The effect of heat stress on skeletal muscle contractile properties

An elevated heat-shock protein (HSP) content protects cells and tissues, including skeletal muscles, from certain stressors. We determined if heat stress and the elevated HSP content that results is correlated with protection of contractile characteristics of isolated fast and slow skeletal muscles when contracting at elevated temperatures. To elevate muscle HSP content, one hindlimb of Sprague–Dawley rats (21–28 days old, 70–90 g) was subjected to a 15 min 42 °C heat-stress. Twenty-four hours later, both extensor digitorum longus (EDL) and soleus muscles were removed, mounted in either 20 °C or 42 °C Krebs-Ringer solution, and electrically stimulated. Controls consisted of the same muscles from the contra-lateral (non-stressed) hindlimbs as well as muscles from other (unstressed) animals. Isolated muscles were twitched and brought to tetanus every 5 min for 30 min. As expected, HSP content was elevated in muscles from the heat-stressed limbs when compared with controls. Regardless of prior treatment, both EDL and soleus twitch tensions were lower at 42 °C when compared with 20 °C. In addition, when incubated at 42 °C, both muscles showed a drop in twitch tension between 5 and 30 min. For tetanic tension, both muscles also showed an increase in tension between 5 and 30 min when stimulated at 20 °C regardless of treatment but when stimulated at 42 °C no change was observed. No protective effect of an elevated HSP content was observed for either muscle. In conclusion, although heat stress caused an elevation in HSP content, no protective effects were conferred to isolated contracting muscles.     

Multiple cryotherapy applications attenuate oxidative stress following skeletal muscle injury

Objectives: To investigate the effects of multiple cryotherapy applications after muscle injury on markers of oxidative stress.

Methods: Following cryolesion-induced skeletal muscle injury in rats, ice was applied at the injured site for 30?minutes, three times per day, on the day of injury, and for 2 days after injury. To determine the effect of the cryotherapy treatment on markers of oxidative stress, biochemical analyses were performed 3, 7, and 14 days after injury.

Results: Compared with non-treated animals, cryotherapy reduced dichlorofluorescein at 7 and 14 days post-injury and thiobarbituric acid reactive substances levels at 3 and 7 days post-injury (P?P?>?0.05), whereas non-treated groups demonstrated lower levels than the control group (P?P?P?=?0.92).

Discussion: Cryotherapy reduced the production of reactive oxygen species after muscle injury, resulting in an attenuated response of the antioxidant system. These findings suggest that using multiple cryotherapy applications is efficient to reduce oxidative stress.     

Diffusion of water in skeletal muscle tissue is not influenced by compression in a rat model of deep tissue injury

Sustained mechanical loading of skeletal muscle may result in the development of a severe type of pressure ulcer, referred to as deep tissue injury. Recently it was shown that the diffusion of large molecules (10–150 kDa) is impaired during deformation of tissue-engineered skeletal muscle, suggesting a role for impaired diffusion in the aetiology of deep tissue injury. However, the influence of deformation on diffusion of smaller molecules on its aetiology is less clear. This motivated the present study designed to investigate the influence of deformation of skeletal muscle on the diffusion of water, which can be measured with diffusion tensor magnetic resonance imaging (MRI). It could be predicted that this approach will provide valuable information on the diffusion of small molecules. Additionally the relationship between muscle temperature and diffusion was investigated. During deformation of the tibialis anterior a decrease of the apparent diffusion coefficient (ADC) was observed (7.2±3.9%). The use of a finite element model showed that no correlation existed between the maximum shear strain and the decrease of the ADC. The ADC in the uncompressed gastrocnemius muscle decreased with 5.9±3.7%. In an additional experiment a clear correlation was obtained between the decrease of the ADC and the relative temperature change of skeletal muscle tissue as measured by MRI. Taken together, it was concluded that (1) the decreased diffusion of water was not a direct effect of tissue deformation and (2) that it is likely that the observed decreased ADC during deformation was a result of a decreased muscle temperature. The present study therefore provides evidence that diffusion of small molecules, particularly oxygen and carbon dioxide, is not impaired during deformation of skeletal muscle tissue.     

Acute heat stress prior to downhill running may enhance skeletal muscle remodeling

Heat shock proteins (HSPs) are chaperones that are known to have important roles in facilitating protein synthesis, protein assembly and cellular protection. While HSPs are known to be induced by damaging exercise, little is known about how HSPs actually mediate skeletal muscle adaption to exercise. The purpose of this study was to determine the effects of a heat shock pretreatment and the ensuing increase in HSP expression on early remodeling and signaling (2 and 48 h) events of the soleus (Sol) muscle following a bout of downhill running. Male Wistar rats (10 weeks old) were randomly assigned to control, eccentric exercise (EE; downhill running) or heat shock + eccentric exercise (HS; 41°C for 20 min, 48 h prior to exercise) groups. Markers of muscle damage, muscle regeneration and intracellular signaling were assessed. The phosphorylation (p) of HSP25, Akt, p70s6k, ERK1/2 and JNK proteins was also performed. As expected, following exercise the EE group had increased creatine kinase (CK; 2 h) and mononuclear cell infiltration (48 h) compared to controls. The EE group had an increase in p-HSP25, but there was no change in HSP72 expression, total protein concentration, or neonatal MHC content. Additionally, the EE group had increased p-p70s6k, p-ERK1/2, and p-JNK (2 h) compared to controls; however no changes in p-Akt were seen. In contrast, the HS group had reduced CK (2 h) and mononuclear cell infiltration (48 h) compared to EE. Moreover, the HS group had increased HSP72 content (2 and 48 h), total protein concentration (48 h), neonatal MHC content (2 and 48 h), p-HSP25 and p-p70s6k (2 h). Lastly, the HS group had reduced p-Akt (48 h) and p-ERK1/2 (2 h). These data suggest that heat shock pretreatment and/or the ensuing HSP72 response may protect against muscle damage, and enhance increases in total protein and neonatal MHC content following exercise. These changes appear to be independent of Akt and MAPK signaling pathways.     

SIRT1 activation by pterostilbene attenuates the skeletal muscle oxidative stress injury and mitochondrial dysfunction induced by ischemia reperfusion injury

Ischemia reperfusion (IR) injury is harmful to skeletal muscles and causes mitochondrial oxidative stress. Pterostilbene (PTE), an analogue of resveratrol, has organic protective effects against oxidative stress. However, no studies have investigated whether PTE can protect against IR-related skeletal muscular injury. In this study, we sought to evaluate the protective effect of PTE against IR-related skeletal muscle injury and to determine the mechanisms in this process. Male Sprague–Dawley rats were pretreated with PTE for a week and then underwent limb IR surgery. The IR injury induced segmental necrosis and apoptosis, myofilament disintegration, thicker interstitial spaces, and inflammatory cell infiltration. Furthermore, mitochondrial respiratory chain activity in the muscular tissue was inhibited, methane dicarboxylic aldehyde concentration and myeloperoxidase activity were up-regulated, and superoxide dismutase was down-regulated after IR. However, these effects were significantly inhibited by PTE in a dose-dependent manner. The mechanism underlying IR injury is attributed to the down-regulation of silent information regulator 1 (SIRT1)-FOXO1/p53 pathway and the increase of the Bax/Bcl2 ratio, Cleaved poly ADP-ribose polymerase 1, Cleaved Caspase 3, which can be reversed with PTE. Furthermore, EX527, an SIRT1 inhibitor, counteracted the protective effects of PTE on IR-related muscle injury. In conclusion, PTE has protective properties against IR injury of the skeletal muscles. The mechanism of this protective effect depends on the activation of the SIRT1-FOXO1/p53 signaling pathway and the decrease of the apoptotic ratio in skeletal muscle cells.     

Engineering vascularized skeletal muscle tissue

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.     

Exercise-induced injury to skeletal muscle

Strenuous or unaccustomed exercise can cause injury to skeletal muscle. This paper reviews our understanding of the mechanisms of exercise-induced injury. Measurements of exercise-induced injury have included muscle soreness, increased serum levels of intracellular enzymes, increased lysosomal enzyme activities, structural changes in muscle fibers, and prolonged decreases in force development that cannot be attributed to fatigue. Injury can be induced by exercise of small muscle groups, which suggests that it involves processes localized in skeletal muscles. Exercise of relatively short duration can result in injury, which indicates that long durations of exercise and associated metabolic changes are not necessary for injury to occur. Exercise that involves lengthening contractions results in greater evidence of muscle injury than exercise involving isometric or shortening contractions. Lengthening contractions are associated with higher levels of force and lower metabolic costs per muscle fiber than isometric or shortening contractions. These results suggest that changes in muscle metabolism are not responsible for exercise-induced injury to skeletal muscle. Exercise-induced injury is more likely the result of mechanical disruption of muscle fibers.     

Morphology of connective tissue in skeletal muscle

The arrangement and distribution of connective tissue in six different skeletal muscles and smooth muscle was examined by scanning electron microscopy. The endomysial arrangement of collagen was similar in all types of muscle and consisted of three components: (1) myocyte-myocyte connectives; (2) myocyte-capillary connectives; and (3) a weave network of collagen intimately associated with the basal laminae of the myocytes. The perimysium of the different muscles was qualitatively similar but quantitatively dissimilar. The perimysium consisted of large tendon-like bundles of interwoven collagen which connected with the dense weave collagen that surrounded groups of muscles. The arrangement of the collagen in the perimysium and endomysium would explain differences in the mechanical properties of the different muscle. The contribution of the connective tissue to mechanical properties of muscle is discussed.     

Cellular responses in exertion-induced skeletal muscle injury

Muscle injury is a common result of muscle exertion caused by overload and over-activity. In this presentation, an attempt was made to discuss models of muscle injury which involve exertion but not excessive strain, although most functional activities of the extremities require some eccentric muscle actions. Muscle injury is characterized by cellular and extracellular matrix responses which appear to be common to all types of muscle trauma - even in the absence of bleeding. Using tenotomy and functional over-load of the rat hindlimb muscles as examples, illustrations of several of these responses are presented and discussed.     

低氧预处理通过上调钙网蛋白表达减轻大鼠心肌细胞氧化应激损伤

本文旨在探讨钙网蛋(calreticulin,CRT)是否参与低氧预处理(hypoxic preconditioning,HPC)对心肌细胞氧化应激损伤的保护及其信号转导过程.将原代培养的Sprague.Dawley乳鼠心肌细胞随机分为8组:氧化应激(H2O2)组、短暂低氧(HPC)组、HPC H202组、SB203580(p38 MAPK特异性抑制剂) HPC H2O2组、干扰心肌细胞CRT表达的反义寡核苷酸(antiscnse oligodeoxynucleotides,AS)组、AS H2O2组、AS HPC H202组和对照组,以细胞存活率、乳酸脱氢酶(1actate dehydrogenase,LDH)漏出及流式细胞术检测细胞损伤情况;采用RT-PCR和Western blot分别检测CRT表达和p38MAPK磷酸化水平.结果表明:(1)HPC可减轻氧化应激损伤,与H202组比较,HPC H2O2组细胞存活率增高18.0%,细胞凋亡率和LDH漏出分别降低19.4%和53.0%(均P<0.05);HPC前以SB203580预孵育可消除HPC保护作用,与HPC H202组相比,SB203580 HPC H2O2组细胞凋亡率和LDH漏出分别增高13.1%和96.0%,存活率降低7.3%(均P<0.05);(2)氧化应激明显上调CRT表达(H202组较对照组高7.1倍,P<0.05);HPC也诱导CRT表达上调(HPC组较对照组高2.4倍,P<0.05),但上调程度较H2O2组低59%(P<0.05);即HPC可减轻氧化应激诱导的CRT过表达:(3)AS干扰CRT表达后,HPC保护作用降低,相关性分析显示HPC诱导的CRT适度表达与细胞存活率呈正相关(r=0.8023,P<0.05);(4)HPC前SB203580预孵育可抑制CRT表达上调(分别较HPC H2O2组和HPC组低75%和53%,均P<0.05).上述结果提示,HPC可能通过p38 MAPK信号途径诱导CRT表达上调,减轻心肌细胞氧化应激损伤.     

Heat stress inhibits skeletal muscle hypertrophy

Heat shock proteins (Hsps) are molecular chaperones that aid in protein synthesis and trafficking and have been shown to protect cells/tissues from various protein damaging stressors. To determine the extent to which a single heat stress and the concurrent accumulation of Hsps influences the early events of skeletal muscle hypertrophy, Sprague-Dawley rats were heat stressed (42 degrees C, 15 minutes) 24 hours prior to overloading 1 plantaris muscle by surgical removal of the gastrocnemius muscle. The contralateral plantaris muscles served as controls. Heat-stressed and/or overloaded plantaris muscles were assessed for muscle mass, total muscle protein, muscle protein concentration, Type I myosin heavy chain (Type I MHC) content, as well as Hsp72 and Hsp25 content over the course of 7 days following removal of the gastrocnemius muscle. As expected, in non-heat-stressed animals, muscle mass, total muscle protein and MHC I content were significantly increased (P < 0.05) following overload. In addition, Hsp25 and Hsp72 increased significantly after 2 and 3 days of overload, respectively. A prior heat stress-elevated Hsp25 content to levels similar to those measured following overload alone, but heat stress-induced Hsp72 content was increased significantly greater than was elicited by overload alone. Moreover, overloaded muscles from animals that experienced a prior heat stress showed a lower muscle mass increase at 5 and 7 days; a reduced total muscle protein elevation at 3, 5, and 7 days; reduced protein concentration; and a diminished Type I MHC content accumulation at 3, 5, and 7 days relative to nonheat-stressed animals. These data suggest that a prior heat stress and/or the consequent accumulation of Hsps may inhibit increases in muscle mass, total muscle protein content, and Type I MHC in muscles undergoing hypertrophy.     

Alanine metabolism in skeletal muscle in tissue culture

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L₆) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P < 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P < 0.0005) as well as medium (r = 0.50, P < 0.025) and intracellular (r = 0.85, P < 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P < 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rather to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.     

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P < 0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P < 0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P < 0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P < 0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.