Heterologous expression of wild-type and mutant β-cardiac myosin changes the contractile kinetics of cultured mouse myotubes

The properties of myosin expressed in muscle are a major determinant of muscle performance. In this study we used a novel approach to examine the functional impact of changes in myosin heavy chain (MHC) isoform expression, as well as the consequences of expressing the mutant MHC implicated in familial hypertrophic cardiomyopathy (FHC). Cultured mouse myoblasts that normally express fast embryonic myosin were untransfected, or stably transfected with a plasmid expressing either wild-type (cWT) or mutant (D778G or G741R) β-cardiac myosin. After differentiation for 5–7 days, cWT or mutant β-cardiac myosin was expressed at 25 % of total myosin in the myotube. We measured time-to-peak shortening (ttp), time for half-relaxation ( t _0.5), the maximum velocity of shortening ( V _max) at 1 Hz stimulation, and the tetanic fusion frequency. Expression of cWT β-cardiac myosin significantly increased ttp and t _0.5 and decreased the fusion frequency compared with untransfected myotubes. However, when we compared myotubes expressing mutant β-cardiac myosin with those expressing cWT β-cardiac myosin, we found that ttp and t _0.5 were significantly decreased, and V _max was increased for the D778G mutant, whereas ttp, t _0.5 and V _max were unchanged for the G741R mutant. The fusion frequency was increased for both mutant myosins. Our data support the conclusion that the impact of the slower myosin isoform dominates when both slow and fast isoforms are present. This work suggests that FHC associated with either D778G or G741R mutation in MHC is an 'energy cost' disease, but that the phenotype of D778G is more severe than that of G741R.     

Differences in sodium voltage-gated channel properties according to myosin heavy chain isoform expression in single muscle fibres

The myosin heavy chain (MHC) isoform determines the characteristics and shortening velocity of muscle fibres. The functional properties of the muscle fibre are also conditioned by its membrane excitability through the electrophysiological properties of sodium voltage-gated channels. Macropatch-clamp is used to study sodium channels in fibres from peroneus longus (PL) and soleus (Sol) muscles (Wistar rats, n = 8). After patch-clamp recordings, single fibres are identified by SDS-PAGE electrophoresis according to their myosin heavy chain isoform (slow type I and the three fast types IIa, IIx, IIb). Characteristics of sodium currents are compared (Student's t test) between fibres exhibiting only one MHC isoform. Four MHC isoforms are identified in PL and only type I in Sol single fibres. In PL, maximal sodium current ( I _max), maximal sodium conductance ( g _Na,max) and time constants of activation and inactivation (τ _m and τ _h ) increase according to the scheme I→IIa→IIx→IIb ( P < 0.05). τ _m values related to sodium channel type and/or function, are similar in Sol I and PL IIb fibres ( P = 0.97) despite different contractile properties. The voltage dependence of activation ( V _a,1/2) shows a shift towards positive potentials from Sol type I to IIa, IIx and finally IIb fibres from PL ( P < 0.05). These data are consistent with the earlier recruitment of slow fibres in a fast-mixed muscle like PL, while slow fibres of postural muscle such as soleus could be recruited in the same ways as IIb fibres in a fast muscle.     

Decreased shortening velocity and altered myosin isoforms in guinea-pig hypertrophic intestinal smooth muscle

The aims of this study were to investigate whether hypertrophy of the small intestinal smooth muscle leads to alterations of myosin isoform composition and shortening velocity and whether possible changes correlate with a change in the sensitivity to ADP of shortening velocity in this tissue. A partial occlusion was introduced in the distal part of the ileum of guinea-pigs. After 2 weeks, the part of the small intestine just proximal of the stenosis was hypertrophied (indicated by a significantly increased cross-sectional area). The most proximal part of the small intestine was used as control, thus enabling comparisons between hypertrophic and normal tissue from the same animal. The outer longitudinal layer of the intestinal wall was gently peeled off and used for biochemistry, RT-PCR and mechanical experiments. The desmin/actin ratio was significantly increased following hypertrophy, although myosin and actin expression were similar in control and hypertrophic tissue. In hypertrophic tissue, the myosin heavy chain mRNA with a 21 base pair insert decreased significantly. The composition of the mRNA encoding the myosin essential light chains changed towards more of the basic type (LC17b). No change in the expression of non-muscle myosin heavy chains A and B was detected. The maximal shortening velocity ( V _max) of maximally activated skinned preparations was significantly lower in the hypertrophic tissue (≈50 % of control). The sensitivity of V _max to ADP was increased in the hypertrophic smooth muscle tissue. We conclude that myosin expression is altered following intestinal hypertrophy and that these alterations affect reactions in the cross-bridge interaction, leading to a slower and more economical contractile function.     

Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human muscles

Maximum shortening velocity ( V ₀) was determined in single fibres dissected from hind limb skeletal muscles of rabbit and mouse and classified according to their myosin heavy chain (MHC) isoform composition. The values for rabbit and mouse V ₀ were compared with the values previously obtained in man and rat under identical experimental conditions. Significant differences in V ₀ were found between fibres containing corresponding myosin isoforms in different species: as a general rule for each isoform V ₀ decreased with body mass. Myosin isoform distributions of soleus and tibialis anterior were analysed in mouse, rat, rabbit and man: the proportion of slow myosin generally increased with increasing body size. The diversity between V ₀ of corresponding myosin isoforms and the different myosin isoform composition of corresponding muscles determine the scaling of shortening velocity of whole muscles with body size, which is essential for optimisation of locomotion. The speed of actin translocation ( V _f) in in vitro motility assay was determined with myosins extracted from single muscle fibres of all four species: significant differences were found between myosin isoforms in each species and between corresponding myosin isoforms in different species. The values of V ₀ and V _f determined for each myosin isoform were significantly correlated, strongly supporting the view that the myosin isoform expressed is the major determinant of maximum shortening velocity in muscle fibres.     

Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening velocity in smooth muscle

The relation between the isoform distribution of the myosin 17 kDa essential light chain (LC₁₇) and the mechanical properties of smooth muscle was investigated. The relative content of the basic (LC_17b) and acidic (LC_17a) isoelectric variants of the 17 kDa myosin light chain was determined in different mammalian smooth muscle tissues. The relative content of LC_17b varied between muscles: rabbit rectococcygeus 0%, rabbit trachea 5%, guinea-pig taenia coli 21%, rat uterus 38%, rabbit aorta 56% and rat aorta 60%. The rate of tension development was determined following photolysis of cagedadenosine triphosphate (ATP) in skinned fibres activated with thiophosphorylation of the regulatory light chains. The half-time for force development was 0.67 s in rabbit rectococcygeus, 1.6 s in rabbit trachea, 1.13 s in guineapig taenia coli and 1.38 s in rabbit aorta. The maximal shortening velocity (v _max) was determined with the isotonic quick release technique in skinned fibre preparations activated with thiophosphorylation. v _max was 0.25 muscle lengths per second (ML/s) in rabbit rectococcygeus, 0.24 ML/s in rabbit trachea, 0.17 ML/s in guinea-pig taenia coli, 0.11 ML/s in rat uterus and 0.03 ML/s in rabbit aorta. The range of variation in v _max between muscles was larger than in the half-time for force development. The inverse relationship between v _max and the relative content of LC_17b in the investigated muscles suggests that the type of essential myosin light chain influences the v _max in smooth muscle.     

The mechanism of the force response to stretch in human skinned muscle fibres with different myosin isoforms

Force enhancement during lengthening of an active muscle, a condition that normally occurs during locomotion in vivo , is attributed to recruitment of myosin heads that exhibit fast attachment to and detachment from actin in a cycle that does not imply ATP splitting. We investigated the kinetic and mechanical features of this cycle in Ca²⁺ activated single skinned fibres from human skeletal muscles containing different myosin heavy chain (MHC) isoforms, identified with single-fibre gel electrophoresis. Fibres were activated by using a new set-up that allows development of most of the tension following a temperature jump from 0–1°C to the test temperature (∼12°C). In this way we could prevent the development of sarcomere non-uniformity and record sarcomere length changes with a striation follower in any phase of the mechanical protocol. We found that: (i) fibres with fast MHC isoforms develop 40–70% larger isometric forces than those with slow isoforms, as a result of both a larger fraction of force-generating myosin heads and a higher force per head; (ii) in both slow and fast fibres, force enhancement by stretch is due to recruitment of myosin head attachments, without increase in strain per head above the value generated by the isometric heads; and (iii) the extent of recruitment is larger in slow fibres than in fast fibres, so that the steady force and power output elicited by lengthening become similar, indicating that mechanical and kinetic properties of the actin–myosin interactions under stretch become independent of the MHC isoform.     

The Smooth Muscle Myosin Seven Amino Acid Heavy Chain Insert's Kinetic Role in the Crossbridge Cycle for Mouse Bladder

The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (−/−) and a heterozygous type (+/−). For skinned muscle, an increase in MgADP or inorganic phosphate (P_i) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and P_i release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca²⁺ concentration in both the +/+ and +/− mouse types. However, the −/− type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the −/− type compared to the +/+ or +/− types. Exogenous P_i led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to P_i release. In addition, the increase in P_i showed similar changes in the +/+ and −/− types whereas in the +/− type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle.     

Caldesmon content of mammalian smooth muscles

Summary In the present study we have used a quantitative immunoblotting method to measure the caldesmon content of a variety of smooth muscles with distinctly different contractile phenotypes. Two tonic vascular smooth muscles and several phasic smooth muscles were examined. The caldesmon, actin and myosin contents of each muscle type were measured. Smooth muscle from large arteries (i.e. bovine aorta and porcine carotid artery) had the lowest caldesmon content and phasic muscles (e.g. rat uterus and guinea pig taenia coli) had the highest. The molar ratio of monomeric caldesmon to monomeric actin was 1205 for the aorta and carotid arteryversus 122–28 for the taenia coli and uterus. The molar ratio of caldesmon to monomeric myosin heavy chain was 19 for the aorta and carotidversus 12 for the uterus and taenia coli. The caldesmon contents of canine trachealis and rabbit ileum were intermediate between these extremes. Evidence was found for the presence of both tissue- and species-specific caldesmon isoforms. The relatively high caldesmon content in rat uterus and guinea pig taenia coli suggests the possibility that the contractile phenotype associated with phasic smooth muscles may be dependent on the presence of caldesmon.     

Inhibition of Contraction and Myosin Light Chain Phosphorylation in Guinea-Pig Smooth Muscle by p21-Activated Kinase 1

The p21-activated protein kinases (PAKs) have been implicated in cytoskeletal rearrangements and modulation of non-muscle contractility. Little, however, is known about the role of the PAK family members in smooth muscle contraction. Therefore, we investigated the effect of the predominant isoform in vascular smooth muscle cells, PAK1, on contraction and phosphorylation of the regulatory light chains of myosin (r-MLC) in Triton-skinned guinea-pig smooth muscle. We also investigated which of the three putative substrates at the contractile apparatus - MLCK, caldesmon or r-MLC - is phosphorylated by PAK1 in smooth muscle tissue. Incubation of Triton-skinned carotid artery and taenia coli from guinea-pig with an active mutant of PAK1 in relaxing solution for 30–60 min resulted in inhibition of submaximal force by about 50 %. The mechanism of inhibition of force was studied in the Triton-skinned taenia coli. In this preparation, inhibition of force was associated with a respective inhibition of r-MLC phosphorylation. In the presence of the myosin phosphatase inhibitor, microcystin-LR (10 μ m ), the rate of contraction and r-MLC phosphorylation elicited at pCa 6.79 were both decreased. Because under these conditions the rate of r-MLC phosphorylation is solely dependent on MLCK activity, this result suggests that the inhibitory effect of PAK1 on steady-state force and r-MLC phosphorylation is due to inhibition of MLCK. In line with this, we found that MLCK was significantly phosphorylated by PAK1 while there was very little ³²P incorporation into caldesmon. PAK1 phosphorylated isolated r-MLC but not those in the skinned fibres or in purified smooth muscle myosin II. In conclusion, these results suggest that PAK1 attenuates contraction of skinned smooth muscle by phosphorylating and inhibiting MLCK.     

Characterization of the cross-bridge force-generating step using inorganic phosphate and BDM in myofibrils from rabbit skeletal muscles

The inhibitory effects of inorganic phosphate (P_i) on isometric force in striated muscle suggest that in the ATPase reaction P_i release is coupled to force generation. Whether P_i release and the power stroke are synchronous events or force is generated by an isomerization of the quaternary complex of actomyosin and ATPase products (AM.ADP.P_i) prior to the following release of P_i is still controversial. Examination of the dependence of isometric force on [P_i] in rabbit fast (psoas; 5-15 °C) and slow (soleus; 15-20 °C) myofibrils was used to test the two-step hypothesis of force generation and P_i release. Hyperbolic fits of force-[P_i] relations obtained in fast and slow myofibrils at 15 °C produced an apparent asymptote as [P_i]∞ of 0.07 and 0.44 maximal isometric force (i.e. force in the absence of P_i) in psoas and soleus myofibrils, respectively, with an apparent K _d of 4.3 m m in both. In each muscle type, the force-[P_i] relation was independent of temperature. However, 2,3-butanedione 2-monoxime (BDM) decreased the apparent asymptote of force in both muscle types, as expected from its inhibition of the force-generating isomerization. These data lend strong support to models of cross-bridge action in which force is produced by an isomerization of the AM.ADP.P_i complex immediately preceding the P_i release step.     

Change in contractile properties of human muscle in relationship to the loss of power and slowing of relaxation seen with fatigue

Slow relaxation from an isometric contraction is characteristic of acutely fatigued muscle and is associated with a decrease in the maximum velocity of unloaded shortening ( V _max) and both these phenomena might be due to a decreased rate of cross bridge detachment. We have compared the change in relaxation rate with that of various parameters of the force–velocity relationship over the course of an ischaemic series of fatiguing contractions and subsequent recovery using the human adductor pollicis muscle working in vivo at approximately 37°C in nine healthy young subjects. Maximal isometric force ( F ₀) decreased from 91.0 ± 1.9 to 58.3 ± 3.5 N (mean ± s.e.m. ). Maximum power decreased from 53.6 ± 4.0 to 17.7 ± 1.2 (arbitrary units) while relaxation rate declined from −10.3 ± 0.38 to −2.56 ± 0.29 s⁻¹. V _max showed a smaller relative change from 673 ± 20 to 560 ± 46 deg s⁻¹ and with a time course that differed markedly from that of slowing of relaxation, showing very little change until late in the series of contractions. Curvature of the force–velocity relationship increased ( a/F ₀ decreasing from 0.22 ± 0.02 to 0.11 ± 0.02) with fatigue and with a time course that was similar to that of the loss of power and the slowing of relaxation. It is concluded that for human muscle working at a normal physiological temperature the change in curvature of the force–velocity relationship with fatigue is a major cause of loss of power and may share a common underlying mechanism with the slowing of relaxation from an isometric contraction.     

Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca²⁺ ( k _act ), and the rate of tension redevelopment following a quick release and restretch ( k _tr ), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (P_i) was abruptly increased ( k _Pi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak-to-strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, k _act or k _tr , suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa₅₀ of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca²⁺ sensitivity and the cooperativity of the force-generating mechanism.     

Contractility and myosin isoform compositions of skeletal muscles and muscle cells from rats treated with thyroid hormone for 0, 4 and 8 weeks

The effects of 4 and 8 weeks of thyroidhormone (3,5,3-triiodothyronine, T3) treatment on skeletalmuscles of young (3--6 months) male Wistar rats were investigatedin the present study. In the slow-twitch soleus, contraction andhalf-relaxation times of the isometric twitch were significantlyshorter in hyperthyroid rats than in the control group, andtwitch duration was shorter in rats treated with T3 for 8 weeksthan for 4 weeks. All single soleus muscle fibres fromhyperthyroid rats co-expressed types I and IIA myosin heavychains (type I/IIA fibres) or type I, IIA and IIX myosin heavychains (type I/IIAX fibres), while only type I MyHC fibres wereisolated from the controls. A significantly higher content oftype IIA myosin heavy chain and fast myosin light chain isoformswas observed in soleus fibres from the 8-week than from 4-week T3group. There was no significant difference in maximum velocity ofunloaded shortening (V0) between type I myosin heavy chain fibresfrom controls (1.12 ± 0.46 muscle lengths s–1, n = 48)and type I/IIA myosin heavy chain fibres from the 4 – (1.09± 0.36 muscle lengths s–1, n = 33) and 8-week (1.03± 0.31 muscle lengths s–, n = 31) groups, but typeI/IIAX fibres from the 8-week T3 group had significantly higherV0 (1.56 ± 0.10, n = 5) than type I from control and typeI/IIA from hyperthyroid rats. In the fast-twitch extensordigitorum longus, neither myosin isoform composition, twitchduration nor V0 was affected by 4 or 8 weeks of T3 exposure. Inconclusion, a dramatic and exposure duration-dependent change inthe contractile speed of the isometric twitch and the expressionof fast myosin isoforms was observed in soleus, but not inextensor digitorum longus, in response to T3 treatment. Long-termT3 treatment had relatively less influence, however, on V0 at thesingle cell level in spite of the dramatic increase in fastmyosin isoforms     

Functional consequence of mutation in rat cardiac troponin T is affected differently by myosin heavy chain isoforms

Cardiac troponin T (cTnT) is an essential component of the thin filament regulatory unit (RU) that regulates Ca²⁺ activation of tension in the heart muscle. Because there is coupling between the RU and myosin crossbridges, the functional outcome of cardiomyopathy-related mutations in cTnT may be modified by the type of myosin heavy chain (MHC) isoform. Ca²⁺ activation of tension and ATPase activity were measured in muscle fibres from normal rat hearts containing α-MHC isoform and propylthiouracil (PTU)-treated rat hearts containing β-MHC isoform. Muscle fibres from normal and PTU-treated rat hearts were reconstituted with two different mutations in rat cTnT; the deletion of Glu162 (cTnT_E162DEL) and the deletion of Lys211 (cTnT_K211DEL). α-MHC and β-MHC isoforms had contrasting impact on tension-dependent ATP consumption (tension cost) in cTnT_E162DEL and cTnT_K211DEL reconstituted muscle fibres. Significant increases in tension cost in α-MHC-containing muscle fibres corresponded to 17% ( P < 0.01) and 23% ( P < 0.001) when reconstituted with cTnT_E162DEL and cTnT_K211DEL, respectively. In contrast, tension cost decreased when these two cTnT mutants were reconstituted in muscle fibres containing β-MHC; by approximately 24% ( P < 0.05) when reconstituted with cTnT_E162DEL and by approximately 17% ( P = 0.09) when reconstituted with cTnT_K211DEL. Such differences in tension cost were substantiated by the mechano-dynamic analysis of cTnT mutant reconstituted muscle fibres from normal and PTU-treated rat hearts. Our observation demonstrates that qualitative changes in MHC isoform alters the nature of cardiac myofilament dysfunction induced by mutations in cTnT.     

Charge immobilization of the voltage sensor in domain IV is independent of sodium current inactivation

Recovery from fast inactivation in voltage-dependent Na⁺ channels is associated with a slow component in the time course of gating charge during repolarization (i.e. charge immobilization), which results from the slow movement of the S4 segments in domains III and IV (S4-DIII and S4-DIV). Previous studies have shown that the non-specific removal of fast inactivation by the proteolytic enzyme pronase eliminated charge immobilization, while the specific removal of fast inactivation (by intracellular MTSET modification of a cysteine substituted for the phenylalanine in the IFM motif, ICM_MTSET, in the inactivation particle formed by the linker between domains III and IV) only reduced the amount of charge immobilization by nearly one-half. To investigate the molecular origin of the remaining slow component of charge immobilization we studied the human cardiac Na⁺ channel (hH1a) in which the outermost arginine in the S4-DIV, which contributes ∼20% to total gating charge ( Q _max), was mutated to a cysteine (R1C-DIV). Gating charge could be fully restored in R1C-DIV by exposure to extracellular MTSEA, a positively charged methanethiosulphonate reagent. The RIC-DIV mutation was combined with ICM_MTSET to remove fast inactivation, and the gating currents of R1C-DIV-ICM_MTSET were recorded before and after modification with MTSEA_o. Prior to MTSEA_o, the time course of the gating charge during repolarization ( off -charge) was best described by a single fast time constant. After MTSEA, the off -charge had both fast and slow components, with the slow component accounting for nearly 35% of Q _max. These results demonstrate that the slow movement of the S4-DIV during repolarization is not dependent upon the normal binding of the inactivation particle.     

Effects of h1-calponin ablation on the contractile properties of bladder versus vascular smooth muscle in mice lacking SM-B myosin

The functional significance of smooth muscle-specific h1-calponin up-regulation in the smooth muscle contractility of SM-B null mice was studied by generating double knockout mice lacking both h1-calponin and SM-B myosin. The double knockout mice appear healthy, reproduce well and do not show any smooth muscle pathology. Loss of h1-calponin in the SM-B null mice bladder resulted in increased maximal shortening velocity ( V _max) and steady-state force generation. The force dilatation pressure, which was decreased in the SM-B null mesenteric vessels, was restored to wild-type levels in the double knockout vessels. In contrast, the half-time to maximal constriction was significantly increased in the double knockout vessels similar to that of SM-B null mice and indicating decreased shortening velocity in the double knockout vessels. Biochemical analyses showed that there is a significant reduction in smooth muscle α-actin levels, whereas h-caldesmon levels are increased in the double knockout bladder and mesenteric vessels, suggesting that these changes may also partly contribute to the altered contractile function. Taken together, our studies suggest that up-regulation of h1-calponin in the SM-B null mice may be necessary to maintain a reduced level of cross-bridge cycling over time in the absence of SM-B myosin and play an important role in regulating the smooth muscle contraction.     

运动在骨骼肌肌纤维类型转变中的作用

骨骼肌纤维类型及其表达的专一蛋白同功型的多样性,是骨骼肌功能和适应性的结构和分子基础.肌球蛋白重链同功型被认为是决定肌纤维快、慢类型的主要因素,已成为区分肌纤维类型和研究肌适应性的分子标志.运动可以导致肌球蛋白重链不同亚型之间的转变.本文就肌球蛋白重链与骨骼肌纤维类型的关系,以及不同运动模式对骨骼肌纤维肌球蛋白重链同功型转变的影响作一综述.     

Cardiac and vasomotor components of the carotid baroreflex control of arterial blood pressure during isometric exercise in humans

We sought to examine the importance of the cardiac component of the carotid baroreflex (CBR) in control of blood pressure during isometric exercise. Nine subjects performed 4 min of ischaemic isometric calf exercise at 20% of maximum voluntary contraction. Trials were repeated with β₁-adrenergic blockade (metoprolol, 0.15 ± 0.003 mg kg⁻¹) or parasympathetic blockade (glycopyrrolate, 13.6 ± 1.5 μg kg⁻¹). CBR function was determined using rapid pulses of neck pressure and neck suction from +40 to −80 mmHg, while heart rate (HR), mean arterial pressure (MAP) and changes in stroke volume (SV, Modelflow method) were measured. Metoprolol decreased and glycopyrrolate increased HR and cardiac output both at rest and during exercise ( P < 0.05), while resting and exercising blood pressure were unchanged. Glycopyrrolate reduced the maximal gain ( G _max) of the CBR-HR function curve (−0.58 ± 0.10 to −0.06 ± 0.01 beats min⁻¹ mmHg⁻¹, P < 0.05), but had no effect on the G _max of the CBR-MAP function curve. During isometric exercise the CBR-HR curve was shifted upward and rightward in the metoprolol and no drug conditions, while the control of HR was significantly attenuated with glycopyrrolate ( P < 0.05). Regardless of drug administration isometric exercise produced an upward and rightward resetting of the CBR control of MAP with no change in G _max. Thus, despite marked reductions in CBR control of HR following parasympathetic blockade, CBR control of blood pressure was well maintained. These data suggest that alterations in vasomotor tone are the primary mechanism by which the CBR modulates blood pressure during low intensity isometric exercise.     

Filament formation in smooth muscle homogenates

Summary To provide more detailed information on the aggregation properties of smooth muscle myosin, we have extended earlier work on the formation of thick filaments when homogenates of guinea-pig taenia coli and chicken gizzard muscle are diluted. In both preparations there is a slow and a fast phase of filament formation. The slow phase, which generally develops over several hours, appears to depend primarily on the ATP concentration while the rapid phase, which develops over 5–15 min, is influenced by the extent of dilution, homogenization conditions, divalent cation concentration, ATP concentration and presence of chicken gizzard tropomyosin. Many of these effects on the rapid phase can be explained by postulating that filament formation only takes place when the ATP concentration is reduced.There are significant differences between the filament populations formed from each muscle, with those from taenia coli being shorter than those from gizzard. Two types of filament are present in preparations from each muscle, the first being characterized by the presence of a central bare zone and cross striations at both ends, whilst the second have cross striations along their entire length; the periodicity of the cross striations appears to be 14.5 nm. The bare zone filaments have an average length and width of 325 nm and 17.6 nm respectively, while the corresponding values for the cross striated filaments are 553 nm and 29.0 nm. The proportion of bare zone to cross striated filaments is 3 : 1 for taenia coli and 1 : 3 for chicken gizzard, which accounts for the difference in average filament length observed between these preparations. The gizzard filaments appear to form more readily than those of taenia coli.     

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures

Summary We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.