The effects of fatigue and oxidation on contractile function of intact muscle fibers and myofibrils isolated from the mouse diaphragm

Abstract The goal of this study was to investigate the effects of repetitive stimulation and the oxidant H2O2 on fatigue of diaphragm intact fibers and in myofibrils measured with different Ca2+ concentrations. Intact fibers were isolated from mice diaphragm, and twitch and tetanic contractions (500 ms duration) were performed at different frequencies of stimulation ranging from 15 Hz to 150 Hz to establish a force-frequency relation before and after a fatigue and recovery protocol, without or after a treatment with H2O2. Fatigue was induced with isometric contractions (500 ms, 40 Hz) evoked every 0.8 seconds, with a total of 625 tetani. After the fatigue, the force recovery was followed by invoking tetanic contractions (500 ms, 40 Hz) every 1 min, with a total duration of 30 min. Individual myofibrils were also isolated from the mouse diaphragm and were tested for isometric contractions before and after treatment with H2O2 and NAC. In a second series of experiments, myofibrils were activated at different pCa (pCa = −log10 [Ca2+]), before and after H2O2 treatment. After 15 minutes of H2O2 treatment, the myofibrillar force was decreased to 54 ± 12% of its control, maximal value, and a result that was reversed by NAC treatment. The force was also decreased after myofibrils were treated with H2O2 and activated in pCa ranging between 4.5 and 5.7. These results suggest that fatigue in diaphragm intact fibers and at the myofibrils level is caused partially by oxidation of the contractile proteins that may be responsible for changing the force in various levels of Ca2+ activation

Characterization of Fiber Types in Different Muscles of the Hindlimb in Female Weanling and Adult Wistar Rats

We analyzed lesser diameter and distribution of fiber types in different skeletal muscles from female Wistar rats using a histoenzymology Myofibrillar Adenosine Tri-phosphatase (mATPase) method. Fragments from muscles were frozen and processed by mATPase in different pH. Adult and weanling rat soleus muscles presented a predominance of type I fibers and larger fiber diameters. In the plantar muscle in adult rats, the type IIB fibers demonstrated greater lesser diameter while in the weanling animals, types I and IIB fibers were larger. The plantar muscle of animals of both ages was composed predominantly of the type IID fibers. The type IID fibers were observed in similar amounts in the lateral gastrocnemius and the medial gastrocnemius muscles. Type IIB fibers showed predominance and presented higher size in comparison with other types in the EDL muscle. The present study shows that data on fiber type distribution and fiber lesser diameter obtained in adult animals cannot always be applied to weanling animals of the same species. Using the mATPase, despite the difficult handling, is an important tool to determine the different characteristics of the specific fibers in the skeletal muscle tissue

Effects of eccentric and concentric training on capillarization and myosin heavy chain contents in rat skeletal muscles after hindlimb suspension

We studied the effects of different protocols of post-disuse rehabilitation on angiogenesis and myosin heavy chain (MHC) content in rat hindlimb muscles after caudal suspension. Thirty female Wistar rats were divided into five groups: (1) Control I, (2) Control II, (3) Suspended, (4) Suspended trained on declined treadmill, and (5) Suspended trained on flat treadmill. Fragments of the soleus and tibialis anterior (TA) muscles were frozen and processed by electrophoresis and immunohistochemistry (CD31 antibody). Hindlimb suspension caused reduction of capillary/fiber (C/F) ratios and contents of MHC type I (MHCI) in the soleus in parallel to increased capillary density. Flat treadmill protocols increased the content of the MHCI isoform. The C/F ratio was increased by concentric training after hypokinesis, but was not modified by eccentric training, which caused a greater reduction of capillary density compared to the other protocols. In the TA muscle, hindlimb suspension caused a non-significant increase in capillary density and C/F ratio with limited changes in MHC. The present data demonstrate that the different training protocols adopted and the functional performance of the muscles analyzed caused specific changes in capillarization and in the content of the various MHC types. (C) 2010 Published by Elsevier GmbH.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo"" (FAPESP)[04/10495-8]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo"" (FAPESP)[04/10496-4]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo"" (FAPESP)[05/51524-3]Fundacao de Apoio ao Ensino. Pesquisa e Assistencia do Hospital das Clinicas (FAEPA, FMRP-USP)CNP

Effects of Moderate–Intensity Physical Training on Skeletal Muscle Substrate Transporters and Metabolic Parameters of Ovariectomized Rats

A deficit of estrogen is associated with energy substrate imbalance, raising the risk of metabolic diseases. Physical training (PT) is a potent metabolic regulator through oxidation and storage of substrates transported by GLUT4 and FAT CD36 in skeletal muscle. However, little is known about the effects of PT on these carriers in an estrogen-deficit scenario. Thus, the aim of this study was to determine the influence of 12 weeks of PT on metabolic variables and GLUT4 and FAT CD36 expression in the skeletal muscle of animals energetically impaired by ovariectomy (OVX). The trained animals swam 30 min/day, 5 days/week, at 80% of the critical load intensity. Spontaneous physical activity was measured biweekly. After training, FAT CD36 and GLUT4 expressions were quantified by immunofluorescence in the soleus, as well as muscular glycogen and triglyceride of the soleus, gluteus maximus and gastrocnemius. OVX significantly reduced FAT CD36, GLUT4 and spontaneous physical activity (p p < 0.01). PT increased soleus glycogen, and OVX decreased muscular triglyceride of gluteus maximus. Therefore, OVX can cause energy disarray through reduction in GLUT4 and FAT CD36 and their muscle substrates and PT prevented these metabolic consequences, masking ovarian estrogen’s absence

Effects of Low-Intensity and Long-Term Aerobic Exercise on the Psoas Muscle of mdx Mice: An Experimental Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a muscle disease characterized by the absence of the protein dystrophin, which causes a loss of sarcolemma integrity, determining recurrent muscle injuries, decrease in muscle function, and progressive degeneration. Currently, there is a need for therapeutic treatments to improve the quality of life of DMD patients. Here, we investigated the effects of a low-intensity aerobic training (37 sessions) on satellite cells, peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1&alpha; protein (PGC-1&alpha;), and different types of fibers of the psoas muscle from mdx mice (DMD experimental model). Wildtype and mdx mice were randomly divided into sedentary and trained groups (n = 24). Trained animals were subjected to 37 sessions of low-intensity running on a motorized treadmill. Subsequently, the psoas muscle was excised and analyzed by immunofluorescence for dystrophin, satellite cells, myosin heavy chain (MHC), and PGC-1&alpha; content. The minimal Feret&rsquo;s diameters of the fibers were measured, and light microscopy was applied to observe general morphological features of the muscles. The training (37 sessions) improved morphological features in muscles from mdx mice and caused an increase in the number of quiescent/activated satellite cells. It also increased the content of PGC-1&alpha; in the mdx group. We concluded that low-intensity aerobic exercise (37 sessions) was able to reverse deleterious changes determined by DMD

Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm) promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin