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Gene expression profiles in skeletal muscle after gene electrotransfer.

ABSTRACT: BACKGROUND: 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. DNA electrotransfer was obtained using a combination of a short high voltage pulse (HV, 1000 V/cm, 100 mus) 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 enzymes. 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 HV+LV pulses alone showed preservation of cell integrity. No difference in the force generation capacity was observed in the muscles 2 weeks after DNA electrotransfer. CONCLUSION: The small and transient changes found in the gene expression profiles are of great importance, as this demonstrates that DNA electrotransfer is safe with minor effects on the muscle host cells. These findings are essential for introducing the DNA electrotransfer to muscle for clinical use. Indeed the HV+LV pulse combination used has been optimised to ensure highly efficient and safe DNA electrotransfer.

SUBMITTER: Hojman P

PROVIDER: S-EPMC1925113 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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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:BACKGROUND: Beta-adrenergic receptor agonists (BA) induce skeletal muscle hypertrophy, yet specific mechanisms that lead to this effect are not well understood. The objective of this research was to identify novel genes and physiological pathways that potentially facilitate BA induced skeletal muscle growth. The Affymetrix platform was utilized to identify gene expression changes in mouse skeletal muscle 24 hours and 10 days after administration of the BA clenbuterol. RESULTS: Administration of clenbuterol stimulated anabolic activity, as indicated by decreased blood urea nitrogen (BUN; P < 0.01) and increased body weight gain (P < 0.05) 24 hours or 10 days, respectively, after initiation of clenbuterol treatment. A total of 22,605 probesets were evaluated with 52 probesets defined as differentially expressed based on a false discovery rate of 10%. Differential mRNA abundance of four of these genes was validated in an independent experiment by quantitative PCR. Functional characterization of differentially expressed genes revealed several categories that participate in biological processes important to skeletal muscle growth, including regulators of transcription and translation, mediators of cell-signalling pathways, and genes involved in polyamine metabolism. CONCLUSION: Global evaluation of gene expression after administration of clenbuterol identified changes in gene expression and overrepresented functional categories of genes that may regulate BA-induced muscle hypertrophy. Changes in mRNA abundance of multiple genes associated with myogenic differentiation may indicate an important effect of BA on proliferation, differentiation, and/or recruitment of satellite cells into muscle fibers to promote muscle hypertrophy. Increased mRNA abundance of genes involved in the initiation of translation suggests that increased levels of protein synthesis often associated with BA administration may result from a general up-regulation of translational initiators. Additionally, numerous other genes and physiological pathways were identified that will be important targets for further investigations of the hypertrophic effect of BA on skeletal muscle.

Project description:BackgroundIn livestock populations the genetic contribution to muscling is intensively monitored in the progeny of industry sires and used as a tool in selective breeding programs. The genes and pathways conferring this genetic merit are largely undefined. Genetic variation within a population has potential, amongst other mechanisms, to alter gene expression via cis- or trans-acting mechanisms in a manner that impacts the functional activities of specific pathways that contribute to muscling traits. By integrating sire-based genetic merit information for a muscling trait with progeny-based gene expression data we directly tested the hypothesis that there is genetic structure in the gene expression program in ovine skeletal muscle.ResultsThe genetic performance of six sires for a well defined muscling trait, longissimus lumborum muscle depth, was measured using extensive progeny testing and expressed as an Estimated Breeding Value by comparison with contemporary sires. Microarray gene expression data were obtained for longissimus lumborum samples taken from forty progeny of the six sires (4-8 progeny/sire). Initial unsupervised hierarchical clustering analysis revealed strong genetic architecture to the gene expression data, which also discriminated the sire-based Estimated Breeding Value for the trait. An integrated systems biology approach was then used to identify the major functional pathways contributing to the genetics of enhanced muscling by using both Estimated Breeding Value weighted gene co-expression network analysis and a differential gene co-expression network analysis. The modules of genes revealed by these analyses were enriched for a number of functional terms summarised as muscle sarcomere organisation and development, protein catabolism (proteosome), RNA processing, mitochondrial function and transcriptional regulation.ConclusionsThis study has revealed strong genetic structure in the gene expression program within ovine longissimus lumborum muscle. The balance between muscle protein synthesis, at the levels of both transcription and translation control, and protein catabolism mediated by regulated proteolysis is likely to be the primary determinant of the genetic merit for the muscling trait in this sheep population. There is also evidence that high genetic merit for muscling is associated with a fibre type shift toward fast glycolytic fibres. This study provides insight into mechanisms, presumably subject to strong artificial selection, that underpin enhanced muscling in sheep populations.

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Gene expression profiles in skeletal muscle after gene electrotransfer.

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Gene expression profiles in skeletal muscle after gene electrotransfer.

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