Sarcoplasmic reticulum of human skeletal muscle: age-related changes and effect of trainirig

The effect of ageing on human skeletal muscle was investigated using needle biopsies from young and aged subjects and from aged subjects trained with different activity patterns. Histochemical staining for myofibrillar ATPase of ageing m. vastus lateralis demonstrated an unchanged fibre type distribution but a selective atrophy of type IIa and type IIb fibres. Analysis of myosin heavy chain (MHC) composition showed that type I MHC increased with ageing (P< 0.05). The relative content of the MHC isoforms correlated with the relative area of the respective fibre types. Sarcoplasmic reticulum (SR) proteins were investigated in muscle extracts by electrophoretic and immunoblotting techniques. When compared to a young control group (28 0.1 years old, n = 7) blots of post-myofibrillar supernatant proteins probed with polyclonal antibodies to the rabbit fast SR Ca-ATPase, a marker of extrajunctional SR, showed that the content of Ca-ATPase was significantly lower (P < 0.05) in the old control group (68 ± 0.5 years old, n= 8). On the other hand the content of calsequestrin (CS), the major intraluminal protein of SR terminal cisternae (TC), and of the 350-kDa ryanodine-binding protein, which is localized in the junctional regions of TC, did not show a concomitant decrease. These results suggest that ageing differentially affects extrajunctional and junctional SR of human skeletal muscle. These age-related changes were not observed within a group of old strength-trained subjects.     

Novel LMNA mutations in patients with Emery‐Dreifuss muscular dystrophy and functional characterization of four LMNA mutations

Mutations in LMNA cause a variety of diseases affecting striated muscle including autosomal Emery‐Dreifuss muscular dystrophy (EDMD), LMNA‐associated congenital muscular dystrophy (L‐CMD), and limb‐girdle muscular dystrophy type 1B (LGMD1B). Here, we describe novel and recurrent LMNA mutations identified in 50 patients from the United States and Canada, which is the first report of the distribution of LMNA mutations from a large cohort outside Europe. This augments the number of LMNA mutations known to cause EDMD by 16.5%, equating to an increase of 5.9% in the total known LMNA mutations. Eight patients presented with either p.R249W/Q or p.E358K mutations and an early onset EDMD phenotype: two mutations recently associated with L‐CMD. Importantly, 15 mutations are novel and include eight missense mutations (p.R189P, p.F206L, p.S268P, p.S295P, p.E361K, p.G449D, p.L454P, and p.W467R), three splice site mutations (c.IVS4 + 1G>A, c.IVS6 ? 2A>G, and c.IVS8 + 1G>A), one duplication/in frame insertion (p.R190dup), one deletion (p.Q355del), and two silent mutations (p.R119R and p.K270K). Analysis of 4 of our lamin A mutations showed that some caused nuclear deformations and lamin B redistribution in a mutation specific manner. Together, this study significantly augments the number of EDMD patients on the database and describes 15 novel mutations that underlie EDMD, which will contribute to establishing genotype–phenotype correlations. Hum Mutat 31:–16, 2011. © 2011 Wiley‐Liss, Inc.     

Theoretical determination of force-length relations of intact human skeletal muscles using the cross-bridge model

The purpose of this study was to determine force-length relations of selected human skeletal muscles, based on the theoretical foundations of the cross-bridge model and to calculate a strength curve for knee extension from these relations. Force-length relations were determined for the rectus femoris, vastus lateralis, vastus medialis, vastus intermedius and gastrocnemius muscles, using sarcomere/ fiber length data form both legs of four cadavers and sarcomere geometry data reported in the literature. It appears that the two-joint muscles investigated in this study are not able to produce force throughout their full anatomical range of motion, whereas the one-joint muscles can. The strength curve for knee extension was determined as the sum of the force-length relations of the individual knee extensor muscles and showed good agreement with experimentally obtained knee extensor strength curves.     

Energy metabolism in different human skeletal muscles during voluntary isometric contractions

Summary The energy turnover in contracting skeletal muscle was studied by measuring the rate of temperature rise during voluntary, isometric contractions and circulatory arrest in M. soleus, M. sacrospinalis and M. biceps brachii in 14 males, by thermoelements inserted in the muscles. A linear relationship between rate of temperature rise and force intensity given as per cent of maximal voluntary contraction (MVC) was demonstrated in biceps (r=0.95), but not so clearly confirmed in soleus (r=0.73). Muscle biopsies were taken from the same muscles and fibre type distribution was determined histochemically by staining for ATPase. The rate of heat production at MVC showed positive correlation to the percentage of fast twitch (FT) fibres in the muscles (r=0.90). Linear extrapolation indicates that the maximal energy turnover in human FT fibres is approximately six times that of slow twitch (ST) fibres during voluntary isometric contractions.This work was submitted by G. Bolstad as a thesis to the University of Bergen in June 1975, in partial fulfilment of the requirements for the degree of Candidatus realium     

LMNA突变体HEK293、C2C12模型的构建及其核纤层蛋白A/C亚细胞定位

目的构建A型核纤层蛋白基因(LMNA)野生型、突变体的融合蛋白真核表达载体及LMNA突变体慢病毒载体,研究其在HEK293、C2C12中表达、核纤层蛋白A/C亚细胞定位及细胞核改变。方法将野生型全长LMNA cDNA克隆入pEGFP-N1质粒,构建LMNA野生型质粒pEGFP-N1-LMNA,以野生型质粒为模板构建c.1117A>G定点突变质粒pEGFP-N1-LMNA-I373V。将构建的2种质粒分别转染HEK293、C2C12,用G418筛选转染的C2C12,荧光显微镜下观察细胞核形态及GFP标记的核纤层蛋白A/C亚细胞定位。以pEGFP-N1-LMNA-I373V为模板,构建pHBLV-h-LMNA-I373V-3*flag-GFP-PURO慢病毒,对慢病毒进行包装与滴度测定。pHBLV-GFP-PURO、pHBLV-LMNA-C1117-3*flag-GFP-PURO分别转染C2C12,免疫荧光染色法观察转染后细胞核形态及核纤层蛋白A/C亚细胞定位的改变。结果构建的pEGFP-N1-LMNA、pEGFP-N1-LMNA-I373V及pHBLV-h-LMNA-I373V-3*flag-GFP-PURO测序与目的基因序列完全一致。pEGFP-N1-LMNA转染的HEK293、C2C12核纤层蛋白A/C均匀表达于核膜下,pEGFP-N1-LMNA-I373V转染的HEK293、C2C12内核纤层蛋白A/C核内异常聚集,呈散点样分布;与HEK293相比,C2C12细胞转染效率明显降低。慢病毒转染C2C12的转染率高,突变体慢病毒转染的细胞核形态异常及核纤层蛋白A/C核内分布异常。结论成功构建2种LMNA突变体真核表达载体及突变体转染的HEK293、C2C12模型,为LMNA突变体导致疾病的机制研究奠定科学基础。     

Doubly heterozygous LMNA and TTN mutations revealed by exome sequencing in a severe form of dilated cardiomyopathy

Familial dilated cardiomyopathy (DCM) is a heterogeneous disease; although 30 disease genes have been discovered, they explain only no more than half of all cases; in addition, the causes of intra-familial variability in DCM have remained largely unknown. In this study, we exploited the use of whole-exome sequencing (WES) to investigate the causes of clinical variability in an extended family with 14 affected subjects, four of whom showed particular severe manifestations of cardiomyopathy requiring heart transplantation in early adulthood. This analysis, followed by confirmative conventional sequencing, identified the mutation p.K219T in the lamin A/C gene in all 14 affected patients. An additional variant in the gene for titin, p.L4855F, was identified in the severely affected patients. The age for heart transplantation was substantially less for LMNA:p.K219T/TTN:p.L4855F double heterozygotes than that for LMNA:p.K219T single heterozygotes. Myocardial specimens of doubly heterozygote individuals showed increased nuclear length, sarcomeric disorganization, and myonuclear clustering compared with samples from single heterozygotes. In conclusion, our results show that WES can be used for the identification of causal and modifier variants in families with variable manifestations of DCM. In addition, they not only indicate that LMNA and TTN mutational status may be useful in this family for risk stratification in individuals at risk for DCM but also suggest titin as a modifier for DCM.     

A method for the determination of the force-length relation of selected in-vivo human skeletal muscles

In this paper a method is presented to determine force-length relations of in-vivo human skeletal muscles. The method is experimental and can be used for selected multi-joint muscles. It contains three basic assumptions: (a) the maximal, isometric force a muscle can exert is constant for a given muscle length, (b) antagonistic muscle activity for the experimental contractions is constant, and (c) resultant joint moments obtained during the experiments are produced by muscular forces exclusively. Experimentally determined force-length relations of intact in-vivo human skeletal muscles have not been determined yet. Application of this method will allow the comparison of actual force-length relations of selected human skeletal muscles to force-length relations used previously. Proposed mechanisms responsible for the force-length characteristics of a muscle, such as the cross-bridge theory, may be critically evaluated. Differences of force-length relations obtained under in vivo and in vitro conditions may be quantified.     

Whole-cell recordings of chloride currents in cultured human skeletal muscle

Chloride currents in human myoballs were investigated with the tight-seal whole-cell recording method in a wide range of membrane voltages (–125 to +145 mV). Two current components having different kinetics could be distinguished. In more than 90% of the myoballs the following results were obtained. At negative potentials, the amplitude of the Cl^– current was small and independent of time. The amplitude of the current increased as the membrane potential was made more positive. At potentials positive to +75mV, the current increased monoexponentially with time. Inactivation occurred only during very long (>3 s) pulses. When such a test pulse was preceded by a conditioning pulse to +60 mV, the current at potentials more than +90 mV was markedly smaller than in the absence of a prepulse, and no activation was provoked by strongly pulses. Recovery from inactivation could only be measured at potentials negative to –40mV. The Cl^– conductance at –85mV was 5.9±3.64 S/cm² (SD; n=10). In about 5% of the myoballs a kinetically different current was visible, characterized by fast inactivation at highly positive potentials. The current amplitudes were substantially larger in such cases, the Cl^– conductance at –85mV being 12.2±9.02 S/cm² (n=4).     

Myostatin and follistatin expression in skeletal muscles of rats with chronic heart failure

Skeletal muscle abnormalities can contribute to decreased exercise capacity in heart failure. Although muscle atrophy is a common alteration in heart failure, the mechanisms responsible for muscle mass reduction are not clear. Myostatin, a member of TGF-β family (transforming growth factor), regulates muscle growth and mass. Several studies have shown a negative correlation between myostatin expression and muscle mass. The aim of this study was to evaluate myostatin expression in skeletal muscles of rats with heart failure. As myostatin gene expression can be modulated by follistatin, we also evaluated its expression. Heart failure was induced by myocardial infarction (MI, n  = 10); results were compared to Sham-operated group ( n  = 10). Ventricular function was assessed by echocardiogram. Gene expression was analyzed by real-time PCR and protein levels by Western blotting in the soleus and gastrocnemius muscles; fibre trophism was evaluated by morphometric analysis. MI group presented heart failure evidence such as pleural effusion and right ventricular hypertrophy. Left ventricular dilation and dysfunction were observed in MI group. In the soleus muscle, cross-sectional area ( P  = 0.006) and follistatin protein levels (Sham 1.00 ± 0.36; MI 0.18 ± 0.06 arbitrary units; P  = 0.03) were lower in MI and there was a trend for follistatin gene expression to be lower in MI group ( P  = 0.085). There was no change in myostatin expression between groups. In gastrocnemius, all MI group parameters were statistically similar to the Sham. In conclusion, our data show that during chronic heart failure, decreased skeletal muscle trophism is combined with unchanged myostatin and reduced follistatin expression.     

Changes in cross-sectional area in human exercising and non-exercising skeletal muscles

This research was performed to study how the cross-sectional area (CSA) changes in the skeletal muscles of exercising (E-leg) and contralateral non-exercising (N-leg) legs and to evaluate to what extent changes in CSA mirror changes in blood flow or extravascular water displacement. Seven healthy volunteers performed plantar flexion exercise at three different exercise intensities for 10 min each. Six plantar flexions followed by a 2-s rest in between allowed repeated measurement of the blood flow to the lower limbs by duplex ultrasonography in the popliteal artery and CSA by magnetic resonance imaging. The CSA was measured using manual planimetry at rest and after 3 and 9 min of the exercise periods. The CSA increased in the E-leg by 4.5% and decreased in the N-leg by −2.4%, from rest to highest exercise intensity. Post-exercise imaging of the E-leg showed a bi-phasic recovery of CSA with a rapid phase followed by a slower phase while the blood flow very rapidly returned almost to basal. The time course of the post-exercise decrease indicated that about 50% of the increase in CSA at the highest exercise intensity might have been a result of extravascular water displacement and 50% of an increase in the vasculature volume related to the flow increase. The CSA reduction in N-leg seems to have been related to vasoconstriction, probably mainly of the capacitance vessels since blood flow was not reduced. Accepted: 23 August 1999     

An allometric analysis of the number of muscle spindles in mammalian skeletal muscles

An allometric analysis of the number of muscle spindles in relation to muscle mass in mammalian (mouse, rat, guinea-pig, cat, human) skeletal muscles is presented. It is shown that the trend to increasing number as muscle mass increases follows an isometric (length) relationship between species, whereas within a species, at least for the only essentially complete sample (human), the number of spindles scales, on average, with the square root rather than the cube root of muscle mass. An attempt is made to reconcile these apparently discrepant relationships. Use of the widely accepted spindle density (number of spindles g(-1) of muscle) as a measure of relative abundance of spindles in different muscles is shown to be grossly misleading. It is replaced with the residuals of the linear regression of ln spindle number against ln muscle mass. Significant differences in relative spindle abundance as measured by residuals were found between regional groups of muscles: the greatest abundance is in axial muscles, including those concerned with head position, whereas the least is in muscles of the shoulder girdle. No differences were found between large and small muscles operating in parallel, or between antigravity and non-antigravity muscles. For proximal vs. distal muscles, spindles were significantly less abundant in the hand than the arm, but there was no difference between the foot and the leg.     

Bilateral eccentric and concentric torque of quadriceps and hamstring muscles in females and males

Summary This study assessed maximum eccentric (ECC) and concentric (CON) torque of quadriceps (QUAD) and hamstring (HAM) muscle groups in healthy females (n=13) and males (n=27). Peak torques (PT) of bilateral muscle actions were recorded at constant angular velocities of 0.52, 1.57 and 2.61 rad·s⁻¹. The QUAD_con and HAM_con PT decreased (p<0.05) with increasing angular velocity. The QUAD_ecc and HAM_ecc PT increased (p<0.05) in females, whereas QUAD_ecc PT decreased (p<0.05) and HAM_ecc PT showed no change in males. In general, ECC PT was higher (p<0.05) than CON PT and QUAD PT was higher (p<0.05) than HAM PT, for any given angular velocity. Males displayed higher (p<0.05) PT than females but when PT were adjusted for body mass the sex differences in QUAD_con and HAM_con were reduced (p<0.05), whereas the differences in QUAD_ecc and HAM_ecc were abolished. The CON and ECC PT were, on average, 60% and 41% greater, respectively, in males than in females. The corresponding differences, when adjusted for body mass, were 23% and 8%. ECC:CON PT for QUAD were higher (p<0.05) in females than in males. CON and ECC HAM:QUAD PT ratio increased (p<0.05), as a function of velocity. This study suggests, that bilateral ECC PT is higher than CON PT and CON HAM:QUAD PT ratio is higher than ECC HAM:QUAD PT ratio. Moreover, females and males display different ECC torque-velocity patterns, whereas CON torquevelocity patterns are similar and females possess greater QUAD_ecc PT relative to QUAD_con PT than males.     

Effects of two high-intensity intermittent training programs interspaced by detraining on human skeletal muscle and performance

Summary The purpose of this study was to investigate the effects of repeated high-intensity intermittent training programs interspaced by detraining on human skeletal muscle and performances. First, nineteen subjects were submitted to a 15-week cycle ergometer training program which involved both continuous and high-intensity interval work patterns. Among these 19 subjects, six participated in a second 15-week training program after 7 weeks of detraining. Subjects were tested before and after each training program for maximal aerobic power and maximal short-term ergocycle performances of 10 and 90 s. Muscle biopsy from the vastus lateralis before and after both training programs served for the determination of creatine kinase (CK), hexokinase, phosphofructokinase (PFK), lactate dehydrogenase (LDH), malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase (HADH) and oxoglutarate dehydrogenase (OGDH) activities. The first training program induced significant increases in all performances and enzyme activities but not in CK. Seven weeks of detraining provoked significant decreases in maximal aerobic power and maximal 90 s ergocycle performance. While the interruption of training had no effect on glycolytic enzyme markers (PFK and LDH), oxidative enzyme activities (HADH and OGDH) declined. These results suggest that a fairly long interruption in training has negligeable effects on glycolytic enzymes while a persistent training stimulus is required to maintain high oxidative enzyme levels in human skeletal muscle. The degree of adaptation observed after the second training program confirms that the magnitude of the adaptive response to exercise-training is limited.     

Oestradiol and SERM treatments influence oestrogen receptor coregulator gene expression in human skeletal muscle cells

Aim: Oestrogen receptors (ER) are present in human skeletal muscle (hSkM) cells; however, the function of the receptor is currently unknown. We investigated the influence of oestradiol and selective ER modulators [tamoxifen (TAM), raloxifene (RAL)] on ER coregulator mRNA expression in hSkM. Methods: Human skeletal muscle cells were treated with 10 nm oestradiol, 5 μm TAM and 10 μm RAL over a 24‐h period. Following the treatment period, mRNA expression was quantified using real‐time PCR to detect changes in ER‐α, ER‐β, steroid receptor coactivator (SRC), silencing mediator for retinoid and thyroid hormone receptors (SMRT), MyoD, GLUT4 and c‐fos. Results: ER‐α mRNA expression increased with all three drug treatments (P < 0.05) while there was no change in mRNA expression of ER‐β in hSkM cells. mRNA expression of SRC increased and SMRT decreased with oestradiol, TAM and RAL in hSkM cells (P < 0.05). Importantly, mRNA expression of MyoD increased with oestradiol and decreased with TAM and RAL in hSkM cells (P < 0.05). mRNA expression of GLUT4 increased with oestradiol and RAL and decreased with TAM in hSkM cells (P < 0.05). Conclusions: These findings are novel in that they provide the first evidence that oestradiol and selective ER modulators influence ER‐α function in hSkM cells. This demonstrates the importance of the ER and alterations in its coregulators, to potentially prevent sarcopenia and promote muscle growth in postmenopausal women using these forms of hormone replacement therapy.     

Effects of age on sarcoplasmic reticulum properties and histochemical composition of fa stand slow-twitch rat muscles

Calcium release activity of sarcoplasmic reticulum and enzyme-histochemical properties were investigated in extensor digitorum longus (e.d.l.) and soleus muscles in young (4 months) and old (24 months) male rats. With age, the caffeine threshold concentration for calcium release from the sarcoplasmic reticulum of soleus skinned muscle fibres showed only minor modifications. On the other hand, in e.d.l. skinned muscle fibres, the caffeine threshold concentration decreased significantly (P < 0.05). The histochemical fibre type composition changed with age both in soleus and in e.d.l. muscles, showing a common transformation toward a more oxidative histochemical profile. In fact, in aged soleus, a significant (P < 0.05) increase was observed of type 1 fibres to represent almost the totality of the muscle fibres (more than 98%), while types 2C and 2A were reduced in proportion. In aged e.d.l. the percentage of type 1 (P < 0.05), 2A and 2X (a recently identified fourth component of the fast-twitch muscle types) fibres increased, with a reduction of type 2B (P < 0.01) fibres. The present results suggest that the changes in contractile properties of aged muscles may be related to the changes not only in fibre composition but also in the mechanism of calcium release from sarcoplasmic reticulum.