Chronic disease, disuse and aging are all causes of muscle atrophy. This fact directly impacts an individual’s ability to maintain an independent life-style. In contrast, stretching and work overload of skeletal muscle induces hypertrophy of skeletal muscle and myotubes in vivo and in vitro, respectively. Compensatory growth in response to an imposed load is an important and well-known biological adaptation of skeletal muscle. However, little is known about the systemic changes in hypertrophying muscle at the molecular level.
In 1967, A.L. Goldberg made the seminal observation that if workload on a muscle is increased suddenly by tenotomy of a synergistic muscle; its wet and dry weight may increase by 30-50% within 6-d. Hypertrophic growth was evident within 24-h and reached maximum levels by 5-d. Furthermore, the rate and extent of skeletal muscle hypertrophy was similar in hypophysectomized and normal animals. On the basis of these studies, it was concluded that increased workload stimulated skeletal muscle growth and that pituitary growth hormone, which is required for normal muscle growth, and thyroid hormones were not essential for skeletal muscle hypertrophy to occur (Goldberg, 1967). As a direct result, many researchers have investigated the biological mechanisms responsible for work overload induced skeletal muscle hypertrophy. This was followed by the additional conclusions that skeletal muscle hypertrophy was independent of insulin, which is required for normal postnatal growth of skeletal muscle (Goldberg, 1968). Skeletal muscle hypertrophy is indistinguishable in male and female rats (Mackova and Hnik, 1972) and it can occur in fasting animals (Goldberg, 1971). In addition, skeletal muscle hypertrophy results in increased amino acid transport (Arvil, 1967), increased satellite cell proliferation (Schiaffino et. al., 1972), increased protein synthesis, decreased protein degradation (Goldberg, 1969), and involves fiber-type switching (Booth and Baldwin, 1996). Whether induced by stretch-overload or stimulation, hypertrophy is accompanied by an increase in total RNA content, which was evident within six hours after tenotomy of a synergistic muscle (Goldberg, 1971). Maximal RNA synthesis was observed three days after the initiation of work overload. De novo synthesis of RNA in response to work overload appears to play an essential role, because treatment of tenotomized animals with actinomycin D, an inhibitor of DNA-dependent RNA synthesis, prevented skeletal muscle hypertrophy (Goldberg and Goodman, 1969), which may be a result of the associated increases in RNA polymerase activities and the rate of RNA synthesis (Carson, 1997).
The hypertrophic signal is intrinsic, as it is primarily the exercised muscle that undergoes hypertrophy and not all the muscles of the limb or the whole body. It appears the hypertrophying skeletal muscle produces autocrine growth factors and that the morphological basis of the mechanism involves the cytoskeleton and the extracellular matrix. The stretching of skeletal muscle results in the activation of intracellular signaling molecules. While there is a body of knowledge that suggests integrins and autocrine growth factors are involved in mechanotransduction (i.e., conversion of a mechanical force into a chemical signal), it is still unclear what the global changes in gene expression pattern are and what intracellular signaling pathways are involved (Figure 1).
Figure 1. A possible model whereby a physical force (i.e. stretch or work overload) is converted into a chemical signal, thereby, regulating gene expression.
Figure 2. In vivo model of stretch induced skeletal muscle hypertrophy
The cellular mechanisms contributing to an increase in skeletal muscle mass, a desired output of the livestock industry, include increased cell size as a result of increased muscle protein accretion, satellite cell proliferation and subsequent fusion of the satellite cells to existing myofibers. Recently, the targeted inactivation of myostatin demonstrated the utility of model organisms in defining a genetic mechanism controlling skeletal muscle growth in livestock (McPherron et al., 1997; McPherron and Lee, 1997; Grobet et al., 1997; Kambadur et al., 1997). There is a body of knowledge that suggests that myostatin inhibits skeletal muscle growth (McPherron et al., 1997; Zhu et al., 2000). In addition, myostatin expression in skeletal muscle increases during muscle atrophy and decreases during the regeneration of skeletal muscle (Carlson et al., 1999; Wehling et al., 2000; Sakuma et al., 2000; Lalani et al., 2000; Gonzalez-Cadavid et al., 1998; Kirk et al., 2000). This correlation suggests that myostatin expression is inversely correlated with the rate of skeletal muscle growth. In contrast to Transforming Growth Factor-b (TGF-b ), which in the presence of serum causes myoblast to become post mitotic (Olson et al., 1986), myostatin inhibits myoblast proliferation and protein synthesis in an autocrine/paracrine manner (Thomas et al.; 2000; Taylor et al.; 2001). However, it is still unclear at a molecular level how myostatin and TGF-b differ in this respect, as they are both members of the TGF super family. Lack of such knowledge is a critical problem, because, until this information becomes available, it will not be possible to develop and effectively evaluate new genetic or therapeutic strategies to specifically inhibit myostatin activity and thereby enhance skeletal muscle growth. Furthermore, myostatin is known to interact with other genes (Casas et al., 2000) to control growth and carcass traits in cattle. However, these interacting genes have not been identified. Thus, we need to define the molecular mechanisms repressing skeletal muscle growth at the molecular level to insure that improvements in the efficiency of lean tissue deposition are made in the future.
There is an increase in skeletal muscle mass and a decrease in body fat in livestock in response to the oral administration of several b -adrenergic agonists (b -AA; for review see Bergen and Merkel, 1991; Mersmann, 1998). While these effects are beneficial to the livestock industry, administration of b -AA can also result in cardiac muscle hypertrophy, which is undesirable. The mode of action of b -AA on gene expression in adipose tissue and cardiac muscle are well documented. In contrast, very little is known about the b -AA activated intracellular signaling pathways in skeletal muscle. Identification of a skeletal muscle specific response to b -AA that was beneficial to lean tissue deposition and not detrimental to cardiac muscle would provide information that is necessary for the development of future growth promotants, which may also have human applications as well.
In cell culture, b -AA increase protein accumulation in skeletal myotubes (Anderson et. al., 1990) and decrease lipid accumulation in adipogenic cells. In growing pigs, oral administration of b -AA results in parallel increases in skeletal muscle mass, protein and RNA content, increased RNA/DNA and protein DNA ratios (Bergen et. al., 1989), increased protein synthesis and decreased protein degradation (Kim and Sainz, 1992), all of which are indicative of skeletal muscle hypertrophy.
Upon binding of an b -AA to the b -adrenergic receptor on adipose cells, the Gs protein ® adenylyl cyclase ® cAMP ® PKA ® CREB intracellular signaling pathway is activated. Thereby regulating adipose-specific gene expression. However, in cardiac muscle, the cAMP ® PKA ® CREB portion of this pathway is incapable of regulating skeletal a -actin gene expression, which is up-regulated in response to b -AA administration (Bishopric et. al., 1992). It has been suggested that b -AA activated intracellular signaling may involve Ca2+ release, MAPK or PI3-kinase activation (Bishopric et. al., 1992; Izevbigie and Bergen, 2000; Morisco et. al., 2000). Although the work cited previously provides convincing evidence that administration of b -AA can alter gene expression, the precise mechanism by which this occurs in skeletal muscle is largely unknown. In addition, because a number of intracellular signaling pathways are activated by b -AA, it is likely that altered intracellular signaling regulates skeletal a -actin gene expression. However, this cannot be established without determining the molecular mechanism whereby b -AA alter the level of gene expression during skeletal muscle hypertrophy. This will require that the specific changes in promoter activity are determined, so the molecular mechanisms of skeletal muscle hypertrophy can be resolved.
It has been estimated that the world population will increase from 6 billion to 8 billion by 2025 (Dyson, 2000). This fact will place a great strain on the world’s food supply. Thus, there is a pressing need to improve the efficiency of food production.
In approximately 30 years, more than 70 million Americans will be over 65 years old, and the 85+ group will be the fastest growing segment of the population. By 60 – 70 years of age, muscle mass of human beings decreases by 25 – 30% (Edstrom and Larsson, 1987). Falling is a serious problem for the elderly, especially for those 85 years and older. Approximately 30% of 65+ year olds sustain a fall, with about half of them falling multiple times. Approximately 10-15% of the time a fall results in serious injuries (Province et. al., 1995) and many of these falls contribute significantly to morbidity and mortality (Robbins et. al., 1989). Buchner et al. (1997) reported that exercise designed to maintain skeletal muscle mass delayed the onset of the first fall, and sped the recovery from fall-related injuries. There are numerous conditions that either result in skeletal muscle atrophy or are the consequence of skeletal muscle atrophy. An example for the latter case is bed-ridden elderly in nursing homes whom develop decubitus ulcers. The cost of skeletal muscle atrophy – medical and emotional – is enormous; not to mention the impact that it has on relatives. In addition, the enlargement of skeletal muscles is of interest to many Americans attempting to improve their athletic ability or physical appearance.
Cardiac Muscle Development
Defining the molecular basis underlying the establishment and maintenance of cardiac muscle differentiation presents a fundamental challenge in the study of developmental biology and molecular genetics, and may eventually lead to an improved understanding of cardiovascular disease. The heart is the first organ formed during embryonic development. Heart development is an elaborate process requiring cell specification, cell differentiation, cell migration, morphogenesis, and interactions among cells from several embryonic origins. Recent studies indicate that the cardiac muscle phenotype is generated through a relatively small number of transcription factors that work in combination to activate downstream of cardiac-specific gene targets. Furthermore, mutations within cardiac-expressed transcription factors have been ascribed to known human genetic disorders. However, it is still unclear what role that transcription factors, which inhibit gene expression, may play during normal heart development. Lack of such knowledge is a critical problem, because, until this information becomes available, it will not be possible to understand in molecular detail the control of gene expression during heart development.
Heart development. The heart is the first functional organ in the developing embryo. It arises from paired regions of dorsolateral mesoderm that are specified by inductive interaction with adjacent tissues. In all vertebrates, the myogenic and endocardial lineages of the heart develop from anterior lateral plate mesoderm and underlying endoderm. Myocardium precursor cells of amphibia and aves are induced by signals which emanate from the organizer or node, an early embryonic patterning center, and then from anterior endoderm (Sater et al., 1989; Sater et al., 1990; Schultheiss et al., 1995; Nascone et al., 1995). Bone morphogenetic proteins appear to have an early role in signaling the formation of mesoderm in embryos. Schultheiss et al. (1997) and Andree et al. (1998) demonstrated that ectopic placement of BMP-2, -4, and -7 medial to the cardiac crescent of chick embryos was capable of inducing Nkx2-5 and GATA4. Furthermore, heart development is perturbed by the inactivation of BMP-2 (Zhang and Bradley, 1996).The primary heart tube forms from the progressive fusion of two cardiac primordia that arise from a region of splanchnic mesoderm found on either side of the anterior-posterior embryonic axis and ventral to the endoderm (Rawles, 1943; Rosenquist and DeHaan, 1966). The heart becomes morphologically distinguishable as a simple tube, which consists of two epithelial layers: the inner endocardium and the outer myocardium (Manasek, 1968). This tubular organ then undergoes a complex series of morphogenetic events to form a looped structure with a common atrium and a common ventricle. The atrium and ventricle then undergo septation and valves develop (Mjaatvedt et. al., 1999; De la Cruz and Markwald, 1998). Based on genetic and evolutionary considerations, it has been suggested that the heart is a modular organ, with individual modules that represent unique and separate innovations, which were added to the heart in a stepwise manner during evolution. This model accounts for the null phenotypes of several heart-expressed gene, in which individual modules were deleted with little effect on overall patterning (Fishman et al., 1997a,b). Thus, an understanding of molecular mechanisms governing embryonic heart formation, and gene expression is indispensable to defining and exploiting the most promising leads for applications to heart repair.
We have reached a critical stage in genetic improvement of cattle. Although significant progress has been made, we are severely disadvantaged in our ability for future improvement by the lack of genomic information and resources. Barendse et al. (1997) and Kappes et al. (1997) reported on the generation of primarily microsatellite-based medium- and high-resolution linkage maps for the bovine genome, respectively. These maps provide a sufficient marker density for genomic scans of populations with segregating quantitative trait loci (QTL) for carcass and growth traits (Elo et. al., 1999; Keele et. al., 1999; Stone et. al., 1999; Casas et. al., 2000). But, we have reached a critical stage in the identification of causal genes that resides within a QTL genomic region. Recently, the identification of myostatin as the double-muscling causal gene has demonstrated the utility of QTL mapping in combination with biological data from model organisms in the identification of candidate genes (Grobet et. al., 1997). These results demonstrate the need to identify bovine genes and understand their function to expedite the identification of causal genes located within an identified QTL interval. A promising approach for rapidly discovering genes is the production and characterization of expressed sequence tags (EST) (Ma et. al., 1998). Currently, there are several large EST sequencing projects involving beef cattle in the United States and throughout the world. To date, these EST projects have generated >150,000 bovine EST. In addition, these projects have greatly increased the number of type I markers on the bovine genetic map (>1000 and increasing daily)(Dr. Tim Smith, Dr. Harris Lewin, personal communications). This approach, in conjunction with radiation hybrid mapping (Band et. al., 2000; Stewart et. al., 1997) has proven invaluable for the development of the bovine and human gene maps. However, knowing where a gene is located in the genome is not enough. With the advent of microarray technology, microarrays have been developed that contain arrayed cDNA or oligonucleotides that can simultaneously evaluate the expression level of thousands of mRNA species. Thus, the technology currently exists to evaluate global changes in mRNA expression. However, the only bovine-specific microarray developed to date is directed toward reproductive tissues (Band et. al., 2001). Granted many of the genes represented on this array are expressed in many different tissues. However, this array will not contain tissue-specific genes of production interest (i.e. hypothalamus, pituitary, skeletal muscle or adipose). Lack of such resources is a critical problem because, until this technology becomes available, it will not be possible to effectively evaluate new strategies at the level of gene expression to enhance beef cattle production. Thus, we need to develop the tools necessary to evaluate the level of mRNA expression associated with various production traits of interest.
Figure 5. Microarray technology