Extracellular potassium concentration and membrane potential in rabbit gastrocnemius muscle during tourniquet ischemia

The relationship between the extracellular potassium concentration (K⁺ e.c.) and the resting membrane potential (MP) of the gastrocnemius muscle was studied in the anesthetized rabbit during a 3-h period of hindleg ischemia and a subsequent 1.5-h period of resumed flow. The K⁺ e.c. was measured on the skeletal muscle surface, using a potassium selective electrode. The MP was recorded with conventional microelectrodes. Small biopsies were taken and analysed for content of ATP and lactate.The lactate content increased fourfold during the ischemic period, but returned to normal values after reflow. No significant changes occurred in the ATP content during the period of the experiment. K⁺ e.c. increased from 3.6±0.2 to 16.1±0.7 mmol/l, and the MP decreased from –90.2±0.6 to –58.5±1.9 mV during the 3-h period of ischemia. After release of the tourniquet there was an initial rapid decrease in K⁺ e.c. to 7.8±0.9 mmol/l during the first 15 min of resumed flow, followed by a slower decrease. The MP increased linearly during the first hour of resumed flow and both variables returned to near normal values 1.5 h after releasing the tourniquet.This work was supported by grants from the Swedish Medical Research Council (project No. 17X-05416), from the Medical Faculty, University of Göteborg and from the Göteborg Medical SocietyPresented in part at the meeting of the Scandinavian Physiological Society, Tammerfors, Finland, May 1981     

Effect of repetitive stimulation on cell volume and its relationship to membrane potential in amphibian skeletal muscle

The effect of electrical stimulation on cell volume, V _c, and its relationship to membrane potential, E _m, was investigated in Rana temporaria striated muscle. Confocal microscope xz-plane scanning and histology of plastic sections independently demonstrated significant and reversible increases in V _c of 19.8±0.62% (n=3) and 27.1±8.62% (n=3), respectively, after a standard stimulation protocol. Microelectrode measurements demonstrated an accompanying membrane potential change, ΔE _m, of +23.6±0.98 mV (n=3). The extent to which this ΔE _m might contribute to the observed changes in V _c was explored in quiescent muscle exposed to variations in extracellular potassium concentration, [K⁺]_e. E _m and V _c varied linearly with log [K⁺]_e and [K⁺]_e, respectively, in the range 2.5–15 mM (R ²=0.99 and 0.96), and these results were used to reconstruct an approximately linear relationship between V _c and E _m (ΔV _c=0.85E _m+68.53; R ²=0.99) and hence derive the ΔV _c expected from the ΔE _m during stimulation. This demonstrated that both the time course and magnitude of the increase and recovery of V _c observed in active muscles could be reproduced by the corresponding [K⁺]_e-induced depolarisation in quiescent muscles, suggesting that the depolarisation associated with membrane activity makes a substantial contribution to the cell swelling during exercise. Furthermore, conditions of Cl⁻ deprivation abolished the relationship between E _m and V _c, supporting a mechanism in which the depolarisation of E _m drives a passive redistribution of Cl⁻ and hence cellular entry of Cl⁻ and K⁺ and an accompanying, osmotically driven, increase in V _c.     

Potassium induced potential changes in rat diaphragm muscle

Summary The effect of different potassium concentrations on the membrane potential and membrane resistance of rat diaphragm muscle fibres was measured by means of a double sucrose gap method and a microelectrode technique. Concentration measurements showed that the muscle fibres gained sodium and lost potassium in the equilibration period.In the absence of external chloride changing the external potassium concentration from 2.8 mM to potassium-free caused a depolarization of the membrane of about 30 mV and a small increase in membrane resistance.This K-dependent potential change (K-response) was reduced by ouabain, K-strophanthin, 2,4-dinitrophenol and cyanide, indicating that an energy requiring process is involved. The temperature dependence of the K-response found is consistent with this assumption. Variation in potassium permeability in the absence and presence of external potassium could account for only 13% of the K-response.The K-response amplitude appeared to depend on the external potassium and the internal sodium concentration. Hyperpolarization of the membrane could not only be produced after readmission of potassium but also after addition of thallium, the latter being more potent. Raising the external chloride concentration resulted in a decrease of the K-response and membrane resistance. The current, generating the Kresponse was shown to be hardly influenced by conditional polarization of the membrane.It is concluded from these results that the Kresponse is mainly due to the operation of an electrogenic sodium pump.     

Intracellular pH during ischemia in skeletal muscle: relationship to membrane potential,extracellular pH,tissue lactic acid and ATP

Intracellular pH (pH_i) was measured with double-barrelled microelectrodes during 4 h of complete tourniquet ischemia in rabbit gastrocnemius muscle (group I). The pH_i was related to extracellular pH (pH_e), membrane potential (E_m), tissue lactic acid (LA) and ATP. A fall in pH_i from 7.00±0.03 to 6.60±0.05 occurred during 4 h of ischemia, with a slight pH-drop (0.07 pH units) during the initial hour and a more pronounced drop of 0.13 pH units during the last hour of ischemia. These changes were paralleled by a considerable decrease in pH_e from 7.30±0.01 to 6.36±0.05 and a sixfold increase of tissue LA. The buffering capacity during the 4 h of ischemia was estimated to 81.9±5.6 mmol H⁺/l×pH. In parallel with the reduction in pH_e, the resting membrane potential decreased from –90 mV and stabilised at around –60 mV after 2 h of ischemia. A less negative cell interior would favour H⁺ extrusion since the E_m-E_H ⁺ gradient was unchanged at about –70 mV during the entire period of ischemia. This could contribute to muscle fiber buffering during ischemia.In another set of experiments (group II) the muscular glycogen reserve was reduced 20 min prior to a 4 h period of ischemia. Thereby an ischemic state was created where ATP levels decreased to 30% of initial, in contrast to the unaltered ATP content in group I. In the low-ATP group II the E_m-E_H ⁺ gradient decreased to 58% of initial and the buffering capacity was slightly but significantly lower (64.6±4 mmol H⁺/l×pH) than in the high-ATP group I. Significant correlation coefficients were obtained between the ATP loss and the E_m-E_H ⁺ gradient (r=0.861,P<0.001). These data suggest that maintenance of the transmembrane H⁺ gradient and to a certain degree the buffer capacity depend on the energy-state of the muscle cell.     

Relation between extracellular [K+], membrane potential and contraction in rat soleus muscle: modulation by the Na+-K+ pump

An increased extracellular K⁺ concentration ([K⁺]₀) is thought to cause muscle fatigue. We studied the effects of increasing [K⁺]₀ from 4 mM to 8–14 mM on tetanic contractions in isolated bundles of fibres and whole soleus muscles from the rat. Whereas there was little depression of force at a [K⁺]₀ of 8–9 mM, a further small increase in [K⁺]₀ to 11–14 mM resulted in a large reduction of force. Tetanus depression at 11 mM [K⁺]_o was increased when using weaker stimulation pulses and decreased with stronger pulses. Whereas the tetanic force/resting membrane potential (E _M) relation showed only moderate force depression with depolarization from –74 to –62 mV, a large reduction of force occurred whenE _M fell to –53 mV. The implications of these relations to fatigue are discussed. Partial inhibition of the Na⁺-K⁺ pump with ouabain (10^–6 M) caused additional force loss at 11 mM [K⁺]₀. Salbutamol, insulin, or calcitonin gene-related peptide all stimulated the Na⁺-K⁺ pump in muscles exposed to 11 mM [K⁺ ₀] and induced an average 26–33% recovery of tetanic force. When using stimulation pulses of 0.1 ms, instead of the standard 1.0-ms pulses, force recovery with these agents was 41–44% which was significantly greater (P < 0.025). Only salbutamol caused any recovery ofE _M (1.3 mV). The observations suggest that the increased Na⁺ concentration difference across the sarcolemma, following Na⁺-K⁺ pump stimulation, has an important role in restoring excitability and force.     

Growth and immobilization effects on sarcomeres: a comparison between gastrocnemius and soleus muscles of the adult rat

The effects of growth and limb immobilization on muscle mass, total physiological cross-section (PC), the number of sarcomeres in series and the length of sarcomere components were investigated in the soleus muscle (SOL) and compared to previously obtained data on gastrocnemius (GM) muscles of rats between age 10 and 16 weeks. For SOL this period of growth was reflected in an increased muscle mass and PC. No such increases were found for GM. In contrast, immobilization caused severe atrophy of fibres of both muscles. Compared to the value at the start of the immobilization, it was found that the fast twitch muscle (GM) atrophied more than the typically slow twitch one (SOL). The number of sarcomeres in series within fibres increased after growth and decreased after immobilization of SOL. For fibres of GM no such changes were observed. Muscle architecture is proposed as an important factor for the explanation of the results concerning the number of sarcomeres in series and those arranged in parallel. Due to the difference in muscle architecture, GM being more pennate than SOL, during growth, it is thought that increases in bone length affect the length of fibres of SOL more than those of GM. During immobilization, atrophy of fibres of GM was sufficient for the muscle length adaptation to meet the muscle length change induced by immobilization but in SOL, atrophy had to be accompanied by decreases in the number of sarcomeres in series to achieve adequate muscle length adaptation.     

Effect of ischemia on contractile and histochemical properties of the rat soleus muscle

Ligature and section of the abdominal aorta results in only minor and temporary functional and metabolic changes in the slow soleus muscle of the rat. A very small decrease in maximal tetanic tension corresponds to a few scattered areas of damaged and necrotic muscle fibres, in which decreased succinic dehydrogenase and loss of phosphorylase activity was observed.A new experimental approach, i.e. ligature and section of the abdominal aorta combined with terminal devascularisation, preserving intact tendons and innervation of the muscle causes maximal muscle ischemia, followed by an almost complete loss of tetanic tension output, marked shortening of contraction time and profound morphological and histochemical changes. The decrease in succinic dehydrogenase and ATPase activities and loss of phosphorylase activity occur in the majority of degenerating muscle fibres except for a thin rim of peripheral fibres during the first 4 days. Subsequently, the contractile properties recover gradually and enzyme activities reappear in the regenerating muscle fibres simultaneously with new revascularisation. Thirty days after the operation all the parameters observed returned to control values.Deceased August 6, 1977     

Lactic acid formation in the working gastrocnemius of the dog

Summary In isolated, supramaximally stimulated, isotonically working gastrocnemii of dogs O₂ consumption and lactic acid (LA) output or uptake were measured according to the Fick principle. Simultaneously LA concentration of the muscle tissue was determined at rest and at different times during work. Within 1.6 min after the onset of work LA concentration of muscle tissue rose from 3.5 at rest to 17.2 (oles per gram wet weight and started to decline slowly at 3.6 min after the onset of work. Resting values were reached after about 30 min of exercise. Maximal LA output amounted to about 1.5 moles per gram per minute during the first minutes of work and decreased slowly until about the 18th minute. LA formation which was calculated from the changes of the LA concentration in muscle tissue and the LA output was 9.3 moles per gram per minute during the first 1.6 min and then declined rapidly reaching zero between the 3rd and the 4th minute. Concentration gradients of LA between muscle tissue and venous blood up to 30 moles per millilitre H₂O were observed, although blood flow was high. It is concluded that LA cannot permeate rapidly out of muscle tissue and that muscle cell membrane is the barrier to the diffusion. Since LA is formed only during the phase of O₂ deficit at the onset of work, there is no evidence for an aerobic glycolysis in skeletal muscle. LA accumulation in muscle cells possibly prevents the organism from metabolic acidosis at the beginning of heavy exercise.This investigation was supported by the Deutsche Forschungsgemeinschaft. 1 [1, 3, 7, 13, 15–17, 28, 29, 35, lit. in 32].     

不同缺血后调适方案对大鼠模型肢体骨骼肌缺血再灌注损伤的影响

背景：近年来不少文献报道缺血后调适在多种组织器官包括心肌和骨骼肌都能激发自身内在的对缺血再灌注损伤的保护作用,而且不同方案引发的保护程度有差异,并表现出种属的特异性。 目的：比较不同缺血后调适方案对大鼠肢体骨骼肌缺血再灌注损伤的保护作用并优选最佳方案。 方法：健康成年雄性SD大鼠随机分为4组(n=9)：缺血再灌注组和不同缺血后调适3个组。各组大鼠均于右侧股动脉进行缺血再灌注实验操作,并分离左侧股动脉作为自身假手术对照,其中缺血再灌注组给予右侧股动脉缺血4 h再灌注24 h;缺血后调适3个组分别于4 h缺血后立即施加4个循环分别为10 s再灌/10 s缺血、30 s再灌/30 s缺血、1 min再灌/1 min缺血的后调适操作,再灌注24 h。各组实验结束后抽血检测乳酸脱氢酶,取样腓肠肌测算湿干质量(湿/干)比值、检测髓过氧化物酶和丙二醛,取样胫前肌电镜观察骨骼肌病理变化。 结果与结论：4个循环的30 s再灌/30 s缺血组湿/干值显著低于缺血再灌注组(P < 0.05),其余2个缺血后调适组与缺血再灌注组差异不明显(P > 0.05)。血浆乳酸脱氢酶、组织髓过氧化物酶和丙二醛值缺血后调适3组均低于缺血再灌注组(P < 0.05),缺血后调适组间未见明显差异。4个循环的30 s再灌/30 s缺血组电镜下骨骼肌线粒体嵴的空泡变性程度、肌原纤维结构清晰程度和细胞核完整性较缺血再灌注组均有明显改善,其余2个缺血后调适组超微结构较缺血再灌注组也有不同程度改善。结果提示缺血后调适对大鼠骨骼肌缺血再灌注损伤有保护作用,4个循环30 s再灌/30 s缺血方案的保护效果最明显,可作为进一步实验研究的基础。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

Alterations in calcium homeostasis reduce membrane excitability in amphibian skeletal muscle

The effects of alterations in intracellular calcium homeostasis on surface membrane excitability were investigated in resting Rana temporaria sartorius muscle. This was prompted by initial results from a fatiguing stimulation protocol study that demonstrated a fibre subpopulation in which action potential generation in response to a standard 1.5 V electrical stimulus failed despite mean membrane potentials [E _m, −69±2.3 mV (n=14)] compatible with spike firing in a control set of quiescent muscle fibres. Intracellular micro-electrode recordings showed a similar reversible loss of excitability, attributable to an increased threshold, despite only small (7.1±1.8 mV) positive changes in E _m after approximately 60-min exposures to nominally 0 Ca²⁺ Ringer solutions in which Ca²⁺ was replaced by Mg²⁺. This effect was not reproduced by addition of Mg²⁺ to the Ringer solution and persisted under conditions of Cl⁻ deprivation. The effects of three pharmacological agents, cyclopiazonic acid (CPA), caffeine and 4-chloro-m-cresol (4-CmC), each known to deplete store Ca²⁺ and increase cytosolic Ca²⁺ through contrasting mechanisms without influencing E _m, were then investigated. All three agents produced a more rapid, but nevertheless still reversible, loss of membrane excitability than in 0 Ca²⁺ Ringer solution alone. This reduction in membrane excitability persisted in fibres studied in solutions containing a normal [Ca²⁺] following prior depletion of store Ca²⁺ using CPA- and 4-CmC-containing solutions. These novel findings suggest that sarcoplasmic reticulum Ca²⁺ content profoundly influences surface membrane excitability, thereby providing a potential mechanism by which spike firing fails in well-polarised fibres during fatigue.The authors Usher-Smith and Xu were equal contributors to this paper.     

A dichtomy of the membrane potential response of rat soleus muscle fibres to low extracellular potassium concentrations

The effects of different extracellular potassium concentrations [K⁺]_o on the resting membrane potential (E_M) of rat soleus muscle fibres was assessed in the absence and presence of 10^–3M ouabain. At concentrations of 3 mM K and below, the fibres could be divided into two significantly different and normally distributed populations on the basis of E_M response: One group responded to a lowering of the [K]_o with a graded hyperpolarization to between –80 and –103 mM. The second fibre population had a less negative E_M (–70 mV) which did not respond to changes in [K]_o between 0 mM and 3 mM. In the [K]_o range, 0 mM to 5.9 mM, the mean E_M of fibres treated with ouabain was only slightly less negative than the E_M of the second fibre population. We conclude that this dichtomy of the mean E_M at low [K]_o reflects the presence of two fibre types with different electrochemical properties.     

Age-related changes in power output during repetitive contractions of rat medial gastrocnemius muscle

Changes in isometric force, power output and relaxation rate have been measured during repetitive tetanic contractions in 2 groups of rats of different ages. During the first 5 contractions there were no differences between a young and mature group. In contrast to isometric force production, which decreased about 3% per contraction, power output initially increased to 108% of the power output in the first contraction. A greater reduction in power output and relaxation rate after the 5th contraction indicated a greater reduction of the cross-bridge cycling rate in the younger rats. ATP, phosphocreatine and lactate concentrations after the last contraction were not different between the age-groups. In contrast IMP production, which has been suggested may play a regulatory role during fatigue was twice as high in the young rats. Judged by isometric force production there is no age-related difference in fatiguability. However, profound differences were observed in power output, which indicates that quantification of fatigue as a loss of isometric force may be seriously misleading when considering the functional status of the muscle for normal dynamic contractions.     

Role of smooth muscle cell membrane potential in neointima formation in arteries of spontaneously hypertensive rats

Based on observations that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) have altered resting potentials as well as abnormal cell proliferation rates, neointima formation after controlled balloon injury was compared in arteries from SHR and Wistar Kyoto rats (WKY). SHR aortic VSMC showed hyperpolarized resting membrane potentials (−93±8 mV) when compared to those from WKY (−61±6 mV). Histomorphometric analysis of cross sections from aortic segments submitted to balloon injury showed reduced neointima formation in SHR (neointima/media ratio: 0.04±0.03) as compared to WKY (0.2±0.1). On the other hand, in injured carotid arteries, neointima formation was more extensive in SHR (neointima/media ratio 5.0±0.9) than in WKY (0.8±0.7), leading in most cases to luminal occlusion. Measurements of VSMC resting potential showed that carotid artery cells from SHR were depolarized with respect to those from WKY (−46±4 vs. −69±5 mV, respectively). The results demonstrate an inverse relationship between VSMC membrane polarization and neointima formation in SHR arteries, suggesting that genetic modifications in SHR determine a dysfunctional cellular physiology that may influence cell proliferation subsequent to injury.     

Effects of skeletal muscle regeneration on the proliferation potential of satellite cells

Skeletal muscle satellite cells are myogenic stem cells that function to repair damaged muscle fibers. Participation of satellite cells in a regeneration response following muscle injury results in a significant reduction in their cumulative proliferation potential. The magnitude of the reduction is proportional to the number of regeneration responses in which the cells participate.     

H2O2对大鼠骨骼肌卫星细胞凋亡和线粒体膜电位的影响及EPO的保护作用

目的: 探讨过氧化氢(H2O2)对大鼠骨骼肌卫星细胞(SMSC)凋亡和线粒体膜电位(MMP)的影响以及促红细胞生成素(EPO)的保护作用。方法: 取体外培养的骨骼肌卫星细胞,随机分为正常对照组、H2O2组、H2O2+EPO组,采用流式细胞仪(FCM)检测细胞凋亡率和MMP的平均荧光强度,Hoechst 33258染色后荧光显微镜观察凋亡细胞的形态学变化。结果: H2O2组凋亡发生率最高,为(22.13±1.79)%,经10、20、40 kU/L浓度的EPO预处理后凋亡率分别为(16.47±2.53)%、(4.97±0.55)%、(2.93±0.47)%;H2O2干预组MMP最低,为9.70±0.09,经10、20、40 kU/L浓度的EPO预处理后MMP分别为12.67±0.32、27.90±0.66、44.53±0.93,对照组凋亡率为(1.93±0.57)%,MMP为51.37±0.64;在H2O2干预组和低剂量EPO预干预组,Hoechst 33258染色呈现明显的凋亡征象。结论: EPO可抑制H2O2诱导的细胞凋亡并稳定线粒体膜电位。     

A post-tetanic decrease of membrane resistance in mammalian skeletal muscle fibres and its antimyotonic effects

Summary The duration of the after-depolarization following an action potential becomes shorter when rat skeletal muscle is stimulated repetitively. This is particularly conspicuous when a muscle has been made myotonic, e.g. by addition of 1 mM 2,4-D to the bathing solution, because in myotonic muscle the duration of the afterdepolarization is longer than in normal muscle. The accelerated decline of afterdepolarization is associated with a temporary decrease of the specific membrane resistance (R _m ) following activity. The decrease ofR _m is more pronounced in myotonic than in nonmyotonic muscle. It becomes greater when activity is increased from 3 to 8 action potentials. In myotonic fibres which on the average have membrane resistances more than twice as large as normal,R _m can thus drop to normal values. The decrease of membrane resistance persists for more than a second after activity. We suggest that this temporary change ofR _m is the basis of the termination of a myotonic burst and for the warm-up phenomenon.This work was supported by the Deutsche Forschungsgemeinschaft.     

Lactate oxidation by skeletal muscle during sustained contraction in vivo

Summary Estimation of lactate oxidation in vivo was carried out by a tracer technique in the muscles of the lower hindleg of the dog during tetani of different duration. The fractional (%) rate of lactate oxidation increased markedly, compared with that of the resting muscle, after the first 2 min of stimulation. Lactate oxidation afforded a large contribution to the total oxygen consumption. Fatigue appeared to play a role in limiting lactate oxidation in the later phase of sustained contraction.     

Enhancement of muscle regeneration in the rat gastrocnemius muscle by low energy laser irradiation

Summary The effect of low-energy laser (He-Ne) irradiation on the rate of skeletal muscle regeneration after partial excision of the rat gastrocnemius muscle was studied using quantitative histological morphometric methods. The injured zones of the experimental rats were subjected to direct He-Ne laser (632.8 nm wavelength) irradiation (6.0 mW for 2.3 min) immediately following injury and once daily thereafter for 5 days. Muscles that were injured as above and subjected to red or room light irradiation served as a control group. The volume fraction (percent of total volume of injured zone) of the mononucleated cells in the injured zone decreased gradually with time after injury, but more rapidly in the laser irradiated muscles than in the control. At 3 days post-injury the myotubes in the laser-irradiated muscles populated a significantly higher percentage (13.9 ± 1.1%) of the injured area than in the control muscles (7.8 ± 1.0%). The volume fraction of the young myofibers in the laser irradiated muscles exceeded 30.6 ± 2.2% and 49.6 ± 5.6% at 8 and 11 days post-injury, respectively, while in control muscles these structures comprised only 9.6 ± 1.0% and 27.2 ± 3.8% of the injured zone at 8 and 11 days after injury, respectively. It is concluded that He-Ne laser irradiation during the regeneration process promotes muscle maturation in the injured zone following partial excision of the rat gastro-cnemius muscle.     

Palmitic acid metabolism in the soleus muscle in vitro in hypo- and hyperthyroid rats

The aim of this study was to establish whether the rate of fatty acid (FA) incorporation and its utilization by the isolated soleus muscle is modified under conditions of thyroid hormone deficit or excess. The rate of palmitic acid (PA) uptake, oxidation and incorporation into intramuscular lipids with increasing PA concentration (0.5–1.5 mM) in the incubation medium were determined. In hypothyroid rats intramuscular triacylglycerol (TG) synthesis was increased, while the rate of PA oxidation to CO₂ and incorporation into mono- and diacylglycerols (MG/DG) and phospholipids (PL) remained unchanged. In rats with triiodothyronine (T₃) excess the rate of all processes studied was enhanced, although the percentage incorporation of PA into different classes of intramuscular lipids was fairly constant and, independently of thyroid state and FA concentration in the medium, was 56–66% for TG, 9-14% for MG/DG and 24–32% for PL. Our results thus indicate that even short-term T₃ excess accelerates the rate of FA uptake and metabolism in the oxidative soleus muscle, whereas in hypothyroid rats only intramuscular TG synthesis is affected.