In sum we report that TNMD

In sum, we report that TNMD/Tnmd is a mechanosensitive gene vital for optimal running performance, proper tendon ionophore to running and maintenance of structurally and functionally integral collagen fibrils.
Materials and Methods
Results
Discussion Mechanical stimuli are important for development, homeostasis and regeneration of musculoskeletal tissues such as bone (Palomares et al., 2009), cartilage (Khoshgoftar et al., 2011) and muscle (Candiani et al., 2010). Muscle-restricted deletion of integrin-linked kinase, the downstream partner of the mechano-receptor integrin β1, leads to muscular dystrophy at the site of myotendinous junctions, a phenotype that is further enhanced by endurance exercise training (Wang et al., 2008). Conversely, mice lacking decorin and undergoing exercise are protected from the onset of osteoarthritis (Gronau et al., 2017). Mechanical stimulation was recognized as indispensable for tendon development and homeostasis (Yang et al., 2005; Galloway et al., 2013). One recent study by Maeda et al., 2011 indicated that in tendons physical forces are translated into biochemical signals via a signaling loop between TGF-β3 growth factor-Smad 2/3 mediators and Scleraxis (Scx) transcriptional activity. In another study, it was discovered that Scx and Osterix antagonistically regulate tensile force-responsive remodeling of periodontal ligaments and alveolar bone (Takimoto et al., 2015). However, the exact involvement of other mechano-sensitive genes and their signaling cascades in tendon tissues remains largely unknown and further efforts in understanding such pathways are fundamental for improving therapeutic strategies for tendinopathies. We have previously described that Tnmd KO mice have a very mild developmental phenotype, consisting of reduced tenocyte density and increased collagen fiber diameter (Docheva et al., 2005). These data suggested that Tnmd is not a master-switch gene, but rather a fine-tuner. For this reason, we decided to examine whether TNMD is a mechanosensitive gene and to challenge control and KO animals by endurance running. Tendons are comprised of abundant ECM, mainly collagen I, and low number of cells (Heinemeier and Kjaer, 2011). A small percentage of these cells are TSPC, a distinctive cell population with common adult stem cell characteristics suggested to modulate tendon homeostasis (Popov et al., 2015; Bi et al., 2007; Kohler et al., 2013). Mechanical loading of TSPC at 4% cyclic uniaxial stretch stimulated their proliferation and differentiation into tenocytes (Zhang and Wang, 2010), promoted the production of collagen I, whilst suppressing inflammatory responses (Yang et al., 2005). We have previously shown that 8% biaxial-loaded human TSPC respond by upregulation of mechano-sensitive genes and production of the proteoglycans fibromodulin and lumican (Popov et al., 2015). Loss of Tnmd transcripts in cultivated human periodontal ligament cells, tenocytes (Itaya et al., 2009; Mazzocca et al., 2011) and in rat tendon fibroblasts has been shown (Jelinsky et al., 2010). We suggest this downregulation results from lack of mechanical stimuli in vitro. Furthermore, a study on the effect of release of tensile strain on engineered human tendon-constructs seeded with tendon fibroblasts reported downregulation of TNMD expression (Bayer et al., 2014). In our study, the PCR and luciferase data on control and mechanically stretched human TSPC showed clearly that TNMD upregulates with mechanical stretching. Interestingly, TSPC transduced with Tnmd promoter, but not undergoing stretching had increased luciferase activity, which we suggest is due to endogenous expression of upstream Tnmd regulators in TSPC such as Scx (Alberton et al., 2012). When subjected to exhaustive tests and endurance running exercise Tnmd-deficient mice exhibited a significantly different performance phenotype, comprised of lower running time, earlier running test failure and worsening of running performance after training regime. These results suggest that Tnmd is important for optimal running performance. In order to exclude side-effects of Tnmd gene KO on mice running performance, we carried out gross morphological assessment of musculoskeletal elements and foot joints revealing no obvious differences between genotypes. Interestingly, we detected a TA myopathy only in the trained KO animals, therefore at present we considered the onset of this pathology secondary to the tendon functional maladaptation to running. The initial study of tenomodulin by Brandau et al., 2001, suggested a low messenger expression of Tnmd in the brain, which so far has not been validated on the protein level. Still, to examine any potential neural side-effects, we performed voluntarily running tests where the animals were not forced to run, and in these tests Tnmd KO animals showed willingness to run, suggesting no profound behavioral differences, but still a tendency for inferior running. Follow up investigations will be undertaken to further study the Tnmd-related TA muscle phenotype as well as possible effects of Tnmd in the neural system.