Source: BOSTON BIOMEDICAL RESEARCH INSTITUTE submitted to
MUSCLE CELL GROWTH AND DEVELOPMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0192323
Grant No.
1999-35206-12074
Project No.
MASR-2002-04005
Proposal No.
2002-04005
Multistate No.
(N/A)
Program Code
42.0
Project Start Date
Nov 1, 2001
Project End Date
Apr 30, 2004
Grant Year
2002
Project Director
Miller, J. B.
Recipient Organization
BOSTON BIOMEDICAL RESEARCH INSTITUTE
(N/A)
WATERTOWN,MA 02478
Performing Department
(N/A)
Non Technical Summary
The experiments will study muscle-forming cells and the regulation of muscle development and growth. Efficient muscle formation is central to successful agricultural production of meat. A protein known as Sca-1 is found only in cells that appear to be a special class of muscle-forming cells termed muscle stem cells. Muscle stem cells are progenitors of the cells which form muscle fibers. We will purify this small group of muscle cells and determine if they are indeed muscle stem cells. Also, we will examine how a group of proteins that regulate cell death affect muscle growth and development. One of these proteins, termed Bcl-2, appears to inhibit cell death and enhance muscle growth. We will examine cellular and molecular mechanisms by which Bcl-2 expression enhances the formation of skeletal muscle. One long-term goal of our work is to identify and purify muscle stem cells. There is currently no method to purify muscle stem cells. Our experiments could greatly increase our understanding of muscle growth. A second goal is to determine how Bcl-2 expression affects the quantitative outcome of myogenesis. These experiments in basic muscle biology will improve our understanding of muscle growth and development and could identify possible methods by which to improve muscle growth in agriculturally important animals. The purpose of these studies is to identify mechanisms that underlie muscle growth.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053840100020%
3053840103020%
3053840104060%
Goals / Objectives
1. Purify the small subset of Sca-1-positive skeletal muscle precursor cells and determine if they have properties of stem cells. 2. Determine if Bcl-2-null cells are less able to form muscle in vivo than wild-type cells. 3. Determine how targeted alteration of Bcl-2 family expression affects muscle development and growth.
Project Methods
1. In the first set of experiments, we propose to isolate the Sca-1-positive subset of muscle cells and determine if they have the properties expected of muscle stem cells. Sca-1-positive cells will be isolated using specific antibodies and magnetic bead selection. Purified cells will be tested using in vitro clonal assays to examine their ability to self-renew and produce progeny that will form muscle. 2. We will use cell transfer assays combined with immunohistology to examine muscle formation by muscle cells from Bcl-2-null and wild-type genotypes. 3. We will use transgenic and knock-out strategies to produce muscles that have specific alterations in expression of cell death regulators of the Bcl-2 family. We will then use quantitative histology to determine how alteration of cell death pathways affects muscle cell growth and development.

Progress 11/01/01 to 04/30/04

Outputs
As part of our work to identify genes expressed within muscle cells, we cloned and characterized expression patterns of two mouse homologs, Cops2/Csn2 and Nrf-1, of Drosophila proteins that appeared to be involved in myogenesis. With collaborators at Yale, we also made Cops2/Csn2 knock-out mice and found that this protein is required for early embryogenesis because it is needed for proper Cyclin E, p21 and p53 expression. As part of our work on development and growth of muscle, we prepared the reviews on muscle stem cells and regulation of muscle function by the Notch/Delta system. Two papers reported much of our work on Bcl-2 family function in muscle. We demonstrated that inactivation of Bcl-2 or Bax produces distinct alterations in muscle in vivo and in satellite cells in vitro. We also showed that Bax inactivation accelerates immortalization of myogenic cells in culture by a mechanism that is, in some cases, distinct from mutations in p16, p19, and p53 that were known to underlie immortalization of non-muscle cells. We also examined possible interconnection of the muscle protein dysferlin, abnormal upregulation of MHC class I, and poor muscle function. This study eliminated MHC class I upregulation as a mechanism behind the poor muscle function in dysferlin-null muscles. In a continuation of work from a previous USDA award, we used transgenic techniques to show that the rate of muscle regeneration can be specifically altered by accelerating expression of the muscle transcription factor MRF4. Additional Preliminary Studies were also completed with whole or partial support from this award.

Impacts
Our new methods for identifying muscle stem cells and determining their cell lineage potential will allow identification of molecular pathways that regulate stem cell function. In addition, our results that Bcl-2 family members can regulate muscle size and function open may a new route to manipulation of muscle formation.

Publications

  • Schaefer, L., Beermann, M. L., and Miller, J. B. (1999). Coding sequence, genomic organization, chromosomal localization, and expression pattern of the signalosome component Cops2: The mouse homologue of Drosophila alien. Genomics 56:310-316.
  • Miller, J. B., Schaefer, L., and Dominov, J. A. (1999). Seeking muscle stem cells. Curr. Top. Dev. Biol. 43, 191-219.
  • Schaefer, L., Engman, H., and Miller, J. B. (2000). Coding sequence, chromosomal location, and expression pattern of Nrf1: the mouse homologue of Drosophila erect wing. Mammalian Genome. 11:104-110.
  • Dominov, J. A., Houlihan-Kawamoto, C. A., Swap, C. J., and Miller, J. B. (2001) Pro- and anti- apoptotic members of the Bcl-2 family in skeletal muscle: A role for Bcl-2 in later stages of myogenesis. Dev. Dynam. 220:18-26.
  • Miller, J. B. (2001). Developmental biology of skeletal muscle. in Disorders of Voluntary Muscle, 7th Edition (eds. D. Hilton-Jones, R. C. Griggs, and G. Karpati) Cambridge Univ. Press (Cambridge, UK) pp. 26-41.
  • Kostek, C. A, Dominov, J. A., and Miller, J. B. (2002) Upregulation of MHC class I expression accompanies, but is not required for, spontaneous myopathy in dysferlin-deficient SJL/J mice. Am. J. Pathol. 160:833-839.
  • Pavlath, G. K., Dominov, J. A., Kegley, K. M., and Miller, J. B. (2003) Regeneration of skeletal muscles with altered timing of expression of the myogenic bHLH factor MRF4. Am. J. Pathol. 162:1685-1691.
  • Lykke-Andersen, K., Schaefer, L., Menon, S., Deng, X.-W., Miller, J. B. and Wei, N. (2003) Disruption of COP9 subunit Csn2 in mouse causes deficient cell proliferation, accumulation of p53 and cyclin E, and early embryonic death. Mol. Cell. Biol. 23:6790-6797.
  • Miller, J. B. and Emerson, C. P. Jr. (2003) Does the road to muscle rejuvenation go through Notch? Sci. Aging Knowl. Environ., Vol. 2003, Issue 48, pp. pe34, 3 December 2003
  • Nowak, J., Malowitz, J., Girgenrath, M., Kostek, C., Kravetz, A., Dominov, J., & Miller, J.B. (2004) Immortalization of myogenic cells does not require loss of p16INK4a, p19ARF, or p53 and is accelerated by inactivation of Bax. BMC Cell Biol. vol. 5, paper 1, pp. 1-14.


Progress 10/01/02 to 09/30/03

Outputs
We have focused on two areas: 1. methods for identifying muscle stem cells and determining their cell lineage potential and 2. effects of Bcl-2 family members on muscle development. Under stem cells, we have developed fluorescence activated cell sorting methods to purify different subsets of muscle-derived stem cells based on expression of cell surface markers such as Sca1, CD45, and CD34. We have further developed methods to measure the proliferation status of these different subsets of cells in developing muscle. Thus, we have been able to identify the proliferation capability of these cells during muscle formation. We have also used several methods to identify the cell lineage potential of the different subsets of muscle stem cells. We have been able to show that the cells are not limited to the classical skeletal muscle cell lineage, but rather are multipotential, with cardiac muscle being a particular outcome. Under Bcl-2 family members, we have produced definite information that Bcl-2 family members can regulate muscle function. For example, we have found that Bax-null muscle fibers are smaller than wild-type muscle fibers and that Bcl-2-null muscles have only about two-thirds as many fast muscle fibers as wild-type. Furthermore, alteration of Bcl-2 or Bax function is able to alter the outcome of particular types of muscle pathology. Thus, these data show that Bcl-2 family members can indeed regulate muscle cell growth and development.

Impacts
Our new methods for identifying muscle stem cells and determining their cell lineage potential will allow identification of molecular pathways that regulate stem cell function. In addition, our results that Bcl-2 family members can regulate muscle size and function open a new route to manipulation of muscle formation.

Publications

  • Pavlath, G. K., Dominov, J. A., Kegley, K. M., and Miller, J. B. (2003) Regeneration of skeletal muscles with altered timing of expression of the myogenic bHLH factor MRF4. Am. J. Pathol. 162:1685-1691.
  • Lykke-Andersen, K., Schaefer, L., Menon, S., Deng, X.-W., Miller, J. B. and Wei, N. (2003) Disruption of COP9 subunit Csn2 in mouse causes deficient cell proliferation, accumulation of p53 and cyclin E, and early embryonic death. Mol. Cell. Biol. 23:6790-6797.
  • Nowak, J.A., Malowitz, J., Girgenrath, M., Kostek, C., Kravetz, A., Dominov, J.A. and Miller, J.B. (2003) Immortalization of myogenic cells is accelerated by inactivation of Bax. (submitted)