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This staff working paper was discussed at the Council's March 2003 meeting. It was prepared by staff solely to aid discussion, and does not represent the official views of the Council or of the United States Government.

Staff Working Paper

Stronger, Long-Lasting Skeletal Muscles through Biotech? 1

Introduction
Our muscles are essential to human life in a variety of ways. They are central components of physical strength and speed, attributes that are admired and celebrated in most human cultures. Our mobility depends on muscles, whether we use them to walk, or when we just use them to turn the wheel of a car or put our foot down on the accelerator or the brake. As a basic component of physical vigor they also play a role in human attractiveness. As such, muscle tone is a major contributor to the “sense of self” developed by each person. Although there are several different types of muscles in the human body, we will concentrate here on processes affecting skeletal muscles.

The strength and fitness of healthy muscles are largely a function of their exercise. In our youth, active use of our muscles in play and in sports strengthens and develops them. At puberty, production of estrogen and testosterone enhances these processes, so that the peak of human muscular development is usually between 20 and 30 years of age. Except for people whose daily work requires much physical exertion, maintaining peak muscular strength and endurance later in life requires regular exercise and fitness training. Some pursue this avidly, while others do not.

The strength and size of human muscles declines by about one-third between the ages of 30 and 80.2 Diminished capacity or loss of a previous ability to do a physical task is a common experience during human aging. The age-related loss of muscle size and strength has been named “sarcopenia”.3 In addition, there are a variety of diseases of muscle tissue (muscular dystrophies), many caused by specific genetic mutations.

We have an increased understanding of how many of the important genes in muscle cells function and are regulated.4 The parallel development of gene therapy techniques for efficient and controlled expression of genes is beginning to open up new possibilities for treating muscular dystrophies as well as maintaining “youthful” muscle size and strength
during the aging process. It is thus timely to begin discussions of the ends to which such increased understanding and power to modify should be put when it comes to human muscles.

As discussed in more detail below, biotechnological approaches to repair and strengthening of diseased and aging skeletal muscles have been demonstrated in experimental animals. The application of these approaches (once they are shown to be safe and effective) to treat human muscular dystrophies clearly falls within current understandings of appropriate therapy. A more difficult judgment is whether we should
extend the application of these approaches to a variety of other situations that are currently “beyond therapy”.

Muscles do not generate human strength and speed in isolation. Muscles need to be physically integrated with, and function harmoniously through their attachments to nerves, tendons, ligaments, and bones. While the focus in this paper is on the activity of muscle cells, we should remain alert to the possibility that biotechnological approaches that strengthen only muscles may lead to imbalances in the interactions with other components of the body, and subsequent malfunction.

Muscles in idealizations of male human form

Muscles play a prominent role in idealizations of male human form. A classical picture of excellence of the youthful male human form is Michelangelo’s sculpture of David, completed around 1504 (see Figure 1). Here the muscles are depicted as well-

Michelangelo's David

Figure 1. Michelangelo's David
(ca. 1504)
i http://www.sculpturegallery.com/sculpture/david.html

Arnold Schwarzenegger

Figure 2. Arnold Schwarzenegger (ca. 1980) ii http://www.schwarzenegger.com/en/athlete/index.asp?sec=athlete


proportioned but without much articulation of individual muscles. The strength and
power of David’s skeletal muscles shine through the marble, and leave us with a mental picture of the classical ideal of muscular development and proportion.

A more contemporary idealization of the male human form is the picture of the modern male body-building champion and actor, Arnold Schwarzenegger (Figure 2). Through specialized weight training and alleged use of anabolic steroids, his muscles (particularly the biceps) have become much larger than those pictured in the statue of David, and the different groups of skeletal muscles are individually articulated.

Although different in proportion and muscle articulation, both the classical and contemporary pictures testify to the importance of muscles in images of male strength and power. Large muscles were also supposed to help males attract females, a point that is still emphasized by males working out in gyms. “I am exercising and increasing the size of my muscles so that the chicks will notice me”.

Interestingly, female body-builders reportedly initially pursued the same path illustrated by the picture of Arnold Schwarzenegger. The result was female body-building champions with smaller but similarly individually developed and articulated skeletal muscles. More recently there has been an “aesthetic” reaction against the resulting female muscle “overdevelopment” and, commercially at least, the more popular and profitable activity today is women’s fitness competitions.

Genetic treatments are not the only biotechnological approach to increasing muscle size and strength. Anabolic steroids are among the most widely used chemical compounds that are used in combination with weight lifting to increase muscle size and strength. Examples of such compounds include methandrostenolone (Dianabo 1), Boldenone (Equip-gan), Stanazolol (Winstrol V) and Drostanolone (Masteron). As information about their effects has diffused throughout American society, they are coming to be used more and more by professional and amateur athletes. Use of some of them is banned by anti-doping organizations. Many (including the ones listed above), are listed as available for sale on the Internet.

Cellular multiplication and differentiation in skeletal muscle

The major cell type present in skeletal muscle fibers is the multinucleated myotube. These fibers arise from the fusion of mononucleated myoblast cells with each other and with pre-existing myotubes. Myoblasts, in turn, are formed by differentiation of a particular stem cell found in muscle tissue, called a satellite cell.5

The multiplication and differentiation of satellite cells into myoblasts is regulated by several specific protein growth factors (primarily insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF)) and also influenced by hormones such as growth hormone, testosterone and estrogen. Secretion of growth hormone by the pituitary acts on the liver to stimulate synthesis and release of IGF-1, which is released into the circulation (Figure 3). In muscle tissue, IGF-1 binds to specific receptors on the surface of satellite cells to stimulate cell multiplication, producing more satellite cells, and differentiation of satellite cells into myoblasts (see Figure 4).

PITUITARY
Down Arrow
Growth Hormone (GH) Secreted
Down Arrow
LIVER
Down Arrow
IGF-1 released
Down Arrow
SKELETAL MUSCLE growth stimulated

Figure 3. Hormone action and muscle growth stimulation

Importantly, a slightly different form of IGF-1 (mIGF-1) is also produced locally in muscle tissue in response to stretching the muscles (exercise). This form is thought to act the same way as circulating IGF-1 does in stimulating satellite cell multiplication and differentiation. However, because mIGF-1 is slightly different in chemical structure from IGF-1 produced in the liver, mIGF-1 apparently does not enter the circulation, so its effects can be restricted to promoting growth and repair of muscle tissue locally.

It is a common human experience that muscle size and strength can be increased by exercise. The number of muscle fibers increases as a consequence of exercise-induced stimulation of the multiplication and differentiation of muscle stem cells. Exercise both transiently damages muscles and causes them to increase in size and strength.

While exercise was previously the only way to do this, biotechnological research and

Skeletal Muscle Fiber Growth and Repair

Figure 4. Schematic diagram of some important processes in skeletal muscle fiber growth and repair.

development have introduced new possibilities. The genes for animal and human IGF-1 have been cloned and their DNA sequences determined. Gene expression vectors have been developed that permit the regulated production of IGF-1 proteins (both the liver and muscle forms) for investigation. So IGF-1 genes can be introduced into cells and experimental animals to determine the effect of enhanced IGF-1 (and/or mIGF-1) production on muscle size and strength.

Loss of muscle size and strength on aging: sarcopenia

With aging, we become more sedentary and use our muscles less. With aging the production of growth hormone and circulating IGF-1 also decreases. There is thus less IGF-1 available to keep the muscles large, and they become smaller and weaker. In addition, aged muscle cells are apparently less responsive to the action of IGF-1 and mIGF-1 6 so that the impact of even vigorous exercise on muscle size and strength diminishes with age. Figure 5 graphically illustrates the appearance of leg muscles as they become smaller and weaker with age (sarcopenia).

As we age, several things change that predispose to the development of sarcopenia. We either reduce the output of, and/or become more resistant to, anabolic stimuli to muscle such as central nervous system input, growth hormone, estrogen, testosterone, dietary protein, physical activity and insulin action. The loss of alpha-motor neuron input to muscle that occurs with age7 is believed to be a critical factor8. Nerve cell-muscle cell connections are critical to maintaining muscle mass and strength.

A loss of muscle size and strength in a significant problem for older persons. While not painful or directly debilitating, sarcopenia is associated with an increased tendency to fall and break bones. Such falls and broken bones are major causes of morbidity among the elderly.

Scarcopenia
This image is from the informative Internet site www.sarcopenia.com.

Figure 5 – Illustrating progressive age-related loss of muscle tissue (sarcopenia) iii

 

Selective stimulation of skeletal muscle growth in experimental animals.

Local injection of regulated exogenous “muscle-specific” IGF-1 Gene
Recombinant viruses, engineered to express a specific foreign gene, are frequently used to stimulate the production of functionally effective amounts of the foreign protein to treat disease. Recombinant viruses created from genetically engineered human
Adenovirus-associated Virus (AAV) have proved to be efficient delivery systems of foreign genes into muscle cells. As AAV is a small virus, only small foreign genes can be used effectively with this virus. Fortunately, the DNA sequence encoding IGF-1 is small enough to function well in AAV-based recombinant viruses.

In experiments described by Barton-Davis and coworkers 9, AAV recombinant viruses containing a rat IGF-1 gene were injected into the anterior compartment of the rear legs of mice containing the extensor digitorum longus (EDL) muscle. The resulting increased IGF-1 production promoted an average increase of about 15% in EDL muscle mass and strength in young adult mice. Strikingly, such injections led to a 27% increase in the strength of the EDL muscles of 24-month (old) mice, thus substantially reducing the decrease in EDL muscle size and strength observed in untreated old mice.

In this study, approximately 1 x 1010 recombinant AAV particles in 100 µliters of fluid were injected into a single small muscle compartment of mice. If such treatments were eventually to be applied to humans, large amounts of recombinant AAV containing the human IGF-1 DNA sequence would be required. Assuming such future treatments were shown to be safe and effective, producing sufficient recombinant AAV to treat millions of dystrophic and aging humans would remain a substantial logistical challenge. However, there may be ways around this logistical problem involving the production and transplantation of human muscle stem cells engineered to produce more IGF-1 (see below).

IGF-1 transgenic mice

The ability to create transgenic mice, in which an appropriately regulated foreign gene is expressed throughout embryonic and adult life, offers another way to assess the biological role(s) of the transgene product. Musaro et al10 introduced a rat mIGF-1 transgene into early stage mouse embryos, where it became integrated with mouse chromosomal DNA. The resulting transgenic mice produce substantial amounts of rat mIGF-1, in addition their production of mouse IGF-1 and mIGF-1.

In these transgenic mice, the rat mIGF-1 transgene was connected to gene expression regulatory elements that restricted production of the rat mIGF-1 protein to muscle tissues containing primarily fast twitch fibers. Embryonic development of these transgenic mice proceeded normally. However, as early as 10 days after birth, enlargement of skeletal muscles where rat mIGF-1 protein was being produced was observed in the transgenic animals compared to the non-transgenic control mice.

Moreover, the skeletal muscle enlargement persisted as the transgenic mice aged.
Muscle size and strength were maximal around six months in unmodified (wild type) mice and decreased as expected by 20 months of age. In contrast, at 20 months the size and strength of skeletal muscle in the rat mIGF-1 transgenic mice remained at essentially the same as at six months.

In previous studies of this type, the IGF-1 transgene was not connected to gene expression regulatory elements that restricted production of mIGF-1 to muscle tissue.
This led to overproduction of IGF-1 in the circulation, and eventually to pathological enlargement of the heart muscle. The growing understanding of muscle physiology at the molecular level coupled with sophisticated genetic engineering has made it possible to enlarge skeletal muscles selectively, without damaging heart muscles in the process.

These and other experimental results stimulate thought about possible extensions of these approaches to humans. Similar procedures might be useful treatments for various diseases of muscle tissue, and well as a possible use in older persons to counteract sarcopenia. However, each of the procedures described above has technical/logistic problems that would need to be overcome before any treatment could be applied on a large scale.

Could these biotechnological approaches be applied to human muscles?

Could the mIGF-1 gene procedures that increase skeletal muscle size and strength in young and old experimental animals be adapted for use in humans? Based on our current understanding, at least three different approaches could be considered. First, one might develop recombinant AAV-based virus vectors containing the human mIGF-1 gene under the control of appropriate regulatory elements that would limit its expression to muscle cells near the site of injection. Alternatively, one might introduce an appropriately regulated mIGF-1 gene into human embryos, as was done in the experiments with mice. Finally, a combined approach might be developed in which one first isolated human muscle stem (satellite) cells and expanded them in vitro, next introduced an appropriately regulated human mIGF-1 gene into those cells in vitro, and finally transplanted the genetically modified satellite cells back into the muscles of the person being treated.

The first approach would be similar to other human gene therapy projects. The appropriately regulated human mIGF-1 gene would be combined with a vector capable of efficient delivery to muscle cells, perhaps AAV. This material could be produced in large volumes, carefully characterized by tests in experimental animals, stored frozen and used as needed. While the logistics of producing the large amounts of recombinant AAV that would be required for treatment of thousands or millions of patients are daunting, in principle this would be possible. The advantages of this approach are 1) that it would develop and use a single, well-characterized biological agent; 2) that treatment could be started very slowly by introducing the recombinant mIGF-1 gene-containing AAV into one muscle at a time and evaluating its effects; 3) that treatment could be stopped immediately if untoward side effects developed. Disadvantages include 1) the possibility that a large number of injections would be necessary to treat each of the large number of human skeletal muscles; 2) the possibility that this would not be an effective treatment for humans who had antibodies to AAV as a consequence of a previous infection.

The second approach is a radical proposal, as it envisions treatment of blastocyst stage human embryos in vitro with a genetic procedure that was intended to change the early development of skeletal muscle size and strength and reduce the rate of loss later in life. This approach shares some advantages with the first approach in that a single biological agent could be prepared and characterized that could treat all embryos; 2) that only a single treatment early in embryonic development would be needed, instead of multiple injections into different muscles. The major disadvantages of this approach are the difficult ethical questions it would raise.

The third approach depends upon the ability to isolate human muscle stem (satellite) cells and expand them in vitro. The isolated human muscle stem cells would then have their mIGF-1 production genetically modified by introducing an appropriately regulated exogenous mIGF-1 gene copy. In theory, this could produce modified muscle stem cells that multiplied continuously in vitro to produce larger numbers of cells, and that differentiated appropriately in vitro. In this case, genetically modified satellite cells would be injected into the aging skeletal muscles. The advantages of this approach include 1) that it would develop and use a single, well-characterized biological agent to modify the muscle stem cells in vitro and 2) the dose of modified stem cells could be varied as necessary to optimize treatment of individual skeletal muscles. The disadvantages include the possibility that a separate preparation of muscle stem cells from each patient to be treated would have to be made in order to get around the immune rejection problem.

Each of these approaches has advantages and disadvantages. Developing any one of them would take a lot of time and money. Before genetic treatments to increase muscle size and strength are tried in humans, the US Food and Drug Administration would require demonstrations that the proposed treatment is safe and effective. This will ensure regulatory oversight of any initial experiments along these lines with humans.

The initial steps in applying to normal humans the kinds of genetic approaches to increasing muscle size and strength described here will likely be performed in the course of using these procedures to treat human muscle diseases. Clinical trials of regulated mIGF-1 gene delivery as a treatment for specific forms of muscular dystrophy may begin within the next several years (Sweeney, personal communication). Data on route of administration, optimal dose and possible side effects will be obtained from these clinical trials. If efficacy is demonstrated and side effects are small, one can imagine that many people, young as well as old, might well be interested in receiving genetic muscle treatments to enhance muscle size and strength. Developing a product for which the eventual potential market is 100% of the human population will be hard to resist.

Some human contexts for future genetic muscle treatments

What would be the human significance of genetic muscle treatments becoming safe, inexpensive and thus potentially widespread in the future? How would it change the physical (and mental) experience of middle and old age? Preventing the decline of skeletal muscle size and strength in older persons would probably decrease the number of their falls and fractures, but would it also decrease the apprehensiveness and growing timidity that frequently accompany old age? Would such application come to blur the distinction between being “young” and being “old”? Would there be any changes in relations between the generations if the young ceased to be physically superior to the old?

How might such future genetic muscle enhancement be used by persons between the ages of say 20 and 50? Given the popularity of body-building and fitness today, one could imagine its use to enhance those activities, both in competitive and non-competitive settings. The commercial and competitive pressures to use genetic muscle treatments to build up, maintain and repair the muscles of competitive professional athletes in all sports would be very strong. Are not pressures to build muscles (even using anabolic steroids) already felt by student athletes in colleges and high schools? Since athletic competition extends down to youth soccer and Little League baseball, would there any place to draw a line against using genetic muscle treatments?

What would be the responsibilities of parents toward their children’s muscle development in a society where genetic muscle treatments were safe, inexpensive and widespread? Should parents allow their children’s muscles to develop “naturally” through age 20? What should they do when daughter Jenny’s soccer coach tells them she would be a stronger player if they got her genetic muscle treatments, or that she won’t make the team unless she gets treated? Would untreated children become stigmatized in a society where many others had genetic muscle treatments?

Should these biotechnological approaches be applied to aged human muscles?

“Sarcopenia” is currently a descriptive term for the loss of muscle size and strength that accompanies aging. It is not yet the name of a human disease. We will have to come to some societal judgment about whether sarcopenia is a disease, and therefore whether biotechnological approaches to treating it are appropriately termed therapies.

Within American society, there is probably a diversity of views about sarcopenia. One view would assert that loss of muscle strength with age is part of the natural human condition, part of the natural trajectory of human life. From this starting point, attempts to delay this process are “unnatural” and therefore suspect. At least, it would be argued, there is no moral requirement that modern medicine move heaven and earth to fix this problem, particularly by expensive, novel biotechnological means.

An alternative view would point to the fact that loss of muscle strength on aging predisposes to falls and broken bones, major sources of morbidity in the elderly. If we could prevent or delay such loss of muscle strength and thereby decrease the frequency of broken bones in the elderly, would not both individuals and society benefit?

Having agreed that sarcopenia puts a name on an important social problem, doesn’t mean that we must be committed to muscle gene injections as the solution. “Old-fashioned” approaches such as diet and exercise are effective at slowing the loss of muscle size and strength during aging. Would it be a good use of brainpower and resources to develop genetic approaches to treating sarcopenia, if in the end all it does is substitute for regular gym visits by older persons for resistance strength training?

Genetic muscle treatments are being evaluated as possible therapies for various dystrophic conditions of skeletal muscle. However, as this paper points out, once such a technology is developed and applied within the medical sphere, there will be substantial pressures to use it in a variety of settings that are currently “beyond therapy”. Genetic muscle treatments that go “beyond therapy” are another example of an emerging bioethical dilemma…should we apply our increasing knowledge of human biology to give future generations of humans biological capabilities that past generations never had?

 

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  1. This discussion owes much to the work (see references 9 and 10 below) of Professor H. Lee Sweeney and his colleagues at the University of Pennsylvania and elsewhere, and to his description and discussion of that work at the September 2002 meeting of the President’s Council on Bioethics.
  2. Tzankoff, S.P., and A.H. Norris, J. Appl. Physiol. 43 (1977): 1001-1006
  3. The term “sarcopenia” was first suggested by I.H. Rosenberg in 1989. It is derived from the Greek words meaning “poverty of flesh”. See I.H. Rosenberg. (1989) Summary Comments, Am J Clin Nutr. 50: 1231-1233.
  4. See, for example Haslett, J.N., et al., “Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle”, Proc. Nat. Acad. Sci. USA, 99 (2002): 15000-15005
  5. Zammit, P.S., and J.R. Beauchamp, “The skeletal muscle satellite cell: stem cell or son of stem cell?”, Differentiation, 68 (2001): 193-204
  6. Owino, V., et al., “Age-related loss of skeletal muscle function and the ability to express the autocrine form of insulin-like growth factor-1 (MGF) in response to mechanical overload”, FEBS Letters, 505 (2001): 259-263
  7. Brown, W.F., “A method for estimating the number of motor units in thenar muscles and the changes in motor unit count with aging”, J Neurol Neurosurg Psych, 35 (1972): 845-852
  8. Roubenoff, R. and V.A. Hughes, “Sarcopenia: Current concepts”, The Journals of Gerontology, Biological and Medical Sciences, Series A, 55A (2000): M716-M724
  9. Barton-Davis, E., et al., “Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function”, Proc. Nat. Acad. Sci. USA, 95 (1998): 15603-15607
  10. Musaro, A., et al., "Localized Igf-1 transgene expression sustains enlargement and regeneration in senescent skeletal muscle", Nature Genetics, 27 (2001): 195-200
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  1. The source for Figure 1 was an Internet site at http://www.sculpturegallery.com/sculpture/david.html.
  2. The source for Figure 2 was http://www.schwarzenegger.com/en/athlete/index.asp?sec=athlete.
  3. These images are from the informative Internet site www.sarcopenia.com.

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