electroporation-of-plasmid-dna-into-mouse-skeletal-muscle

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

Penn State College of Medicine

The role of bone-derived factors in regulation of skeletal muscle function is an important emerging aspect of research into bone-muscle crosstalk. Implications for this area of research are far reaching and include understanding skeletal muscle weakness in cancer, osteoporosis, cachexia, rare diseases of bone, and aging.

The Role of TGFβ in Bone-Muscle Crosstalk.

Tumor cell interactions with the bone microenvironment are vital for the establishment and progression of bone metastases. Recently in Cancer Cell, Wang et al. (2018) showed that cells of the osteoblast lineage are critical for the delivery of calcium to tumor cells through gap junctions, pointing toward potential therapies for bone metastases.

A "Connexin" Responsible for the Fatal Attraction of Cancer to Bone.

Carboplatin is a chemotherapy drug used to treat solid tumors but also causes bone loss and muscle atrophy and weakness. Bone loss contributes to muscle weakness through bone-muscle crosstalk, which is prevented with the bisphosphonate zoledronic acid (ZA). We treated mice with carboplatin in the presence or absence of ZA to assess the impact of bone resorption on muscle. Carboplatin caused loss of body weight, muscle mass, and bone mass, and also led to muscle weakness as early as 7 days after treatment. Mice treated with carboplatin and ZA lost body weight and muscle mass but did not lose bone mass. In addition, muscle function in mice treated with ZA was similar to control animals. We also used the anti-TGFβ antibody (1D11) to prevent carboplatin-induced bone loss and showed similar results to ZA-treated mice. We found that atrogin-1 mRNA expression was increased in muscle from mice treated with carboplatin, which explained muscle atrophy. In mice treated with carboplatin for 1 or 3 days, we did not observe any bone or muscle loss, or muscle weakness. In addition, reduced caloric intake in the carboplatin treated mice did not cause loss of bone or muscle mass, or muscle weakness. Our results show that blocking carboplatin-induced bone resorption is sufficient to prevent skeletal muscle weakness and suggests another benefit to bone therapy beyond bone in patients receiving chemotherapy. © 2019 American Society for Bone and Mineral Research.

Zoledronic Acid Improves Muscle Function in Healthy Mice Treated with Chemotherapy.

Prolonged residence of mice in spaceflight is a scientifically robust and ethically ratified model of muscle atrophy caused by continued unloading. Under the Rodent Research Program of the National Aeronautics and Space Administration (NASA), we assayed the large-scale mRNA and metabolomic perturbations in the quadriceps of C57BL/6j male mice that lived in spaceflight (FLT) or on the ground (control or CTR) for approximately 4 weeks. The wet weights of the quadriceps were significantly reduced in FLT mice. Next-generation sequencing and untargeted mass spectroscopic assays interrogated the gene-metabolite landscape of the quadriceps. A majority of top-ranked differentially suppressed genes in FLT encoded proteins from the myosin or troponin families, suggesting sarcomere alterations in space. Significantly enriched gene-metabolite networks were found linked to sarcomeric integrity, immune fitness, and oxidative stress response; all inhibited in space as per in silico prediction. A significant loss of mitochondrial DNA copy numbers in FLT mice underlined the energy deprivation associated with spaceflight-induced stress. This hypothesis was reinforced by the transcriptomic sequencing-metabolomics integrative analysis that showed inhibited networks related to protein, lipid, and carbohydrate metabolism, and adenosine triphosphate (ATP) synthesis and hydrolysis. Finally, we discovered important upstream regulators, which could be targeted for next-generation therapeutic intervention for chronic disuse of the musculoskeletal system. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Gene-Metabolite Network Linked to Inhibited Bioenergetics in Association With Spaceflight-Induced Loss of Male Mouse Quadriceps Muscle.

What is the central question of this study? Cachexia causes severe changes in skeletal muscle metabolism and function and is a key predictor of negative outcomes in cancer patients: what are the changes in whole animal energy metabolism and mitochondria in skeletal muscle? What is the main finding and its importance? There is decreased whole animal energy expenditure in mice with cachexia. They displayed highly dysmorphic mitochondria and mitophagy in skeletal muscle.

Bone metastases induce metabolic changes and mitophagy in mice.

Chemotherapy is an essential treatment to combat solid tumours and mitigate metastasis. Chemotherapy causes side effects including muscle wasting and weakness. Regulated in Development and DNA Damage Response 1 (REDD1) is a stress-response protein that represses the mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1), and its expression is increased in models of muscle wasting. The aim of this study was to determine if deletion of REDD1 is sufficient to attenuate chemotherapy-induced muscle wasting and weakness in mice.

Loss of REDD1 prevents chemotherapy-induced muscle atrophy and weakness in mice.

Cancer cachexia is a wasting disorder associated with advanced cancer that contributes to mortality. Cachexia is characterized by involuntary loss of body weight and muscle weakness that affects physical function. Regulated in DNA damage and development 1 (REDD1) is a stress-response protein that is transcriptionally upregulated in muscle during wasting conditions and inhibits mechanistic target of rapamycin complex 1 (mTORC1). C2C12 myotubes treated with Lewis lung carcinoma (LLC)-conditioned media increased REDD1 mRNA expression and decreased myotube diameter. To investigate the role of REDD1 in cancer cachexia, we inoculated 12-wk-old male wild-type or global REDD1 knockout (REDD1 KO) mice with LLC cells and euthanized 28 days later. Wild-type mice had increased skeletal muscle REDD1 expression, and REDD1 deletion prevented loss of body weight and lean tissue mass but not fat mass. We found that REDD1 deletion attenuated loss of individual muscle weights and loss of myofiber cross-sectional area. We measured markers of the Akt/mTORC1 pathway and found that, unlike wild-type mice, phosphorylation of both Akt and 4E-BP1 was maintained in the muscle of REDD1 KO mice after LLC inoculation, suggesting that loss of REDD1 is beneficial in maintaining mTORC1 activity in mice with cancer cachexia. We measured Foxo3a phosphorylation as a marker of the ubiquitin proteasome pathway and autophagy and found that REDD1 deletion prevented dephosphorylation of Foxo3a in muscles from cachectic mice. Our data provide evidence that REDD1 plays an important role in cancer cachexia through the regulation of both protein synthesis and protein degradation pathways. Cancer cachexia is a debilitating and lethal consequence of many advanced cancers. REDD1, a negative regulator of mTORC1 activity, is an emerging target in cachexia. Our data show that skeletal muscle REDD1 expression is increased in LLC-induced cancer cachexia. Mice lacking REDD1 have attenuated skeletal muscle atrophy that is likely due to maintaining both protein synthesis and inhibiting protein degradation.

Article	Year
The Role of TGFβ in Bone-Muscle Crosstalk. Current osteoporosis reports\| PubMed ID: 28161871	2017
A "Connexin" Responsible for the Fatal Attraction of Cancer to Bone. Cell metabolism\| PubMed ID: 30625309	2019
Zoledronic Acid Improves Muscle Function in Healthy Mice Treated with Chemotherapy. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research\| PubMed ID: 31614017	2020
Gene-Metabolite Network Linked to Inhibited Bioenergetics in Association With Spaceflight-Induced Loss of Male Mouse Quadriceps Muscle. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research\| PubMed ID: 32511780	2020
Bone metastases induce metabolic changes and mitophagy in mice. Experimental physiology\| PubMed ID: 33369797	2021
Loss of REDD1 prevents chemotherapy-induced muscle atrophy and weakness in mice. Journal of cachexia, sarcopenia and muscle\| PubMed ID: 34664403	2021
REDD1 deletion attenuates cancer cachexia in mice. Journal of applied physiology (Bethesda, Md. : 1985)\| PubMed ID: 34672766	2021

David L. Waning

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