BACKGROUND
in vivo gene transfer to skeletal muscle is a promising strategy for treatment of muscular disorders and for systemic delivery of therapeutic proteins.
transgene expression has been reported following intramuscular injection of naked plasmid dna  <cit> , yet the clinical use is limited due to low efficiency and large variation.
by combining intramuscular plasmid injection with local application of electric pulses a 200-fold increase in transfection efficiency with high reproducibility has been achieved in vivo  <cit> .
this proves that dna electrotransfer can be an efficient and feasible way of introducing genes into tissues.
dna electrotransfer allows high production of systemic delivered proteins e.g.
erythropoietin  <cit>  and cytokines  <cit>  with expression detected more than a year after treatment in rodents  <cit> .
the high level of gene expression in muscle after dna electrotransfer is a consequence of plasmid distribution, membrane permeabilisation and plasmid electrophoresis  <cit> .
recently efforts have been made to optimise the electric pulses for gene transfer, resulting in a combination of a short high voltage  pulse for membrane permeabilisation, followed by a long low voltage  pulse for electrophoretic transport of plasmid towards the cell membrane.
in skeletal muscle and skin the hv + lv pulse combination has led to increased marker gene expression  <cit> .
dna electrotransfer is moving rapidly towards clinical use.
in fact, electroporation in combination with chemotherapy  has been used in the clinic for several years now  <cit> , showing encouraging results for anti-tumour treatment with good tolerability for the patients  <cit> .
however the biological response to electroporation and dna electrotransfer in vivo remains poorly defined.
rubenstrunk et al studied the expression of stress related genes after dna electrotransfer, and found no significant variation between treated and non-treated muscles  <cit> .
furthermore, studies have shown that transient changes in force generation, muscular ion content and atp levels were observed following dna electrotransfer  <cit> .
yet no thorough description of the transcriptional changes caused by dna electrotransfer has been reported.
in this first comprehensive microarray analysis covering the entire murine genome, we examine the transcriptional changes underlying the muscular response to dna electrotransfer by evaluating the gene expression profiles of mice tibialis cranialis muscles  <dig> hrs,  <dig> hrs and  <dig> weeks after dna electrotransfer using oligonucleotide microarrays and quantitative pcr .
furthermore we performed histology and physiological tests such as force generation measurements and reflex and motor testing to support our findings.
RESULTS
the effect of dna electrotransfer on gene expression profiles
for precise description of the transcriptional changes following dna electrotransfer, total rna was extracted and pooled from  <dig> muscles excised  <dig> hrs,  <dig> hrs and  <dig> weeks after treatment and gene expression profile analysis was performed.
statistical analysis was employed by comparing electroporated  and non-ep groups, groups either injected or not with plasmid, or groups at the different time points.
inferential statistics
by two-way anova testing with a significance level of p =  <dig> ,  <dig> genes were found to be differentially expressed between ep and non-ep groups,  <dig> genes were differentially expressed in groups with or without intramuscular plasmid injection, and  <dig> genes were differentially expressed across time.
to further filter the gene lists, unpaired t-test with  <dig> permutations accepting a false discovery rate  less than 5% and significance levels of p =  <dig>  were performed.
in the ep vs. non-ep groups, the unpaired t-test revealed that  <dig> of the  <dig> genes were significantly differentially expressed  of which  <dig> were up-regulated and  <dig> down-regulated.
unpaired t-test among the groups injected with plasmid or not, revealed that  <dig> genes were significantly differentially expressed  with  <dig> genes up-regulated and  <dig> down-regulated.
unpaired t-test among genes differing across time resulted in  <dig> genes with a false discovery rate of  <dig> %, therefore the significance level was changed to p ≤  <dig> , resulting in  <dig> differentially expressed genes and a fdr of 0%.
descriptive statistics and unsupervised clustering
electroporated versus non-electroporated muscles
in an unsupervised cluster analysis, the ep groups clustered significantly  together as depicted from the sample correlation matrix plot in fig.
<dig>  no correlation within the ep or non-ep clusters, as to whether the groups had received plasmid injection or at which time following treatment the muscles had been excised, was established .
performing gene ontology annotation  on the cluster analysis revealed that rna encoding proteins assembling the cytoskeleton e.g.
microtubule-associated protein  <dig> , cadherin-like  <dig>  and sarcospan  were down-regulated in the electroporated groups.
yet,  <dig> weeks after treatment the expression of these genes in the dna+hv+lv group had returned to control levels .
furthermore expression of genes involved in the metabolism e.g.
phosphoenolpuryvate carboxykinase  and dipeptidase  were also down-regulated following electroporation, indicating that the general catabolism might be depressed.
ephrin , which is involved in neural development and cell-cell interaction among vascular endothelial cells were found to be up-regulated in all ep groups.
plasmid vs. no plasmid injection
the plasmid injected groups clustered significantly together  in the sample correlation matrix analysis with clear separation from the non-injected groups .
interestingly the lda plot revealed that within the plasmid injected groups, two apparent sub-clusters of ep and non-ep groups were found.
likewise among the non-injected groups, the ep groups formed a distinct sub-cluster .
go annotation showed that the expression of genes involved in intracellular trafficking e.g.
sentrin , nuclear transport factor  <dig>  and adp-ribosylation factor-like 10c  were down-regulated in the plasmid injected groups.
in addition polymerase ii  was also down-regulated in plasmid injected groups, indicating the transcription also was depressed.
furthermore the er ca2+ binding protein reticulocalbin  and ca2+ release modulator fk <dig> binding protein  were both down-regulated following plasmid injection, which might be important for controlling the ca2+ homeostasis.
indeed we have previously shown that dna injection especially in combination with electroporation affects the intracellular level of ca2+ .
time
groups within each of the  <dig> time points clustered significantly together  regardless of the treatment.
the lda plot revealed that groups from  <dig> and  <dig> hrs clustered closely together, while the control and  <dig> weeks groups formed individual clusters .
at  <dig> hrs the chloride channel ka  is up-regulated in all groups compared to all other time point.
at this initial time point after treatment and membrane permeabilisation, the ion homeostasis across the membrane might still be disturbed, stimulating the expression of the cl-channel.
at  <dig> hrs and in the egt-treated group at  <dig> hrs, the transducer of erbb <dig> , was up-regulated.
up-regulation of this inhibitor of proliferation might indicate disturbances in the muscles, which limit the proliferation potential transiently.
compared to the control groups, the cl- channel activator kruppel-like factor  <dig>  and the k+ voltage-gated channel  were down-regulated at  <dig> hrs,  <dig> hrs and  <dig> weeks, indicating the ion homeostasis might be disturbed longer than the initial phase.
similarly the solute carrier family  <dig> protein , which transports creatine into the muscle cells and is essential for energy consumption, was also down-regulated at all time points compared to the control groups.
at  <dig> and  <dig> hrs the transcription activator host cell factor c <dig> , the regulator of polymerase ii ccr4-not transcription complex  and the polyadenylation element binding protein  were down-regulated compared to the control and  <dig> weeks groups; indicating that the protein synthesis apparatus might be negatively affected within the first  <dig> hrs after treatment.
q-pcr validation of differentially expressed genes
the gene expression levels were validated by q-pcr for  <dig> selected genes .
these measurements confirmed the level of mrna determined by microarray analysis.
for example transducer of erbb <dig>  was in the array analysis found to be up-regulated at  <dig> hours after all treatments, indeed q-pcr also showed that the transducer of erbb <dig> was up-regulated at  <dig> hours e.g.
in the case of dna+hv+lv treatment by 6-fold.
similarly the expression of the solute carrier family  <dig> , responsible for creatine transport, was verified by q-pcr to be down-regulated at  <dig> and  <dig> hours.
eleven genes were selected for q-pcr validation of their expression level.
the genes were normalised to β-microglobulin and down and up-regulation in expression compared to the control muscles were determined.
histological changes in tibialis cranialis muscles after dna electrotransfer
preparations of muscles slides were stained with he and examined for morphological changes like integrity of fibres, central nuclei and fainting striation pattern.
furthermore, cd <dig> and cd <dig> staining were performed on parallel slides to verify infiltration of mononuclear cells.
at  <dig> hrs, no histological changes were observed for the control, dna injected and electroporated muscles .
contrarily after dna+hv+lv treatment, loss of fibre structure and fainting striation pattern was observed in 15% of the muscle.
after  <dig> hrs, no changes were observed in the control and dna injected muscles, while the electroporated muscles exhibited fainting striation pattern in a few muscle fibres.
in the dna+hv+lv treated muscles, structural changes with loss of cell integrity and striation pattern were observed in 15% of the fibres, in addition limited intra-fiber infiltration of mononuclear cells was also observed.
three weeks after treatment, the dna+hv+lv treated muscles had around 5% of the fibres containing central nuclei, indicating regenerating myocytes.
in the electroporated muscles single cells with central nuclei were also detectable, while the control and dna injected muscles showed no signs of histological changes.
no loss in muscle performance after dna electrotransfer
the reflex and motor function of mice were tested by assessing the running and seizing patterns.
thus, mice were allowed to run on the top of the cage, their tails were gently manually lifted, and it was observed whether they could grasp the bars with normal force.
similarly, spontaneous motoric movement was observed in the cage.
fig.
<dig> depicts the average score, short duration films of the test results can be found in additional file  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  at  <dig> hours the mice had not fully awoken from the anaesthesia and could therefore not be tested.
at  <dig> hours no effects were observed in the control and dna injected mice, while slight impairments were observed in a couple of the electroporated and dna+hv+lv treated mice.
from  <dig> days after treatment no functional impairments in the seizing and running patterns were observed in any of the mice.
the effects of dna electrotransfer on the force generating capacity were tested in rat edl muscles  <dig> weeks after treatment and depicted in fig.
<dig>  rat edl muscles were chosen for this measurements as mice ta cannot be used in our set-up.
the force generating capacity in the dna electrotransferred and electroporated muscles did not differ significantly from the control muscles.
discussion
dna electrotransfer to skeletal muscle is highly efficient and holds great promise for future treatment of monogenetic disorders, protein deficiency syndromes, cancers, etc.
for electric pulsing of muscle in vivo trains of long low field strength pulses have typically been employed  <cit> .
however studies have shown that the electric pulse-mediated gene transfer might consist of two components; a component, which permeabilises the membrane, and an electrophoretic component, which can facilitate the transport of plasmid dna across the membrane  <cit> .
using combination pulses, a short high voltage permeabilising pulse followed by a long low voltage electrophoretic pulse, less intense pulsing parameters are needed for obtaining the same expression levels, which reduces the discomfort from the pulsing regiment.
indeed in this study we confirm these observations as little changes in the gene expression profiles are observed.
moreover we find less histological changes than previously described after dna electrotransfer with trains of long, low field strength pulses  <cit> .
in addition we find no reductions in muscle performance  <dig> weeks after treatment, indicating that no major muscle regeneration is required after dna electrotransfer.
array analysis, clustering and time aspect
due to the expenses of the study, the replica number was low in this study, however this was accommodated by designing our statistical analysis as a multiple factor analysis.
during the inferential statistics we performed an anova test, which gives an efficient and comprehensive evaluation despite the low replicate number in this set-up  <cit> .
moreover, permutation test and unpaired t-tests were performed using stringent level of significance to correctly identify genes that were differentially expressed.
furthermore extensive descriptive statistics were performed, which supported the findings of differentially expressed genes.
unsupervised cluster analyses showed clear separations between ep and non-ep muscles and dna injected or non-injected muscles, indicating that the differentially transcribed genes in these clusters were highly characteristic for the given treatment.
indeed some of the changes we observed were in line with previous reported changes following dna electrotransfer to cells.
not surprisingly the gene expression profiles at  <dig> and  <dig> hrs were relatively comparable, while the gene expression profiles after  <dig> weeks were more similar to the control muscles.
this indicates that the observed changes after dna electrotransfer are transient, followed by a return to the naïve state of the muscle.
the most striking point about the expression profiles is that no genes involved in stress, immune responses, cell death or muscle regeneration were found to be differentially expressed, demonstrating that egt is non-toxic.
indeed guided supervised cluster analysis looking at cell homeostasis, ion homeostasis and immune response ontologies showed no significant changes in the gene expression profiles.
dna electrotransfer-induced transcriptional changes
based on previous in vitro studies certain effects of electroporation and plasmid injection would have been expected.
electroporation of cells is known to cause cytoskeleton rearrangements and electro-conformational changes of ion channels, while dna injection is associated in cytotoxic effects on the cells.
changes in cytoskeleton-associated proteins
during the application of an electric field across cells, electro-compressive forces exert pressure on the membrane leading to elongation of cells.
during this elongation phase the pressure on the membrane enlarges, resulting in the development of membrane destabilisations, which allow for entry of plasmids  <cit> .
formation of microvilli and blebs associated with deformation of the cytoskeleton has been observed immediately after pulsing  <cit> .
in fact, the same phenomenon is observed in cyclic stretch-induced gene transfer, where cytoskeleton rearrangements are involved in mediating the gene transfer  <cit> .
in this study we found down-regulation of cytoskeleton proteins after electro-pulsing, corresponding to the reported deformations of the cytoskeleton after pulsing.
interestingly, studies have shown that muscle contractile function returns to normal within  <dig> hours after egt with the hv+lv pulse combination , indicating that the cytoskeleton rearrangements only have a minor effect on the muscle function.
electro-conformational changes of ion channels
during electro-pulsing, voltage-dependent ion channels are particularly sensitive to the external electric field due to their intrinsic voltage sensors.
high intensity electric fields cause structural changes, also known as electro-conformational changes, which especially affect the moveable charge particles in the channel  <cit> .
chen et al have shown that the movement of charge particles in the delayed rectifier k+ channels was reduced after applying an external transmembrane potential of  <dig> mv  <cit> .
this applied membrane potential is considerably larger than the  <dig> –  <dig> mv required for lipid bilayer breakdown.
at lower than  <dig> mv field intensities no current changes demonstrating conformational changes were observed  <cit> .
in our study we found down-regulation of the k+ voltage-gated channel.
although our applied electric field does not reach amplitude of  <dig> mv at the membrane level, weaker field strengths might be sufficient to induce electro-conformational changes of ion channels in muscle fibres, as these cells are larger.
this suggests that electro-conformational changes might play a role in the regulation of voltage-gated channels after dna electrotransfer in vivo.
effects of plasmid injection
intramuscular injection of plasmid can be associated with toxicity in the target tissue  <cit> .
hartikka et al  observed that muscle damage after dna electrotransfer with super-coiled endotoxin-free plasmid was dna dependent.
when introducing  <dig> μg of plasmid into the quadriceps muscle, they observed lesions with necrotic myofibres and infiltration of inflammatory cells.
after injection of vehicle and electric pulses delivery no such lesions were observed.
we have used a low concentration of plasmid, which might explain why we observed little infiltration at our histological evaluation and no effects on the transcriptional level.
intracellular plasmid dna transport
unexpectedly we found that proteins involved in intracellular transport were down-regulated following dna injection.
after entry into muscle fibres plasmid dna must be transported to the nuclei for transcription, but as the cytoplasmic latticework is quite condense, it is unlikely that this transport occur by simple diffusion  <cit> .
in hela and cos cells it has been shown that plasmid dna has a limited half-life  in the cytosol due to degradation by cytoplasmic nucleases  <cit> , meaning that efficient transport to the nucleus is required.
in fact studies have shown that the cytoplasmic movement of dna towards the nucleus in cells is facilitated by the microtubular network  <cit> .
the down-regulated intracellular transport proteins are not directly involved in these transport pathways, but they belong to a group of proteins, which might be a limiting factor in efficient gene transfer.
this could therefore be of importance for the transfection efficacy.
CONCLUSIONS
dna electrotransfer by hv+lv pulses demonstrates small and transient adverse effects on the muscle.
this is highly important for introduction in the clinical setting, where numerous applications of this technology are envisaged.
examples of interesting pre-clinical experiments with gene transfer to muscle tissue include correction of anaemia  <cit>  or β-thalassemia  <cit>  by electrotransfer of epo.
furthermore dna electrotransfer can also be used to target the muscle itself, either for research purposes or for correction of muscle anomalies e.g.
muscular dystrophies.
in this regard, it is important that the gene transfer procedure itself imposes minimal effects on the target tissue.
in conclusion dna electrotransfer using the hv+lv pulse combination is safe with only small changes in the expression of cytoskeletal and intracellular transport proteins, while most importantly no detrimental changes in muscle performance or gene expression profiles of proteins involved in cell death, inflammation or muscle regeneration was verifiable both shortly and long after treatment.
histology did, however, revealed some cells with morphological changes after  <dig> and  <dig> hrs and few cells containing central nuclei after  <dig> weeks.

 in vivo gene transfer to skeletal muscle is a promising strategy for treatment of muscular disorders and for systemic delivery of therapeutic proteins. transgene expression has been reported following intramuscular injection of naked plasmid dna, yet the clinical use is limited due to low efficiency and large variation. by combining intramuscular plasmid injection with local application of electric pulses a 200-fold increase in transfection efficiency with high reproducibility has been achieved in vivo. this proves that dna electrotransfer can be an efficient and feasible way of introducing genes into tissues. dna electrotransfer allows high production of systemic delivered proteins e.g. erythropoietin and cytokines with expression detected more than a year after treatment in rodents. the high level of gene expression in muscle after dna electrotransfer is a consequence of plasmid distribution, membrane permeabilisation and plasmid electrophoresis. recently efforts have been made to optimise the electric pulses for gene transfer, resulting in a combination of a short high voltage pulse for membrane permeabilisation, followed by a long low voltage pulse for electrophoretic transport of plasmid towards the cell membrane. in skeletal muscle and skin the hv + lv pulse combination has led to increased marker gene expression. dna electrotransfer is moving rapidly towards clinical use. in fact, electroporation in combination with chemotherapy has been used in the clinic for several years now, showing encouraging results for anti-tumour treatment with good tolerability for the patients. however the biological response to electroporation and dna electrotransfer in vivo remains poorly defined. rubenstrunk et al studied the expression of stress related genes after dna electrotransfer, and found no significant variation between treated and non-treated muscles. furthermore, studies have shown that transient changes in force generation, muscular ion content and atp levels were observed following dna electrotransfer. yet no thorough description of the transcriptional changes caused by dna electrotransfer has been reported. in this first comprehensive microarray analysis covering the entire murine genome, we examine the transcriptional changes underlying the muscular response to dna electrotransfer by evaluating the gene expression profiles of mice tibialis cranialis muscles hrs, hrs and weeks after dna electrotransfer using oligonucleotide microarrays and quantitative pcr. furthermore we performed histology and physiological tests such as force generation measurements and reflex and motor testing to support our findings.  the effect of dna electrotransfer on gene expression profiles for precise description of the transcriptional changes following dna electrotransfer, total rna was extracted and pooled from muscles excised hrs, hrs and weeks after treatment and gene expression profile analysis was performed. statistical analysis was employed by comparing electroporated and non-ep groups, groups either injected or not with plasmid, or groups at the different time points. inferential statistics by two-way anova testing with a significance level of p =, genes were found to be differentially expressed between ep and non-ep groups, genes were differentially expressed in groups with or without intramuscular plasmid injection, and genes were differentially expressed across time. to further filter the gene lists, unpaired t-test with permutations accepting a false discovery rate less than 5% and significance levels of p = were performed. in the ep vs. non-ep groups, the unpaired t-test revealed that of the genes were significantly differentially expressed of which were up-regulated and down-regulated. unpaired t-test among the groups injected with plasmid or not, revealed that genes were significantly differentially expressed with genes up-regulated and down-regulated. unpaired t-test among genes differing across time resulted in genes with a false discovery rate of %, therefore the significance level was changed to p ≤, resulting in differentially expressed genes and a fdr of 0%. descriptive statistics and unsupervised clustering electroporated versus non-electroporated muscles in an unsupervised cluster analysis, the ep groups clustered significantly together as depicted from the sample correlation matrix plot in fig. no correlation within the ep or non-ep clusters, as to whether the groups had received plasmid injection or at which time following treatment the muscles had been excised, was established. performing gene ontology annotation on the cluster analysis revealed that rna encoding proteins assembling the cytoskeleton e.g. microtubule-associated protein, cadherin-like and sarcospan were down-regulated in the electroporated groups. yet, weeks after treatment the expression of these genes in the dna+hv+lv group had returned to control levels. furthermore expression of genes involved in the metabolism e.g. phosphoenolpuryvate carboxykinase and dipeptidase were also down-regulated following electroporation, indicating that the general catabolism might be depressed. ephrin, which is involved in neural development and cell-cell interaction among vascular endothelial cells were found to be up-regulated in all ep groups. plasmid vs. no plasmid injection the plasmid injected groups clustered significantly together in the sample correlation matrix analysis with clear separation from the non-injected groups. interestingly the lda plot revealed that within the plasmid injected groups, two apparent sub-clusters of ep and non-ep groups were found. likewise among the non-injected groups, the ep groups formed a distinct sub-cluster. go annotation showed that the expression of genes involved in intracellular trafficking e.g. sentrin, nuclear transport factor and adp-ribosylation factor-like 10c were down-regulated in the plasmid injected groups. in addition polymerase ii was also down-regulated in plasmid injected groups, indicating the transcription also was depressed. furthermore the er ca2+ binding protein reticulocalbin and ca2+ release modulator fk binding protein were both down-regulated following plasmid injection, which might be important for controlling the ca2+ homeostasis. indeed we have previously shown that dna injection especially in combination with electroporation affects the intracellular level of ca2+. time groups within each of the time points clustered significantly together regardless of the treatment. the lda plot revealed that groups from and hrs clustered closely together, while the control and weeks groups formed individual clusters. at hrs the chloride channel ka is up-regulated in all groups compared to all other time point. at this initial time point after treatment and membrane permeabilisation, the ion homeostasis across the membrane might still be disturbed, stimulating the expression of the cl-channel. at hrs and in the egt-treated group at hrs, the transducer of erbb, was up-regulated. up-regulation of this inhibitor of proliferation might indicate disturbances in the muscles, which limit the proliferation potential transiently. compared to the control groups, the cl- channel activator kruppel-like factor and the k+ voltage-gated channel were down-regulated at hrs, hrs and weeks, indicating the ion homeostasis might be disturbed longer than the initial phase. similarly the solute carrier family protein, which transports creatine into the muscle cells and is essential for energy consumption, was also down-regulated at all time points compared to the control groups. at and hrs the transcription activator host cell factor c, the regulator of polymerase ii ccr4-not transcription complex and the polyadenylation element binding protein were down-regulated compared to the control and weeks groups; indicating that the protein synthesis apparatus might be negatively affected within the first hrs after treatment. q-pcr validation of differentially expressed genes the gene expression levels were validated by q-pcr for selected genes. these measurements confirmed the level of mrna determined by microarray analysis. for example transducer of erbb was in the array analysis found to be up-regulated at hours after all treatments, indeed q-pcr also showed that the transducer of erbb was up-regulated at hours e.g. in the case of dna+hv+lv treatment by 6-fold. similarly the expression of the solute carrier family, responsible for creatine transport, was verified by q-pcr to be down-regulated at and hours. eleven genes were selected for q-pcr validation of their expression level. the genes were normalised to β-microglobulin and down and up-regulation in expression compared to the control muscles were determined. histological changes in tibialis cranialis muscles after dna electrotransfer preparations of muscles slides were stained with he and examined for morphological changes like integrity of fibres, central nuclei and fainting striation pattern. furthermore, cd and cd staining were performed on parallel slides to verify infiltration of mononuclear cells. at hrs, no histological changes were observed for the control, dna injected and electroporated muscles. contrarily after dna+hv+lv treatment, loss of fibre structure and fainting striation pattern was observed in 15% of the muscle. after hrs, no changes were observed in the control and dna injected muscles, while the electroporated muscles exhibited fainting striation pattern in a few muscle fibres. in the dna+hv+lv treated muscles, structural changes with loss of cell integrity and striation pattern were observed in 15% of the fibres, in addition limited intra-fiber infiltration of mononuclear cells was also observed. three weeks after treatment, the dna+hv+lv treated muscles had around 5% of the fibres containing central nuclei, indicating regenerating myocytes. in the electroporated muscles single cells with central nuclei were also detectable, while the control and dna injected muscles showed no signs of histological changes. no loss in muscle performance after dna electrotransfer the reflex and motor function of mice were tested by assessing the running and seizing patterns. thus, mice were allowed to run on the top of the cage, their tails were gently manually lifted, and it was observed whether they could grasp the bars with normal force. similarly, spontaneous motoric movement was observed in the cage. fig. depicts the average score, short duration films of the test results can be found in additional file at hours the mice had not fully awoken from the anaesthesia and could therefore not be tested. at hours no effects were observed in the control and dna injected mice, while slight impairments were observed in a couple of the electroporated and dna+hv+lv treated mice. from days after treatment no functional impairments in the seizing and running patterns were observed in any of the mice. the effects of dna electrotransfer on the force generating capacity were tested in rat edl muscles weeks after treatment and depicted in fig. rat edl muscles were chosen for this measurements as mice ta cannot be used in our set-up. the force generating capacity in the dna electrotransferred and electroporated muscles did not differ significantly from the control muscles. discussion dna electrotransfer to skeletal muscle is highly efficient and holds great promise for future treatment of monogenetic disorders, protein deficiency syndromes, cancers, etc. for electric pulsing of muscle in vivo trains of long low field strength pulses have typically been employed. however studies have shown that the electric pulse-mediated gene transfer might consist of two components; a component, which permeabilises the membrane, and an electrophoretic component, which can facilitate the transport of plasmid dna across the membrane. using combination pulses, a short high voltage permeabilising pulse followed by a long low voltage electrophoretic pulse, less intense pulsing parameters are needed for obtaining the same expression levels, which reduces the discomfort from the pulsing regiment. indeed in this study we confirm these observations as little changes in the gene expression profiles are observed. moreover we find less histological changes than previously described after dna electrotransfer with trains of long, low field strength pulses. in addition we find no reductions in muscle performance weeks after treatment, indicating that no major muscle regeneration is required after dna electrotransfer. array analysis, clustering and time aspect due to the expenses of the study, the replica number was low in this study, however this was accommodated by designing our statistical analysis as a multiple factor analysis. during the inferential statistics we performed an anova test, which gives an efficient and comprehensive evaluation despite the low replicate number in this set-up. moreover, permutation test and unpaired t-tests were performed using stringent level of significance to correctly identify genes that were differentially expressed. furthermore extensive descriptive statistics were performed, which supported the findings of differentially expressed genes. unsupervised cluster analyses showed clear separations between ep and non-ep muscles and dna injected or non-injected muscles, indicating that the differentially transcribed genes in these clusters were highly characteristic for the given treatment. indeed some of the changes we observed were in line with previous reported changes following dna electrotransfer to cells. not surprisingly the gene expression profiles at and hrs were relatively comparable, while the gene expression profiles after weeks were more similar to the control muscles. this indicates that the observed changes after dna electrotransfer are transient, followed by a return to the naïve state of the muscle. the most striking point about the expression profiles is that no genes involved in stress, immune responses, cell death or muscle regeneration were found to be differentially expressed, demonstrating that egt is non-toxic. indeed guided supervised cluster analysis looking at cell homeostasis, ion homeostasis and immune response ontologies showed no significant changes in the gene expression profiles. dna electrotransfer-induced transcriptional changes based on previous in vitro studies certain effects of electroporation and plasmid injection would have been expected. electroporation of cells is known to cause cytoskeleton rearrangements and electro-conformational changes of ion channels, while dna injection is associated in cytotoxic effects on the cells. changes in cytoskeleton-associated proteins during the application of an electric field across cells, electro-compressive forces exert pressure on the membrane leading to elongation of cells. during this elongation phase the pressure on the membrane enlarges, resulting in the development of membrane destabilisations, which allow for entry of plasmids. formation of microvilli and blebs associated with deformation of the cytoskeleton has been observed immediately after pulsing. in fact, the same phenomenon is observed in cyclic stretch-induced gene transfer, where cytoskeleton rearrangements are involved in mediating the gene transfer. in this study we found down-regulation of cytoskeleton proteins after electro-pulsing, corresponding to the reported deformations of the cytoskeleton after pulsing. interestingly, studies have shown that muscle contractile function returns to normal within hours after egt with the hv+lv pulse combination, indicating that the cytoskeleton rearrangements only have a minor effect on the muscle function. electro-conformational changes of ion channels during electro-pulsing, voltage-dependent ion channels are particularly sensitive to the external electric field due to their intrinsic voltage sensors. high intensity electric fields cause structural changes, also known as electro-conformational changes, which especially affect the moveable charge particles in the channel. chen et al have shown that the movement of charge particles in the delayed rectifier k+ channels was reduced after applying an external transmembrane potential of mv. this applied membrane potential is considerably larger than the – mv required for lipid bilayer breakdown. at lower than mv field intensities no current changes demonstrating conformational changes were observed. in our study we found down-regulation of the k+ voltage-gated channel. although our applied electric field does not reach amplitude of mv at the membrane level, weaker field strengths might be sufficient to induce electro-conformational changes of ion channels in muscle fibres, as these cells are larger. this suggests that electro-conformational changes might play a role in the regulation of voltage-gated channels after dna electrotransfer in vivo. effects of plasmid injection intramuscular injection of plasmid can be associated with toxicity in the target tissue. hartikka et al observed that muscle damage after dna electrotransfer with super-coiled endotoxin-free plasmid was dna dependent. when introducing μg of plasmid into the quadriceps muscle, they observed lesions with necrotic myofibres and infiltration of inflammatory cells. after injection of vehicle and electric pulses delivery no such lesions were observed. we have used a low concentration of plasmid, which might explain why we observed little infiltration at our histological evaluation and no effects on the transcriptional level. intracellular plasmid dna transport unexpectedly we found that proteins involved in intracellular transport were down-regulated following dna injection. after entry into muscle fibres plasmid dna must be transported to the nuclei for transcription, but as the cytoplasmic latticework is quite condense, it is unlikely that this transport occur by simple diffusion. in hela and cos cells it has been shown that plasmid dna has a limited half-life in the cytosol due to degradation by cytoplasmic nucleases, meaning that efficient transport to the nucleus is required. in fact studies have shown that the cytoplasmic movement of dna towards the nucleus in cells is facilitated by the microtubular network. the down-regulated intracellular transport proteins are not directly involved in these transport pathways, but they belong to a group of proteins, which might be a limiting factor in efficient gene transfer. this could therefore be of importance for the transfection efficacy.  dna electrotransfer by hv+lv pulses demonstrates small and transient adverse effects on the muscle. this is highly important for introduction in the clinical setting, where numerous applications of this technology are envisaged. examples of interesting pre-clinical experiments with gene transfer to muscle tissue include correction of anaemia or β-thalassemia by electrotransfer of epo. furthermore dna electrotransfer can also be used to target the muscle itself, either for research purposes or for correction of muscle anomalies e.g. muscular dystrophies. in this regard, it is important that the gene transfer procedure itself imposes minimal effects on the target tissue. in conclusion dna electrotransfer using the hv+lv pulse combination is safe with only small changes in the expression of cytoskeletal and intracellular transport proteins, while most importantly no detrimental changes in muscle performance or gene expression profiles of proteins involved in cell death, inflammation or muscle regeneration was verifiable both shortly and long after treatment. histology did, however, revealed some cells with morphological changes after and hrs and few cells containing central nuclei after weeks. 
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. gene transfer by electroporation to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. the biological significance of the results was assessed by gene annotation and supervised cluster analysis. 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 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. we have therefore investigated transcriptional changes through gene expression profile analyses, morphological changes by histological analysis, and physiological changes by force generation measurements. however little is known about the effects of dna electrotransfer on muscle fibres. dna electrotransfer was obtained using a combination of a short high voltage pulse followed by a long low voltage pulse; a pulse combination optimised for efficient and safe gene transfer. no difference in the force generation capacity was observed in the muscles weeks after dna electrotransfer. 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. the effects on muscle fibres were transient as the expression profiles 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.
 gene transfer by electroporation 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 followed by a long low voltage pulse; a pulse combination optimised for efficient and safe gene transfer. muscles were transfected with green fluorescent protein and excised at hours, hours or weeks after treatment.  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 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 weeks after dna electrotransfer.  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. 