**6. 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.

#### **Figure 4.**

*Highly marbled loin muscle shows a pattern of fat arborization and invasion with adipocytes predominantly in the perimysium, between muscle fascicles. Note extensive vascularization centrally within the fat. M508 (wy63 ak25 dx13), MSA MB 1100, DOF 471. See also Figure 1.*

**111**

**Figure 7.**

*Figures 1, 4, and 5.*

*Interspecies Translation: Bovine Marbling to Human Muscular Dystrophy*

*Histological section of Sacrocaudalis dorsalis medialis of a highly marbled, high Wagyu content (88%) steer M508 (wy63 ak25 dx13), showing variation of fiber size, with the presence of rounded fibers, internal nuclei, abundant perimysial connective tissue, and considerable adipose tissue. Formalin-fixed H & E, MSA MB 1100, DOF 471. CYO lab number Ch18/110G. See also Figure 1 for macroscopic* 

*Histological section of Sacrocaudalis dorsalis medialis of a highly marbled, high Wagyu content (75%) heifer M621 (wy75 dx25). Field selected to show eosinophilic rounded fibers of variable size, with abundant perimysial connective tissue in their proximity. Formalin-fixed H & E, MSA MB 820, DOF 471. CYO lab* 

*Histological section of Sacrocaudalis dorsalis medialis of a highly marbled, high Wagyu content steer (88%) (wy63 ak25 dx13), showing aggressive adipose invasion, with abundant perimysial connective tissue and the generation of island-like areas of fibers with evident architectural changes including shrinkage of fibers as the front advances. Formalin-fixed H & E, MSA MB 1100, DOF 471. CYO lab number Ch18/110G. See also* 

*number Ch18/109Y. See also Figure 2 for macroscopic features such as coarse marbling.*

*DOI: http://dx.doi.org/10.5772/intechopen.82685*

**Figure 5.**

*comparison.*

**Figure 6.**

*Interspecies Translation: Bovine Marbling to Human Muscular Dystrophy DOI: http://dx.doi.org/10.5772/intechopen.82685*

#### **Figure 5.**

*Muscular Dystrophies*

Calpain-3 (easier to assess on immunoblots than sections)

**6. Histopathological approach**

*Protein accumulations and deficits in dystrophy.*

**Table 3.**

the study of Dubowitz et al. [15].

The substantial range of changes found in the human dystrophies is illustrated in

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.

**Absent protein Dystrophy type Gene location**

LAMP-2 Danon disease LAMP2 Xq24 **Accumulating protein Dystrophy type Gene location**

Myosin Myosin storage myopathy MYH7 Hosa 14q11 Myotilin Myotilin-related myofibrillar myopathy MYOT Hosa 5q31.2 Desmin Desmin myopathy DES Hosa 2q35

*(Adapted from [15] Table 6.3 dystrophy related protein changes detectable with immunohistochemistry).*

SERCA 1 Brody disease *ATP2A1 Hosa 16p11.2*

dystrophy, limb-girdle dystrophy 2Q

Emerin X-Linked emery-dreifuss muscular dystrophy

Plectin Epidermolysis bullosa with muscular

Actin Congenital actin myopathy/nemaline myopathy

Limb-girdle muscular dystrophy 2A CAPN3 Hosa 15q15.1 Bota 10

EMD Hosa Xq28

PLEC Hosa 8q24.3

ACTA1 Hosa 1q24.13 TPM3 Hosa 1q23

SEPN1 Hosa 1p36

*Highly marbled loin muscle shows a pattern of fat arborization and invasion with adipocytes predominantly in the perimysium, between muscle fascicles. Note extensive vascularization centrally within the fat. M508 (wy63* 

**110**

**Figure 4.**

*ak25 dx13), MSA MB 1100, DOF 471. See also Figure 1.*

*Histological section of Sacrocaudalis dorsalis medialis of a highly marbled, high Wagyu content (88%) steer M508 (wy63 ak25 dx13), showing variation of fiber size, with the presence of rounded fibers, internal nuclei, abundant perimysial connective tissue, and considerable adipose tissue. Formalin-fixed H & E, MSA MB 1100, DOF 471. CYO lab number Ch18/110G. See also Figure 1 for macroscopic comparison.*

#### **Figure 6.**

*Histological section of Sacrocaudalis dorsalis medialis of a highly marbled, high Wagyu content (75%) heifer M621 (wy75 dx25). Field selected to show eosinophilic rounded fibers of variable size, with abundant perimysial connective tissue in their proximity. Formalin-fixed H & E, MSA MB 820, DOF 471. CYO lab number Ch18/109Y. See also Figure 2 for macroscopic features such as coarse marbling.*

#### **Figure 7.**

*Histological section of Sacrocaudalis dorsalis medialis of a highly marbled, high Wagyu content steer (88%) (wy63 ak25 dx13), showing aggressive adipose invasion, with abundant perimysial connective tissue and the generation of island-like areas of fibers with evident architectural changes including shrinkage of fibers as the front advances. Formalin-fixed H & E, MSA MB 1100, DOF 471. CYO lab number Ch18/110G. See also Figures 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.

#### **Figure 8.**

*Histological section of Sacrocaudalis dorsalis medialis of a highly marbled, high Wagyu content steer (63%), M129 (wy63 dx13). Higher power selected to illustrate variability of fiber size, affinity for eosin, and the presence of intracytoplasmatic and interstitial nuclei. Formalin-fixed H & E, MSA MB 880, DOF 481. CYO lab number Ch18/135Z.*

#### **Figure 9.**

*Examples of adipocyte intrusion in human muscular dystrophy. (a) Case of limb-girdle muscular dystrophy showing most fibers surrounded by endomysial connective tissue with some adipocytes (\*) ([15], Figure 11.4b). (b) From the deltoid muscle of a patient with ophthalmoplegia associated with a MYH2 mutation showing fatty infiltration, mild fiber atrophy, fibers with internal nuclei, an irregular myofibrillar network, and lobulated fibers ([15], Figure 15.27). (c) From the quadriceps of a patient with facioscapulohumeral dystrophy at 42 years showing pronounced proliferation of connective tissue and fat with a wide variation of muscle cell size and many internal nuclei ([15], Figure 14.1b). (d) Low power view of a biopsy from a case of congenital muscular dystrophy showing only islands of fibers in a vast amount of adipose tissue ([15], Figure 4.30).*

**113**

*Interspecies Translation: Bovine Marbling to Human Muscular Dystrophy*

Some forms of human dystrophy have very similar histopathology, for example, congenital myopathies as illustrated by Dubowitz et al. [15] and reproduced here in

*Muscle samples taken from carcasses where steatosis was observed macroscopically at slaughter. Fat infiltration occurs within the muscle fascicle, there are few adipocytes within the perimysium. Used with permission from [17].*

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

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

In spite of similarities in pathology and genomics, there is more to learn before

This research was supported by the CY O'Connor ERADE Village Foundation as publication number MS1803. The authors **acknowledge the earlier contributions of J F Williamson and** would like to thank JR Dawkins and Melaleuka Stud for

Collectively, the authors associated with the CY O'Connor ERADE Village Foundation have an interest in the work described in this manuscript as it forms

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.

*DOI: http://dx.doi.org/10.5772/intechopen.82685*

**Figure 9**.

**Figure 10.**

post-mortem.

than invasion [1].

**7. Conclusion**

**Acknowledgements**

**Conflict of interest**

access to the cattle used in this study.

part of the foundation's intellectual property.

*Interspecies Translation: Bovine Marbling to Human Muscular Dystrophy DOI: http://dx.doi.org/10.5772/intechopen.82685*

#### **Figure 10.**

*Muscular Dystrophies*

secondary loss of myocytes.

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

*Histological section of Sacrocaudalis dorsalis medialis of a highly marbled, high Wagyu content steer (63%), M129 (wy63 dx13). Higher power selected to illustrate variability of fiber size, affinity for eosin, and the presence of intracytoplasmatic and interstitial nuclei. Formalin-fixed H & E, MSA MB 880, DOF 481. CYO* 

*Examples of adipocyte intrusion in human muscular dystrophy. (a) Case of limb-girdle muscular dystrophy showing most fibers surrounded by endomysial connective tissue with some adipocytes (\*) ([15], Figure 11.4b). (b) From the deltoid muscle of a patient with ophthalmoplegia associated with a MYH2 mutation showing fatty infiltration, mild fiber atrophy, fibers with internal nuclei, an irregular myofibrillar network, and lobulated fibers ([15], Figure 15.27). (c) From the quadriceps of a patient with facioscapulohumeral dystrophy at 42 years showing pronounced proliferation of connective tissue and fat with a wide variation of muscle cell size and many internal nuclei ([15], Figure 14.1b). (d) Low power view of a biopsy from a case of congenital muscular* 

*dystrophy showing only islands of fibers in a vast amount of adipose tissue ([15], Figure 4.30).*

**112**

**Figure 9.**

**Figure 8.**

*lab number Ch18/135Z.*

*Muscle samples taken from carcasses where steatosis was observed macroscopically at slaughter. Fat infiltration occurs within the muscle fascicle, there are few adipocytes within the perimysium. Used with permission from [17].*

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].

### **7. 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.

### **Acknowledgements**

This research was supported by the CY O'Connor ERADE Village Foundation as publication number MS1803. The authors **acknowledge the earlier contributions of J F Williamson and** would like to thank JR Dawkins and Melaleuka Stud for access to the cattle used in this study.

### **Conflict of interest**

Collectively, the authors associated with the CY O'Connor ERADE Village Foundation have an interest in the work described in this manuscript as it forms part of the foundation's intellectual property.

*Muscular Dystrophies*
