The effect of melatonin treatment on doxorubicin-induced skeletal muscle atrophy within a cancer model

Thesis (PhD)--Stellenbosch University, 2018.ENGLISH ABSTRACT: Background and Aim: Skeletal muscle atrophy is a major concern in patients suffering with malignancy. Chemotherapeutic agents, such as doxorubicin (DOX), can further exacerbate this loss of skeletal muscle. Although many cancer patients on chemotherapeutic agents suffer from this condition, there are no therapies routinely used to moderate muscle atrophy. The aim of the study was to investigate whether melatonin (MLT) can attenuate doxorubicin‐induced skeletal muscle and myotube atrophy in an in vivo rodent model of breast cancer as well as in an in vitro model of DOXinduced myotoxicity respectively. The safe and cost‐effective role of melatonin as a possible therapy to limit the burden of doxorubicin‐induced muscle toxicity in cancer patients serves as rationale for the in vivo study and the in vitro study allows for the exploration of more invasive mechanistic aspects using the cell lines, which would not be possible when viewing excised tissue. Methods: Female Sprague‐Dawley rats were inoculated with LA7 cancer cells and were randomly assigned to six groups: Control, Tumour control (TCON), Vehicle control (VEH), MLT, DOX and DOX + MLT (DM). Prophylactic treatment of MLT (6 mg/kg) was administered in drinking water daily and rats received three intraperitoneal injections of DOX (4 mg/kg, 3 times at 3‐day intervals). Following sacrifice blood samples (whole blood counts) and skeletal muscle tissue were collected for histological, immunoblot, antioxidant capacity and immunofluorescence analyses. Furthermore, C2C12 myoblasts grown to confluency and differentiated into myotubes were pretreated with MLT (50 nM) for 48h followed by DOX treatment (0.8 μM) for 24h. The effect of MLT treatment on C2C12 myotube diameter, mitochondrial reactive oxygen species (mtROS) production, sirtuin levels and autophagy activity was then assessed. Results: DOX treatment significantly reduced animal weight (279.1 ± 21.34 g vs. 222.2 ± 20.40 g, p˂0.0001) compared to DM weight (281.5 ± 7.11 g vs. 284.0 ± 6.53 g) and gastrocnemius muscle weight (1.4 ± 0.13 g vs. 0.99 ± 0.076 g, p˂0.0001) and cross sectional area (CSA), while increasing markers of muscle degradation compared to MLT treated groups. Serum myoglobin levels were significantly elevated in the DOX group compared to the DM group (572.6 ± 444.19 ng/mL vs. 218.2 ± 83.66 ng/mL, p˂0.0001); while, white & red blood cell counts (WBC & RBC) were significantly decreased in the DOX group compared to the MLT treated groups respectively (2.06 ± 1.59 x 109L‐1 vs. 4.13 ± 1.56 x 109L‐1 & 4.00 ± 1.52 x 1012L‐1 vs. 5.66 ± 1.03 x 1012L1, p˂0.0001). Furthermore, MLT treatment significantly increased intramuscular antioxidant capacity, mitochondrial biogenesis and satellite cell number. In vitro DOX treatment resulted in increased myotube atrophy, mitochondrial ROS levels and these effects were significantly reduced with MLT pre‐treatment. Discussion: The improvement in animal weight, muscle to body weight ratio, muscle CSA as well as the reduction in myoglobin levels in the treatment groups compared to the DOX group indicate that MLT protects against DOX‐induced atrophy. Moreover, MLT pre‐treatment improved circulating levels of WBC & RBC compared to the DOX only group and attenuated skeletal muscle atrophy by reducing cell apoptosis and increasing satellite cell number suggesting that MLT assists with muscle repair. The in vitro study indicated that DOX‐induced myotube atrophy was preceded by increases in mitochondrial ROS. Conclusion: Results indicate that pre‐treatment with exogenous MLT protects against skeletal muscle wasting induced by DOX in a pre‐cachectic tumour‐bearing rat model.AFRIKAANSE OPSOMMING: Agtergrond en Doel: Skeletspier atrofie is ‘n groot bekommernis van pasiënte wat aan kanker ly. Chemoterapie soos doxorubicin (DOX) kan skeletspierverlies nog verder vererger. Alhoewel heelwat kankerpasiënte wat chemoterapie kry, hierdie toestand onder lede het, is daar nog geen terapie wat normaalweg toegedien word om dit te verlig nie. Die doel van hierdie studie was dus om te bepaal of melatonien (MLT) doxorubicin‐geïnduseerde skeletspieratrofie in ‘n in vivo rotmodel van borskanker sowel as in ‘n in vitro model van DOX‐geïnduseerde miotoksisiteit kan teenwerk. Die veilige en koste effektiewe rol van melatonien as ‘n moontlik terapeutiese middel om doxorubicin‐geïnduseerde spiertoksisiteit in kankerpasiënte te verminder, dien as rasionaal vir die in vivo studie en die in vitro studie is gedoen om meganistiese aspekte van die werking van die terapie meer in diepte te ondersoek. Metodes: Vroulike Sprague‐Dawley rotte is met LA7 selle geïnokuleer en is in ses groepe verdeel: Kontrole, Tumor Kontrole (TCON), Oplosmiddel Kontrole (VEH), MLT, DOX en DOX + MLT (DM). Die rotte is profilakties met MLT (6 mg/kg), wat daagliks in hul drinkwater toegedien is, behandel. Die rotte het ook drie intraperitoneale DOX (4 mg/kg, 3 keer met 3 dae intervalle) inspuitings ontvang. Na die eksperimentele tydperk, is bloedmonsters (heelbloedtellings) en skeletspier versamel vir histologie, immunoblot, antioksidant kapasiteit en immunofluoresensie bepalings. Verder is C2C12 mioblaste gedifferensieer in miobuise en met MLT (50 nM) behandel vir 48 uur voordat 0.8 M DOX toegedien is vir 24 uur. Die effek van MLT in C2C12 miobuise is bepaal deur die miobuis deursnit, mitokondriale reaktiewe suurstof spesie (mtROS) produksie, sirtuinvlakke en autofagie aktiwiteit te bestudeer. Resultate: DOX behandeling het die gewig van die diere insiggewend verminder (279.1 ± 21.34 g vs. 222.2 ± 20.40 g, p˂0.0001) asook die gastrocnemius spiermassa (1.4 ± 0.13 g vs. 0.99 ± 0.076 g, p˂0.0001) en deursnitarea (CSA) in vergelyking met die DM groep (281.5 ± 7.11 g vs. 284.0 ± 6.53 g), terwyl merkers van atrofie verhoog is in vergelyking met die MLT groepe. Serum myoglobien vlakke is insiggewend in die DOX groep verhoog in vergelyking met die DM groep (572.6 ± 444.19 ng/mL vs. 218.2 ± 83.66 ng/mL, p˂0.0001); terwyl die wit‐ en rooibloedseltellings (WBS & RBS) weer insiggewend verlaag is wanneer dit met die MLT groepe vergelyk is (2.06 ± 1.59 x 109L‐1 vs. 4.13 ± 1.56 x 109L‐1 & 4.00 ± 1.52 x 1012L‐1 vs. 5.66 ± 1.03 x 1012L‐1, p˂0.0001). Verder het MLT behandeling die intramuskulêre antioksidant kapasiteit, mitokondriale biogenese en aantal satellietselle insiggewend verhoog. In vitro DOX behandeling het miobuis atrofie asook mitokondriale ROS vlakke verhoog, terwyl hierdie effekte insiggewend verlaag is in die groepe wat vooraf met melatonien behandel is. Bespreking: Die toename in die diere se gewig, spier tot liggaamsgewig ratio en spierdeursnitarea asook die verlaging in mioglobienvlakke in die MLT behandelde groepe in vergelyking met die DOX groepe, bewys dat MLT die liggaam teen DOX‐geïnduseerde atrofie beskerm. Verder het MLT behandeling ook die sirkulerende vlakke van WBS & RBS verhoog in vergelyking met die DOX behandeling, terwyl apoptose verminder het en satellietselle toegeneem het, wat aanduidend is van MLT se rol in spierherstel. Die in vitro studie dui aan dat DOX‐geïnduseerde miobuis atrofie voorafgegaan is deur ‘n toename in mitokondriale ROS. Gevolgtrekking: Resultate dui aan dat vooraf behandeling met eksogene MLT, DOX‐geïnduseerde skeletspieratrofie teenwerk in ‘n tumor‐draende rot model

Low-Volume Squat Jump Training Improves Functional Performance Independent of Myofibre Changes in Inactive Young Male Individuals

An investigation into the histological changes in skeletal muscle fibres and jump performance indicators after 8 weeks of plyometric squat jump training was conducted. Healthy inactive participants (n = 13; age: 21.5 ± 1.7 year.; height: 173.6 ± 10.7 cm; weight: 68.5 ± 18.4 kg; BMI 22.4 ± 3.8 kg/m2) were recruited, where eight participants completed plyometric squat jump training and five control participants refrained from performing any jumping activities. Blood samples, vastus lateralis muscle biopsies and functional testing (peak and average power, peak and average velocity, maximal jump height) were collected/recorded 10 days prior to and 3 days after the training/rest period. Participants completed 1644 squat jumps over an 8-week training period of 24 sessions with a progressive increase in the number of squat jumps. The trained group significantly increased their jumping average and peak power (mean increases in average power: 16.7 ± 1.2% and peak power: 8.2% ± 0.1) and velocity (mean increases in average velocity: 13.7 ± 0.1% and peak velocity: 5.2% ± 0.03), resulting in a 25% improvement in vertical jump height. No muscle morphological changes in terms of the cross-sectional area (CSA) or muscle-fibre-type transition were observed after the plyometric training. Improvements in the functional performance indicators following training may more likely be explained by sarcomere ultrastructural adaptation, which did not directly affect myosin heavy chain or CSA

Low-Volume Squat Jump Training Improves Functional Performance Independent of Myofibre Changes in Inactive Young Male Individuals

An investigation into the histological changes in skeletal muscle fibres and jump performance indicators after 8 weeks of plyometric squat jump training was conducted. Healthy inactive participants (n = 13; age: 21.5 ± 1.7 year.; height: 173.6 ± 10.7 cm; weight: 68.5 ± 18.4 kg; BMI 22.4 ± 3.8 kg/m2) were recruited, where eight participants completed plyometric squat jump training and five control participants refrained from performing any jumping activities. Blood samples, vastus lateralis muscle biopsies and functional testing (peak and average power, peak and average velocity, maximal jump height) were collected/recorded 10 days prior to and 3 days after the training/rest period. Participants completed 1644 squat jumps over an 8-week training period of 24 sessions with a progressive increase in the number of squat jumps. The trained group significantly increased their jumping average and peak power (mean increases in average power: 16.7 ± 1.2% and peak power: 8.2% ± 0.1) and velocity (mean increases in average velocity: 13.7 ± 0.1% and peak velocity: 5.2% ± 0.03), resulting in a 25% improvement in vertical jump height. No muscle morphological changes in terms of the cross-sectional area (CSA) or muscle-fibre-type transition were observed after the plyometric training. Improvements in the functional performance indicators following training may more likely be explained by sarcomere ultrastructural adaptation, which did not directly affect myosin heavy chain or CSA

Muscle damage and adaptation in response to plyometric jumping

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 assistanc

Low-Volume Squat Jump Training Improves Functional Performance Independent of Myofibre Changes in Inactive Young Male Individuals

An investigation into the histological changes in skeletal muscle fibres and jump performance indicators after 8 weeks of plyometric squat jump training was conducted. Healthy inactive participants (n = 13; age: 21.5 ± 1.7 year.; height: 173.6 ± 10.7 cm; weight: 68.5 ± 18.4 kg; BMI 22.4 ± 3.8 kg/m2) were recruited, where eight participants completed plyometric squat jump training and five control participants refrained from performing any jumping activities. Blood samples, vastus lateralis muscle biopsies and functional testing (peak and average power, peak and average velocity, maximal jump height) were collected/recorded 10 days prior to and 3 days after the training/rest period. Participants completed 1644 squat jumps over an 8-week training period of 24 sessions with a progressive increase in the number of squat jumps. The trained group significantly increased their jumping average and peak power (mean increases in average power: 16.7 ± 1.2% and peak power: 8.2% ± 0.1) and velocity (mean increases in average velocity: 13.7 ± 0.1% and peak velocity: 5.2% ± 0.03), resulting in a 25% improvement in vertical jump height. No muscle morphological changes in terms of the cross-sectional area (CSA) or muscle-fibre-type transition were observed after the plyometric training. Improvements in the functional performance indicators following training may more likely be explained by sarcomere ultrastructural adaptation, which did not directly affect myosin heavy chain or CSA