Intramuscular connective tissue contributes to the background toughness of meat, which is mainly synthesized by intramuscular fibroblasts. Recent studies show that adipocytes and fibroblasts are derived from a common pool of mesenchymal progenitor cells during the early embryonic development. Due to the bipotent developmental potential of these progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which provides an opportunity to improve marbling and tenderness of meat, thus the overall palatability.
Meat quality is determined by flavor, tenderness, juiciness, color, nutritional value and others. Tender meat, which contains more intramuscular fat and less connective tissue is demanded by consumers. Meat tenderness is determined by both the myofibrillar effects and the presence and cross-linking of connective tissue. Myofibrillar contribution to toughness can be partially addressed by aging carcasses, which results in the fragmentation of myofibrils primarily due to proteolysis by calpains (Koohmaraie and Geesink, Reference Koohmaraie and Geesink2006). On the other hand, postmortem aging is ineffective in improving the tenderness of a meat with high collagen content, due to the resistance of collagen to proteolysis. Thus, meat toughness due to connective tissue is called the ‘background toughness’ of meat (Nishimura, Reference Nishimura2010). Consistently, the longissimus muscle in beef cattle contains low collagen and is tenderer while beef from limb muscles possesses higher collagen content and is tougher (McCormick, Reference McCormick1999; Dubost et al., Reference Dubost, Micol, Meunier, Lethias and Listrat2013a). In addition, the cross-linking of collagen has even greater influence on meat toughness (McCormick, Reference McCormick1994). Because during cooking, collagen is gelatinized, which is hampered due to the presence of cross-linking, contributing to the toughness of meat from old animals (Dubost et al., Reference Dubost, Micol, Picard, Lethias, Andueza, Bauchart and Listrat2013b). The detailed effects of connective tissue structure, collagen cross-linking, and their impacts on meat tenderness have been previous reviewed (Purslow, Reference Purslow, Archile-Contreras and Cha2014).
Intramuscular connective tissue is mainly derived from fibroblasts, which are generated through fibrogenesis, a process referring to the generation of fibroblasts and their synthesis of proteins and other components composing the connective tissue. Fibrogenesis is active during the whole life of animals, particularly during the early developmental stage in utero; connective tissues synthesized inside fetal muscle form primordial perimysium and epimysium of muscle bundles at late gestation (Du et al., Reference Du, Yan, Tong, Zhao and Zhu2010). In humans, fibrosis refers to a state of excessive deposition of collagen and other extracellular matrix proteins, which is often elicited by a pathological condition and becomes noticeable during the recovery period (Liu and Pravia, Reference Liu and Pravia2010). Lysyl oxidase is a rate limiting enzyme catalyzing cross-linking of collagen fibrils (Borg et al., Reference Borg, Klevay, Gay, Siegel and Bergin1985; Huang et al., Reference Huang, Zhao, Yan, Zhu, Long, McCormick, Ford, Nathanielsz and Du2012b). Available studies demonstrated that the content and cross-linking of collagen are frequently correlated to each other, but the turnover of collagen reduces cross-linking (Archile-Contreras et al., Reference Archile-Contreras, Cha, Mandell, Miller and Purslow2010), a process increasing tenderness (Hill, Reference Hill1967; Archile-Contreras et al., Reference Archile-Contreras, Mandell and Purslow2011; Purslow et al., Reference Purslow2012).
Intramuscular fat is considered part of the intramuscular connective tissue, and intramuscular adipogenesis is inseparable from fibrogenesis due to closely related developmental origins. However, knowledge regarding regulatory mechanisms, or specific and effective manipulations to augment progenitor cell differentiation to a particular lineage, such as adipogenesis, remains poorly defined. The intent of this review is to provide an overview of current knowledge regarding intramuscular collagen deposition and associated marbling development, and discuss possible mechanisms regulating mesenchymal progenitor cell differentiation focusing on fibrogenesis, and their impacts on muscle growth and meat quality.
Intramuscular connective tissue structure
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).
Development of connective tissue
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.
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).
Connective tissue and muscle development
Satellite cells are critical for muscle growth and regeneration. They are wedged between the basal lamina and the plasma membrane (sarcolemma) of skeletal muscle fibers. Extracellular matrix together with growth factors and cytokines sequestered inside and those secreted by interstitial cells, forms the niche environment needed for satellite cell quiescence, activation, migration, myogenic differentiation and muscle development (Rhoads et al., Reference Rhoads, Fernyhough, Liu, McFarland, Velleman, Hausman and Dodson2009; Dodson et al., Reference Dodson, Hausman, Guan, Du, Rasmussen, Poulos, Mir, Bergen, Fernyhough, McFarland, Rhoads, Soret, Reecy, Velleman and Jiang2010; Murphy et al., Reference Murphy, Lawson, Mathew, Hutcheson and Kardon2011; Urciuolo et al., Reference Urciuolo, Quarta, Morbidoni, Gattazzo, Molon, Grumati, Montemurro, Tedesco, Blaauw, Cossu, Vozzi, Rando and Bonaldo2013).
Muscle regeneration involves extensive proliferation and myogenic differentiation of satellite cells. Shortly after muscle injury, both satellite cells and non-myogenic progenitor cells are activated and proliferate; non-myogenic progenitor cells stimulate satellite cell proliferation and facilitate muscle regeneration (Joe et al., Reference Joe, Yi, Natarajan, Le Grand, So, Wang, Rudnicki and Rossi2010; Murphy et al., Reference Murphy, Lawson, Mathew, Hutcheson and Kardon2011). In addition, intramuscular fibroblasts particularly promote slow myogenesis, thus affecting muscle fiber type composition and overall maturation during muscle development (Mathew et al., Reference Mathew, Hansen, Merrell, Murphy, Lawson, Hutcheson, Hansen, Angus-Hill and Kardon2011). Extracellular component, collagen VI, regulates satellite cell self-renewal and differentiation (Urciuolo et al., Reference Urciuolo, Quarta, Morbidoni, Gattazzo, Molon, Grumati, Montemurro, Tedesco, Blaauw, Cossu, Vozzi, Rando and Bonaldo2013). Besides, other components of extracellular matrix, such as proteoglycan, regulate proliferation and differentiation of satellite cells (Zhang et al., Reference Zhang, Nestor, McFarland and Velleman2007). Decorin, a small leucine-rich proteoglycan, traps TGFβ to regulate satellite cell activation and muscle growth (Li et al., Reference Li, McFarland and Velleman2006 and Reference Li, McFarland and Velleman2008).
Extracellular matrix also interacts with a number of growth factors, including TGFβ, hepatocyte growth factor, fibroblast growth factor 2, myostatin and others to either promote or inhibit muscle growth (Yamaguchi et al., Reference Yamaguchi, Mann and Ruoslahti1990; Rapraeger et al., Reference Rapraeger, Krufka and Olwin1991; Allen et al., Reference Allen, Sheehan, Taylor, Kendall and Rice1995; Miura et al., Reference Miura, Kishioka, Wakamatsu, Hattori, Hennebry, Berry, Sharma, Kambadur and Nishimura2006; Kishioka et al., Reference Kishioka, Thomas, Wakamatsu, Hattori, Sharma, Kambadur and Nishimura2008). Table 2 lists selected growth factors known to interact with extracellular matrix and regulate muscle growth.
EGF=epithelial growth factor; FGF-2=fibroblast growth factor-2; HGF/SF=hepatocyte growth factor/scatter factor; IGF=insulin growth factor; PDGF-BB=platelet-derived growth factor-BB; SDF-1=stromal-derived factor-1; TGFβ=transforming growth factor β.
Intramuscular connective tissue regulates muscle growth and development, and also is the site for intramuscular fat (marbling) deposition. The abundance and cross-linking of intramuscular connective tissue contribute to the background toughness of meat. Connective tissue is mainly synthesized by intramuscular fibroblasts. Non-myogenic mesenchymal progenitor cells are the common source of fibroblasts and adipocytes. Strengthening progenitor cell formation and proliferation enhances both intramuscular adipogenesis and fibrogenesis, while enhancing progenitor differentiation to adipogenesis reduces fibrogenesis, resulting in the overall improvement of marbling and tenderness of meat. Fibrogenesis is mainly regulated by the TGFβ signaling pathway, and a number of factors affect connective tissue deposition via altering TGFβ signaling. Extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment to regulate myogenic differentiation of satellite cells and muscle growth. Despite rapid progress in our understanding of mechanisms regulating fibrogenesis, many questions remain on the synthesis of intramuscular connective tissue and the role of extracellular matrix in muscle development, which warrants further studies.
This project was supported by Agriculture and Food Research Initiative Competitive Grant No. 2015-67015-23219 from the USDA National Institute of Food and Agriculture, and NIH R01 HD067449.