Rate of isometric tension development in relation to calcium binding of skinned muscle fibres

Isolated muscle fibres from the sartorius or semitendinosus muscles of frogs were mechanically skinned and kept in a relaxed state in a medium containing Mg-ATP and EGTA. When subjected to a rapid increase in internal calcium ion concentration tension rose relatively slowly in comparison to the time course of establishment of the new calcium concentration. Stiffness measurements made during the rise of tension yielded the same stiffness to tension ratio as that observed at steady state force. The linear force extension curve of the activated fibres (T₁-curves) measured at various moments during the rise of tension extrapolated to zero tension intersected the base line at the same length (–0.8% L_o). This suggests that the extent of myosin interaction increases with the same time course as tension.The rate of tension development accompanying a Ca-jump was strongly increased by an increase in calcium ion concentration and there was a linear relationship between the logarithm of the rate tension development and pCa. The rate of recovery of tension following a large quick release > 2% L_o was not calcium sensitive, and occurred at a rate more than an order of magnitude faster than the corresponding calcium activation in the range of pCa's studied. We suggest that the slowness of tension development accompanying a rapid calcium activation reflects slow reactions occurring after a single Ca-ion has bound to a myofilament binding site and does not reflect the slowness of actin and myosin interaction.This work was supported by a grant from the Deutsche Forschungs-gemeinschaft Gu 160/3. One of us (P.J.G.) was supported by the Royal Society European Science Exchange Programme     

Compared properties of the contractile system of skinned slow and fast rat muscle fibres

Chemically skinned fibres from soleus and plantaris rat muscles were used to compare the contractile properties of slow and fast muscles. The maximal isometric tension appeared larger in plantaris than in soleus fibres. The apparent Ca²⁺ threshold for activation was lower in slow than in fast fibres while Ca²⁺ concentrations required to obtain either the maximal tension or half maximal tension (pCa₅₀) were lower in fast than in slow fibres. This apparent difference in Ca²⁺ sensitivity will be discussed. As could be expected from other studies, a faster force development in plantaris than in soleus fibres occurred. However, one interesting new result showed that in soleus, the kinetics of the tension development estimated by the t _max parameter were slightly dependent on the Ca²⁺ concentration whereas the t ₅₀ parameter changed significantly with the Ca²⁺ concentration. In plantaris, both t _max and t ₅₀ parameters were found to depend strongly on the Ca²⁺ concentration. Finally, the plantaris muscle showed a greater caffeine sensitivity than the soleus muscle. All the results suggested that the Ca-regulatory mechanism in the slow fibres was essentially different from that in the fast fibres.     

Inhibition of TnI-TnC interaction and contraction of skinned muscle fibres by the synthetic peptide TnI [104–115]

Circular dichroism was used to study the induction of helix in TnC or TnI-TnC by the TnI peptide [104–115] at various Ca²⁺ concentrations. The increase in negative ellipticity and pCa²⁺ values for the peptide-TnC complex, indicates that binding of the peptide to TnC, induces a small helical conformational change in TnC. This results in an increase in the Ca²⁺ binding constant and the pCa₅₀ value required to induce 50% of Ca²⁺-dependent helix in TnC. The introduction of the peptide to a preformed mixture of TnI-TnC resulted in an increase in negative ellipticity and a decrease in the pCa₅₀ and the apparent Ca²⁺ binding constant towards the values obtained for the TnI peptide-TnC complex and away from those of TnI-TnC. This demonstrates that the TnI peptide can successfully compete with TnI for TnC and thereby inhibit the TnI-TnC interaction. The addition of the TnI peptide to skinned rabbit psoas or porcine cardiac fibres resulted in the inhibition of the force development and a decrease in the pCa₅₀ values required for 50% Ca²⁺ activation. The magnitude of the inhibition of tension development and the shift in the Ca²⁺ sensitivity for skinned cardiac muscle fibres was approximately half that observed with skeletal muscle fibres. In view of the CD findings, these skinned fibre results can be accounted for by the peptide inhibiting the TnI interaction with TnC. However, it is possible that the TnI peptide also has a direct inhibitory effect on TM-actin. Mastoparan, another TnC binding peptide, also inhibited the tension development in skinned skeletal and cardiac muscle fibres, but was much less efficient than the TnI peptide.Abbreviations used Boc N-tert-butyloxycarbonyl - Tn troponin - TM tropomyosin - TnI troponin I - TnC troponin C - HPLC high performance liquid chromatography - DIEA diisopropylethylamine - TFA trifluoroacetic acid - DCC dicyclohexylcarbodiimide - 2-Cl-Z 2-chlorobenzyloxycarbonyl - Tos 4-toluenesulfonyl - EGTA ethylene glycol bis(-amino ethyl ether) N,N,N,N-tetraacctic acid - DTE 1,2-ethanedithiol - ME -mercaptoethanol - S1 myosin subfragment 1 - acto-S1 actin and myosin subfragment This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie, and by grants from the Medical Research Council of Canada, the Alberta Heart Foundation and a studentship (J. V. E.) from the Alberta Heritage Foundation for Medical Research     

Stretch-induced increase in the Ca2+ sensitivity of myofibrillar ATPase activity in skinned fibres from pig ventricles

The increase in force development in the heart with increase in end-diastolic pressure (Frank-Starling mechanism) has been ascribed to an increase in contractile responsiveness of the myofibrils to calcium. We now show that this calcium sensitization is also associated with an increase in calcium responsiveness of the myofibrillar ATPase. Thus, at submaximal Ca activation (pCa 6.0), the ATPase activity of skinned fibres from pig right ventricles is increased from 57.9±4.4% to 70.6±4.4% of the maximal Ca²⁺ activation of ATPase by stretching (by 15%l _o). At maximal Ca²⁺ activation, ATPase was barely altered by stretching. The relationship between ATPase activity of skinned trabecula of pig right ventricle and ATPase-Ca²⁺ concentrations is shifted (by 0.1 pCa unit) to higher pCa values after a stretch-induced increase of the sarcomere length from 2.1 μm to 2.4 μm. The relationship between force and pCa was affected in a similar way by extension. This increased calcium sensitivity is, however, not associated with an alteration in the relationship between ATPase activity and force development (tension cost). In accordance with Brenner's hypothesis, we propose therefore that stretch activation of ATPase is associated with an increase in the apparent rate constant of crossbridge attachment rather than with a decrease in the apparent rate constant of crossbridge detachment.     

Effects of halothane on functionally skinned rabbit soleus muscle fibers: A correlation between tension transient and45Ca release

The mechanism(s) involved in the halothane-induced increase in skeletal muscle contraction was studied using functionally skinned soleus muscle fibers from rabbits: For the tension study, single functionally skinned fibers were individually mounted on two pairs of forceps, with one end attached to a photodiode tension transducer. Ca²⁺-activated tension development of the contractile proteins, and Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients were studied. To measure the amount of calcium, skinned fibers at 0.1 g/ml were used and 0.075 Ci⁴⁵Ca/ml was spiked in the solution 3 (pCa 6.5 and 1 mM [EGTA]) which promoted rapid loading of Ca²⁺. Halothane (1–3%) did not change the [Ca²⁺]-tension relationship; 2 and 3% halothane reduced the maximum Ca²⁺-activated tension by 6–7%. Halothane (1–3%) added to the solution 3, reduced⁴⁵Ca uptake by 3, 22 and 23%; however, the subsequent caffeine-induced tension transient and⁴⁵Ca release were increased by 10–40%. During the release phase only halothane increased both caffeine-induced tension transient and⁴⁵Ca release by 20–60%. The effects of halothane on the tension transient and on the⁴⁵Ca release were comparable. There was no dose-response relationship to the effects of halothane on the above parameters. It is concluded that halothane affects the SR by increasing its membrane permeability to Ca²⁺, resulting in an increase in myoplasmic [Ca²⁺] and thus in the twitch tension in skeletal muscle.     

Effect of perchlorate on calcium release in skinned fibres stimulated by ionic substitution and caffeine

We have determined the effects of the perchlorate ion on the contracture of skinned (sarcolemma removed) skeletal muscle fibres, stimulated either by ionic substitution or caffeine. Calcium release was monitored in single cells by measuring the peak height of tension transients. Perchlorate significantly sensitizes fibres to activation by ionic substitution, a manipulation that is thought to trigger calcium release via the normal physiological pathway. Adding 0.8 mM perchlorate to the solutions shifted the curve relating the magnitude of ionic substitution to the level of activation leftward, such that smaller stimuli were needed to produce a contracture of a given height. Perchlorate could also trigger a contracture directly. Exposing fibres to 1.0 mM perchlorate caused contractures averaging 60% of bracketing controls. In contrast to contractures stimulated by ionic substitution, those triggered by caffeine were unaffected by perchlorate. Since caffeine is thought to act directly on the sarcoplasmic reticulum to cause calcium release, these results suggest that perchlorate enhances activation in skinned fibres by interacting with transverse tubular membranes.     

Calcium sensitivity and myofibrillar protein isoforms of rat skinned skeletal muscle fibres

We investigated the calcium sensitivity for tension generation of different fibre types and the possible correlation between calcium sensitivity and the presence of distinct regulatory protein and myosin light chain (MLC) isoforms in rat skinned skeletal muscle fibres. Fibre types 1, 2A and 2B were identified by electrophoretic analysis of myosin heavy chain (MHC) isoforms. Fibres showing more than one MHC isoform were discarded. Type 1 fibres from the soleus showed a higher pCa (–log₁₀ [Ca], where [ ] denotes concentration) threshold and a lower slope of pCa/tension curve than type 2 extensor digitorum longus (EDL) fibres; between type 2 fibres, type 2B showed the higher slope of pCa/tension curve. Type 1 fibres from different muscles showed similar calcium sensitivities when containing only the slow set of regulatory proteins and MLC; when both slow and fast isoforms were present, calcium sensitivity shifted toward fast type fibre values. Type 2A fibres from different muscles showed a similar calcium sensitivity, independently of the set (purely fast or mixed) of regulatory proteins and MLC. It is suggested that when both fast and slow isoforms of regulatory proteins and of MLC are present in a muscle fibre, calcium sensitivity is dictated mainly by the fast isoforms.     

The effect of exercise on the contractile properties of single skinned fast- and slow-twitch skeletal muscle fibres from the adult rat

The effects of long-term endurance exercise on the contractile properties of single skinned muscle fibres from adult rats, were investigated. Adult (4-month-old) male rats were subjected to a 16-week, high-intensity endurance swimming programme, where animals carried a load (corresponding to 2% of body wt), during all 2-h training sessions. At the conclusion of the training period, muscle fibres isolated from the extensor digitorum longus (EDL), and soleus (SOL), could be classified into distinct classes or fibre types on the basis of their Ca²⁺- and Sr²⁺-activated contractile characteristics. The fast-twitch EDL comprised two fibre populations, while the slow-twitch SOL was found to be composed of three distinct fibre types. Endurance swimming modified the contractile characteristics of fibres from both the EDL and SOL, but exerted greater influence on those of the SOL. This was illustrated by significant increases in the sensitivity to Ca²⁺ and Sr²⁺, and a lower threshold for contraction by these activating ions, in the exercised group. Not one of the total of 272 fibres sampled, exhibited mixed fast- and slow-twitch contractile characteristics, often associated with exercise-induced fibre type transformations. Thus, high-intensity endurance swimming induced changes in some single muscle fibre contractile properties of adult rats, but did not cause major changes in fibre type distribution.     

Venous drainage of the soleus muscle

Soleus veins have been implicated as the site for deep venous thrombosis initiation. Detailed anatomic knowledge is required for the early diagnosis using non-invasive ultrasound techniques. In the present work, we describe the anatomy of the veins that emerge from the ventral face of the soleus muscle. Twenty-eight soleus muscles were dissected and 543 veins were found. The number of veins per leg ranged from 7 to 38. The distribution of these veins per quadrant ranged from 0 to 12. The greatest number of veins occurred at the superior lateral quadrant. Most soleus veins mainly drained into the posterior tibial and fibular veins. The mean length of the soleus veins ranged from 0.907 to 2.804 cm. We conclude that there is a wide variability in the distribution of soleus veins through the soleus muscle and their quadrants. The majority of the soleus veins drain into the tibial and fibular veins.     

恒河猴掌长肌的应用解剖

目的　评价恒河猴掌长肌的实验应用价值。方法　对 30侧恒河猴掌长肌进行解剖 ,测量其肌腹、肌腱的长度、宽度、厚度 ,并对其肌门进行定位。结果　掌长肌肌腹、肌腱均细长 ,且血管、神经变异少。结论　掌长肌是作为骨骼肌游离移植的良好供肌     

Force and force transients in skeletal muscle fibres of the frog skinned by freeze-drying

A freeze-drying method is described by which single skinned skeletal muscle fibres or fibre bundles can readily be obtained. Skinned fibre segments of the ileofibularis and semitendinous muscles of the frog — activated by means of a rapid increase in the Ca-concentration — showed very stable and reproducible contractions. Complete activation occurred at a Ca-concentration of 1.6·10^–6 M and the mid-point of the pCa-tension curve occurred at 6.3·10^–7 M. Addition of phosphate (10^–2 M) had a depressing effect on the speed of the Ca-activated tension development as well as on the maximum tension reached.Addition of caffeine (10^–2 M) had no effect on the tension generation, indicating that the sarcoplasmic reticulum, if present, was not active. The force responses due to rapid length changes applied to the Ca-activated fibre preparations were found to be qualitatively similar to the force responses on intact tissue. This skinning technique might be employed on human biopsies, enabling the measurement of physiological parameters such as for example force and shortening velocity.     

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Thapsigargin has been reported to inhibit ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca²⁺ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca²⁺ content of the SR. The SR was first depleted of Ca²⁺ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 M was required to inhibit Ca²⁺ loading by 50%. In contrast, another SR Ca²⁺ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 M and total inhibition at 50 M. When cyclopiazonic acid (100 M) was applied after, rather than during, Ca²⁺ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 M), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca²⁺ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.     

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 M, ryanodine slightly but significantly increased the submaximal Ca²⁺-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine 1 M decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and activity-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 M and 5 M for SL and AM, respectively, following ryanodine administration in the release phase, and 100 M and 30 M, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca²⁺ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca²⁺ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.     

Alteration of calcium sensitivity of skinned frog skeletal muscle fibres by inositol triphosphate and calmodulin antagonists

We investigated the influence of inositol triphosphate (IP₃), trifluoperazine (TFP), and perhexiline on the calcium sensitivity of freeze-dried frog semitendinosus muscle fibres. Further, the effect of IP₃ on calcium release from the sarcoplasmic reticulum (SR) of frog semitendinosus fibres skinned by saponin was studied. IP₃ decreased the calcium sensitivity of freeze-dried frog skeletal muscle fibres and failed to induce a calcium release from SR of saponin-skinned fibres. Freeze-dried frog skeletal muscle fibres were strongly sensitized for calcium by TFP and perhexiline.     

Topography of commissural fibers of the prefrontal cortex in the rhesus monkey

Summary The topography of commissural fibers of the prefrontal cortex was studied in the rhesus monkey using autoradiography. Commissural fibers originating in the medial prefrontal and the caudal orbital regions course through the anterior portion of the genu and the rostrum of the corpus callosum, while those from the arcuate concavity travel at the rostral border of the body of the corpus callosum. Fibers emanating from the peri-principalis region occupy an intermediate position in the genu of the corpus callosum.     

恒河猴实验感染庚型肝炎病毒的实验研究

目的研究庚型肝炎病毒（ＨＧＶ）在恒河猴中的实验感染状态。方法用一名ＨＧＶＲＮＡ阳性、ＨＢＶ、ＨＣＶ均阴性的健康献血员血浆实验感染２只恒河猴，并取第一代猴感染后６周的血再感染１只第二代恒河猴，然后用以第二代猴感染６周后血继续感染２只第三代恒河猴。分别用逆转录聚合酶链反应（ＲＴ－ｎＰＣＲ）检测受感染猴血清中的ＨＧＶＲＮＡ，并每周抽血测定血清中丙氨酸转氨酶（ＡＬＴ）。结果感染１周后猴血清ＨＧＶＲＮＡ阳转，最长持续阳性２８周以上。不同感染个体血清ＡＬＴ水平有明显差异，其中１号猴有短期轻度升高，５号猴血清ＡＬＴ较长时间在１００Ｕ／Ｌ以上。肝活检发现，感染后１６周猴肝组织出现明显的病毒性肝炎样病理改变。进一步对该献血员血浆和感染后猴血清中的ＨＧＶ５’端部分非编码区基因ＰＣＲ产物进行测序，结果显示感染用献血员血浆和猴血清中ＨＧＶ序列与国外株ＨＧＵ４４４０２的同源性分别为９８３３％和９５８３％；与ＨＧＵ３６３８０株的同源性分别为９２５０％和８９１７％；感染猴血清中ＨＧＶ序列与献血员ＨＧＶ序列同源性为９５８３％。结论恒河猴对ＨＧＶ敏感，可以做为实验模型动物     

Functional differences in type-I fibres from two slow skeletal muscles of rabbit

The present study addressed the question of whether the slow fibres of mammalian skeletal muscle, containing the myosin heavy chain MHCI (type-I fibres), are a functionally homogeneous population. We compared various properties of Ca²⁺-activated, skinned, type-I fibres from the soleus and semitendinosus muscles of a rabbit. Soleus type-I fibres showed significantly faster kinetics of stretch activation, measured as the time-to-peak of the stretch-induced, delayed force increase, t₃, than semitendinosus fibres (1239±438 ms, n=136, vs. 1600±409 ms, n=208 respectively) (means±SD, 22 °C). Similarly, the speed of unloaded shortening at 15 °C was faster in soleus than in semitendinosus fibres [0.79±0.16 fibre lengths (FL) s^–1, n=44, vs. 0.65±0.15 FL s^–1, n=35 respectively]. The kinetics of stretch activation were more temperature sensitive in semitendinosus than in soleus fibres. Finally, the generation of steady-state isometric force was more sensitive to Ca²⁺ in semitendinosus than in soleus fibres: [pCa₅₀ (–log [Ca²⁺] for half-maximal activation) at 22 °C: 6.29±0.15, n=28, vs. 6.19±0.10, n=18 respectively]. These results suggest strongly that there is no functional homogeneity within type-I fibres of different muscles. The observed differences might reflect the existence of more than one functionally different slow myosin heavy chain isoforms or other modifications of contractile proteins.     

Temporal relationship between force,ATPase activity,and myosin phosphorylation during a contraction/relaxation cycle in a skinned smooth muscle

The temporal relationship between myosin phosphorylation, contractile force and ATPase activity was studied in skinned preparations from the guinea-pig Taenia coli. When free Calcium concentration ([Ca²⁺]) was increased from pCa (–log[Ca²⁺]) 9 to pCa 4.5 at low calmodulin concentration (0.05 M), ATPase activity and myosin light-chain phosphorylation rose quickly, while the increase in force and stiffness was delayed. The time-course of tension increase was faster at higher calmodulin concentrations (5 M), although the maximal level of phosphorylation was unchanged. Lowering the calcium concentration from pCa 4.5 to pCa 9 at the plateau of contraction caused a rapid decrease in ATPase activity and in myosin phosphorylation, while force and stiffness decayed more slowly. The force decay could be accelerated by inorganic phosphate. These results suggest that, during contraction, force may be produced actively by phosphorylated and ATP-splitting cross-bridges, but may be maintained by dephosphorylated cross-bridges which cycle slowly. However, force could also be modulated by calmodulin and inorganic phosphate in a manner not involving an alteration in the extent of myosin phosphorylation.     

Shortening velocity and force/pCa relationship in skinned crab muscle fibres of different types

Single fibres of three different types, which had been characterized histochemically with regard to differences in myofibrillar adenosine triphosphatase (ATPase) activity and its pH stability, were microdissected from freeze dried preparations of the closer muscle in walking legs of the crab Eriphia spinifrons. Shortening velocities were determined in slack tests and under constant load conditions in maximally Ca²⁺-activated skinned muscle fibres. Force/pCa relationships were also measured for the different types of fibres. Compared with data on vertebrate muscles, all crab muscle fibres required large length changes to reach zero force and showed low Ca²⁺ sensitivity for isometric force generation. The length/time relationship obtained from slack tests had a biphasic course. Maximal velocity of filament sliding differed in the three types of fibres investigated. The filament sliding of type IV fibres was about 3 times faster than that of type I fibres. The values obtained for type II fibres ranged in between. These data are positively correlated with myofibrillar ATPase activity determined histochemically. Ca²⁺ sensitivity of force generation was lowest in the fast type IV fibres. It was high in the slow type I and the faster contracting type II fibres. Ca²⁺ sensitivity in crab muscle seems not to be correlated with speed of shortening.