Effect of an endurance training-overtraining protocol on rat muscular oxidative capacity

Overtraining may outcomes for functional overreaching (FOR), a short term decline in performance that leads eventually to an improvement in performance after recovery, or nonfunctional overreaching (NFOR) when performance decline may be reversed only by a longer regenerative period. Recently we developed a training-overtraining protocol for rats with increased workload: eight weeks of daily exercise sessions, followed by three weeks of increasing daily training frequency (2, 3 and 4 times) with decreasing recovery time between sessions (4, 3 and 2h), characterized by analyses of performance before training (T1) and after the 4th(T2), 8th(T3), 9th(T4), 10th(T5) and 11th(T6) training weeks. All rats showed significantly increased performance at T4 and eight rats constitute the trained group (Tr). After T6, two groups were distinguishable by differences in the slope (α) of a line fitted to the individual performances at T4, T5 and T6: NFOR: α\u3c-15.05Kgm and FOR: α≥-15.05Kgm. Our goal was to verify the muscle’s oxidative capacity of Tr, FOR and NFOR groups. Skeletal muscle mitochondrial complexes I, IV and citrate synthase (CS) activity were quantified through Histochemical-staining-BN-PAGE and spectrophotometer analysis, respectively. The area of complexes I and IV was expressed relative to the area for comassie-stained complex V. Significant decreases were found in complex IV (17.2±5.84) and CS activity (22.7±3.21U/gwet) in NFOR group when compared with Tr (35.39±11.33, 32.8±5.24U/gwet) and FOR (28.9±11.37, 31.8±2.75U/gwet). There is a relationship between NFOR and the reduction of muscle oxidative capacity. Thus, aerobic tests should be performed in training routine to monitor performance alterations preventing these situations

Effect of Body Weight Variation on Swimming Exercise Workload in Rats With Constant and Size-Adjusted Loads

In swimming animal models, weights are added according to some percentage of body weight (%BW) or as a constant load (CL) to equalize the workload of each animal or to reduce the time in swimming-to-exhaustion endurance tests. The objective of the present study was to evaluate the effect of body weight variation on swimming exercise workload through the reliability analysis of swimming-to-exhaustion endurance tests. We examined the reliability by comparing the mean time to exhaustion (TEx) in trials performed on the 30th, 60th, 90th, 120th and 150th days of life of Wistar rats using three %BW and CL workloads (4%, 6% and 8% and 7 g, 11 g and 15 g, respectively). We also examined the within-subject variation of TEx over three trials of a CL test (15 g) within one week (when variability in body weight is minimal). The rats’ body density was maintained during growth (mean (SD) 1.031 (0.026) g/ml – 1.026 (0.005) g/ml) despite their significant increase in body weight (mean(SD) 109.05(13.80) g - 442.92(29.39) g). Thus, the absolute loads in longitudinal %BW tests increased gradually, causing a decrease in TEx under all workloads. The CV confidence limits for TEx in CL tests showed high within subjects variation (17.1-111%) compared to the body weight variation (0.4-2.8%). We conclude that load adjustment based on %BW does not adequately equate to the workload between rats of different sizes. The methodology also showed high within-subject variation between trials (not related to body mass changes) that compromises the significance of small effects

Padronização de um modelo de indução ao overreaching em ratos diagnosticado atraves da queda de desempenho e da razão glutamina/glutamato plasmatica

Atletas overtreinados apresentam redução de sua capacidade fisiológica funcional, entretanto os mecanismos biológicos responsáveis por tal acometimento ainda precisam ser melhor elucidados. Experimentos em modelo animal justificam-se quando a utilização de seres humanos é dificultada tendo em vista os impactos fisiológicos causados por um protocolo de indução ao overtraining. A avaliação da queda de desempenho é tida como o padrão ouro no diagnóstico do overtraining e recentemente foi sugerido que atletas bem adaptados a um protocolo de treinamento apresentariam uma razão Glutamina/Glutamato (Gm/Ga) plasmática de 3.58 a 5.88, enquanto atletas overtreinados apresentariam valores abaixo de 3.58. OBJETIVOS: Desenvolvimento de um modelo animal de indução ao overtraining em esteira avaliado através da queda de desempenho e da razão plasmática. MÉTODOS: 33 ratos machos da raça Wistar foram submetidos a 11 semanas de treinamento na esteira. Durante 8 semanas, os exercícios tiveram cargas crescentes de treinamento sendo realizados diariamente (Fase 1 Treinamento Adaptativo TA1 e TA2). Nas 3 semanas posteriores a carga de treinamento alcançada ao final da 8ª semana foi mantida, sendo a freqüência de exercícios aumentada (2,3,4 vezes/dia) com uma redução no tempo de recuperação entre as sessões (4,3,2 horas), respectivamente. Oito testes de desempenho até a exaustão foram realizados: Antes do início do protocolo de treinamento (teste 0), após a 4ª semana de treinamento (teste 1), 8ª (teste 2), 9a (teste 3), 10a (teste 4) e 11a (teste 5). Dois testes adicionais foram realizados após uma e duas semanas de repouso completo (testes 6 e 7). Dois grupos de animais foram determinados através da análise da variação angular da reta obtida pelos resultados dos testes de desempenho (3, 4 e 5). Grupo Alto Desempenho (AD H > 0) (N=13) e Grupo Baixo Desempenho (BD H < 0) (N=12). Dezesseis ratos controle sedentários (CO) e oito ratos treinados (T) representando a fase 1 também foram analisados. RESULTADOS: O grupo BD apresentou uma queda persistente de desempenho após duas semanas de repouso quando comparado o teste 7 (148.9 ± 51.7 Kg.m) ao teste 3 (304.4 ± 119.7 Kg.m), possuindo ainda valores de desempenho similares aos do grupo controle (CO) no teste 7 (150.6 ± 97.8 Kg.m). O grupo AD manteve os valores encontrados de desempenho após o teste 3 (337 ± 144.6 Kg.m) até o teste 7 (297.5 ± 118.7 Kg.m). O grupo T apresentou níveis maiores de desempenho (478.9 ± 62.8 Kg.m) quando comparados ao grupo controle (CO) (175.6 ± 78.5 Kg.m). A razão Gm/Ga foi significativamente maior nos grupos T e AD (4.5 ± 1.7 e 4.5 ± 0.9) quando comparados aos grupos CO e BD (2.8 ± 0.5 e 3.1 ± 0.2). CONCLUSÕES: Esse modelo controlado e reprodutível de indução ao overtraining em modelo animal pode ser útil a fim de se explicar as hipóteses específicas dos mecanismos causadores da queda de desempenho induzidos por um desbalanço entre exercício e recuperação.Overtrained athletes show reduction of functional physiological capacity and its biological mechanisms still need to be elucidated. Experimental animal models are justified when it would not be appropriate to use human subjects for studies of exercises impact. The evaluation of the performance decrement represents the gold standard for diagnosis of overtraining (OT) and recently it was suggested that athletes with acceptable performances would present a plasma Glutamine/Glutamate (Gm/Ga) ratio of 3.58 to 5.88, while overtrained athletes would present values below 3.58. PURPOSE: Development of an animal model of OT characterized by performance decrement and plasma Gm/Ga ratio. METHODS: 33 male Wistar rats were submitted to 11 weeks of running exercise. During 8 weeks, the exercises had increasing loads and were performed daily (Phase 1 - Adaptive Training). In the next 3 weeks, the load achieved at the 8th week was maintained and the daily frequency was increased (2, 3, 4 times) with a reduction in the recovery time between the sessions (4, 3, 2 hours). Eight performance tests until exhaustion were performed: before the beginning (1), after the 4th (2), 8th (3), 9th (4), 10th (5), 11th (6) weeks of training and after one and two weeks of complete resting (7 and 8). Two groups were detected by least square fit using tests 4, 5 and 6: Not Overtrained (NOTr - HX0) (N=13) and Overtrained (OTr - H<0) (N=12). A sedentary control group (CO; N=16) and a trained group (T; N=8), representing Phase1 were also analyzed. RESULTS: OTr showed persisted performance decrement after 2 weeks of rest in test 8 (148.9 ± 51.7 Kg.m) compared with test 4 (304.4 ± 119.7 Kg.m) (p<0,05) and closer to CO performance in test 8 (150.6 ± 97.8 Kg.m). NOTr maintained the performance after test 4 (337 ± 144.6 Kg.m) until test 8 (297.5 ± 118.7 Kg.m). T group showed higher performance level (478.9 ± 62.8 Kg.m) than CO (175.6 ± 78.5 Kg.m) (p<0,01) in test 4. Plasma Gm/Ga was significantly higher in T and NOTr (4.5 ± 1.7 and 4.5 ± 0.9) than in the CO and OTr groups (2.8 ± 0.5 and 3.1 ± 0.2) (p<0,01). CONCLUSION: This controlled and reproducible animal model of OT could be useful to test specific hypotheses to explain the causative mechanisms of performance decrement induced by an imbalance between the exercise and recovery time

Analysis of mitochondrial function and dynamics in rats subjected to a treadmill overtraining protocol

Orientador: Denise Vaz de MacedoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O overtraining (OT) é um processo contínuo de treinamento intensificado, caracterizado por um desequilíbrio entre as cargas de esforço físico e o tempo de descanso entre as sessões, no sentido de aumentos no primeiro. Um período de OT pode gerar dois estados diferenciados de desempenho: manutenção ou ligeiro aumento, denominado de functional overreaching (FOR), ou queda no desempenho previamente adquirido, caracterizado como non functional overreaching (NFOR). O estado NFOR pode ser revertido com um período regenerativo maior, de dias ou semanas. Nosso grupo de pesquisa desenvolveu um protocolo de OT para ratos com duração de 11 semanas, onde após um período de 8 semanas com treinos diários são realizadas mais três semanas de treinamento aumentando-se o número de sessões diárias (2, 3 e 4) com diminuição no tempo de pausa entre as sessões (4, 3 e 2h). O protocolo é capaz de discriminar ratos nos estados FOR e NFOR ao final da 11ª semana, após um momento em que todos os animais exibiram aumentos de desempenho (9ª semana). A seleção dos grupos FOR e NFOR é feita pelos resultados em 6 testes incrementais até a exaustão realizados ao longo do protocolo. OBJETIVOS: Investigar a hipótese de uma deficiência na função e dinâmica mitocondrial como uma das possíveis causas desencadeadoras da queda de desempenho no estado NFOR. MÉTODOS: Analisamos nos ratos FOR, NFOR e controle (sem treinamento) a integridade muscular por meio de análises histoquímica e hemograma (Neutrófilos, Linfócitos, WBC e RBC) nos diferentes grupos de animais. Analisamos as taxas de liberação de H2O2 e da produção de O2- por mitocôndrias isoladas para detectar possíveis alterações na geração de EROs nos diferentes grupos de animais. A função mitocondrial foi avaliada por meio da quantificação do consumo de oxigênio e produção de ATP em mitocôndrias isoladas do músculo gastrocnêmio. Analisamos os processos da dinâmica mitocondrial, pela quantificação de vias relacionadas a síntese (biogênese) e a degradação (autofagia) mitocondrial. Para a análise da biogênese mitocondrial quantificamos a expressão do marcador proteico PGC1-? bem como a expressão do estimulador de duplicação do mtDNA, TFAM. Para avaliar a ocorrência dos processos autofágicos analisamos a expressão dos marcadores protéicos de autofagia e fluxo autofágico, mTOR, LC3 (razão LC3-II/LC3-I) e p62. RESULTADOS: Não foram encontradas diferenças significativas entre os grupos nas análises histoquímica do músculo e no hemograma. Por outro lado houve aumento significativo na produção de O2- e H2O2 associado a uma diminuição no consumo de O2 mitocondrial e uma tendência de diminuição no conteúdo de ATP nos ratos NFOR quando em comparação aos ratos FOR. O grupo NFOR também apresentou uma diminuição significativa nos processos de autofagia, observados pelos menores valores encontrados da razão LC3-II/LC3-I e pelo acumulo de p62, mesmo com a mTOR inibida em ambos os grupos. Já o grupo FOR exibiu fluxo autofágico aumentado.Com relação à biogênese mitocondrial, ambos os grupos apresentaram efeito parecido: aumentos na PGC-1?. Entretanto, somente o grupo FOR apresentou diminuição significativa nos níveis de TFAM. A impossibilidade da ligação da TFAM ao mtDNA, sugere queda na sinalização da biogênese no grupo NFOR. O conjunto dos dados sugere que uma atenuação da função, possivelmente por problemas na dinâmica mitocondrial está na gênese do estado NFOR. Estes dados são originais e fornecem um caminho alternativo e promissor para entender a queda de desempenho nesse estadoAbstract: INTRODUCTION: Overtraining (OT) is a continuous process of intense training, characterized by an imbalance between the levels of physical effort and time to rest between sessions. An overtraining period can generate two different states of performance: maintenance or slight increase, called functional overreaching (FOR), or drop in performance, characterized as non-functional overreaching (NFOR). The NFOR state can only be reversed by a regenerative period of days or weeks. Our research group has developed a training-overtraining protocol for rats with 11 weeks duration where after a period of 8 weeks with daily workouts the last three weeks are made increase in daily sessions (2, 3 and 4) with a decrease in the rest time between sessions (4, 3 and 2 hours). The protocol is able to discriminate FOR and NFOR states at the end of 11th week, after a period that all animals exhibited performance increases (9th week). The selection of FOR groups and NFOR is done through performance results in six incremental test to exhaustion performed throughout the protocol. OBJECTIVES: Investigate the possibility of a mitochondrial function and dynamics deficiency as a possible cause of performance decrease in NFOR state. METHODS: We analyzed in FOR, NFOR and CO (without training) the muscle integrity through immunohistochemical analysis and blood count (neutrophils, lymphocytes, WBC and RBC) in different groups of animals. We analyze the H2O2 release rates and the O2- production by isolated mitochondria to detect possible changes in the generation of ROS in the different groups of animals. The mitochondrial function was evaluated by oxygen consumption and by ATP content in isolated mitochondria of the gastrocnemius muscle of animals. To analyze the mitochondrial dynamics processes, we quantified the synthesis (biogenesis) and degradation (autophagy) mitochondrial pathways. For the analysis of mitochondrial biogenesis we quantify PGC1-? and TFAM expression. To evaluate the occurrence of autophagic processes we analyzed the expression of autophagy markers; mTOR, LC3 (LC3-II reason / LC3-I) and p62. RESULTS: There were no significant differences between groups in immunohistochemistry and blood analysis. On the other hand there was a significant increase in the levels of O2- and H2O2 associated with a decrease in the mitochondrial O2 consumption and a decreasing trend in the ATP content when compared NFOR to FOR rats. The NFOR group also showed a significant decrease in autophagy processes, observed by the lowest values of LC3-II/LC3-I reason and the accumulation of p62, although there was inhibition of mTOR like the FOR group. With respect to mitochondrial biogenesis, both groups showed similar effect: increases in PGC-1?. However only the FOR group showed a significant decrease in the levels of TFAM. The impossibility of NFOR's TFAM link to mtDNA suggests a biogenesis signaling drop. These results suggests that the attenuation of mitochondrial function, possibly by mitochondrial dynamics problems is the genesis of NFOR state. These data are unique and provide an alternative and promising path to understand the performance drop in this stateDoutoradoBioquimicaDoutor em Biologia Funcional e Molecular141707/2011-9CNP

Study of the relation ship between oxidative damage, antioxidant defense system and mitochondrial activity in rats induced to a treadmill overtraining protocol

Orientadores: Denise Vaz de Macedo, Rodrigo HohlDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O overtraining e um processo continuo de treinamento intenso que pode gerar um declínio curto no desempenho, denominado de overreaching funcional (FOR), o qual conduz eventualmente a uma melhoria no desempenho apos um breve período de recuperação (dias-semanas); ou um declínio mais prolongado no desempenho, caracterizado como overreaching nao funcional (NFOR), que somente e revertido por um período regenerativo mais longo (semanas-meses). Nos desenvolvemos um protocolo de treino-overtraining para ratos que induz aos estados FOR e NFOR apos um período de adaptação ao treinamento e aumento de desempenho (Tr). O desempenho dos animais foi avaliado através de 6 testes ao longo do protocolo, testes estes que serviram para separarmos nossos grupos experimentais. Nosso objetivo foi investigar a hipótese do estresse oxidativo, relacionado com alterações na atividade mitocondrial, como uma das possíveis causas desencadeadoras da queda de desempenho durante a instalação do overtraining. Para isso analisamos a atividade das enzimas antioxidantes catalase (CAT), glutationa redutase (GR) e superóxido dismutase (SOD), dos marcadores de produção de espécies reativas de oxigênio (EROs), xantina oxidase (XO) e ataque oxidativo por meio da analise das substâncias que reagem ao acido tiubarbiturico (TBARs), alem da atividade da enzima citrato sintase (CS) e dos complexos mitocondriais da cadeia de transporte de elétrons, complexo I e IV. Analisamos ainda, em busca de possíveis biomarcadores, a capacidade antioxidante sanguínea por meio das analises do FRAP (ferric reducing ability of plasma), albumina e acido úrico. A peroxidação lipídica no plasma foi obtida por meio da analise do malondialdeido (MDA) e o marcador de lesão muscular utilizado foi a creatina quinase sanguínea (CK). Os resultados estão apresentados por meio de boxplots com seus respectivos intervalos de confiança (95%) que dão a significância ou não de diferenças entre as analises. Os resultados encontrados apontaram para diminuições significativas na atividade do complexo IV e CS somada a atividades aumentadas das enzimas antioxidantes CAT e SOD no grupo NFOR, quando em comparação aos grupos Tr e FOR, respectivamente. Alem disso, os marcadores de peroxidação lipídica TBARs e MDA encontraram-se significativamente aumentados no grupo NFOR quando em comparação aos grupos CO, Tr e FOR. Estes dados sugerem, que a queda de desempenho no estado NFOR pode estar relacionada a diminuição da capacidade oxidativa devido a instalação de um quadro de estresse oxidativo mitocondrial, entretanto os mecanismos que causam este processo ainda precisam ser esclarecidos.Abstract Overtraining is a continuous process with possible outcomes of functional-overreaching (FOR) and nonfunctional-overreaching (NFOR). FOR is characterized by a short-term decline in performance that leads eventually to an improvement in performance after a short recovery (daysweeks). NFOR is a decline in performance that can be reversed by a longer regenerative period (weeks-months). We developed an animal model of training-overtraining that led Wistar rats to FOR and NFOR states after a training adaptation period where they achieved increased performance (Tr group). The animal performance was evaluated through six tests during the protocol. These tests have served to separate our experimental groups. Our objective was to investigate the oxidative stress-related changes in mitochondrial activity hypothesis as a possible cause of the performance drop during the overtraining installation. We analyzed the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) activity, ROS generator xanthine oxidase (XO) activity and oxidative damage indicator through the analysis of substances which react to thiubarbituric acid (TBARS) production. In addiction we analyzed citrate syntase (CS) enzyme activity and mitochondrial complexes of the electron transport chain (complex I and IV) activity. We also analyzed, for potencial biomarkers, the antioxidant capacity of blood through the FRAP (ferric reducing ability of plasma), albumin and uric acid analysis. Lipid peroxidation in plasma was obtained by malondialdehyde (MDA) and the muscle damage marker used was blood creatine kinase (CK). The results are presented by boxplots with their respective confidence intervals (95%) giving a significance or not of differences between analysis. The results pointed to significant decreases in complex IV and CS activity plus an increased of antioxidant enzymes SOD and CAT activities in NFOR group when compared to Tr and FOR groups respectively. Furthermore, the lipid peroxidation TBARS and MDA markers were found significantly increased in NFOR group when compared to CO, Tr and FOR. These data suggest that the performance drop in the NFOR state may be related to decreased oxidative capacity due to a mitochondrial oxidative stress installation. However the mechanisms involved in this process yet to be clarified.MestradoBioquimicaMestre em Biologia Funcional e Molecula

Interaction between Overtraining and the Interindividual Variability May (Not) Trigger Muscle Oxidative Stress and Cardiomyocyte Apoptosis in Rats

Severe endurance training (overtraining) may cause underperformance related to muscle oxidative stress and cardiomyocyte alterations. Currently, such relationship has not been empirically established. In this study, Wistar rats (n=19) underwent eight weeks of daily exercise sessions followed by three overtraining weeks in which the daily frequency of exercise sessions increased. After the 11th training week, eight rats exhibited a reduction of 38% in performance (nonfunctional overreaching group (NFOR)), whereas eleven rats exhibited an increase of 18% in performance (functional overreaching group (FOR)). The red gastrocnemius of NFOR presented significantly lower citrate synthase activity compared to FOR, but similar to that of the control. The activity of mitochondrial complex IV in NFOR was lower than that of the control and FOR. This impaired mitochondrial adaptation in NFOR was associated with increased antioxidant enzyme activities and increased lipid peroxidation (in muscle and plasma) relative to FOR and control. Cardiomyocyte apoptosis was higher in NFOR. Plasma creatine kinase levels were unchanged. We observed that some rats that presented evidence of muscle oxidative stress are also subject to cardiomyocyte apoptosis under endurance overtraining. Blood lipid peroxides may be a suitable biomarker for muscle oxidative stress that is unrelated to severe muscle damage