Since Tnmd KO mice harbor very mild

Since Tnmd KO mice harbor very mild developmental changes, including interesting ultrastructural phenotype characterized by irregular and thicker collagen I fibrils when examined by electron microscopy (Docheva et al., 2005), we hypothesized possible structural and biomechanical alterations of the tendon tissue on nano-level. To pinpoint such we implicated AFM topography and force indentation analyses, a technology that has been largely used in cartilage research, demonstrating solid data on nano-structural and -biomechanical assessment of cartilage pathologies (Gronau et al., 2017; Prein et al., 2016; Stolz et al., 2009). AFM of collagen I fibers in sedentary and trained Achilles tendons confirmed these previous observations and additionally revealed that the fibers are significantly stiffer in the KO than in WT tendons. Moreover, while fiber size increased and stiffness decreased with training in control mice, the KO tendons were non-responsive. The ability of tendons to stretch and recoil is important to save energy during locomotion for economic force generation (Alexander, 1991). However, at the same time they must not undergo irreversible deformation. Physiological stretching of tendons in vivo decreases crimp numbers close to 50% in order to reduce the degree of fibril undulation (Franchi et al., 2007). This is more important for tendons than muscle, since muscle can elongate by almost 10% while in meclofenamate the whole tendon unit can only lengthen by 3–4% (Elsalanty et al., 2007). Vilarta et al., observed more aligned and intensely packed fibrils in Achilles tendons of rats after exercise (Vilarta and Vidal Bde, 1989). Stiffening of human tendons was observed in aging or diabetic individuals due to increased pathological cross-linking of collagen fibrils (Shadwick, 1990; Couppe et al. 2016). Stiffness of tendons varies with age, sex, physical activity and fatigue (Kubo et al., 2001b; Kubo et al., 2001a; Kubo et al., 2001c; Reeves, 2006). Interestingly, different types of tendons modulate their stiffness to each other, for example in endurance running, tendons in the knee joints become stiffer and in planter joints softer to guarantee optimal running performance (Kubo et al., 2015). Supplementary Fig. 7 depicts the explanation of this phenomenon. When subjected to the same amount of mechanical load, stiffer tendons extend less and in turn store less energy. In contrast, tendons that are more elastic extend further, store more energy and can therefore prevent premature rupturing. In endurance training the tendon spring softens; the softer the spring, the more elastic potential energy can be stored and less restoring force is needed resulting in more economic running. A rise in blood-flow (Langberg et al., 1998), collagen-turnover (Langberg et al., 2000), glucose uptake (Hannukainen et al., 2005) and altered regulation of matrix metalloproteases (Koskinen et al., 2004) are detected in tendons during exercise. Tendons also adapt differently depending on the nature of the exercise. For example, Svensson et al. (2016) used resistance training protocol (building up strength, anaerobic endurance and size of skeletal muscles; involving fast-twitch muscle fibers) that resulted in tendon stiffening. In contrast, Wood and Brooks (2016) reported endurance running (involving slow-twitch fibers) led to more elastic tendon tissues in tendons of old mice, which is in line with our results. Endurance running in humans leads to softening of Achilles tendons (Ooi et al., 2015) and tendons of young mice adjusted to treadmill running by alterations in collagen fiber diameter, distribution, cross-sectional area and number (Michna, 1984; Michna and Hartmann, 1989; Majima et al., 2003). It will be of great importance in subsequent studies to investigate more precisely the structural-functional relationship between tendon tissue and both muscle fiber types by applying different exercise protocols as well as to couple AFM data with whole tendon biomechanical tests.