Optimizing Plasmid-Based Gene Transfer for Investigating Skeletal Muscle Structure and Function

Department of Physiology, The University of Melbourne, Melbourne, VIC 3010, Australia.
Molecular Therapy (Impact Factor: 6.23). 05/2006; 13(4):795-803. DOI: 10.1016/j.ymthe.2005.09.019
Source: PubMed


Intramuscular injection of naked plasmid DNA is a less cytotoxic alternative to viral vectors for delivering genetic material to skeletal muscle in vivo. However, the low efficiency of plasmid-based gene transfer limits its potential therapeutic efficacy and/or its use for many experimental applications. Current strategies to enhance transfection efficiency (i.e., electroporation) can cause significant muscle damage, confounding physiological assessments such as muscle contractility. Optimizing protocols to limit damage is critical for accurate physiological, biochemical, and molecular measurements. Following extensive testing, we developed an electroporation protocol that enhances transfection efficiency in skeletal muscles without causing muscle damage. Pretreating mouse tibialis anterior muscles with hyaluronidase and electroporation at 75 V/cm (using 50% vol/vol saline as a vehicle for plasmid DNA) resulted in 22 +/- 5% of the muscle fibers expressing a reporter gene. This protocol did not compromise contractile function of skeletal muscles assessed at both the intact (whole) muscle and the cellular (single fiber) level. Furthermore, ectopic expression of insulin-like growth factor I to levels that induced muscle fiber hypertrophy without causing tissue damage or compromising muscle function highlights the therapeutic potential of these methods for myopathies, muscle wasting disorders, and other pathophysiologic conditions.

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Available from: Jonathan D Schertzer, Apr 16, 2014
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    • "Following the plasmid injection, muscles were stimulated with a low voltage electrical current using two-paddle electrodes [12] that delivered 6 pulses at 86 V/cm for 20 ms with an interpulse interval of 200 ms (Electro Square Porator ECM 830, BTX). This is a low-dose electroporation protocol that does not induce damage [13], [14], [15]. At day 25 post-inoculation, TA muscles were removed from both control and C26 tumor-bearing mice, weighed, and either snap-frozen in liquid nitrogen for biochemical analysis or mounted on tongue depressors, embedded in tissue-freezing medium, and frozen in liquid nitrogen-cooled isopentane for histochemical analysis. "
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    ABSTRACT: Existing data suggest that NF-kappaB signaling is a key regulator of cancer-induced skeletal muscle wasting. However, identification of the components of this signaling pathway and of the NF-κB transcription factors that regulate wasting is far from complete. In muscles of C26 tumor bearing mice, overexpression of dominant negative (d.n.) IKKβ blocked muscle wasting by 69% and the IκBα-super repressor blocked wasting by 41%. In contrast, overexpression of d.n. IKKα or d.n. NIK did not block C26-induced wasting. Surprisingly, overexpression of d.n. p65 or d.n. c-Rel did not significantly affect muscle wasting. Genome-wide mRNA expression arrays showed upregulation of many genes previously implicated in muscle atrophy. To test if these upregulated genes were direct targets of NF-κB transcription factors, we compared genome-wide p65 binding to DNA in control and cachectic muscle using ChIP-sequencing. Bioinformatic analysis of ChIP-sequencing data from control and C26 muscles showed very little p65 binding to genes in cachexia and little to suggest that upregulated p65 binding influences the gene expression associated with muscle based cachexia. The p65 ChIP-seq data are consistent with our finding of no significant change in protein binding to an NF-κB oligonucleotide in a gel shift assay, no activation of a NF-κB-dependent reporter, and no effect of d.n.p65 overexpression in muscles of tumor bearing mice. Taken together, these data support the idea that although inhibition of IκBα, and particularly IKKβ, blocks cancer-induced wasting, the alternative NF-κB signaling pathway is not required. In addition, the downstream NF-κB transcription factors, p65 and c-Rel do not appear to regulate the transcriptional changes induced by the C26 tumor. These data are consistent with the growing body of literature showing that there are NF-κB-independent substrates of IKKβ and IκBα that regulate physiological processes.
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    • "Each TA muscle was injected with 30 lL of vector (25 lg of vector, 5 lg of GFP), and 5 mm electrodes (BTX Gentrodes, Harvard Apparatus , Holliston, MA, USA) were applied to either side of injection site. Three 20-ms pulses of 100 mV were applied using an ECM-830 electroporator (Harvard Apparatus) according to a protocol previously reported to cause only moderate damage to the tissue (Schertzer et al. 2006); polarity of electrodes was reversed and electroporation was repeated. Skin was closed over muscle using wound clips, and 1 lL of buprenorphine was injected subcutaneously for pain relief. "
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    • "However, bioenergetic analyses of permeabilized muscle fibres only require ∼2 mg (wet weight) of muscle. This raises the possibility that muscle fibers will be selected that have not been transfected with MFN-2, as it is well known that in mature mammalian muscle not all muscle fibres are necessarily transfected [27]. Therefore we first needed to establish a methodology that could identify muscle fibres that over-expressed MFN-2, so we could select these fibers for bioenergetic analyses. "
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    ABSTRACT: The role of mitofusin-2 (MFN-2) in regulating mitochondrial dynamics has been well-characterized in lower order eukaryotic cell lines through the complete ablation of MFN-2 protein. However, to support the contractile function of mature skeletal muscle, the subcellular architecture and constituent proteins of this tissue differ substantially from simpler cellular organisms. Such differences may also impact the role of MFN-2 in mature mammalian muscle, and it is unclear if minor fluctuations in MFN-2, as observed in response to physiological perturbations, has a functional consequence. Therefore, we have transiently transfected MFN-2 cDNA into rat tibialis anterior muscle to determine the effect of physiolgically relevant increases in MFN-2 protein on mitochondrial bioenergetics. Permeabilized muscle fibres generated from muscle following MFN-2-transfection were used for functional assessments of mitochondrial bioenergetics. In addition, we have further established a novel method for selecting fibre bundles that are positively transfected, and using this approach transient transfection increased MFN-2 protein ∼2.3 fold in selected muscle fibres. However, this did not alter maximal rates of oxygen consumption or the sensitivity for ADP-stimulated respiration. In addition, MFN-2 over-expression did not alter rates of H(2)O(2) emission. Altogether, and contrary to evidence from lower order cell lines, our results indicate that over-expressing MFN-2 in healthy muscle does not influence mitochondrial bioenergetics in mature mammalian skeletal muscle.
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