Análises morfofuncionais, bioquímicas e moleculares de modelo experimental de miopatia associada à estatina
Arquivos
Data
2017-03-06
Autores
Andrade, Pamela Vieira de 
Orientadores
Silva, Helga Cristina Almeida da
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: As estatinas atualmente são consideradas os agentes mais úteis para o tratamento da hipercolesterolemia. Nos últimos anos, aumentaram os relatos sobre efeitos colaterais importantes no músculo esquelético, com mialgia, aumento dos valores séricos de creatinoquinase (CK) e até rabdomiólise após uso de altas doses dessas medicações. O reconhecimento precoce da miopatia induzida pelas estatinas e a descontinuação das estatinas é fundamental na prevenção da miopatia e diminuição de possíveis sequelas. Objetivos: 1. Estudar o efeito da estatina sobre a contração e contratura muscular em ratos expostos a baixas e altas concentrações da sinvastatina 2. Correlacionar essas alterações com a morfologia, bioquímica e proteômica da fibra muscular e 3. Analisar o uso do bloqueador de canal de liberação de cálcio (dantrolene) no presente modelo. Material e Métodos: O estudo foi realizado com 50 ratos machos Wistar, divididos em 5 grupos: A - Controle; B - Estatina baixa dose (5 mg/kg/dia); C - Estatina alta dose (20 mg/kg/dia); D – Sem estatina, com dantrolene (5 mg/Kg); E – Estatina alta dose (20 mg/kg/dia) e dantrolene (5 mg/Kg). Os animais dos grupos B, C e E receberam a sinvastatina diariamente, diluída com carboximetilcelulose (CMC) 0,5%, via gavagem, por dois meses; os do grupo A e D receberam apenas o veículo (CMC), via gavagem, por dois meses; os dos grupos D e E receberam ainda dantrolene (5 mg/Kg) via subcutânea por dois meses. Os animais foram pesados semanalmente, sendo anotado o consumo da dieta líquida e sólida diariamente; a cada sete dias foram realizados exames físico e neurológico. Após dois meses, os animais foram anestesiados para a retirada dos músculos vastos laterais, gastrocnêmios e sóleos. Com os vastos laterais foram realizados testes de estimulação elétrica in vitro em presença de halotano e cafeína - nos quais as curvas de contração e contratura de cada teste foram analisadas - e teste de estimulação tetânica pré e pós-isquemia - referido como grau de contração após cada estímulo elétrico. Com os gastrocnêmios e os sóleos foi realizado estudo morfológico (histologia e morfometria). O gastrocnêmio foi empregado ainda para estudo bioquímico, feito pela quantificação do malonaldeído (MDA), e análise proteômica por espectrometria de massas. Resultados: Na estimulação elétrica in vitro na presença de cafeína, houve aumento significativo nos valores de contração nas concentrações de 1; 1,5; 2; 3, 4 e 32 mM de cafeína do grupo C em relação aos outros grupos. Não houve diferença significativa da contração em resposta a diferentes concentrações de halotano e a diferentes estimulações tetânicas pré e pós-isquemia, bem como não houve diferença no grau de contratura após vários estímulos. Houve aumento significativo na produção de MDA no grupo C em relação ao grupo B. O estudo morfológico qualitativo mostrou, na reação do Tricrômico de Gomori, pequenos aglomerados fucsinofílicos grosseiros na periferia das fibras musculares, vistos mais uniformemente no grupo C. Na análise morfométrica do músculo gastrocnêmio, pela coloração de hematoxilina/eosina, houve diminuição significante da área transversa das fibras musculares no grupo C em relação aos grupos D e E. Na análise proteômica houve aumento significativo da expressão das proteínas glicolíticas nos grupos tratados com estatina. Conclusão: Nossos dados mostraram que, apesar dos animais não apresentarem miopatia clínica evidente, doses de estatina de 20 mg/Kg/dia aumentaram a contração muscular após estímulo com cafeína, provocaram alterações morfológicas mitocondriais com atrofia do músculo gastrocnêmio e aumentaram a peroxidação lipídica no músculo.
Introduction: Statins are considered the most useful agents for the treatment of hypercholesterolemia. In recent years, increased the reports of significant side effects in skeletal muscle, with myalgia, increased serum creatine kinase (CK), and even rhabdomyolysis following the use of high doses of these medications. The early recognition of statin-induced myopathy and the discontinuation of statins are fundamental in the prevention of myopathy and the reduction of possible sequels. Objectives: To study the effect of statin on muscle contraction and contracture in rats exposed to low and high concentrations of simvastatin 2. To correlate these alterations with the morphology, biochemistry and proteomics of muscle fiber and 3. To analyze the use of the calcium channel blocker (Dantrolene) in the present model. Material and Methods: The study was performed with 50 male Wistar rats, divided into 5 groups: A - Control; B - Statin low dose (5 mg / kg / day); C – Statin high dose (20 mg / kg / day); D - No statin, with dantrolene (5 mg / kg); E - Statin high dose (20 mg / kg / day) and dantrolene (5 mg / kg). The animals of groups B, C and E received simvastatin daily, diluted with carboxymethylcellulose (CMC) 0.5%, via gavage, for two months; group A and D received only vehicle (CMC), via gavage, for two months; groups D and E were also given dantrolene (5 mg / kg) subcutaneously for two months. The animals were weighed weekly, being recorded the consumption of the liquid and solid diet daily; physical and neurological exams were performed every seven days. After two months, the animals were anesthetized for withdrawal of vastus lateralis, gastrocnemius and soleus muscles. With the vastus lateralis, in vitro electrical stimulation tests were performed in the presence of halothane and caffeine - in which the contraction and contraction curves of each test were analyzed - and tetanic stimulation test pre and post-ischemia - referred to as degree of contraction after each electrical stimulus. Morphological study (histology and morphometry) was performed with the gastrocnemius and the soleus. The gastrocnemius was also used for biochemical study, made by the quantification of malonaldehyde (MDA), and proteomic analysis by mass spectrometry. Results: In the electrical stimulation in vitro in the presence of caffeine, there was a significant increase in the values of contraction in the concentrations of 1; 1.5; 2; 3, 4 and 32 mM caffeine in group C in relation to the other groups. There was no significant difference in contraction at different halothane concentrations and in different tetanic stimulation pre and post- ischemia. There was a significant increase in the production of MDA in group C in relation to group B. The qualitative morphological study showed, in the reaction of Gomori's trichrome, small coarse fucsinophilic clusters at the periphery of the muscle fibers, seen more uniformly in group C. In the morphometric analysis of the gastrocnemius muscle, by hematoxylin / eosin staining, there was a significant decrease in the cross-sectional area of muscle fibers in group C in relations to groups D and E. In the proteomic analysis there was a significant increase in the expression of glycolytic proteins in the groups treated with statin. Conclusion: Our data showed that although the animals did not present evident clinical myopathy, statin doses of 20 mg /kg/ day increased muscle contraction after caffeine stimulation, caused mitochondrial morphological alterations with gastrocnemius muscle atrophy and increased lipid peroxidation in the muscle.
Introduction: Statins are considered the most useful agents for the treatment of hypercholesterolemia. In recent years, increased the reports of significant side effects in skeletal muscle, with myalgia, increased serum creatine kinase (CK), and even rhabdomyolysis following the use of high doses of these medications. The early recognition of statin-induced myopathy and the discontinuation of statins are fundamental in the prevention of myopathy and the reduction of possible sequels. Objectives: To study the effect of statin on muscle contraction and contracture in rats exposed to low and high concentrations of simvastatin 2. To correlate these alterations with the morphology, biochemistry and proteomics of muscle fiber and 3. To analyze the use of the calcium channel blocker (Dantrolene) in the present model. Material and Methods: The study was performed with 50 male Wistar rats, divided into 5 groups: A - Control; B - Statin low dose (5 mg / kg / day); C – Statin high dose (20 mg / kg / day); D - No statin, with dantrolene (5 mg / kg); E - Statin high dose (20 mg / kg / day) and dantrolene (5 mg / kg). The animals of groups B, C and E received simvastatin daily, diluted with carboxymethylcellulose (CMC) 0.5%, via gavage, for two months; group A and D received only vehicle (CMC), via gavage, for two months; groups D and E were also given dantrolene (5 mg / kg) subcutaneously for two months. The animals were weighed weekly, being recorded the consumption of the liquid and solid diet daily; physical and neurological exams were performed every seven days. After two months, the animals were anesthetized for withdrawal of vastus lateralis, gastrocnemius and soleus muscles. With the vastus lateralis, in vitro electrical stimulation tests were performed in the presence of halothane and caffeine - in which the contraction and contraction curves of each test were analyzed - and tetanic stimulation test pre and post-ischemia - referred to as degree of contraction after each electrical stimulus. Morphological study (histology and morphometry) was performed with the gastrocnemius and the soleus. The gastrocnemius was also used for biochemical study, made by the quantification of malonaldehyde (MDA), and proteomic analysis by mass spectrometry. Results: In the electrical stimulation in vitro in the presence of caffeine, there was a significant increase in the values of contraction in the concentrations of 1; 1.5; 2; 3, 4 and 32 mM caffeine in group C in relation to the other groups. There was no significant difference in contraction at different halothane concentrations and in different tetanic stimulation pre and post- ischemia. There was a significant increase in the production of MDA in group C in relation to group B. The qualitative morphological study showed, in the reaction of Gomori's trichrome, small coarse fucsinophilic clusters at the periphery of the muscle fibers, seen more uniformly in group C. In the morphometric analysis of the gastrocnemius muscle, by hematoxylin / eosin staining, there was a significant decrease in the cross-sectional area of muscle fibers in group C in relations to groups D and E. In the proteomic analysis there was a significant increase in the expression of glycolytic proteins in the groups treated with statin. Conclusion: Our data showed that although the animals did not present evident clinical myopathy, statin doses of 20 mg /kg/ day increased muscle contraction after caffeine stimulation, caused mitochondrial morphological alterations with gastrocnemius muscle atrophy and increased lipid peroxidation in the muscle.
Descrição
Citação
ANDRADE, Pâmela Vieira de. Análises morfofuncionais, bioquímicas e moleculares de modelo experimental de miopatia associada à estatina. São Paulo, 2017. [127] p. Tese (Doutorado em Neurologia/Neurociências) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo, 2017.