Skeletal muscle atrophy is a common, debilitating consequence of muscle disuse, malnutrition, critical illness, musculoskeletal conditions, neurological disease, cancer, and organ failure. Despite its prevalence, little is known about the molecular pathogenesis of this devastating condition due in large part to an incomplete understanding of the molecular mechanisms that drive the atrophy process. In previous studies, we identified the transcription factor ATF4 as a critical mediator of skeletal muscle atrophy. We found that ATF4 is necessary and sufficient for skeletal muscle atrophy during limb immobilization. However, ATF4 mKO mice were only partially protected from skeletal muscle atrophy during limb immobilization, indicating the existence of another pro-atrophy factor that acts independently of the ATF4 pathway. Using mouse models, we identify p53 as this ATF4-independent factor. We show that skeletal muscle atrophy increases p53 expression in skeletal muscle fibers. In addition, overexpression of p53 causes skeletal muscle atrophy. Further, p53 mKO mice are partially resistant to muscle atrophy during limb immobilization. Taken together, these data indicate that like ATF4, p53 is sufficient and required for skeletal muscle atrophy during limb immobilization. Importantly, overexpression of p53 induces muscle atrophy in the absence of ATF4, whereas ATF4-mediated muscle atrophy does not require p53. Furthermore, overexpression of p53 and ATF4 induces greater muscle atrophy than p53 or ATF4 alone. Moreover, skeletal muscle lacking both p53 and ATF4 is more resistant to skeletal muscle atrophy than muscle lacking either p53 or ATF4 alone. Taken together, these data indicate that p53 and ATF4 mediate distinct and additive mechanisms to skeletal muscle atrophy. However, the precise mechanism by which p53 and ATF4 cause skeletal muscle atrophy remained unclear. Using genome-wide expression arrays, we identify p21 as a skeletal muscle mRNA that is highly induced by p53 and ATF4 during limb immobilization. Further, overexpression of p21 causes skeletal muscle atrophy. In addition, p21 is required for muscle atrophy due to limb immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as critical and complementary mediators of skeletal muscle atrophy during limb immobilization, and discover p21 as an essential downstream mediator of the p53 and ATF4 pathways.

Skeletal muscle (SM) atrophy complicates many illnesses, diminishing quality of life and increasing disease morbidity, health resource utilization and health care costs. In animal models of muscle atrophy, loss of SM mass results predominantly from ubiquitin-mediated proteolysis and ubiquitin ligases are the key enzymes that catalyze protein ubiquitination. We have previously shown that ubiquitin ligase Nedd4-1 is up-regulated in a rodent model of denervation-induced SM atrophy and the constitutive expression of Nedd4-1 is sufficient to induce myotube atrophy in vitro, suggesting an important role for Nedd4-1 in the regulation of muscle mass. In this study we generate a Nedd4-1 SM specific-knockout mouse and demonstrate that the loss of Nedd4-1 partially protects SM from denervation-induced atrophy confirming a regulatory role for Nedd4-1 in the maintenance of muscle mass in vivo. Nedd4-1 did not signal downstream through its known substrates Notch-1, MTMR4 or FGFR1, suggesting a novel substrate mediates Nedd4-1’s induction of SM atrophy.

In the present study Fourier Transform Infrared Spectroscopy was used to examine the effects of streptozotocin-induced diabetes mellitus on the structural components of slow- and fast-twitch rat skeletal muscles, at molecular level. Diabetes mellitus is a chronic disorder of carbohydrate, fat and protein metabolism, which is characterized by hyperglycemia caused by a defective or deficient insulin secretory response. The effect of diabetes is seen on a variety of tissues leading to important secondary complications such as kidney failure, liver dysfunction, cardiac disorders, etc. Skeletal muscle is one of the major tissues determining carbohydrate and lipid metabolism in the body / therefore, is one of the target tissues of diabetes. The two main types of muscle fibers are type I (slow-twitch) and type II (fast-twitch) fibers / having different structural organization and metabolic features. The FTIR spectra revealed a considerable decrease in lipid and protein content of diabetic skeletal muscles, indicating an increased lipolysis and protein breakdown or decreased protein synthesis. Moreover changes in protein structure and conformation were observed. In diabetes, muscle membrane lipids were more ordered and the amount of unsaturated lipids was decreased possibly due to lipid peroxidation. Diabetes caused a decrease in the content of nucleic acids, especially RNA, and hydrogen bonded phospholipids in the membrane structures of skeletal muscles. In all of the spectral parameters investigated slow-twitch muscle was more severely affected from diabetes. Thus, FTIR spectroscopy appears to be a useful method to evaluate the effect of diabetes on skeletal muscle tissues at molecular level.

Congenital myopathies are a rare and heterogeneous group of diseases. They are primarily characterised by skeletal muscle weakness and disease-specific pathological features. They harshly limit ordinary life and in severe cases, these myopathies are associated with early death of the affected individuals. The congenital myopathies investigated in this thesis are nemaline myopathy and myofibrillar myopathy. These diseases are usually caused by missense mutations in genes encoding myofibrillar proteins, but the exact mechanisms by which the point mutations in these proteins cause the overall weakness remain mysterious. Hence, in this thesis two different nemaline myopathy-causing actin mutations and one myofibrillar myopathy-causing myosin-mutation found in both human patients and mouse models were used to investigate the cascades of molecular and cellular events leading to weakness. I performed a broad range of functional and structural experiments including skinned muscle fibre mechanics, small-angle X-ray scattering as well as immunoblotting and histochemical techniques. Interestingly, according to my results, point mutations in myosin and actin differently modify myosin binding to actin, cross-bridge formation and muscle fibre force production revealing divergent mechanisms, that is, gain versus loss of function (papers I, II and IV). In addition, one point mutation in actin appears to have muscle-specific effects.  The presence of that mutant protein in respiratory muscles, i.e. diaphragm, has indeed more damaging consequences on myofibrillar structure than in limb muscles complexifying the pathophysiological mechanisms (paper II). As numerous atrophic muscle fibres can be seen in congenital myopathies, I also considered this phenomenon as a contributing factor to weakness and characterised the underlying causes in presence of one actin mutation. My results highlighted a direct muscle-specific up-regulation of the ubiquitin-proteasome system (paper III). All together, my research work demonstrates that mutation- and muscle-specific mechanisms trigger the muscle weakness in congenital myopathies. This gives important insights into the pathophysiology of congenital myopathies and will undoubtedly help in designing future therapies.

Skeletal muscle (SM) atrophy complicates many illnesses, diminishing quality of life and increasing disease morbidity, health resource utilization and health care costs. In animal models of muscle atrophy, loss of SM mass results predominantly from ubiquitin-mediated proteolysis and ubiquitin ligases are the key enzymes that catalyze protein ubiquitination. We have previously shown that ubiquitin ligase Nedd4-1 is up-regulated in a rodent model of denervation-induced SM atrophy and the constitutive expression of Nedd4-1 is sufficient to induce myotube atrophy in vitro, suggesting an important role for Nedd4-1 in the regulation of muscle mass. In this study we generate a Nedd4-1 SM specific-knockout mouse and demonstrate that the loss of Nedd4-1 partially protects SM from denervation-induced atrophy confirming a regulatory role for Nedd4-1 in the maintenance of muscle mass in vivo. Nedd4-1 did not signal downstream through its known substrates Notch-1, MTMR4 or FGFR1, suggesting a novel substrate mediates Nedd4-1’s induction of SM atrophy.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The aim of the study was to investigate skeletal muscle changes induced by an acute bout of plyometric exercise before and after plyometric training. The study consisted of an acute study and training intervention study. The acute study, investigated whether direct evidence of ultrastructural damage and identification of indirect factors were more evident in subjects presenting with rhabdomyolysis. Moreover the training intervention study investigated whether plyometric training would protect the muscle from ultrastructural damage and rhabdomyolysis. During the acute intervention, twenty six healthy untrained individuals completed an acute bout of plyometric exercise (10 x 10 squat-jumps, 1 min rest). After, thirteen subjects continued with the training intervention. Eight of these subjects completed 8 weeks of plyometric jump training, while five subjects were instructed to rest from physical activity for 8 weeks. Seven days after the final training session the training and rest group repeated a second acute bout of plyometric exercise. Acute Study: Creatine kinase (CK) activity increased significantly following the single bout of plyometric exercise in all subjects (baseline: 129 to day 4: 5348 U/l). This was accompanied by an increase in perceived pain, C-reactive protein (CRP) a marker of inflammation as well as white blood cells (WBCs). Electron micrographs of muscle biopsies taken 3 days post exercise showed evidence of ultrasructural damage and membrane damage was apparent by immunofluorescence by the loss of dystrophin staining. A stretch of the c-terminus of titin was observed by immunogold, and western blot analysis indicated an increase in calpain-3 autolysis. Based on individual CK responses (CK range: 153-71,024 U/L at 4days after exercise) the twenty six subjects were divided into two groups, namely the high (n=10) and low responders (n=16). Training intervention: Following training the trained group did not experience: a rise of CK activity (110.0 U/l), perceived pain, CRP, WBCs, Z-line streaming, a stretch of titin or calpain-3 activation; while in the control group only two subjects presented with Z-line streaming. The results indicate that high responders have a more pronounced inflammatory response compared to low responders after eccentric exercise, therefore more WBCs and more specifically neutrophils are recruited to damaged areas resulting in greater membrane damage by respiratory burst in high responders. This damage can be limited with training by remodelling sarcomeric proteins via calpain activation resulting in the stable assembly of proteins in the sarcomere preventing the release of proteins. / AFRIKAANSE OPSOMMING: Die doel van die studie was om skeletspier veranderinge wat teweeggebring is deur voor en na afloop van akute pleometriese oefening, te ondersoek. Die studie bestaan uit ‘n akute intervensie en ‘n oefeningsintervensie gedeelte. Die akute intervensie het ondersoek ingestel na die direkte bewyse van ultrastrukturele skade en identifikasie van indirekte faktore meer sigbaar is in proefpersone wat met rhabdomiolose presenteer. Meerso het die oefningsintervensie die moontlikheid dat pleometriese oefening die spier van ultrastrukturele skade en rhabdomiolose beskerm, ondersoek. Tydens die akute intervensie is 26 gesonde ongeoefende individue die akute pleometriese oefeningsessie (10 x 10 hurkspronge, 1 min rus) voltooi. Hierna het 13 proefpersone voortgegaan met die oefeningsintervensie. Agt van hierdie proefpersone het agt weke pleometriese sprongsessie oefeninge voltooi, terwyl vyf proefpersone gevra is om vir 8 weke geen oefeninge te doen nie. Sewe dae na afloop van die finale oefeningssessie het die oefening en kontrole groep in ‘n tweede herhaalde akute pleometriese oefeningsessie deelgeneem. Akute intervensie: kreatienkinase (KK) aktiwiteit het betekenisvol verhoog na die enkel pleometriese oefeningsessie in all proefpersone (basislyn: 129 tot op dag vier: 5348 U/l). Hierdie is vergesel met ‘n toename in die persepsie van pyn, c-reaktiewe proteïen (CRP) ‘n merker van inflammasie sowel as witbloedselle (WBS). Elektronmikrograwe van spierbiopsies wat geneem is drie dae na afloop van die oefeninge, het tekens van ultrastrukturele skade en membraanskade getoon wat ook deur immunofluoresensie duidelik warneembaar was deur die verlies van distrofienverkleuring. ‘n Verrekking van die c-terminus van titin is ook waargeneem deur middel van immunogold. Westernblot analyse het ‘n toename in calpain-3 outolise getoon. Gegrond op individuele KK response (KK grense: 153-71,024 U/L na vier dae post oefening) is 26 proefpersone verdeel in twee groepe naamlik ‘n hoë (n=10) en lae responders (n=16). Oefeningintervensie:: Na oefening het die geoefende groep nie ‘n toename in KK aktiwiteit getoon nie (KK aktiwiteit (110.0 U/l)), pynervaring, CRP, WBS, Z-lynstroming, ‘n strekking van titin of calpain-3 aktivering; terwyl in die kontrole groep daar slegs twee proefpersone met Z-lynstroming geïdentifiseer is. Die resultate wyse daarop dat hoë responders ‘n meer uitgesproke inflammatoriese reaksie toon vergeleke met die lae responders na afloop van essentriese oefening. Daar word dus meer WBS en spesifiek meer neutrofiele na beskadigde areas gelokaliseer wat in grootter membraanskade deur respiratoriese inspanning in die hoë responders. Hierdie skade kan beperk word deur oefening waardeur hermodulering van sarkomeriese proteïene via calpain aktivering tot stabiele rangskiking van proteïene in die sarcomere lei en daardeur proteïen vrystelling verhinder. / The NRF for financial assistance

Despite the normally robust regenerative capacity of muscle tissue, extensive soft tissue damage often results in a functional loss that cannot be restored using classic reconstruction techniques. Although implanted biomaterials are capable of mechanically transmitting force generated from the remaining tissue, cellular repopulation, reinnervation and revascularization of the injured area is necessary to achieve complete functional restoration. Using an in vivo tissue engineering model, a 1.0 x 1.0 cm portion of the lateral gastrocnemius (LGAS) of Lewis rats was removed and replaced with a muscle-derived extracellular matrix (ECM). Constructs were seeded with bone marrow-derived (BMSCs) or adipose-derived stem cells (ADSCs) and the peroneal nerve was relocated over the implanted ECM. Creation of the defect resulted in a functional impairment of the LGAS, only capable of producing 85.1 ± 4.1% of the force generated in the contralateral LGAS following ECM implantation. A significant increase in specific tension (SPo) was seen in all groups following the nerve relocation procedure when compared to their corresponding cellular treatment without nerve relocation (p < 0.05). Histological quantification revealed significant increases in cellular content and blood vessel density in the top and bottom regions of ECM implants seeded with BMSCs (p < 0.05). The nerve relocation procedure significantly increased these same variables within the middle region of the ECM when compared to all groups lacking this treatment (p < 0.05). The presence of regenerating myofibers was immunofluorescently confirmed using antibodies against desmin, myosin heavy chain and laminin, while their developmental state was substantiated by the presence of central nuclei. These data corroborate a therapeutic effect of BMSCs on skeletal muscle regeneration within the ECM implant that was not seen following ADSC injection. Furthermore, the nerve relocation procedure stimulated an increased cellular and vascular growth within the middle region of the construct, likely the cause of improved functional output. / text

Skeletal muscle protein synthesis is regulated by the mammalian target of rapamycin (mTOR) signaling pathway. The first objective was to optimize the methodological procedures for assessing mTOR signaling in horses. The response of mTOR signaling (P-Akt Ser473, P-S6K1 Thr389, P-rpS6 Ser235/26 & 240/244, and P-4EBP1 Thr37/46 by Western blotting techniques) to meal consumption was determined at three gluteal muscle biopsy depths (6, 8, and 10 cm), and the repeatability of the contralateral side at 8 cm during 5 days of repeated biopsies. There was no effect (P > 0.05) of sampling side or biopsy depth on mTOR signaling in mature horses. During repeated biopsies there was an increase (P < 0.05) in downstream (P-S6K1 Thr389, P-rpS6 Ser235/236 & 240/244 and P-4EBP1 Thr389) mTOR signaling in response to feeding. The second objective was to characterize alterations in mTOR signaling throughout the equid lifespan. Adolescent horses (yearlings and two year olds) studied in the postprandial had a lowered (P < 0.05) activation of downstream mTOR signaling with aging. There was a lower (P < 0.05) abundance of P-S6K1 Thr389 in aged horses (23.5 years old) than in mature horses (11 years old) during the post-absorptive state. The final objective was to assess mTOR signaling during acute and chronic inflammation. Acute inflammation occurred during 5 days of repeated biopsies, and chronic inflammation is characteristic of the aged. During acute inflammation, characterized by increased muscle mRNA expression of inflammatory cytokines, there was an increase (P < 0.05) in downstream mTOR signaling. Chronic inflammation resulted in a decrease (P < 0.05) in the abundance of P-S6K1 Thr389. Phenylbutazone was administered to reduce (P < 0.05) acute and chronic inflammation in muscle. Phenylbutazone administration during acute inflammation reduced (P < 0.05) the activation of downstream mTOR signaling and trended to increase (P = 0.09) P-S6K1 Thr389 abundance during chronic inflammation. Whole-body protein synthesis determined using isotope infusion techniques increased (P < 0.05) when chronic inflammation was reduced due to phenylbutazone administration. This research provides new standards for muscle biopsy collection when examining mTOR signaling, and insight into management and feeding practices for adolescent and aging horses.

Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordination of myogenesis as well as angiogenesis. While these processes are often studied independently, recent evidence from our lab has shown that the resident adult stem cell population within skeletal muscle, called satellite cells, begins secreting soluble growth factors likely to contribute to the proangiogenic response. The overall aim of this study is to investigate the role of pro-angiogenic factors secreted by satellite cells during skeletal muscle regeneration. Results from the study indicate that Hepatocyte Growth Factor (HGF) is a critical protein for the proangiogenic effect of satellite cells. It was also shown that in hypoxic environments, such as those seen in an injury state, it appears that satellite cells decrease their proangiogenic effect if oxygen levels fall below a threshold level. This decrease in pro-angiogenic effect in the hypoxic environment appears to be due to the decrease in HGF expression and protein secretion and is not compensated for by the increase in Vascular Endothelial Growth Factor secretion also seen in the hypoxic response. Furthermore, the regulation of HGF in these hypoxic conditions appears to be in part due to increased levels of hypoxia inducible factor, which are acting on the hypoxia response element site found on the HGF promoter. In the last set of experiments, this injury response was further investigated as the effect of satellite cell mediated angiogenesis was examined in the disease state of muscular dystrophy. Here, we also observed a reduction in angiogenesis from media conditioned by satellite cells from dystrophic muscle compared to healthy muscle. Overall, this study further strengthens the case for satellite cells as important mediators of the angiogenic response in regenerating muscle and may serve as a potential site for therapeutic intervention in the future.

Cold-water immersion (CWI) is a common recovery modality used to facilitate restoration of pre-exercise muscle force generation and soreness following high-intensity exercise. Although it is commonly used by athletes and commonly studied in sport science, evidence is equivocal regarding its efficacy. We compared 4 CWI protocols (10 or 30 minutes at 10 or 20°C) of different durations and temperatures with passive rest for their effects on drop jump and squat jump height, inflammation (IL-6, IL-10, IL-8, MPO, IL-1β, TNFα, IFNγ, GM-CSF, IL-2), and ratings of soreness/impairment following high-intensity intermittent sprint-exercise. CWI for 10 minutes at 10°C promoted restoration of force generation, while CWI for 30 minutes at 10°C was associated with lower ratings of soreness/impairment, but higher plasma IL-8 and MPO at 2 hours post-exercise. Overall, minor functional benefits of CWI for 10 minutes at 10°C were observed, while longer duration CWI protocols may increase post-exercise inflammation.