Organization of intramuscular connective tissue

All connective tissues (cartilage, bone, blood and interstitial tissue) possess three common components: cells, fibers and ground substance. Extracellular matrix tissue refers to a major portion of intramuscular connective tissues surrounding muscle fibers and other cells, which is composed of collagen, elastin, fibronectin, proteoglycans, and other ground substance components (Purslow, Reference Purslow, Archile-Contreras and Cha2014). Embedded in extracellular matrix and connective tissue, there are abundant fibroblasts, adipocytes, immune cells, preadipocytes, mesenchymal progenitor cells, and other stromal vascular cells. Connective tissue and associated proteins organize muscle structure, connect muscle fibers to the bone for locomotion, and also mediate muscle growth and development (Sanes, Reference Sanes2003; Jenniskens et al., Reference Jenniskens, Koevoet, de Bart, Weinans, Jahr, Verhaar, DeGroot and van Osch2006). The connective tissues surrounding each muscle fiber, termed endomysium, comprised two layers. The inner layer, termed basal lamina, is a 50 to 100 nm thick layer surrounding the sarcolemma, which connects muscle fibers to extracellular niche environment and regulates myogenesis (Wang et al., Reference Wang, Liu, Tsai, Chen, Chang, Tsai, Leu, Zhen, Chai, Chung, Chua, Yen and Yip2014), and muscle growth (Velleman, Reference Velleman1999). Outside of the endomysium, a thin layer of connective tissue, which integrates into thicker layers between muscle bundles, termed perimysium, and surrounding each muscle, termed epimysium. These connective tissues connect muscle fibers and bundles together, and maintain muscle integrity. Intramuscular adipocytes, blood vessels and nerves are integrated into the connective tissue matrix of the muscle.

Connective tissue structure

Collagen is the major component of connective tissue. There are a number of different types of collagens, which are derived from more than 30 genes (Myllyharju and Kivirikko, Reference Myllyharju and Kivirikko2004; Veit et al., Reference Veit, Kobbe, Keene, Paulsson, Koch and Wagener2006; Soderhall et al., Reference Soderhall, Marenholz, Kerscher, Ruschendorf, Esparza-Gordillo, Worm, Gruber, Mayr, Albrecht, Rohde, Schulz, Wahn, Hubner and Lee2007). However, in muscle, types I and III collagen are dominant (Light et al., Reference Light, Champion, Voyle and Bailey1985). The ratio of type I to III may be altered depending on muscle types, locations and animal ages (Listrat et al., Reference Listrat, Picard and Geay1999). In mature bovine muscles, type I collagen is more abundant in perimysium, but type III collagen levels are enriched in the endomysium (Mayne and Sanderson, Reference Mayne and Sanderson1985). In rats, during aging, the proportion of type I collagen increased, while type III collagen decreased (Kovanen and Suominen, Reference Kovanen and Suominen1989); an increase in type I collagen was also observed in the intramuscular connective tissue of beef cattle at around 6 months of age (Listrat et al., Reference Listrat, Picard and Geay1999). Up to now, most studies about connective tissue in muscle have been focused on types I and III collagens (Sato et al., Reference Sato, Ando, Kubota, Origasa, Kawase, Toyohara, Sakaguchi, Nakagawa, Makinodan, Ohtsuki and Kawabata1994; Sato et al., Reference Sato, Sakuma, Ohtsuki and Kawabata1997; Duarte et al., Reference Duarte, Paulino, Das, Wei, Serao, Fu, Harris, Dodson and Du2013).

Each collagen molecule contains three helical polypeptide chains, which are interwined. At both ends, however, non-helical regions termed telopeptide regions are found. Lysyl oxidase is a critical enzyme regulating collagen cross-linking (Siegel and Fu, Reference Siegel and Fu1976; Siegel et al., Reference Siegel, Fu and Chang1976). Lysyl oxidase oxidizes lysine or hydroxylysine in the non-helical portions of collagen molecules to aldehydes, which then react with neighboring collagen molecules to form divalent bonds. Therefore, the presence of lysine and hydroxylysine in the non-helical regions is critical in determining cross-linking development (Robins, Reference Robins2007). The degree of collagen cross-linking differs in animals of different breeds. In our study with Wagyu and Angus cattle, we found that the collagen content and cross-linking are higher in Wagyu, which correlates with less soluble collagen content (Duarte et al., Reference Duarte, Paulino, Das, Wei, Serao, Fu, Harris, Dodson and Du2013). We also observed that early nutrition affects collagen content and cross-linking in sheep (Huang et al., Reference Huang, Yan, Zhu, McCormick, Ford, Nathanielsz and Du2010). In addition, collagens of different muscle types have various degrees of cross-linking, with the collagen in longissimus muscle having less cross-linking than biceps muscle (Dubost et al., Reference Dubost, Micol, Meunier, Lethias and Listrat2013a), correlated with meat tenderness. Collagen cross-linking is a slow process, which increases as animals age, and the high degree of cross-linking is one of the primary reasons for the toughness of meat from old animals. On the other hand, collagens undergo consistent turnover, albeit slower than other proteins. Because newly synthesized collagens do not contain cross-linking, factors that enhance collagen turnover, reduce cross-linking and improve meat tenderness (Purslow, Reference Purslow, Archile-Contreras and Cha2014). Indeed, cross-linking was reduced and soluble collagen content was raised in compensatory growing pigs (Kristensen et al., Reference Kristensen, Therkildsen, Riis, Sørensen, Oksbjerg, Purslow and Ertbjerg2002). Collagen turnover, or remodeling, is regulated by metalloproteinases (Woessner, Reference Woessner1991; Murphy, Reference Murphy2010). The expression of metalloproteinases and their inhibitors, the tissue inhibitors of metalloproteinases, are regulated by a number of factors (Clark et al., Reference Clark, Swingler, Sampieri and Edwards2008), such as inflammation and oxidative stress, which affect cross-linking and meat tenderness (Purslow, Reference Purslow, Archile-Contreras and Cha2014).

Fibrogenic cells and adipocytes share common progenitor cells

During early skeletal muscle development, mesenchymal stem cells first diverge to either myogenic or non-myogenic lineages. Myogenic progenitors further develop into muscle fibers and satellite cells, whereas non-myogenic progenitor cells develop into the stromal-vascular fraction of mature skeletal muscle in which resides adipocytes, fibroblasts and resident mesenchymal progenitor cells (Du et al., Reference Du, Huang, Das, Yang, Duarte, Dodson and Zhu2013). These non-myogenic progenitors have adipogenic and fibrogenic capacity, as well as osteogenic and chondrogenic potential (Joe et al., Reference Joe, Yi, Natarajan, Le Grand, So, Wang, Rudnicki and Rossi2010; Wosczyna et al., Reference Wosczyna, Biswas, Cogswell and Goldhamer2012). These cells are mainly located in the stromal-vascular fraction of skeletal muscle and are distinct from satellite cells (Joe et al., Reference Joe, Yi, Natarajan, Le Grand, So, Wang, Rudnicki and Rossi2010; Uezumi et al., Reference Uezumi, Ikemoto-Uezumi and Tsuchida2010). Platelet-derived growth factor receptor α (PDGFRα) is a reliable marker for separating these cells, and Sca-1⁺CD34⁺ appears to label the same cell population (Joe et al., Reference Joe, Yi, Natarajan, Le Grand, So, Wang, Rudnicki and Rossi2010; Uezumi et al., Reference Uezumi, Ikemoto-Uezumi and Tsuchida2010, Reference Uezumi, Ito, Morikawa, Shimizu, Yoneda, Segawa, Yamaguchi, Ogawa, Matev, Miyagoe-Suzuki, Takeda, Tsujikawa, Tsuchida, Yamamoto and Fukada2011 and Reference Uezumi, Fukada, Yamamoto, Takeda and Tsuchida2014).

The notion that mesenchymal progenitor cells as the common sources of adipogenic and fibrogenic cells are further proven by the co-expression of PDGFRα with fibrogenic markers (Murphy et al., Reference Murphy, Lawson, Mathew, Hutcheson and Kardon2011), or PDGFRα with adipogenic markers (Yang et al., Reference Yang, Liang, Rogers, Zhao, Zhu and Du2013). Transcription factor 4 (TCF4), also known as transcription factor 7-like 2 (Tcf7l2), was first found to be related with limb development by interacting with Wnt signaling pathway (Cho and Dressler, Reference Cho and Dressler1998). Subsequent studies demonstrate TCF4 as a fibrogenic marker (Kardon et al., Reference Kardon, Harfe and Tabin2003; Mathew et al., Reference Mathew, Hansen, Merrell, Murphy, Lawson, Hutcheson, Hansen, Angus-Hill and Kardon2011). A portion of TCF4⁺ fibroblasts also express PDGFRα (Murphy et al., Reference Murphy, Lawson, Mathew, Hutcheson and Kardon2011), showing the intrinsic relationship between mesenchymal progenitor cells and TCF4⁺ fibroblasts. Similarly, in our previous studies, we detected the co-expression of PDGFRα with ZFP423, a marker of adipogenic commitment (Yang et al., Reference Yang, Liang, Rogers, Zhao, Zhu and Du2013). The lack of TCF4+ and ZFP423 co-expressed cells show the divergence of the fibrogenic and adipogenic lineages during progenitor differentiation.

Mechanisms regulating fibrogenesis

Transforming growth factor (TGF)-β is the most important profibrogenic cytokine (Liu and Pravia, Reference Liu and Pravia2010). TGF superfamily contains several structurally related subfamilies, including TGF-β, bone morphogenetic proteins and activin. Three isoforms of TGF-β have been identified, which are TGF-β1, TGF-β2 and TGF-β3. The TGF-β1 isoform is primarily expressed in endothelial cells, fibroblasts, hematopoietic cells and smooth muscle cells; TGF-β2 mainly exists in epithelial cells and neurons; and TGF-β3 is specifically expressed in mesenchymal cells (Ghosh et al., Reference Ghosh, Murphy, Turner, Khwaja, Halka, Kielty and Walker2005). All TGF-β isoforms activate down-stream SMAD signaling (Attisano and Wrana, Reference Attisano and Wrana1996; Letterio and Roberts, Reference Letterio and Roberts1998). The SMAD family contains five receptor-regulated SMAD (R-SMAD 1, 2, 3, 5 and 8), a common SMAD (Co-SMAD 4), and two inhibitor SMAD (I-SMAD 6 and 7) (Moustakas et al., Reference Moustakas, Souchelnytskyi and Heldin2001). The ligand, TGF-β, first binds to TGF-β receptor II (TβRII), which then recruits and activates TβRI. Then SMAD2 and SMAD3 are phosphorylated and subsequently bind to SMAD4 (Suwanabol et al., Reference Suwanabol, Kent and Liu2011), and the resulting SMAD complex is translocated into the nucleus where it binds to SMAD-specific binding elements of target genes, thereby activating the expression of fibrogenic genes including procollagen and enzymes catalyzing collagen cross-linking (Massague and Chen, Reference Massague and Chen2000). As an anti-inflammatory cytokine, TGF-β signaling is enhanced by inflammation (Bhatnagar et al., Reference Bhatnagar, Panguluri, Gupta, Dahiya, Lundy and Kumar2010; Voloshenyuk et al., Reference Voloshenyuk, Hart, Khoutorova and Gardner2011), while inhibited by anti-inflammatory factors (Wang et al., Reference Wang, Dumont and Rudnicki2012).

Connective tissue growth factor (CTGF) is a crucial switch to regulate downstream fibrotic progress (Grotendorst, Reference Grotendorst1997; Leask et al., Reference Leask, Denton and Abraham2004). CTGF is a member of CCN family, which are cysteine rich proteins. CTGF gene expression is induced by TGF-β-activated Smad3 binding to its promoter region (Denton and Abraham, Reference Denton and Abraham2001; Holmes et al., Reference Holmes, Abraham, Sa, Shiwen, Black and Leask2001). Then, CTGF directly stimulates fibroblast proliferation and ECM deposition (Shi-Wen et al., Reference Shi-Wen, Leask and Abraham2008; Morales et al., Reference Morales, Cabello-Verrugio, Santander, Cabrera, Goldschmeding and Brandan2011). Wingless/int (Wnt) signaling pathway plays a crucial role in cell fate commitment (Dorsky et al., Reference Dorsky, Moon and Raible1998; Ross et al., Reference Ross, Hemati, Longo, Bennett, Lucas, Erickson and MacDougald2000), and synergizes with TGF-β signaling to promote connective tissue synthesis and fibrosis (Brack et al., Reference Brack, Conboy, Roy, Lee, Kuo, Keller and Rando2007; Zhou et al., Reference Zhou, Liu, Kahn, Ann, Han, Wang, Nguyen, Flodby, Zhong, Krishnaveni, Liebler, Minoo, Crandall and Borok2012; Cisternas et al., Reference Cisternas, Vio and Inestrosa2014).

Ski/sno family includes ski and sno, which has four distinct isoforms SnoN, SnoN2, SnoA and Snol (Nomura et al., Reference Nomura, Sasamoto, Ishii, Date, Matsui and Ishizaki1989; Pearson-White, Reference Pearson-White1993; Pelzer et al., Reference Pelzer, Lyons, Kim and Moreadith1996). Ski/sno family acts as negative regulators of TGF-β1 pathway by functioning on the downstream signal molecules R-smad/Co-smad complex (Luo, Reference Luo2004; Deheuninck and Luo, Reference Deheuninck and Luo2009; Jahchan and Luo, Reference Jahchan and Luo2010), thus reducing connective tissue deposition.

MicroRNAs regulate cell differentiation through inhibiting the expression of target genes. MiR-101a inhibits fibrosis by targeting the TβRI on cardiac fibroblasts (Zhao et al., Reference Zhao, Wang, Liao, Zeng, Li, Hu, Liu, Meng, Qian, Zhang, Guan, Feng, Zhou, Du and Chen2015). High glucose increases the activity of transcriptional co-activator p300, which subsequently enhances the activity of TGFβ pathway by inducing Smad2 acetylation (Bugyei-Twum et al., Reference Bugyei-Twum, Advani, Advani, Zhang, Thai, Kelly and Connelly2014). Besides, ERK5, one of the MAPK family members, is a critical regulator in TGF-β1-induced lung fibrosis by enhancing Smad3 acetylation (Kim et al., Reference Kim, Lim and Woo2013). A number of cytokines and growth factors, which are involved in the regulation of fibrogenesis are listed in Table 1.

Table 1 Factors enhancing and decreasing intramuscular fibrogenesis

CTGF=connective tissue growth factor; FGF-2=basic fibroblast growth factor; MMPs=matrix metablloproteinase; PDGF=platelet-derived growth factor; TGFβ=tumor growth factor β; TIMP=tissue inhibitor of metalloproteinase; Wnts=wingless and ints.

Antagonistic effects of adipogenesis on fibrogenesis

Because fibrogenesis and adipogenesis are considered as a competitive process, enhancing adipogenesis reduces fibrogenesis. Adipogenesis can be separated into two steps, the commitment of progenitors to preadipocytes, and the differentiation of preadipocytes to mature adipocytes. Quite recently, Zfp423 was identified as the key regulator committing progenitors to preadipocytes; in addition, Zfp423 promotes the expression of peroxisome proliferator-activated receptor γ, the crucial transcription factor inducing the conversion of preadipocytes to adipocytes (Gupta et al., Reference Gupta, Arany, Seale, Mepani, Ye, Conroe, Roby, Kulaga, Reed and Spiegelman2010; Gupta et al., Reference Gupta, Mepani, Kleiner, Lo, Khandekar, Cohen, Frontini, Bhowmick, Ye, Cinti and Spiegelman2012). Importantly, in cattle mesenchymal progenitor cells, the expression of Zfp423 is negatively correlated with TGF-β1 expression, indicating the mutual exclusion of adipogenesis and fibrogenesis (Huang et al., Reference Huang, Das, Yang, Zhu and Du2012a).