Semi-Annual Status Report of NASA-Ames Research Grant No. NAG2-414 

Growth Factor Involvement in Tension-Induced 

Skeletal Muscle Growth 

- 

Herman H. Vandenburgh, Ph.D. 

Department of Pathology 

Brown University Program in Medicine 

Providence, RI 02912 

and 

Department of Pathology and Laboratory ffedicine 

The Miriarn Hospital 

Providence, RI 02906 

Period covered by review: September, 1986 - !.larch, 1987 

Correspondence Address: 

Dr. H.N. Vandenburgh 

Dept. Laboratory Medicine 

164 Summit hve. 

Providence, RI 02906 

tel. no. (401)-274-3700 x4254 

(hASA-CR-180201) GliOLTH EAC?Cli INVCLYEBENT N87- 1657 7 

II IdhSICN-INEUCEL SKELETAL CC2CIE GliOWlH 

Semiannual Statcs gepcrt, Seg, 1S86 - Har, 12E7 (Brcvn Uriw.) 8 F CSCL 06P Unclas 

G3/52 43614 
2 

Table of Contents 

I Description of Research 

I1 Accomplishments 

I11 Significance of Accomplishments 

IV Publications 

V References 

VI Appendix 

3-5 

5-6 
3 

Description of Resea.rch 

Long-term manned space travel will require a better understanding of 

skeletal muscle atrophy which results from the microgravity found in 

space. Astronaut strength and dexterity must be maintai.ned for normal 

mission operations, for emergency situations, and for the rigors of 

reentry into the earth's atmosphere. A biochemical understanding of how 

gravity, by increasing the tension on muscle cells, helps to maintain 

muscle size and strength, should ultimately allow pharmacological 

intervention to prevent muscle atrophy in microgravity. Because of the 

limited availability of performing experiments in space, a number of 

earth-based model systems have been designed to study the relationship of 

muscle tension to muscle growth (1). These models include the casting of 

animal's limbs such that the limb muscles are held under increased 

(stretched) or decreased (shortened) tension; placing weights on limb 

muscles to increase their load; and suspending an animal's hinquarters 

above the ground in order to reduce skeletal muscle use. 

Our laboratory has developed several tissue culture model systems for 

studying the relationship of tension to skeletal muscle growth (2,3). 

Tissue culture cells have been extensively used for studying basic 

biochemical growth processes in other cell types and allows easier 

manipulation of experimental conditions than whole animals. Cultured 

cells have been especially helpful in studying the role of growth factors 

in normal and pathological cell growth (4). We are utilizing one of our 

tissue culture model systems to determine the role played by exogenous and 

endogenous growth factors in tension-induced skeletal muscle growth. 

Briefly, this model involves isolating muscle cells from 12 day old chick 

embryos and growing them on a highly elastic substratum 
4 

material in a humidified C02 incubator at 37OC (3). The muscle cells, 

growing in a complex medium, initially proliferate, and then fuse to form 

multinucleated, spontaneously contractile myotubes in a manner very 

similar to growth and differentiation -- in vivo (5). Thes'e cells actively 

synthesize actin and myosin and incorporate them into cross-striated 

myofibrillar material. These -- in vitro differentiated, "embryonic" 

myofibers, can be subjected to different patterns of mechanical activity 

with our "Mechanical Cell Stimulator" which simulates in vivo muscle 

activity (Figure 1, Appendix). The activity patterns used to stimulate 

muscle growth are generated by an Apple IIe computer. In effect, we can 

subject muscle fibers in a defined in vitro environment to different types 

of "exercise" programs, and study their growth respose on a molecular 

level. The results obtained using these avian muscle should be applicable 

to mammalian muscle, since most basic molecular mechanisms utilized 

by Nature are identical or very similar in different classes of 

-- 

vertebrates. 

The specific goal of this project is to determine the role played by 

exogenous and endogenous growth factors in mechanical activity-induced 

growth of cultured skeletal muscle. IJe have shown previously that 

increased tension on these cells in a complex medium stimulates their 

growth (2). These factors include the medium growth factors found in 

serum and chicken embryo extract supplements that the cells are grown in, 

as well as endogenous growth factors produced by the muscle tissue itself 

(6,7). The importance of endogenously synthesized and secreted 

prostaglandins to mechanically-stimulated muscle growth is being studied. 
5 

By understanding the interactions between growth factors and 

mechanically-stimulated growth, the mechanisms by which gravity-induced 

tension are transduced into the biochemical alterations leading to muscle 

growth will be more clearly defined, and methods for prevention of muscle 

loss from disused muscles in microgravity can be developed. 

Accomplishments 

The first hypothesis being tested on our grant is whether 

extracellular serum factors present in the growth medium are essential for 

mechanical stimulation of skeletal muscle growth - in vitro (Hypothesis I). 

The protocol being used is to allow the avian muscle cells to 

differentiate into spontaneously contractile "embryonic" myofibers in the 

Cell Growth Chamber of our Mechanical Cell Stimulator in complete culture 

medium (with 10% horse serum, 52 chicken embryo extract). Once developed, 

the cells are switched to medium with or without added growth factors. 

Repetitive mechanical stimulation of the cells is then initiated and at 

varying times, groups of cells are removed for determination of their 

growth rate by measuring protein:DNA ratios. Mechanically-stimulated 

growth of the muscle cells in medium containing added growth factors is 

compared to mechanically-stimulated growth in defined medium without any 

added growth factors. If the stimulated growth rates are identical in the 

two groups, then mechanically-stimulated muscle growth in our in vitro 

model system would be independent of exogenous growth factors and only 

dependent on internal cellular processes. Conversely, if by removing 

medium growth factors, mechanically-stimulated muscle growth is 

significantly reduced, then the interaction of as yet undefined exogenous 
6 

growth factors will be essential, and their role examined more closely. 

When we began these studies we immediately found that there was a 

I 

significant increase in the cultured "embryonic" myofibers spontaneous 

contractile activity when they were placed into defined medium in the 

absence of serum or chick embryo extract. These contractions were so 

rigorous that within 12-24 hours in defined medium, the myofibers would 

contract off the Substratum and degenerate. This obviously made the 

experiments outlined in the above paragraph difficult to run. We 

therefore spent a significant period of time attempting to find a method 

for attaching the contractile cells more strongly to the substratum. We 

found that by embedding the already formed myofibers in a collagen gel 

(collagel), combined with a perimeter nylon support ring, the myofibers 

could be maintained attached to the substratum and under active tension 

for 7-14 days in defined medium without serum or embryo extract (8,9). 

This new culturing technique thus allows us to continue with our 

mechanical stimulation experiments in examining the role played by 

exogenous growth factors in the tension-induced growth process. An 

additional benefit of using this collagel technique on the cultured 

myofibers is that they are able to differentiate into myofibers which are 

more "neonatal-like" rather than "embryonic" (9). These "neonatal" 

myofibers have subsarcolemnal nuclei, a more complete external lamina cell 

coat, and a more extensively organized myofibrils. This significant 

advance in muscle cell culturing should make the results of our 

growth-factor studies more directly applicable to in vivo studies using 

adult muscle cells. 
7 

Significance of Accomplishments: 

New muscle tissue culture techniques have been developed to grow 

embryonic skeletal myofibers which are able to differentiate into more 

adult-like myofibers. Studies on mechanical stimulation of cultured 

muscle cell growth will now be more directly applicable to 

mechanically-induced growth in adult muscle, and lead to better models for 

understanding muscle tissue atrophy caused by disuse in the microgravity 

of space. 

Publications 

1. See Reference 8 

2. See Reference 9 
8 

1. 

2. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 

References 

Vandenburgh, H.H. Motion into Mass: How does tension stimulate muscle 

growth? Med. Sci. Sports Exercise, In Press. 

Vandenburgh, H.H.; Kaufman, S. An vitro model for stretch-induced 

hypertrophy of skeletal muscle. Science 203: 265-268, 1979. 

Vandenburgh, H.H. A computerized model system for studying the effects 

of mechanical activity on cell growth - in vitro. - In Vitro 

abstract, in press (copy in Appendix). 

Rozengurt, E. Early signals in the mitogenic response. Science 234: 

161-166, 1986. 

Coleman, J.R.; Coleman, A.W. Muscle differentiation and macromolecular 

synthesis. J. Cell. Physiol. 72: 19-34, 1965. 

Vandenburgh, 1I.H. Cell shape and growth regulation in skeletal muscle: 

exogenous versus endogenous factors. J. Cell. Physiol. 116: 

363-371, 1983. 

Summers, P.J.; Ashmore, C.R.; Less, Y.B.; Ellis, S. Stretch-induced 

grolfth in chicken wing muscles: ??ole of soluble growth-promoting 

factors. J. Cell. Physiol. 125: 288-294, 1985. 

Vandenburgh, 1I.H.; Karlisch, P.; Farr, L. Maintenance of highly 

contractile tissue-cultured avian skeletal myotubes in collagen 

gel. Vitro abstract, 1937 (copy in Appendix). 

Vandenburgh, 1I.H.; Rarlisch, P.; Farr, L. Maintenance of highly 

contractile tissue-cultured avian skeletal myotubes in collagen 

gel. & Vitro, in revision, 1987 (copy in Appendix).