Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse skeletal muscle after gene transfer by electroporation vs. Controls

ABSTRACT: Abstract; Background: Gene transfer by electroporation (electro gene transfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of electro gene transfer on muscle fibres. We have therefore investigated transcriptional changes through gene expression profile analyses, as well as morphological changes evaluated by histological analysis. Electro gene transfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 @s) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment. Results: Differentially expressed genes were investigated by microarray analysis, and descriptive statistics were performed to evaluate the effects of 1) electroporation, 2) DNA injection, and 3) time after treatment. The biological significance of the results was assessed by gene annotation and supervised cluster analysis. Generally, electroporation caused down-regulation of structural proteins e.g. sarcospan and catalytic en-zymes such as phosphoenolpuryvate carboxykinase. Injection of DNA induced down-regulation of intracellular transport proteins e.g. sentrin. The effects on muscle fibres were transient as the expression profiles 3 weeks after treatment were closely related with the control muscles. Most interestingly, no changes in the expression of proteins involved in inflammatory responses or muscle regeneration was detected, indicating limited muscle damage and regeneration. Histological analysis revealed structural changes with loss of cell integrity and striation pattern in some fibres after DNA+HV+LV treatment, while electroporation alone caused minor loss of striation pattern but preservation of cell integrity. Conclusion: The small and transient changes found in the gene expression profiles are of great importance, as this demonstrates that electro gene transfer is safe with minor effects on the muscle host cells. These findings are essential for introducing the electro gene transfer to muscle for clinical use. Indeed the HV+LV pulse combination used have been optimised to ensure highly efficient and safe electro gene transfer. Experiment Overall Design: The mice did recieve to their tibialis cranialis muscle either No tretment/control (CTRL), Electroporation only (EP), Plasmid injection only (DNA) or Plasmid DNA and in vivo Electro gene transfer (EP+DNA). Experiment Overall Design: Four hrs, 48 hrs and 3 weeks after treatment the mice were euthanized and the expression profile of the treated muscles were analysed. Experiment Overall Design: The following number of mice were included: Experiment Overall Design: CTRL, 3 mice, Experiment Overall Design: EP at 4 hrs, 1 mouse, Experiment Overall Design: EP at 48 hrs, 1 mouse, Experiment Overall Design: EP at 3 weeks, 1 mouse, Experiment Overall Design: DNA at 4 hrs, 1 mouse, Experiment Overall Design: DNA at 48 hrs, 1 mouse, Experiment Overall Design: DNA at 3 weeks, 1 mouse, Experiment Overall Design: EP+DNA at 4 hrs, 2 mice, Experiment Overall Design: EP+DNA at 48 hrs, 1 mouse, Experiment Overall Design: EP+DNA at 3 weeks, 1 mouse. Experiment Overall Design: A total number of mice 13.

ORGANISM(S): Mus musculus

SUBMITTER: John Zibert

PROVIDER: E-GEOD-6686 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Gene expression profiles in skeletal muscle after gene electrotransfer.

Hojman Pernille P Zibert John R JR Gissel Hanne H Eriksen Jens J Gehl Julie J

BMC molecular biology 20070629

<h4>Background</h4>Gene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have therefore investigated transcriptional changes through gene expression profile analyses, morphological changes by histological analysis, and physiological changes by force generation measurements. ...[more]

PMID: 17598924

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Project description:Aims/hypothesis: While lipid deposition in skeletal muscle is considered to be involved in obesity-associated insulin resistance, neutral intramyocellular lipid (IMCL) accumulation per se does not necessarily induce insulin resistance. We previously demonstrated that overexpression of the lipid droplet coat protein perilipin 2 augments intramyocellular lipid content while improving insulin sensitivity. Another member of the perilipin family, perilipin 5 (PLIN5), is predominantly expressed in oxidative tissues like skeletal muscle. Here we investigated the effects of PLIN5 overexpression M-bM-^@M-^S in comparison with effects of PLIN2 M-bM-^@M-^S on skeletal muscle lipid levels, gene expression profiles and insulin sensitivity. Methods: Gene electroporation was used to overexpress PLIN5 in tibialis anterior muscle of rats fed a high fat diet. Eight days after electroporation, insulin-mediated glucose uptake in skeletal muscle was measured by means of a hyperinsulinemic euglycemic clamp. Electron microscopy, fluorescence microscopy and lipid extractions were performed to investigate IMCL accumulation. Gene expression profiles were obtained using microarrays. Results: TAG storage and lipid droplet size increased upon PLIN5 overexpression. Despite the higher IMCL content, insulin sensitivity was not impaired and DAG and acylcarnitine levels were unaffected. In contrast to the effects of PLIN2 overexpression, microarray data analysis revealed a gene expression profile favoring FA oxidation and improved mitochondrial function. Conclusions/interpretation: Both PLIN2 and PLIN5 increase neutral IMCL content without impeding insulin-mediated glucose uptake. As opposed to the effects of PLIN2 overexpression, overexpression of PLIN5 in skeletal muscle promoted expression of a cluster of genes under control of PPARM-NM-1 and PGC1M-NM-1 involved in FA catabolism and mitochondrial oxidation. Rats received a high fat diet for 3 weeks; 2 weeks after start of the diet intervention Plin5 (OXPAT) or Plin2 (ADRP) were overexpressed in either the right or left tibialis anterior muscle. One week later pooled tibialis anterior muscle samples were analysed on microarrays.

Project description:Mice were subjected to 50 eccentric contractions (EC) or 50 isometric contractions (IC) using a non-invasive model, and then sacrificed 48 hours later. RNA from the tibialis anterior of 4 animals were pooled and then split into two groups for hybridization onto two separate Affymetrix MGU74Av2 chips. Control samples were contralateral to the exercised legs, and were only subjected to enough contractions to measure isometric torque. Eccentric contractions (ECs), in which a muscle is forced to lengthen while activated, result in muscle injury and, eventually, muscle strengthening and prevention of further injury. Although the mechanical basis of eccentric contraction-induced injury has been studied in detail, muscle's biological response is less well characterized. This study presents the development of a minimally-invasive model of EC injury in the mouse, follows the time course of torque recovery after an injurious bout of ECs, and uses Affymetrix microarrays to compare the gene expression profile 48 hours after ECs to both isometrically stimulated muscles and contralateral muscles. Torque dropped by about 55% immediately after the exercise bout, and recovered to initial levels 7 days later. 36 known genes were upregulated after ECs compared to contralateral and isometrically stimulated muscles, including five muscle specific genes: muscle LIM protein (MLP), Muscle Ankyrin Repeat Proteins (MARP 1 and 2; also known as cardiac ankyrin repeat protein and Arpp/Ankrd2, respectively), Xin, and Myosin Binding Protein H. The time courses of MLP and MARP expression after the injury bout (determined by quantitative real-time polymerase chain reaction) indicate that these genes are rapidly induced, reaching a peak expression level of 6-11 times contralateral values 12-24 hours after the EC bout and returning to baseline within 72 hours. Very little gene induction was seen after either isometric activation or passive stretch, indicating that the MLP and MARP genes may play an important and specific role in the biological response of muscle to EC-induced injury. Keywords = mouse tibialis anterior eccentric contraction muscle

Dataset Information

Transcription profiling of mouse skeletal muscle after gene transfer by electroporation vs. Controls

Publications

Gene expression profiles in skeletal muscle after gene electrotransfer.

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