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Stronger, Long-Lasting Skeletal
Muscles through Biotech? 1 Introduction 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 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 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-
proportioned but without much articulation of individual muscles.
The strength and 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 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
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). 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.
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 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. 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. 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? 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? “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? _______________
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