Electrical pulse stimulation (EPS) has been utilized to investigate how the cellular response changes during exercise. By this way it is simulated an in vivo contraction. With this method, it is possible to obtain a model to study human skeletal muscle in vitro. By using this method, it is possible to analyse how the myotubes react in different conditions in vitro. In this work, it is described how the myotubes, derived from C2C12 myoblasts, react to different electrical pulse stimulation, and different stimuli length. It was analysed the expression of specific gene markers important for myotubes to maintain intracellular homeostasis during the contraction and how their expression changes during the time and with different types of EPS. The markers were analysed through Real Time PCR technique. We analysed changes in mRNA expression of Activating transcription factor 3 (ATF3), interleukin 6 (IL6), interleukin 8 (IL8), C-X-C motif chemokine ligand 5 (CxCl5), extracellular signal-regulated kinase 1/2 (Erk1/2), phospholemman (FXYD1), dysadherin (FXYD5), Nuclear receptor of the subfamily 4 group A 3 (Nra4a3), Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Ppargc1-α), Secreted protein acidic and rich in cysteine (SPARC), sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1 (SERCA1) and vascular endothelial growth factor A (VEGFA). Basing on gene expression, we selected some proteins to evaluate their concentration in the medium in relationship to EPS stimuli. During contraction, myotubes release specific proteins that could act in an autocrine, paracrine and endocrine way to regulate different intracellular processes. Protein secretion depends on the expression of the mRNA. The release of these proteins was analysed firstly using the Bradford assay that permits to show the concentration of all proteins in the medium. Then, ELISA assay was used to evaluate if EPS may modify IL6 secretion. Since myoblast differentiation create a disorganized array of myotubes we engineered myoblast seeding to allow the production of oriented myotubes. To this purpose, cells were plated on a specific pattern of extracellular proteins created through or the oxygen etching or microcontact printing techniques. This last technique is used to print a specific protein of interest (an adhesive protein) on a substrate. The adhesive protein used for this work were the Poly-L-Lysine and the Gelatine. C2C12 cells were grown according to the shape created with a specific stamp. We created stamps permitting to obtain a pattern of lines of proteins where the cells were able to attach, grow and differentiate. Oxygen etching allows the construction of the same pattern described for the microcontact printing. In this work it was optimized pattering techniques to obtain a good protein deposition that allows cell organization and differentiation in oriented myofibers. Moreover, we analysed the advantages and the disadvantages of these two methods used to produce patterned proteins.

Electrical pulse stimulation (EPS) has been utilized to investigate how the cellular response changes during exercise. By this way it is simulated an in vivo contraction. With this method, it is possible to obtain a model to study human skeletal muscle in vitro. By using this method, it is possible to analyse how the myotubes react in different conditions in vitro. In this work, it is described how the myotubes, derived from C2C12 myoblasts, react to different electrical pulse stimulation, and different stimuli length. It was analysed the expression of specific gene markers important for myotubes to maintain intracellular homeostasis during the contraction and how their expression changes during the time and with different types of EPS. The markers were analysed through Real Time PCR technique. We analysed changes in mRNA expression of Activating transcription factor 3 (ATF3), interleukin 6 (IL6), interleukin 8 (IL8), C-X-C motif chemokine ligand 5 (CxCl5), extracellular signal-regulated kinase 1/2 (Erk1/2), phospholemman (FXYD1), dysadherin (FXYD5), Nuclear receptor of the subfamily 4 group A 3 (Nra4a3), Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Ppargc1-α), Secreted protein acidic and rich in cysteine (SPARC), sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1 (SERCA1) and vascular endothelial growth factor A (VEGFA). Basing on gene expression, we selected some proteins to evaluate their concentration in the medium in relationship to EPS stimuli. During contraction, myotubes release specific proteins that could act in an autocrine, paracrine and endocrine way to regulate different intracellular processes. Protein secretion depends on the expression of the mRNA. The release of these proteins was analysed firstly using the Bradford assay that permits to show the concentration of all proteins in the medium. Then, ELISA assay was used to evaluate if EPS may modify IL6 secretion. Since myoblast differentiation create a disorganized array of myotubes we engineered myoblast seeding to allow the production of oriented myotubes. To this purpose, cells were plated on a specific pattern of extracellular proteins created through or the oxygen etching or microcontact printing techniques. This last technique is used to print a specific protein of interest (an adhesive protein) on a substrate. The adhesive protein used for this work were the Poly-L-Lysine and the Gelatine. C2C12 cells were grown according to the shape created with a specific stamp. We created stamps permitting to obtain a pattern of lines of proteins where the cells were able to attach, grow and differentiate. Oxygen etching allows the construction of the same pattern described for the microcontact printing. In this work it was optimized pattering techniques to obtain a good protein deposition that allows cell organization and differentiation in oriented myofibers. Moreover, we analysed the advantages and the disadvantages of these two methods used to produce patterned proteins.

Electric Pulse Stimulation of Myotubes as an In Vitro Exercise Model: gene expression changes after acute or chronic EPS

DE NAPOLI, COSIMO
2021/2022

Abstract

Electrical pulse stimulation (EPS) has been utilized to investigate how the cellular response changes during exercise. By this way it is simulated an in vivo contraction. With this method, it is possible to obtain a model to study human skeletal muscle in vitro. By using this method, it is possible to analyse how the myotubes react in different conditions in vitro. In this work, it is described how the myotubes, derived from C2C12 myoblasts, react to different electrical pulse stimulation, and different stimuli length. It was analysed the expression of specific gene markers important for myotubes to maintain intracellular homeostasis during the contraction and how their expression changes during the time and with different types of EPS. The markers were analysed through Real Time PCR technique. We analysed changes in mRNA expression of Activating transcription factor 3 (ATF3), interleukin 6 (IL6), interleukin 8 (IL8), C-X-C motif chemokine ligand 5 (CxCl5), extracellular signal-regulated kinase 1/2 (Erk1/2), phospholemman (FXYD1), dysadherin (FXYD5), Nuclear receptor of the subfamily 4 group A 3 (Nra4a3), Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Ppargc1-α), Secreted protein acidic and rich in cysteine (SPARC), sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1 (SERCA1) and vascular endothelial growth factor A (VEGFA). Basing on gene expression, we selected some proteins to evaluate their concentration in the medium in relationship to EPS stimuli. During contraction, myotubes release specific proteins that could act in an autocrine, paracrine and endocrine way to regulate different intracellular processes. Protein secretion depends on the expression of the mRNA. The release of these proteins was analysed firstly using the Bradford assay that permits to show the concentration of all proteins in the medium. Then, ELISA assay was used to evaluate if EPS may modify IL6 secretion. Since myoblast differentiation create a disorganized array of myotubes we engineered myoblast seeding to allow the production of oriented myotubes. To this purpose, cells were plated on a specific pattern of extracellular proteins created through or the oxygen etching or microcontact printing techniques. This last technique is used to print a specific protein of interest (an adhesive protein) on a substrate. The adhesive protein used for this work were the Poly-L-Lysine and the Gelatine. C2C12 cells were grown according to the shape created with a specific stamp. We created stamps permitting to obtain a pattern of lines of proteins where the cells were able to attach, grow and differentiate. Oxygen etching allows the construction of the same pattern described for the microcontact printing. In this work it was optimized pattering techniques to obtain a good protein deposition that allows cell organization and differentiation in oriented myofibers. Moreover, we analysed the advantages and the disadvantages of these two methods used to produce patterned proteins.
2021
Electric Pulse Stimulation of Myotubes as an In Vitro Exercise Model: gene expression changes after acute or chronic EPS
Electrical pulse stimulation (EPS) has been utilized to investigate how the cellular response changes during exercise. By this way it is simulated an in vivo contraction. With this method, it is possible to obtain a model to study human skeletal muscle in vitro. By using this method, it is possible to analyse how the myotubes react in different conditions in vitro. In this work, it is described how the myotubes, derived from C2C12 myoblasts, react to different electrical pulse stimulation, and different stimuli length. It was analysed the expression of specific gene markers important for myotubes to maintain intracellular homeostasis during the contraction and how their expression changes during the time and with different types of EPS. The markers were analysed through Real Time PCR technique. We analysed changes in mRNA expression of Activating transcription factor 3 (ATF3), interleukin 6 (IL6), interleukin 8 (IL8), C-X-C motif chemokine ligand 5 (CxCl5), extracellular signal-regulated kinase 1/2 (Erk1/2), phospholemman (FXYD1), dysadherin (FXYD5), Nuclear receptor of the subfamily 4 group A 3 (Nra4a3), Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Ppargc1-α), Secreted protein acidic and rich in cysteine (SPARC), sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1 (SERCA1) and vascular endothelial growth factor A (VEGFA). Basing on gene expression, we selected some proteins to evaluate their concentration in the medium in relationship to EPS stimuli. During contraction, myotubes release specific proteins that could act in an autocrine, paracrine and endocrine way to regulate different intracellular processes. Protein secretion depends on the expression of the mRNA. The release of these proteins was analysed firstly using the Bradford assay that permits to show the concentration of all proteins in the medium. Then, ELISA assay was used to evaluate if EPS may modify IL6 secretion. Since myoblast differentiation create a disorganized array of myotubes we engineered myoblast seeding to allow the production of oriented myotubes. To this purpose, cells were plated on a specific pattern of extracellular proteins created through or the oxygen etching or microcontact printing techniques. This last technique is used to print a specific protein of interest (an adhesive protein) on a substrate. The adhesive protein used for this work were the Poly-L-Lysine and the Gelatine. C2C12 cells were grown according to the shape created with a specific stamp. We created stamps permitting to obtain a pattern of lines of proteins where the cells were able to attach, grow and differentiate. Oxygen etching allows the construction of the same pattern described for the microcontact printing. In this work it was optimized pattering techniques to obtain a good protein deposition that allows cell organization and differentiation in oriented myofibers. Moreover, we analysed the advantages and the disadvantages of these two methods used to produce patterned proteins.
EPS
EXPRESSION
Myotubes
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12608/11463