Interspecies Translation: Bovine Marbling to Human Muscular Dystrophy

There are interesting similarities and differences when comparing the histopathology of bovine marbling and human muscular dystrophy. At the simplest level, both conditions are characterized by genetically controlled and more or less inexorable replacement of muscle fibers with fat cells. At issue is whether an improved understanding of these two processes can lead to better outcomes for patients. There are many forms of dystrophy that differ in their genetics and their histopathology. There are also many forms of “marbling” ranging from the coarse to fine, epimysial, perimysial to endomysial and even to total replacement or steatosis. A detailed examination of marbling will provide a framework for further investiga-tion of human dystrophy. Ultimately, the many genetic factors involved can be addressed through a better understanding of the metabolic pathways involved in marbling.


Introduction
The purpose of this review is to compare the genetics and histopathology of bovine marbling and human muscular dystrophy. Surprisingly, in spite of similarities, the literature suggests that marbling is a function of extreme adipogenesis whereas dystrophy is a consequence of fundamental defects in muscle itself. In fact, completely independent studies, as summarized here, reveal that similar genes have been implicated in some selected situations. Further, it is clear that the histopathology of some forms of dystrophy can resemble some forms of bovine marbling.

Marbling
Marbling is the term used to describe the presence of macroscopically visible fat within muscle (Figures 1 and 2). Coarse marbling refers to white areas of fat through and around muscle bundles, generally as continuous bands arising from the subcutaneous adipose tissue. By contrast, fine or "snowflake" marbling is characterized by more even white flecks resulting in pink rather than red muscle.

Interspecies translation
Interspecies translation from cattle to man has unrecognized potential. Firstly, cattle are close to humans in evolutionary time and fall within that window of 50-100 million years of separation (or last common ancestor) which is characterized by very similar proteins but vastly different regulations of expression. The same window may explain the fact that the two species have synergized over some 40,000 years of contact and at least 7000 years of domestication. As one example, infections can be similar and, in some cases, are transmissible from one to the other, but close exposure to cattle is generally innocuous implying some form of immunity. As for example in the case of pox and tuberculosis. We argue that cattle are both relevant and relatively safe for translational studies.   Secondly, domestic cattle are well maintained, closely observed, and very well understood. There are huge databases and DNA banks which have been in existence for 50 years. Innumerable breeds can be compared often under different environmental conditions. Many of these breeds have been closed for hundreds of years and then intentionally crossed with each other. There is great potential for meaningful studies of population genetics and family and haplotype associations and, even more so, for structure-function genomics. Metabolic and inflammatory pathways are relatively well understood and are supported by inestimable funding available to ensure future supplies of meat, milk, cheese, butter, leather, and fertilizer.
Thirdly, cattle are plentiful and even more so than humans. Because the generation time and life expectancy are much shorter, there are excellent opportunities to study and treat genetically determined diseases prospectively [2].

Other instances of translation
White muscle disease or selenium/vitamin E deficiency occurs quite commonly in livestock raised on leached soils. The pathology resembles dystrophy in some respects. A mutation in the selenoprotein N gene (SEPN1) is responsible for some types of congenital muscular dystrophies and myopathies [3]. Kakulas [4] demonstrated that dystrophy-like changes explained the weakness observed in quokkas on Rottnest Island. Importantly, the condition could be corrected by treating the deficiency raising the possibility that human dystrophies could be reversible if the basic defect could be corrected.

Genomic approach
The term genome is used here to refer to the architecture of DNA sequences, whereas others have come to use the term in the context of single-nucleotide polymorphisms wherever they occur. The difference is fundamental to the discovery of gene clusters with coherent cis and trans interactions between conserved sequences known as ancestral haplotypes [5][6][7][8][9]. Many studies have shown that the SNP approach in livestock and humans fails to identify these critical sequences and can be misleading at best [10]. SNPs are neutral markers of parentage rather than functionally important [11].
One major benefit of ancestral haplotypes as opposed to SNPs is that it is possible to use interspecies translation. During mammalian evolution, polymorphic frozen blocks have diverged to some extent although the functionally important sequences tend to be conserved.
As shown in Figure 3 and Table 1, there are similarities between genomic regions on Hosa 17 and Bota 19. Although there have been architectural changes such as insertions and transversions, the gene content has been preserved.
Bota 19 was chosen as the reference because of its critical role in determining the degree of marbling between individuals of a breed, F1 crosses and between breeds [5,[12][13][14].
Hosa 17 was chosen for comparison because it contains some of the same genes such as TCAP. Further analysis revealed an extraordinary degree of preservation or synteny in spite of an evolutionary separation time of at least 50 million years and therefore millions of generations. Implicit is that there are functional reasons for similarities in genomic architecture.

Gene location Description
Human muscular dystrophy Meat quality trait   Table 1 for more information about these genes. Yet further analysis suggests some explanations for the co-location of similar genes. Irrespective of cis and trans interactions between the protein products, there is evidence of co-regulation (see, e.g., SREBP). In this context, we conclude that, although products and their regulating transcription factors are preserved, separation has permitted the insertion of species-specific elements, which control the quantitative differences between humans and cattle.
Importantly, as shown in Figure 3 and Table 1, Hosa 17 contains multiple candidates for involvement in human muscular dystrophy. There is even more complexity in explaining the multiple candidates as shown in Tables 2 and 3.
Thus, syntenic analysis has suggested a novel approach to identification of operative elements in marbling and in some forms of dystrophy. The alpha-7 integrin is a specific cellular receptor for the basement membrane proteins laminin-1, laminin-2, and laminin-4. The alpha-7 subunit is expressed mainly in skeletal and cardiac muscles and may be involved in differentiation and migration processes during myogenesis Congenital muscular dystrophy EMD Xq28 Emerin is found along the nuclear rim of many cell types and is a member of the nuclear lamina-associated protein family Plectin-1 is one of the largest polypeptides known and is believed to provide mechanical strength to cells and tissues by acting as a cross-linking element of the cytoskeleton Epidermolysis bullosa with muscular dystrophy Limb-girdle type 2Q

Histopathological approach
The substantial range of changes found in the human dystrophies is illustrated in the study of Dubowitz et al. [15].
We are fortunate in having histological muscle samples from cattle with degrees of marbling [14]. Some of these changes are illustrated in Figures 4-8 from three animals (M508, M621, and M129) fed a standard ration for 471, 443, and 481 days respectively. The macroscopic measure of marbling (MSA MB) ranged from high to moderate (1100, 920 and 820, respectively) as expected in high content Wagyu (88, 75, and 63%, respectively). A common feature is the invasion of adipose tissue between intact muscle fascicles (Figure 4). For the most part, the process extends along the perimysium leading to variation in fiber size, staining of myofibers (Figures 5 and 6), and the formation of residual islands of myofibers (Figure 7), which suggests an explanation for fine (see Figure 1) rather than coarse (see Figure 2) marbling; fine is due to more aggressive invasion reflecting quantitative differences in gene regulation.    1, 4, and 5.
In some fields, there are collections of nuclei including intracytoplasmic (Figure 8).
These observations have led us to the conclusion that the extent and type of marbling is a function of the aggressive extension of the advancing adipocytes with secondary loss of myocytes.   Some forms of human dystrophy have very similar histopathology, for example, congenital myopathies as illustrated by Dubowitz et al. [15] and reproduced here in Figure 9.
As in human dystrophies, there can be different degrees depending upon the muscle group and the field selected. Here, we focus on Sacrocaudalis dorsalis medialis, because it is convenient to biopsy, whereas the loin can only be accessed readily post-mortem. Accordingly, it will be possible to undertake detailed time course studies so as to monitor sequential changes and eventually responses to therapy. Future studies should also address bovine steatosis. The pathology [16,17] is different from marbling. Adipocytes occur within rather than around fascicles (Figure 10) suggesting that the process may be a function of differentiation of stem cells, rather than invasion [1].

Conclusion
In spite of similarities in pathology and genomics, there is more to learn before precise translation is possible. However, there are strong indications that such approaches could have important implications for human dystrophies and other muscle diseases. Moreover, a better understanding of the control factors and signals responsible for determining the relative proportions of muscle and adipose tissue in bovine muscles, and how they are coordinated, is fundamental and will be crucial to understanding more fully the significance of adipose tissue replacement in human dystrophies and to developing new therapeutic strategies for these diseases.