulation of glucose homeostasis. Muscle activity is closely regulated to adapt to different demands and shows a plasticity that relies on both transcriptional activity and nerve stimuli. These two processes, both dependent on depolarization of the plasma membrane, have so far been regarded as separated and independent processes due to a lack of evidence of common protein partners or molecular mechanisms. In this study, we reveal intimate functional interactions between the process of excitation-induced contraction and the process of excitation-induced transcriptional activity in skeletal muscle. We show that the plasma membrane voltagesensing protein Ca V 1.1 and the ATP-releasing channel Pannexin-1 (Panx1) regulate each other in a reciprocal manner, playing roles in both processes. Specifically, knockdown of Ca V 1.1 produces chronically elevated extracellular ATP concentrations at rest, consistent with disruption of the normal control of Panx1 activity. Conversely, knockdown of Panx1 affects not only activation of transcription but also Ca V 1.1 function on the control of muscle fiber contraction. Altogether, our results establish the presence of bidirectional functional regulations between the molecular machineries involved in the control of contraction and transcription induced by membrane depolarization of adult muscle fibers. Our results are important for an integrative understanding of skeletal muscle function and may impact our understanding of several neuromuscular diseases.
Frontiers in Endocrinology
Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, an... more Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, and energy metabolism. Although it is released mainly from the liver, in recent years it has been shown that it is a “myokine”, synthesized in skeletal muscles after exercise and stress conditions through an Akt-dependent pathway and secreted for mediating autocrine and endocrine roles. To date, the molecular mechanism for the pathophysiological regulation of FGF21 production in skeletal muscle is not totally understood. We have previously demonstrated that muscle membrane depolarization controls gene expression through extracellular ATP (eATP) signaling, by a mechanism defined as “Excitation-Transcription coupling”. eATP signaling regulates the expression and secretion of interleukin 6, a well-defined myokine, and activates the Akt/mTOR signaling pathway. This work aimed to study the effect of electrical stimulation in the regulation of both production and secretion of skeletal muscle FGF...
HAL (Le Centre pour la Communication Scientifique Directe), Nov 21, 2018
Schizophrenia (SZ) is a complex neuropsychiatric disorder, affecting 1% of the world population. ... more Schizophrenia (SZ) is a complex neuropsychiatric disorder, affecting 1% of the world population. Long-standing clinical observations and molecular data have pointed out a possible vascular deficiency that could be acting synergistically with neuronal dysfunction in SZ.As SZ is a neurodevelopmental disease, the use of human induced pluripotent stem cells (hiPSC) allows disease biology modeling retaining the patient’s unique genetic signature. Previously, we reported a VEGF-A signaling impairment in SZ-hiPSC derived neural lineages leading to a decreased angiogenesis. Here, we present a functional characterization of SZ-derived brain microvascular endothelial-like cells (BEC), the counterpart of the neurovascular crosstalk, revealing an intrinsically defective Blood-Brain Barrier (BBB) phenotype. Transcriptomic assessment of genes related to endothelial function among three control (Ctrl BEC) and five schizophrenia patients derived BEC (SZP BEC), revealed that SZP BEC have a distincti...
PLOS ONE, 2015
<p>Muscle fibers were isolated, loaded with DCF (30min) and stimulated with exogenous ATP. ... more <p>Muscle fibers were isolated, loaded with DCF (30min) and stimulated with exogenous ATP. A, representative traces of DCF fluorescence under control or stimulated with ATP in the absence or presence of BIM (5μM). B, muscle cells were stimulated with ATP and the slope of fluorescence was analyzed (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>) (n = 3, *p<0.05). C, muscle fibers were isolated and transfected with HyPer plasmid, 24h post transfection the cells were stimulated in the presence of PMA, BIM or Rotterin (Rotl) as indicated in the graph, maximal fluorescence was plotted (n = 5), *p<0.05, **p<0.01.</p
During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxi-dase (NOX2)... more During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxi-dase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydro-fluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and sura-min; antagonists of pannexin channel and purinergic receptors respectively. In addition, transien...
Introduction: Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that induces g... more Introduction: Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that induces glucose uptake in both primary myotubes and C2C12 myoblasts. However, the cellular mechanism involved and its role in adult skeletal muscle fibers is poorly understood. Material and Methods: Male mice were used at 6-8 weeks of age. The glucose uptake was evaluated in single living fibers from flexor digitorum brevis muscle. To determine glucose uptake, we used the phosphorylable, non-metabolizable fluorescent glucose analog 2-NBDG (300 µM) that has been used to monitor glucose uptake in single living cells. Results: FGF21 induces a dose-response effect, increasing glucose uptake in isolated skeletal muscle fibers. This effect is prevented by the use of either Cytochalasin B (5 µM) or Indinavir (100 µM), both antagonists of GLUT4 activity. The use of PI3K inhibitors such as Wortmannin (100 nM) and LY294002 (50 µM) prevents the FGF21-dependent glucose uptake. In fibers electroporated with t...
Nucleic Acids Research, 2005
Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating th... more Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor bPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNAmodified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.
Journal of General Physiology, 2021
One of the most important functions of skeletal muscle is to respond to nerve stimuli by contract... more One of the most important functions of skeletal muscle is to respond to nerve stimuli by contracting. This function ensures body movement but also participates in other important physiological roles, like regulation of glucose homeostasis. Muscle activity is closely regulated to adapt to different demands and shows a plasticity that relies on both transcriptional activity and nerve stimuli. These two processes, both dependent on depolarization of the plasma membrane, have so far been regarded as separated and independent processes due to a lack of evidence of common protein partners or molecular mechanisms. In this study, we reveal intimate functional interactions between the process of excitation-induced contraction and the process of excitation-induced transcriptional activity in skeletal muscle. We show that the plasma membrane voltage-sensing protein CaV1.1 and the ATP-releasing channel Pannexin-1 (Panx1) regulate each other in a reciprocal manner, playing roles in both processe...
Frontiers in Physiology, 2021
The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle... more The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle fibers and regulated by the extracellular ATP/IP3/IP3R pathway has been related to muscle plasticity. A regulation of muscular tropism associated with the MCU has also been described. However, the role of transient cytosolic calcium signals and signaling pathways related to muscle plasticity over the regulation of gene expression of the MCU complex (MCU, MICU1, MICU2, and EMRE) in adult skeletal muscle is completely unknown. In the present work, we show that 270 0.3-ms-long pulses at 20-Hz ES (and not at 90 Hz) transiently decreased the mRNA levels of the MCU complex in mice flexor digitorum brevis isolated muscle fibers. Importantly, when ATP released after 20-Hz ES is hydrolyzed by the enzyme apyrase, the repressor effect of 20 Hz on mRNA levels of the MCU complex is lost. Accordingly, the exposure of muscle fibers to 30 μM exogenous ATP produces the same effect as 20-Hz ES. Moreover,...
Diabetologia, 2021
A Correction to this paper has been published: https://doi.org/10.1007/s00125-021-05451-1
Exercise produces oxidants from a variety of intracellular sources, including NADPH oxidases (NOX... more Exercise produces oxidants from a variety of intracellular sources, including NADPH oxidases (NOX) and mitochondria. Exercise-derived ROS are beneficial, and the amount and location of these ROS are important to avoid muscle damage associated with oxidative stress. We discuss here some of the evidence that involves ROS production associated with skeletal muscle contraction and the potential oxidative stress associated with muscle contraction. We also discuss the potential role of H2O2 produced after NOX activation in the regulation of glucose transport in skeletal muscle. Finally, we propose a model based on evidence for the role of different populations of mitochondria in skeletal muscle in the regulation of ATP production upon exercise. The sub-sarcolemmal population of mitochondria has the enzymatic and metabolic components to establish a high mitochondrial membrane potential when fissioned at rest but lacks the capacity to produce ATP; calcium entry to the mitochondria will further increase the metabolic input. Upon exercise, sub-sarcolemmal mitochondria will fuse to intermyofibrillar mitochondria and will transfer the membrane potential to them. These mitochondria are rich in ATP synthase and will subsequentially produce the ATP needed for muscle contraction in long-term exercise. These events will optimize energy use and minimize mitochondria ROS production.
The Royal Society of Chemistry eBooks, Oct 8, 2015
Cells have developed a variety of mechanisms to keep free calcium ion concentrations at very low ... more Cells have developed a variety of mechanisms to keep free calcium ion concentrations at very low levels in the cytosol. These mechanisms allow transient increases in cell calcium concentrations to be used as signals to trigger a variety of cellular processes, gene expression being one of them. Skeletal muscle relies on nerve activity both for contraction and also for the expression of genes related to pathways that include survival and the plastic changes required for adaptation to exercise. A particular pathway that involves Cav1.1 as a voltage sensor for nerve activity, pannexin-1 channels to release ATP to the extracellular milieu, purinergic P2Y receptors to link the signal via G protein to PI3 kinase and phospholipase C, will finally give rise to slow, long-lasting calcium transients in the nuclear region that can be linked to either expression or repression of a variety of genes. This mechanism appears to be the basis of fast to slow muscle fiber transition.
The FASEB Journal, Apr 1, 2019
Diabetologia, Aug 25, 2020
Aims/hypothesis Disrupted intracellular Ca 2+ handling is known to play a role in diabetic cardio... more Aims/hypothesis Disrupted intracellular Ca 2+ handling is known to play a role in diabetic cardiomyopathy but it has also been postulated to contribute to obesity-and type 2 diabetes-associated skeletal muscle dysfunction. Still, there is so far very limited functional insight into whether, and if so to what extent, muscular Ca 2+ homeostasis is affected in this situation, so as to potentially determine or contribute to muscle weakness. In differentiated muscle, force production is under the control of the excitationcontraction coupling process: upon plasma membrane electrical activity, the Ca V 1.1 voltage sensor/Ca 2+ channel in the plasma membrane triggers opening of the ryanodine receptor Ca 2+ release channel in the sarcoplasmic reticulum (SR) membrane. Opening of the ryanodine receptor triggers the rise in cytosolic Ca 2+ , which activates contraction while Ca 2+ uptake by the SR ATPase Ca 2+pump promotes relaxation. These are the core mechanisms underlying the tight control of muscle force by neuronal electrical activity. This study aimed at characterising their inherent physiological function in a diet-induced mouse model of obesity and type 2 diabetes. Methods Intact muscle fibres were isolated from mice fed either with a standard chow diet or with a high-fat, high-sucrose diet generating obesity, insulin resistance and glucose intolerance. Properties of muscle fibres were investigated with a combination of whole-cell voltage-clamp electrophysiology and confocal fluorescence imaging. The integrity and density of the plasma membrane network (transverse tubules) that carries the membrane excitation throughout the muscle fibres was assessed with the dye Di-8-ANEPPS. Ca V 1.1 Ca 2+ channel activity was studied by measuring the changes in current across the plasma membrane elicited by voltage-clamp depolarising pulses of increasing amplitude. SR Ca 2+ release through ryanodine receptors was simultaneously detected with the Ca 2+-sensitive dye Rhod-2 in the cytosol. Ca V 1.1 voltage-sensing activity was separately characterised from the properties of intra-plasma-membrane charge movement produced by short voltage-clamp depolarising pulses. Spontaneous Ca 2+ release at rest was assessed with the Ca 2+-sensitive dye Fluo-4. The rate of SR Ca 2+ uptake was assessed from the time course of cytosolic Ca 2+ recovery after the end of voltage excitation using the Ca 2+-sensitive dye Fluo-4FF. The response to a fatigue-stimulation protocol was determined from the time course of decline of the peak Fluo-4FF Ca 2+ transients elicited by 30 trains of 5-ms-long depolarising pulses delivered at 100 Hz. Results The transverse tubule network architecture and density were well preserved in the fibres from the obese mice. The Ca V 1.1 Ca 2+ current and voltage-sensing properties were also largely unaffected with mean values for maximum conductance and maximum amount of charge of 234 ± 12 S/F and 30.7 ± 1.6 nC/μF compared with 196 ± 13 S/F and 32.9 ± 2.0 nC/μF in fibres from mice fed with the standard diet, respectively. Voltage-activated SR Ca 2+ release through ryanodine receptors also exhibited very similar properties in the two groups with mean values for maximum rate of Ca 2+ release of 76.0 ± 6.5 and 78.1 ± 4.4 μmol l-1 ms-1 , in fibres from control and obese mice, respectively. The response to a fatigue protocol was also largely unaffected in fibres from the obese mice, and so were the rate of cytosolic Ca 2+ removal and the spontaneous Ca 2+ release activity at rest.
Journal of Molecular Endocrinology, Oct 1, 2020
Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that is considered a myokine... more Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that is considered a myokine playing a role in a variety of endocrine functions, including regulation of glucose transport and lipid metabolism. Although FGF21 has been associated with glucose metabolism in skeletal muscle cells, its cellular mechanism in adult skeletal muscle fibers glucose uptake is poorly understood. In the present study, we found that FGF21 induced a dose−response effect, increasing glucose uptake in skeletal muscle fibers from the flexor digitorum brevis muscle of mice, evaluated using the fluorescent glucose analog 2-NBDG (300 µM) in single living fibers. This effect was prevented by the use of either cytochalasin B (5 µM) or indinavir (100 µM), both antagonists of GLUT4 activity. The use of PI3K inhibitors such as LY294002 (50 µM) completely prevented the FGF21-dependent glucose uptake. In fibers electroporated with the construct encoding GLUT4myc-eGFP chimera and stimulated with FGF21 (100 ng/mL), a strong sarcolemmal GLUT4 label was detected. This effect promoted by FGF21 was demonstrated to be dependent on atypical PKC-ζ, by using selective PKC inhibitors. FGF21 at low concentrations potentiated the effect of insulin on glucose uptake but at high concentrations, completely inhibited the uptake in the presence of insulin. These results suggest that FGF21 regulates glucose uptake by a mechanism mediated by GLUT4 and dependent on atypical PKC-ζ in skeletal muscle.
The Journal of General Physiology, Oct 12, 2021
One of the most important functions of skeletal muscle is to respond to nerve stimuli by contract... more One of the most important functions of skeletal muscle is to respond to nerve stimuli by contracting. This function ensures body movement but also participates in other important physiological roles, like regulation of glucose homeostasis. Muscle activity is closely regulated to adapt to different demands and shows a plasticity that relies on both transcriptional activity and nerve stimuli. These two processes, both dependent on depolarization of the plasma membrane, have so far been regarded as separated and independent processes due to a lack of evidence of common protein partners or molecular mechanisms. In this study, we reveal intimate functional interactions between the process of excitation-induced contraction and the process of excitation-induced transcriptional activity in skeletal muscle. We show that the plasma membrane voltagesensing protein Ca V 1.1 and the ATP-releasing channel Pannexin-1 (Panx1) regulate each other in a reciprocal manner, playing roles in both processes. Specifically, knockdown of Ca V 1.1 produces chronically elevated extracellular ATP concentrations at rest, consistent with disruption of the normal control of Panx1 activity. Conversely, knockdown of Panx1 affects not only activation of transcription but also Ca V 1.1 function on the control of muscle fiber contraction. Altogether, our results establish the presence of bidirectional functional regulations between the molecular machineries involved in the control of contraction and transcription induced by membrane depolarization of adult muscle fibers. Our results are important for an integrative understanding of skeletal muscle function and may impact our understanding of several neuromuscular diseases.
Frontiers in Endocrinology
Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, an... more Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, and energy metabolism. Although it is released mainly from the liver, in recent years it has been shown that it is a “myokine”, synthesized in skeletal muscles after exercise and stress conditions through an Akt-dependent pathway and secreted for mediating autocrine and endocrine roles. To date, the molecular mechanism for the pathophysiological regulation of FGF21 production in skeletal muscle is not totally understood. We have previously demonstrated that muscle membrane depolarization controls gene expression through extracellular ATP (eATP) signaling, by a mechanism defined as “Excitation-Transcription coupling”. eATP signaling regulates the expression and secretion of interleukin 6, a well-defined myokine, and activates the Akt/mTOR signaling pathway. This work aimed to study the effect of electrical stimulation in the regulation of both production and secretion of skeletal muscle FGF...
HAL (Le Centre pour la Communication Scientifique Directe), Nov 21, 2018
Schizophrenia (SZ) is a complex neuropsychiatric disorder, affecting 1% of the world population. ... more Schizophrenia (SZ) is a complex neuropsychiatric disorder, affecting 1% of the world population. Long-standing clinical observations and molecular data have pointed out a possible vascular deficiency that could be acting synergistically with neuronal dysfunction in SZ.As SZ is a neurodevelopmental disease, the use of human induced pluripotent stem cells (hiPSC) allows disease biology modeling retaining the patient’s unique genetic signature. Previously, we reported a VEGF-A signaling impairment in SZ-hiPSC derived neural lineages leading to a decreased angiogenesis. Here, we present a functional characterization of SZ-derived brain microvascular endothelial-like cells (BEC), the counterpart of the neurovascular crosstalk, revealing an intrinsically defective Blood-Brain Barrier (BBB) phenotype. Transcriptomic assessment of genes related to endothelial function among three control (Ctrl BEC) and five schizophrenia patients derived BEC (SZP BEC), revealed that SZP BEC have a distincti...
PLOS ONE, 2015
<p>Muscle fibers were isolated, loaded with DCF (30min) and stimulated with exogenous ATP. ... more <p>Muscle fibers were isolated, loaded with DCF (30min) and stimulated with exogenous ATP. A, representative traces of DCF fluorescence under control or stimulated with ATP in the absence or presence of BIM (5μM). B, muscle cells were stimulated with ATP and the slope of fluorescence was analyzed (<i>see</i><i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129882#sec002" target="_blank">material and method</a></i>) (n = 3, *p<0.05). C, muscle fibers were isolated and transfected with HyPer plasmid, 24h post transfection the cells were stimulated in the presence of PMA, BIM or Rotterin (Rotl) as indicated in the graph, maximal fluorescence was plotted (n = 5), *p<0.05, **p<0.01.</p
During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxi-dase (NOX2)... more During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxi-dase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydro-fluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and sura-min; antagonists of pannexin channel and purinergic receptors respectively. In addition, transien...
Introduction: Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that induces g... more Introduction: Fibroblast growth factor 21 (FGF21) is a pleiotropic peptide hormone that induces glucose uptake in both primary myotubes and C2C12 myoblasts. However, the cellular mechanism involved and its role in adult skeletal muscle fibers is poorly understood. Material and Methods: Male mice were used at 6-8 weeks of age. The glucose uptake was evaluated in single living fibers from flexor digitorum brevis muscle. To determine glucose uptake, we used the phosphorylable, non-metabolizable fluorescent glucose analog 2-NBDG (300 µM) that has been used to monitor glucose uptake in single living cells. Results: FGF21 induces a dose-response effect, increasing glucose uptake in isolated skeletal muscle fibers. This effect is prevented by the use of either Cytochalasin B (5 µM) or Indinavir (100 µM), both antagonists of GLUT4 activity. The use of PI3K inhibitors such as Wortmannin (100 nM) and LY294002 (50 µM) prevents the FGF21-dependent glucose uptake. In fibers electroporated with t...
Nucleic Acids Research, 2005
Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating th... more Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor bPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNAmodified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.
Journal of General Physiology, 2021
One of the most important functions of skeletal muscle is to respond to nerve stimuli by contract... more One of the most important functions of skeletal muscle is to respond to nerve stimuli by contracting. This function ensures body movement but also participates in other important physiological roles, like regulation of glucose homeostasis. Muscle activity is closely regulated to adapt to different demands and shows a plasticity that relies on both transcriptional activity and nerve stimuli. These two processes, both dependent on depolarization of the plasma membrane, have so far been regarded as separated and independent processes due to a lack of evidence of common protein partners or molecular mechanisms. In this study, we reveal intimate functional interactions between the process of excitation-induced contraction and the process of excitation-induced transcriptional activity in skeletal muscle. We show that the plasma membrane voltage-sensing protein CaV1.1 and the ATP-releasing channel Pannexin-1 (Panx1) regulate each other in a reciprocal manner, playing roles in both processe...
Frontiers in Physiology, 2021
The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle... more The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle fibers and regulated by the extracellular ATP/IP3/IP3R pathway has been related to muscle plasticity. A regulation of muscular tropism associated with the MCU has also been described. However, the role of transient cytosolic calcium signals and signaling pathways related to muscle plasticity over the regulation of gene expression of the MCU complex (MCU, MICU1, MICU2, and EMRE) in adult skeletal muscle is completely unknown. In the present work, we show that 270 0.3-ms-long pulses at 20-Hz ES (and not at 90 Hz) transiently decreased the mRNA levels of the MCU complex in mice flexor digitorum brevis isolated muscle fibers. Importantly, when ATP released after 20-Hz ES is hydrolyzed by the enzyme apyrase, the repressor effect of 20 Hz on mRNA levels of the MCU complex is lost. Accordingly, the exposure of muscle fibers to 30 μM exogenous ATP produces the same effect as 20-Hz ES. Moreover,...
Diabetologia, 2021
A Correction to this paper has been published: https://doi.org/10.1007/s00125-021-05451-1