**5.2. Effect of unloading on the synthesis of collagen**

Several quantitative and qualitative changes in the intramuscular connective tissue contribute to the deteriorated function and biomechanical properties of the immobilized skeletal muscle [6,11,51]. Muscle and tendon collagen and the connective tissue network are known to respond to altered levels of physical activity [51,55]. In contrast to physical loading, immobilization of rat limb leads to a decrease in the activities of collagen synthesizing enzymes both in skeletal muscle and tendon [20,56].

Along with the increased amount of intramuscular connective tissue, the number of capillaries decreases dramatically [57,58]. Each capillary is surrounded by a dense layer of the connective tissue fibres, isolating the capillary from the adjacent muscle fibre, which disturbs the blood supply of the muscle fibres and further increases the muscle fibre atrophy [57,58].

During immobilization, the normal three-dimensional orientation of the collagen fibres is disrupted. The normal orientation constitutes of the fibres running parallel to the muscle fibres on their surface, preventing muscle cells from over-elongation and –contraction. In addition, thin perpendicular fibres connect adjacent muscle fibres to each other [7,10,11]. As a result of decreased loading, the number of longitudinal fibres increases, the crimp angel of the collagen decreases and this diminishes the ability of the muscle to elongate [2,11] and because of that the skeletal muscle shows significantly decreased tensile strength [1,3]. In addition to changes in collagen abundance, alterations in the degree of collagen cross-linking would have a profound effect on the mechanical properties of skeletal muscle, causing a decrease in muscle stiffness [10,17].

In contrast to physical loading, immobilization leads to the decrease in the enzyme activities of collagen biosynthesis, which suggests that the biosynthesis of the collagen network decreases as a result of reduced muscular and tendinous activity [8,56]. The rate of the total collagen synthesis depends mostly on the overall protein balance of the tissue, but it seems to be positively affected by stretch in both muscle and tendon [55].

Collagen expression during immobilization has been shown to be at least partially downregulated at the pretranslational level [55]. Although the relative amount of the connective tissue increases during immobilization, the gene expression of type I and III collagens decreases during the first three days of immobilization [20]. The content of type IV collagen was also reduced as a result of immobilization [23]. The activities of prolyl 4-hydroxylase (P 4-H) and galactosylhydroxylysyl glucosyltransferase (GGT) decrease from the first three days of immobilization up to at least three weeks, suggesting decreased collagen biosynthesis during that time [20,59]. The degradation of collagens has been found to be enhanced during immobilization, as the expression of both MMP-2 and MMP-9 increased after 30 days of immobilization [30]. The quantity of TIMP-1 was also increased after 30 days of immobilization [30].

The collagen concentration increases when expressed both as a function of muscle dry weight or muscle cross-sectional area, but this increase in muscle collagen is primarily due to the muscle atrophy induced by immobilization [13].

#### **5.3. Effect of unloading on the contractile apparatus of skeletal muscle**

As the skeletal muscle is a highly plastic tissue, the conditions associated with the disuse are accompanied by adaptation. A period of time without weight bearing cause modifications of structure and the function of skeletal muscles, of which atrophy and a slow-to-fast transition are the most prominent [60,61]. Many animal models such as the kind limb suspension, immobilization in shortened and lengthened position, spaceflight and denervation show that the removal of a mechanical load produces atrophy and contractile alterations, more evident in the slow muscle soleus than in fast muscles as extensor digitorum longus [60,62,63]. Besides space flight and bed rest experiments, long periods of muscle disuse in relation to a disease or traumatic injuries of the joints or of the bones are relatively common experience for human beings [62,63]. The inactivity causes only small increases in contractile speed and myofibrillar adenosine triphosphatase (ATPase) activity and slight elevations in the percentage of the fast type myosin heavy chain (MyHC) isoforms in fast-twitch muscles, as compared to slow-twitch muscle [64,65].

#### **5.4. Slow-to-fast transition in skeletal muscle during unloading**

tissue fibres, isolating the capillary from the adjacent muscle fibre, which disturbs the blood

During immobilization, the normal three-dimensional orientation of the collagen fibres is disrupted. The normal orientation constitutes of the fibres running parallel to the muscle fibres on their surface, preventing muscle cells from over-elongation and –contraction. In addition, thin perpendicular fibres connect adjacent muscle fibres to each other [7,10,11]. As a result of decreased loading, the number of longitudinal fibres increases, the crimp angel of the collagen decreases and this diminishes the ability of the muscle to elongate [2,11] and because of that the skeletal muscle shows significantly decreased tensile strength [1,3]. In addition to changes in collagen abundance, alterations in the degree of collagen cross-linking would have a profound effect on the mechanical properties of skeletal muscle, causing a decrease in muscle

In contrast to physical loading, immobilization leads to the decrease in the enzyme activities of collagen biosynthesis, which suggests that the biosynthesis of the collagen network decreases as a result of reduced muscular and tendinous activity [8,56]. The rate of the total collagen synthesis depends mostly on the overall protein balance of the tissue, but it seems to

Collagen expression during immobilization has been shown to be at least partially downregulated at the pretranslational level [55]. Although the relative amount of the connective tissue increases during immobilization, the gene expression of type I and III collagens decreases during the first three days of immobilization [20]. The content of type IV collagen was also reduced as a result of immobilization [23]. The activities of prolyl 4-hydroxylase (P 4-H) and galactosylhydroxylysyl glucosyltransferase (GGT) decrease from the first three days of immobilization up to at least three weeks, suggesting decreased collagen biosynthesis during that time [20,59]. The degradation of collagens has been found to be enhanced during immobilization, as the expression of both MMP-2 and MMP-9 increased after 30 days of immobilization [30]. The quantity of TIMP-1 was also increased after 30 days of immobilization

The collagen concentration increases when expressed both as a function of muscle dry weight or muscle cross-sectional area, but this increase in muscle collagen is primarily due to the

As the skeletal muscle is a highly plastic tissue, the conditions associated with the disuse are accompanied by adaptation. A period of time without weight bearing cause modifications of structure and the function of skeletal muscles, of which atrophy and a slow-to-fast transition are the most prominent [60,61]. Many animal models such as the kind limb suspension, immobilization in shortened and lengthened position, spaceflight and denervation show that the removal of a mechanical load produces atrophy and contractile alterations, more evident in the slow muscle soleus than in fast muscles as extensor digitorum longus [60,62,63]. Besides space flight and bed rest experiments, long periods of muscle disuse in relation to a disease or

**5.3. Effect of unloading on the contractile apparatus of skeletal muscle**

be positively affected by stretch in both muscle and tendon [55].

muscle atrophy induced by immobilization [13].

supply of the muscle fibres and further increases the muscle fibre atrophy [57,58].

56 Composition and Function of the Extracellular Matrix in the Human Body

stiffness [10,17].

[30].

The different response can be explained, considering the fact that skeletal muscles in different parts of the body are subjected to different patterns of recruitment and activity [66,67]. The anti-gravitational Sol muscle is recruited for prolonged periods at a moderate level of intensity, whereas the extensor digitorum longus muscle is less frequently recruited, performing short, high-force contractions [68,69]. It is commonly known that inactivity affects the functional and biochemical properties of antigravity muscles, causing a significant decrease in both contrac‐ tion and relaxation times [66,70] and a significant increase in the maximal shortening velocity and myofibrillar ATPase activity [71]. The above-mentioned changes are considered to be a result of the gene expression, especially the genes involved in the fibre type transformation [71–73]. The co-ordinated changes in the gene expression are particularly apparent for myosin and consequently the disuse induces a slow-to-fast transition, as reflected by an increase in fast MyHC isoforms at the expense of slow MyHC in the Sol and a fast-to-faster MyHC shift in the GM muscle [74,75]. An increase in fast MyLC isoforms, an increased proportion of fast troponin subunits and hybrid fibres co-expressing fast and slow MyHC and MyLC appears during slow-to-fast transition in Sol muscle [64,71,76]. The fibre type transition results in a change in muscle metabolism, fuel use, and more fatigable muscle [77]. Several histochemical analyses have also suggested that the functional changes in immobilized muscles are due to an increase in fast-twitch IIa fibres [64]. In addition to above-mentioned facts, the increased sarcoplasmic reticulum calcium-ATPase activity and the preferential loss of thin filaments all contribute to faster contractile properties of the Sol muscle [78]. The increased shortening velocity may be an attempt to compensate for the loss of power generating capacity during unloading caused by weakening [79].

Muscle disuse is often accompanied by increased fatigability, which is caused by the reduced oxidative capacity of disused muscles [65,80,81]. Capillary loss and reduction in blood flow might contribute to the increased fatigability by an impaired supply of energy substrates and oxygen to the muscle [82].

#### **5.5. Formation of muscle atrophy during unloading**

While immobilization at shortened length induces atrophy, immobilization in lengthened position induces hypertrophy, which is largely attributable to addition of sarcomeres in the longitudinal direction [26,83]. Immobilization in the shortened position, e.g. hindlimb suspension, induces preferential transcription of fast MyHC isoforms, reminiscent of the slowto-fast transition observed in other models of disuse [26,84]. The disuse atrophy is character‐ ized by the loss of muscle mass and decrease of muscle diameter. In the case of muscle atrophy, there some noticeable changes in the muscle cell at the cellular level including sarcomere dissolution and endothelial degradation, a marked reduction in the number of mitochondria, the accumulation of the connective tissue, the elimination of apoptotic myonuclei and a decrease in capillary density [85–87]. Selective susceptibility of fibre types to immobilization seems to exist, while the red muscle fibres show the greatest atrophy. The decreased synthesis of protein and increased protein degradation are characteristic features to muscle atrophy. At least half of the total muscle protein is myofibrillar protein, and this fraction is lost at a faster rate than other muscle proteins during atrophy [84]. Three major proteolytic systems to skeletal mass protein loss are the cytosolic calcium-dependent calpain system, the lysosomal proteases and the ATP-dependent ubiquitin-proteasome system, which work as partners during muscle proteolysis rather than one system being used exclusively during atrophy [88]. Recent advances in cellular biology show the oxidative stress to be an important regulator of pathways leading to muscle atrophy during periods of disuse, increasing the expression of the key components of the proteasome proteolytic system. This proteolytic system is a prominent contributor to protein breakdown in skeletal muscle during periods of inactivity [37,84,86].

### **5.6. Effect of unloading on the skeletal muscle MyHC composition**

Prolonged periods of time spent with a diminished or no-weight bearing have a deleterious effect on skeletal muscle with the decreased protein synthesis, the loss of muscle mass and alterations of biochemical parameters [74,77]. The main findings confirmed that the proportion of slow MyHC isoforms decreased and the proportion of MyHC fast isoforms increased in consequence of altered functional conditions [46,64,89].

Clinical observations show that the atrophy of the skeletal muscle occurs as a result of immobilization and is caused by the changed functional conditions in the muscular system. Skeletal muscle function depends on the intact proprioceptive activity, motor innervation, mechanical load, and joint mobility. If one of these factors is altered, the muscle will undergo adaptation. As an increased muscular activity leads to the enhancement of the structures involved in contraction, inactivity or disuse is followed by the reduction of the muscle mass [8,77].

Alterations of biochemical parameters and changes at the ultrastructural level of the contractile apparatus are considered to be characteristic of atrophied muscles. The effect of disuse on the skeletal muscle depends on the fibre type composition of the muscle. The degenerative changes in disused muscles at the ultrastructural level have been shown to be most severe in slow oxidative muscle fibres. It is suggested that the most vulnerable muscles were antigravity muscles crossing a single joint [90].

The properties of muscle contraction which depend on the MyHC isoform composition decrease in atrophied skeletal muscle [46,89,91]. Contractile activity can induce differential expression of myosin protein isoforms in skeletal muscle. MyHC composition has an important regulatory role in myosin ATPase activity and muscle fibre shortening velocity [92]. A prolonged activity causes alterations in the MyHC composition. A decrease in the mechanical load stimulates the conversion of slow myosin in muscles of mixed fibre type composition, whereas a decrease in the weight-bearing load results in a decrease in slow myosin content [46,92].

Comparing the changes of proportion of MyHC isoforms in the contractile apparatus during unloading among human subjects and experimental animals, we can see that they are similar in their direction and amplitude. In conclusion, the adaptation of the mammalian skeletal muscles to the unloading depends on the contractile and metabolic characteristics of skeletal muscle and is not dependent on the species of the mammal. As the certain connections exist between the contractile and metabolic characteristics of skeletal muscle it is understandable why the specific atrophy causes the decrease of the main function of the skeletal muscle.
