**2. Overview of Skeletal Muscle Structure**

Before considering the structural changes that drive mechanical load-induced growth, we want to ensure that the reader appreciates the basic structural design of skeletal muscle. One of the easiest ways to appreciate this design is to consider skeletal muscle as a hierarchy of contractile machinery that is visible at the macroscopic level (viewable without magnification), followed by the microscopic level (viewable with standard microscopy), and finally the ultrastructural level (viewable with high resolution microscopy). Below we will provide a brief overview of the primary components that are found at each of these levels. For excellent illustrations and more comprehensive discussions on this topic, the reader is referred to the following reviews [24–26].

At the macroscopic level, it can be noted that skeletal muscles are connected to bones via tendinous attachments and enact their contractile function by providing movement and articulation of the skeletal system. Moreover, as illustrated in Figure 1, skeletal muscles are surrounded by an outer layer of connective tissue called the epimysium, and underneath the epimysium are bundles of myofibers (i.e., fascicles) that are surrounded by another layer of connective tissue called the perimysium [24]. In most skeletal muscles, the fascicles, and their associated myofibers, are not directly aligned with the longitudinal axis of the muscle, but instead are offset at an angle called the pennation angle (Figure 2) [27].

**Figure 1.** (**A**) Illustration of skeletal muscle structure copied with permission under the Creative Commons Attribution 4.0 International license and adapted for this review, available online: https: //openstax.org/books/anatomy-and-physiology/pages/10-2-skeletal-muscle (accessed on 6 January 2020) [28]. (**B**) Cross-section of a mouse plantaris muscle that was subjected to immunohistochemistry for the identification of Type IIA (cyan), and Type IIB (magenta) myofibers as well as laminin to identify the endomysium (white). (**C**) Cross-section of a mouse plantaris muscle that was subjected to immunohistochemistry for the identification of dystrophin to identify the inner boundary of the sarcolemma (white) and nuclei (green). (**D**) Cross-section of a mouse plantaris muscle that was subjected to electron microscopy to highlight the sarcoplasmic reticulum (SR) that surrounds individual myofibrils as well as the mitochondria (Mito) that run between the myofibrils. (**E**) Higher magnification of the boxed region in D reveals the presence of the thick and thin myofilaments.

At the microscopic level, a cross-section of skeletal muscle will reveal the presence of individual myofibers (Figure 1A,B). The myofibers are multinucleated cells that are encased by a layer of connective tissue called the endomysium, and they are surrounded by interstitial cells such as fibroblasts, immune cells, pericytes and fibro-adipogenic progenitors (Figure 1A–C) [29,30]. Furthermore, another important class of cells, called satellite cells, resides between the endomysium and the plasma membrane of the myofibers (i.e., the sarcolemma) [31]. The endomysium is physically coupled to the sarcolemma, and everything the resides underneath the sarcolemma is typically referred to as the sarcoplasm.

**Figure 2.** Various pennation angle arrangements of the fascicles/myofibers in skeletal muscle: (**A**) fusiform, (**B**) unipennate, (**C**) bipennante, (**D**) multipennate. Blue lines indicate the plane for the anatomical cross-sectional area (CSA) (i.e., the CSA that runs perpendicular to the longitudinal axis of the muscle), and green lines indicate the plane for physiological CSA (i.e., the CSA that runs perpendicular to the longitudinal axis of the fascicles/myofibers). Adapted under the Creative Commons Attribution-Share Alike 3.0 Unported license from original work by Uwe Gille (Available online: https://creativecommons.org/licenses/by-sa/3.0/deed.en (accessed on 6 January 2020).

The gelatinous sarcoplasm contains the primary ultrastructural elements of the myofiber, and as illustrated in Figure 1, an examination at the ultrastructural level reveals that ≈80% of the sarcoplasm is filled with an in-parallel array of rod-like structures called myofibrils [32–36]. The myofibrils are composed of a long in-series array of force-generating elements called sarcomeres and are surrounded by a mitochondrial reticulum and a membranous structure called the sarcoplasmic reticulum [37,38]. The sarcomeres within the myofibrils enact their function through the active sliding of thick and thin myofilaments, and in a longitudinal view, it can be seen that the sarcomeres consist of regions called the Z-disc, the I-band which contains the thin (actin) myofilaments, and the A-band which contains the thick (myosin) myofilaments [39,40]. It should also be noted that within a given species, the optimal/resting length of sarcomeres (2.0–2.5 μm) is highly conserved, and alterations in this length can profoundly influence force production [41–43].

When considering the structure of skeletal muscle, it is also essential to recognize that all myofibers are not created equal. For instance, some types of myofibers are heavily reliant on oxidative metabolism, exhibit a slow contractile speed and are resistant to fatigue. In contrast, other types of myofibers rely on anaerobic glycolytic metabolism, exhibit a fast contractile speed and rapidly fatigue when stimulated to contract [40]. Different fiber types are typically grouped according to the predominant isoform of the myosin heavy chain that they express, and these isoforms include Type I (slow oxidative), Type IIA (fast oxidative), and Type IIB (fast glycolytic) fibers [44]. It is also important to point out that humans do not express the Type IIB myosin isoform, but instead express a very similar (yet slightly slower) Type IIX myosin isoform [45]. However, since this differentiation was only solidified in 1990 s [46–48], some older studies with human subjects used the Type IIB classification, while other studies have grouped Type IIB and Type IIX fibers together as a similar fiber type [49]. This use of IIB and IIX myosin labeling has led to some confusion when comparing earlier muscle growth studies to current studies, so it is important to keep this distinction in mind when relating fiber type-specific adaptations across the current body of literature.

#### **3. Mechanical Load-Induced Growth of Skeletal Muscle at the Macroscopic Level**

#### *3.1. Whole Muscle*

At the whole muscle level, mechanical load-induced growth can be mediated by an increase in the length and/or an increase in the cross-sectional area (CSA) of the muscle. Growth resulting from an increase in length is referred to as longitudinal growth, and it can occur in response to a variety of different perturbations. For instance, during development, the length of muscles can more than double from birth to the termination of bone growth [50–52]. Longitudinal growth can also be induced in adults by placing muscles in a chronically stretched state [53]. As a case in point, it has been shown that immobilizing a rat lower hindlimb in a fully dorsiflexed position can lead to a >20% increase in the length of the soleus muscle [54]. Likewise, limb-lengthening procedures can lead to a >20% increase in muscle length [55,56]. Indeed, even some of the more extreme models of mechanical load-induced growth can lead to an increase in muscle length [23,57]. For example, surgical removal of the gastrocnemius and soleus muscles (i.e., synergist ablation) is a commonly used rodent model for stimulating mechanical load-induced growth, and it has been reported that this can lead to a 13% increase length of the plantaris muscle [57,58]. Thus, it is clear that both adolescent and adult skeletal muscles can undergo longitudinal growth.

Although skeletal muscle is capable of undergoing longitudinal growth, most models of mechanical loading do not lead to notable alterations in whole muscle length [18,59]. Instead, mechanical load-induced growth is usually driven by an increase in the CSA of the muscle (also known as radial growth). For example, in humans, 8-16 weeks of resistance exercise will generally produce a 5–30% increase in whole muscle CSA but no change in muscle length [16,59–66]. Interestingly, the magnitude of increase in CSA is often greater than the increase that is observed for muscle volume/mass. An excellent example of this paradox was reported by Roman et al. (1993), whom reported that 12 weeks of resistance exercise led to a 14% increase in the volume of the elbow flexors, but the CSA at the mid-belly increased by 23% [60]. Importantly, however, the magnitude of the increase is CSA got progressively smaller towards the proximal and distal ends of the muscle, which explained why the muscle volume only increased by 14%. Simply put, the study by Roman et al. (1993) demonstrated that the radial growth response was not evenly distributed along the length of the muscle. Indeed, regional differences in the magnitude of radial growth have been reported in several animal and human-based studies, and in our opinion, this phenomenon represents an often overlooked aspect of the mechanical load-induced growth response [13,59,64,67–72].
