Source: IOWA STATE UNIVERSITY submitted to
MOLECULAR MECHANISMS REGULATING SKELETAL MUSCLE GROWTH AND DIFFERENTIATION (NC131)
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0221057
Grant No.
(N/A)
Project No.
IOW05257
Proposal No.
(N/A)
Multistate No.
NC-1131
Program Code
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2010
Grant Year
(N/A)
Project Director
Selsby, J. T.
Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
Animal Science
Non Technical Summary
How skeletal muscle cells control which genes are being expressed is currently unknown. In this project, we will determine the extent to which the PGC-1alpha pathway impacts gene expression in skeletal muscle cells.
Animal Health Component
10%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053510102020%
3053840102080%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3510 - Swine, live animal; 3840 - Laboratory animals;

Field Of Science
1020 - Physiology;
Goals / Objectives
Determine molecular mechanisms that control gene expression in skeletal muscle.
Project Methods
The precise molecular mechanisms that regulate gene expression in skeletal muscle have yet to be fully characterized. Further, how these mechanisms may change with development is currently unknown. The Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha) has been recently tied to expression of not only oxidative genes, but also genes associated with slow skeletal muscle including utrophin and type I myosin heavy chain. We will determine the extent to which post-natal PGC-1alpha over-expression impacts expression of myosin heavy chain isoforms as well as expression of oxidative proteins and mitochondrial function. The PGC-1alpha gene will be cloned and inserted into an expression vector ultimately used for the production of a recombinant adeno-associated virus (AAV) serotype 6. Virus will be injected into single mouse hind limbs while contralateral limbs receive a sham injection with empty vector. Groups of mice will be injected with virus as neonates, at three weeks of age, and at six months of age to determine the extent to which development has on the efficacy of PGC-1alpha to regulate gene expression. Myosin heavy chain expression will be determined through immunohistological techniques. Briefly, antibody specific for type I or type II muscle will be applied to sections of gastrocnemius and soleus. We will determine the relative and absolute contribution of each isoform to total myosin heavy chain expression. Additionally, these results will be confirmed through gel electrophoresis experiments where the cytoskeletal fraction of homogenate is separated by mass on gels. Oxidative protein expression will be determined through western blotting for various protein targets. Mitochondrial expression will be reported as a ratio of mitochondrial DNA to nuclear DNA based on the ratio genes expressed only once in each genome. Following these experiments, a similar strategy will be applied both up and down stream of PGC-1alpha. It will allow us to determine the extent to which contractile properties can be separated from metabolic properties. In production agriculture, for example, it may be beneficial to express one set of genes without the other. Moreover, in diseased muscle, independent expression and regulation of these gene sets may allow therapeutic advances. Specifically, genes for Sirt-1, MTFa, NRF-1, NRF-2, and ERR alpha will be cloned into an expression vector like above. We will also following the injection regimen as above. Dependent variables will also be similar.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: We have been working to understand the extent to which PGC-1alpha gene transfer can alter gene expression in skeletal muscle. PGC-1alpha is a transcriptional co-activator that can drive expression of both slow/NMJ proteins as well as oxidative proteins. In dystrophin-deficient skeletal muscle, utrophin replacement has been incredibly effective in the mouse model of the disease. Further, in these muscles mitochondrial dysfunction has been noted. Importantly, one of the genes activated by PGC-1alpha gene transfer is utrophin. Hence, PGC-1alpha pathway activation may be able to correct two aspects of dystrophic pathology through utrophin induction and expression of oxidative proteins. We have shown that neonatal gene transfer protects skeletal muscle from acute eccentric injury. We are working to understand associated changes leading to this protection. To that end we have performed a proteomic study where we identified approximately 100 proteins that were dysregulated. This is important because it provides insight regarding acute dystrophin-deficiency, akin to a toddler either before or immediately after initial diagnosis. To better understand differential protein expression we performed a microRNA analysis and discovered that more than 50 microRNA's were differently regulated between dystrophic muscle and dystrophic muscle over-expressing PGC-1alpha. We are currently working to understand the relationship between microRNA expression and protein expression in these muscles. We have also shown that PGC-1alpha gene transfer can be used to rescue dystrophic skeletal muscle. This is an important step as patients with Duchenne muscular dystrophy are diagnosed with an active pathology. Hence, showing disease prevention is useful, but showing disease rescue provides a much more realistic scenario to test the power of an intervention. Research findings have been presented at several conferences including FASEB '09 and '10. A presentation was also made at the New Directions in Skeletal Muscle Biology meeting held in 2010. Further, a publication from this work is in review and several others are in preparation. PARTICIPANTS: Several people contributed to this project:Delphine Gardan-Salmon, Post doc; Katrin Hollinger, Graduate Student; Jenna Dixon, Undergrad; Alyona Avdonina, Undergrad; Lauren Gealow, Undergrad; Steven Lonergan, Ph.D., Collaborator. This project involved two dimensional differential in-gel electrophoresis, which is a proteomics technique that Dr. Lonergan performs routinely in his lab. He was kind enough to teach it to Delphine and Jenna. Also, regulation of protein expression by miroRNA's is an emerging area. We all learned about their function in performance of this work. Finally, as histological evaluation was our primary means of determining the extent to which PGC-1alpha gene transfer benefitted dystrophin-deficient muscle it was necessary for me to train Delphine, Katrin, and Lauren in histological techniques. TARGET AUDIENCES: Researchers interested in the study of muscle physiology or Duchenne muscular dystrophy. Parents and families of boys with Duchenne muscular dystrophy. Clinicians caring for DMD patients. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The purpose of the project is to improve our understanding of gene regulation in skeletal muscle. We have made significant strides by showing the broad affects of PGC-1alpha gene transfer. Specifically, we are performing PGC-1alpha gene transfer in the mouse model for Duchenne muscular dystrophy. Our rationale for this choice was that not only can we learn about gene regulation in skeletal muscle but we can simultaneously make contributions to developing a potential therapy or intervention for this disease. Collectively we have shown that PGC-1alpha gene transfer can prevent disease onset, prevent acute muscle injury, and rescue muscle from typical decline. We have also collected biochemical data that addresses the underlying mechanism of PGC-1alpha's protective effects. These data provide overwhelming evidence that PGC-1alpha gene transfer has great potential as an effective therapeutic approach for DMD.

Publications

  • No publications reported this period