Rabbit Soleus Research Articles

Reconsidering assumptions in the analysis of muscle fibre cross-sectional area.

Cross-sectional area (CSA) is a fundamental variable in characterizing muscle mechanical properties. Typically, the CSA of a single muscle fibre is assessed by measuring either one or two diameters, and assuming the cross-section is either circular or elliptical in shape. However, fibre cross-sections have irregular shapes. The accuracy and precision of CSAs determined using circular and elliptical shape assumptions are unclear for mammalian skinned muscle fibres. Second harmonic generation imaging of skinned rabbit soleus fibres revealed that the circular assumption overstated real CSA by 5.3±25.9% whereas the elliptical assumption overstated real CSA by 2.8±6.9%. A preferred rotational alignment can bias the circular assumption, as real CSA was overstated by 22.1±24.8% when using the larger fibre diameter and understated by 11.4±13% when using the smaller fibre diameter. With 73% lower variable error and reduced bias, the elliptical assumption is superior to the circular assumption when assessing the CSA of skinned mammalian fibres.

Influence of caffeine on the maximal isometric and concentric force produced by skinned fibers

Caffeine is one of the most famous and widely used ergogenic drugs, especially by athletes to improve sports performance. Caffeine is known to enhance muscle contraction by facilitating Ca2+ release from the sarcoplasmic reticulum. While the effect of caffeine on the cross-bridge dynamics has also investigated, the results is controversial. Therefore, the purpose of this study was to examine the influence of caffeine on cross-bridge dynamics using skinned fiber preparations from rabbit soleus (N = 19 in total). We performed isometric contractions at an average sarcomere length of 2.4 μm; thereafter, skinned fibers were shortened by 20% of the fiber length at a velocity of 0.1 mm/s (slow shortening) or 0.5 mm/s (fast shortening). The contractions were performed under both normal and caffeine-containing activating solution conditions to compare the isometric, slow concentric, and fast concentric forces between conditions. The isometric force did not differ between normal and caffeine-containing activating solution conditions. Similarly, the concentric forces obtained during the slow and fast shortening trials did not differ between conditions. We also measured the stiffness and the rate of force redevelopment (kTR) during the isometric contraction phase and found that these values were not different between normal and caffeine conditions. Based on these results, we conclude that the influence of caffeine on cross-bridge dynamics is negligible, and the ergogenic effect of caffeine, from the view of muscle contractility, is by facilitating Ca2+ release, as suggested in previous studies, and not by modulating the cross-bridge dynamics.

Age-dependent mechanical and microstructural properties of the rabbit soleus muscle

During growth there are serious changes in the skeletal muscles to compensate for the changed requirements in terms of body weight and size. In this study, the age-dependent (between 21 and 100 days) mechanical and microstructural properties of rabbit soleus muscle tissue were investigated. For this purpose, morphological properties (animal mass, soleus muscle mass, tibial length) were measured at 5 different times during aging. On the other hand, fibre orientation-dependent axial and semi-confined compression experiments were realised. In addition, the essential components (muscle fibres, extracellular matrix, remaining components), dominating the microstructure of muscle tissue, were analysed. While the mechanical results show hardly any age-dependent differences, the morphological and microstructural results show clear age-dependent differences. All morphological parameters increase significantly (animal mass by 839.2%, muscle mass 1050.6%, tibial length 233.6%). In contrast, microstructural parameters change differently. The percentage of fibres (divided into slow-twitch (ST) and fast-twitch (FT) fibres) increases significantly (137.6%), while the proportions of the extracellular matrix and the remaining components (48.2% and 46.1%) decrease. At the same time, the cross-sectional area of the fibres increases significantly (697.9%). The totality of this age-dependent information provides a deeper understanding of age-related changes in muscle structure and function and may contribute to successful development and validation of growth models in the future. Statement of significanceThis article reports the first comprehensive data set on age-dependent morphological (animal mass, soleus muscle mass, tibial length), mechanical (axial and semi-confined compression), and microstructural (muscle fibres, extracellular matrix, remaining components) properties of the rabbit soleus muscle. On the one hand, the results of this study contribute to the understanding of muscle mechanics and thus to understanding of load transfer mechanisms inside the muscle tissue during growth. On the other hand, these results are relevant to the fields of constitutive formulation of age-dependent muscle tissue.

The stretch-shortening cycle effect is prominent in the inhibited force state

It has been suggested that residual force enhancement (RFE) contributes to the work enhancement observed in stretch-shortening cycles (SSC). Based on recent findings that RFE was preserved in the reduced force state, one may speculate that the SSC effect may be preserved in the reduced force state as well. The purpose of this study was to examine the magnitude of the SSC effect in inhibited skeletal muscle force states. Normal and inhibited force conditions were analyzed using skinned rabbit soleus fibres (N = 18). The inhibited force condition was achieved by adding 2,3-Butanedione monoxime into the activating solution. For both conditions, a SSC test and a pure shortening test were performed. In the SSC tests, fibres were activated at an average sarcomere length of 2.4 μm, and then stretched to 3.0 μm. Immediately after the end of the stretch, fibres were shortened to 2.4 μm. In the pure shortening tests, fibres were activated at an average sarcomere length of 3.0 μm and then shortened to 2.4 μm. The relative increase in mechanical work in the shortening phase of the SSC compared to the pure shortening condition was defined as the SSC effect index, and the magnitude of the SSC effect was compared between the normal and the inhibited force condition. The SSC effect was greater in the inhibited compared to the normal force condition (p < 0.001). We conclude that the SSC effect is at least in part preserved in the reduced force state.

The effects of inorganic phosphate on contractile function of slow skeletal muscle fibres are length-dependent

Muscle operates across a wide range of sarcomere lengths. Inorganic phosphate (Pi) diminishes force output of striated muscle, with greater influence at short relative to long sarcomere lengths in fast skeletal and cardiac muscle fibres. The purpose of this study was to fill a gap in the literature regarding the length-dependent effects of Pi on contractile function of slow skeletal muscle fibres. Permeabilized slow skeletal muscle fibres from rabbit soleus were assessed at average sarcomere lengths of 2.0, 2.4, or 2.8 μm, with and without 20 mM Pi added to activating solutions (22±1 °C). The magnitude of Pi-induced reductions in peak force (43 ± 7% at 2.0 μm, 38 ± 7% at 2.4 μm, and 31 ± 8% at 2.8 μm) and peak stiffness (41 ± 9% at 2.0 μm, 36 ± 8% at 2.4 μm, and 26 ± 9% at 2.8 μm) were length dependent. Peak stiffness was less affected by Pi than peak force. Pi diminished the Ca2+-sensitivity of the force-pCa and stiffness-pCa relationships to a greater extent at 2.8 μm than 2.0 μm. Comparable results were obtained from a cooperative model of Ca2+ and myosin binding to regulated actin. In conclusion, Pi is more detrimental to the peak force output of slow skeletal muscle fibres held at short relative to long sarcomere lengths, whereas Pi has a greater effect on the Ca2+-sensitivity of force production at long relative to short sarcomere lengths. Stiffness data suggest that Pi-induced reductions in force are primarily due to fewer bound cross-bridges, with a lesser contribution attributable to lower average force per cross-bridge.

Orthophosphate increases the efficiency of slow muscle-myosin isoform in the presence of omecamtiv mecarbil

Omecamtiv mecarbil (OM) is a putative positive inotropic tool for treatment of systolic heart dysfunction, based on the finding that in vivo it increases the ejection fraction and in vitro it prolongs the actin-bond life time of the cardiac and slow-skeletal muscle isoforms of myosin. OM action in situ, however, is still poorly understood as the enhanced Ca2+-sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca2+. Here we show, by combining fast sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus, that the depressant effect of OM on the force per attached motor is reversed, without effect on the ATPase rate, by physiological concentrations of inorganic phosphate (Pi) (1-10 mM). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients, whenever [Pi] increases with heart-beat frequency.

A simple geometrical model accounting for 3D muscle architectural changes across muscle lengths

Muscle architecture parameters change when the muscle changes in length. This has multiple effects on the function of the muscle, e.g. on force production and on contraction velocity. Here we present a versatile geometrical model that predicts changes in muscle architecture as a consequence of length changes of the muscle on the basis of the known architecture at a given muscle length. The model accounts for small changes in aponeuroses’ dimensions relative to changes in fascicle length and keeps muscle volume constant. We evaluate the model on the rabbit soleus muscle by comparing model predictions of fascicle lengths and pennation angles with experimental data. For this, we determined the internal architecture of the soleus muscle at different muscle belly lengths (67.8 mm at 35° ankle angle and 59.3 mm at 80° ankle angle). The long and the short soleus muscle exhibited mean fascicle lengths and pennation angles of 20.8 ± 1.3 mm, 4 ± 2° and 13.5 ± 1 mm, 10 ± 4°, respectively. The model predicted reasonable mean fascicle lengths and pennation angles for the long and short soleus that differed only by 1 mm and 1° from the measured data, respectively. Differences between predicted and measured distributions seem to stem from interindividual variability in muscle architecture. Even if the proposed approach has been used for the soleus muscle, which is relatively simple in architecture, it is not restricted to homogeneous unipennate architectures.

In Situ Characterization of the Working Stroke of the Slow and Fast Isoforms of Muscle Myosin

Muscle performance depends on the myosin heavy chain (MHC) isoform expressed in the myosin II motor. Stiffness measurements at sarcomere level in skinned fibres from either soleus or psoas muscles of rabbit have recently indicated that the stiffness and the force of the slow myosin isoform (soleus), are three times smaller than those of the fast isoform (psoas) (Percario et al. J Physiol 596:1243, 2018). Here the investigation on the mechano-kinetic features of the two isoforms in situ is extended to the comparison of the size and speed of the working stroke, measured on the isotonic velocity transient elicited by stepwise drops in force of different sizes superimposed on the plateau force (T0) of maximally Ca2+ activated skinned fibres from either muscle (sarcomere length 2.4 μm, temperature 12°C, 4% dextran T-500). The analysis of the early rapid shortening, due to the synchronisation of the working stroke in the attached motors shows that at high load (0.8 T0) the size of the working stroke is smaller in the slow isoform (2 nm) than in the fast isoform (5 nm), while it is similar at low load (7-8 nm at 0.2 T0). The rate of the working stroke increases by ∼6 times with the decrease in load from 0.8 to 0.2 T0, but, at any load, is one order of magnitude slower in the slow isoform than in the fast isoform. A reduced size of the working stroke of the slow isoform, together with the smaller force per motor, opens the question on the mechanism that can explain the high efficiency of the slow muscle with respect to the fast muscle. Supported by MIUR-PRIN 2010R8JK2X and Telethon GGP12282 (Italy).

Influence of muscle length on the stretch-shortening cycle in skinned rabbit soleus

Muscle force generated during shortening is instantaneously increased after active stretch. This phenomenon is called as stretch-shortening cycle (SSC) effect. It has been suggested that residual force enhancement contributes to the SSC effect. If so, the magnitude of SSC effect should be larger in the longer muscle length condition, because the residual force enhancement is prominent in the long muscle length condition. This hypothesis was examined by performing the SSC in the short and long muscle length conditions. Skinned fibers obtained from rabbit soleus (N = 20) were used in this study. To calculate the magnitude of SSC effect, the SSC trial (isometric-eccentric-concentric-isometric) and the control trial (isometric-concentric-isometric) were conducted in the short (within the range of 2.4 to 2.7 μm) and long muscle (within the range of 3.0 to 3.3 μm). The magnitude of SSC effect was calculated as the relative increase in the mechanical work attained during the shortening phase between control and SSC trials. As a result, the magnitude of SSC effect was significantly larger in the long (176.8 ± 18.1%) than in the short muscle length condition (157.4 ± 8.5%) (p < 0.001). This result supports our hypothesis that the magnitude of SSC effect is larger in the longer muscle length condition, possibly due to the larger magnitude of residual force enhancement.

Effects of adrenaline on contractility and endurance of isolated mammalian soleus with different calcium concentrations.

The β-adrenergic receptor stimulation improves endurance in fast twitch muscles and these effects are sensitive to extracellular Ca2+ influx. Present study is aimed to determine the effects of adrenaline, with different concentrations of extracellular Ca2+[Formula: see text], on the contractility and endurance of slow twitch muscles during high frequency stimulations (HFS). Isolated soleus of rabbit was electrically stimulated (strength; 50Hz, duration; 0.5ms) in the presence (Test) of adrenaline (1 × 10-7mM) or without adrenaline (CTL). Fatigue was induced with HFS (80Hz) for the duration of 20s. Contractions were recorded through isometric transducer connected with Powerlab. Kreb's buffer was used with three compositions: standard with 2.5mM Ca2+ (Ca-S), Ca2+ free buffer (Ca-F) and buffer with raised Ca2+ i.e., 10mM (Ca-R). Muscles endurance was assessed by measuring the decline in tetanic tension in the terms of percentage (%Pmax) and rate of decline in tetanic tension (dP/dt). During 20s, %Pmax showed reduction of only 10% in Ca-S. This decline was enhanced in Ca-F (50%) and reduced in Ca-R (6%). Effect of adrenaline was observed only in Ca-F where %Pmax was about 20% greater in Test than CTL. These effects were not observed in both Ca-S and Ca-R during 20s. However, when duration of stimulation was increased to 120 or 150s in Ca-S and Ca-R respectively, decline in %Pmax was less in Test as compared to CTL. Thus, [Formula: see text] plays protective role against fatigue during continuous HFS in slow twitch muscles. In addition, adrenaline improves the muscles endurance during fatiguing contraction but these effects are not mediated through [Formula: see text] influx.

Influence of stretch magnitude on the stretch-shortening cycle in skinned muscle fibres.

The mechanical work attained during muscle fibre shortening is increased by prior stretching. Recently, we suggested that residual force enhancement (RFE) may contribute to this enhanced work. RFE can be changed reliably by changing the stretch magnitude. Therefore, the purpose of this study was to examine the effect of stretch magnitude, and by association RFE, on the mechanics of the stretch-shortening cycle (SSC) in skinned skeletal muscle fibres. Three tests were performed using skinned rabbit soleus fibres (N=18). The first test was a pure shortening contraction in which fibres were activated and then shortened from an average sarcomere length of 3.3 μm to 3.0 μm. The second test was a SSC in which fibres were activated and stretched from 3.0 μm to 3.3 μm, and then shortened to 3.0 μm. The third test was a SSC in which fibres were activated and stretched from 2.4 μm to 3.3 μm, and then shortened to 3.0 μm. The mechanical work during shortening and the force maintained 15 s after the end of shortening were determined. The relative increase in mechanical work with respect to the pure shortening condition was greater for the large than for the small stretch condition (P<0.001). Similarly, the relative increase in force 15 s after the end of shortening was greater for the large than for the small stretch condition (P=0.043). We conclude that increasing the magnitude of stretch results in an increase in mechanical work and increased force at steady state following the stretch, probably because of the greater RFE.

Residual Force Enhancement Is Attenuated in a Shortening Magnitude-dependent Manner.

The isometric force attained after active stretch is greater than that attained in a purely isometric contraction. This property is referred to as residual force enhancement (RFE). Although RFE is thought to contribute to the enhanced force and power in stretch-shortening cycles (SSCs), it is unclear whether shortening that occurs after active stretch eliminates the RFE induced by active stretch. Therefore, we evaluated the influence of shortening on RFE. Skinned rabbit soleus fibers (N = 43) were used for all tests. Residual force enhancement was compared between the stretch-only condition and the SSC condition. In the SSC conditions, shortening magnitudes were either 1% or 12.5% of fiber length. The final muscle length where RFE was evaluated was 3 μm for all trials. In the SSCs with 12.5% shortening, the isometric force before and after the SSC was also compared. Residual force enhancement was similar between the stretch only (7.9% ± 2.7%) and the SSC with 1% shortening condition (7.1% ± 2.9%) (P = 0.316), whereas RFE was smaller in the SSC with 12.5% shortening (3.5% ± 2.4%) than the stretch-only condition (8.4% ± 2.5%) (P < 0.001). The isometric forces after SSCs (0.437 ± 0.103 mN) were greater than those measured before the SSC (0.422 ± 0.104 mN) (P = 0.016). Residual force enhancement was preserved when the shortening magnitude was small and was reduced when the shortening magnitude was large. Although RFE was attenuated by the 12.5% shortening, RFE was still observed, suggesting that RFE can contribute to SSCs.

An In-Situ Study of the Modulation of the Mechano-Kinetic Parameters of the Slow Isoform of Muscle Myosin II by the Heart Drug Omecamtiv Mecarbil

Omecamtiv Mecarbil (OM), a small molecule that binds to the catalytic domain of the myosin isoforms of both cardiac and slow muscles, increases actin affinity and promotes Pi release and, in myocells, increases Ca2+-sensitivity of force production (Malik et al. Science 331:1439, 2011; Nagy et al. Br J Pharmacol 172:4506, 2015). Despite the interest for OM as an activator of the myosin motor to understand motor-related myopathies and devise therapies (Spudich Biophys J 106:1236, 2014), the details of OM effects on force generation are unclear. We used fast sarcomere-level mechanics in demembranated rabbit soleus fibers to define the effect of OM (1 µM) on the force and stiffness of the half-sarcomere (hs) following Ca2+-activation. The hs-stiffness was analysed with a model that accounts for the contribution of the filament compliance and provides the estimate of the stiffness of the motors (e, which depends on the number of actin-attached motors). At saturating [Ca2+], OM depresses the isometric force by ∼50% by reducing the strain in the motors, without effects on e. Both the force-pCa and e-pCa relations are shifted to the left in OM with respect to the control (pCa50 is increased by 0.4 units) and their steepness is reduced by 50%. Thus the effect of OM on the force-pCa relation is fully accounted for by that on the e-pCa relation. We conclude that OM has a dual effect: it increases the Ca2+-sensitivity of the motor binding to actin, while it reduces the structural change associated to force generation. In the heart the internal [Ca2+] is below saturation and OM acts as a potentiator by increasing the number of motors. Supported by ECRF and Telethon.

Residual Force Enhancement is Attenuated by Shortening in a Magnitude-Dependent Manner

The isometric force attained after active stretch is greater than that attained in a purely isometric contraction at the corresponding muscle length. This property is referred to as residual force enhancement (RFE). Although RFE is considered to contribute to the force/work enhancement in stretch-shortening cycles, it is unclear whether shortening that occurs after active stretch eliminates the influence of RFE. Therefore, we evaluated the influence of shortening on the magnitude of RFE. Skinned fibers of rabbit soleus (N = 45) were used for all experiments. The first test consisted of an isometric reference contraction at an average sarcomere length of 3.0 μm. The second test consisted of a typical way to induce RFE. This was achieved by active stretching of single fibres from an average sarcomere length of 2.7 to 3.0 μm. The third test consisted of a stretch shortening cycle. RFE was induced by active stretch from 3.0 to 3.3μm. Immediately after stretching, fibers were shortened to 3.0 μm (12.5% of the fiber length) in 0.5 ms. Force attained 15 s after the active stretch was used to evaluate the magnitude of RFE. Compared to the isometric reference condition, force was significantly greater in the normal RFE condition (108.4 ± 2.5%) and in the stretch-shortening cycle condition (RFE 103.5 ± 2.4%) (p< 0.001). This difference in the magnitude of RFE disappeared when the magnitude of shortening was decreased from 12.5% to 1% of the fiber length (p = 0.316). In conclusion, RFE is attenuated by shortening following active stretching. This attenuation effect depends on the magnitude of shortening and decreases with decreasing shortening magnitudes. The molecular events underlying these history-dependent force effects remain unknown.

Mechanical parameters of the molecular motor myosin II determined in permeabilised fibres from slow and fast skeletal muscles of the rabbit.

The different performance of slow and fast muscles is mainly attributed to diversity of the myosin heavy chain (MHC) isoform expressed within them. In this study fast sarcomere-level mechanics has been applied to Ca2+ -activated single permeabilised fibres isolated from soleus (containing the slow myosin isoform) and psoas (containing the fast myosin isoform) muscles of rabbit for a comparative definition of the mechano-kinetics of force generation by slow and fast myosin isoforms in situ. The stiffness and the force of the slow myosin isoform are three times smaller than those of the fast isoform, suggesting that the stiffness of the myosin motor is a determinant of the isoform-dependent functional diversity between skeletal muscles. These results open the question of the mechanism that can reconcile the reduced performance of the slow MHC with the higher efficiency of the slow muscle. The skeletal muscle exhibits large functional differences depending on the myosin heavy chain (MHC) isoform expressed in its molecular motor, myosin II. The differences in the mechanical features of force generation by myosin isoforms were investigated in situ by using fast sarcomere-level mechanical methods in permeabilised fibres (sarcomere length 2.4μm, temperature 12°C, 4% dextran T-500) from slow (soleus, containing the MHC-1 isoform) and fast (psoas, containing the MHC-2X isoform) skeletal muscle of the rabbit. The stiffness of the half-sarcomere was determined at the plateau of Ca2+ -activated isometric contractions and in rigor and analysed with a model that accounted for the filament compliance to estimate the stiffness of the myosin motor (ε). ε was 0.56±0.04 and 1.70±0.37pNnm-1 for the slow and fast isoform, respectively, while the average strain per attached motor (s0 ) was similar (∼3.3nm) in both isoforms. Consequently the force per motor (F0 =εs0 ) was three times smaller in the slow isoform than in the fast isoform (1.89±0.43 versus 5.35±1.51pN). The fraction of actin-attached motors responsible for maximum isometric force at saturating Ca2+ (T0,4.5 ) was 0.47±0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29±0.08), so that F0 in slow fibres was decreased by only 53%. The lower stiffness and force of the slow myosin isoform open the question of the molecular basis of the higher efficiency of slow muscle with respect to fast muscle.

ПРЕДОТВРАЩЕНИЕ АТРОФИИ ТЕНОТОМИРОВАННОЙ КАМБАЛОВИДНОЙ МЫШЦЫ КРОЛИКА ПРИ ДЕНЕРВАЦИИ. НЕЙРОТРОФИЧЕСКОЕ ВЛИЯНИЕ

In the 1930—1940s R. G. Leibson and A. G. Ginеtsinsky with colleagues carried out the experiments were it was shown that the changes in tenotomized rabbit soleus muscle are completely eliminated if the nerve is cut at the same time. The authors concluded that atrophy from inactivity is not a passive process, but it is an active result of neural influences which are carried out by the ventral roots of sciatic nerve. The nature of these influences has not been studied and nervous control is not clear. The present study was carried out on 2-month old Californian rabbits to confirm the described phenomenon and to investigate the assumed neurotrophic mechanisms. The morphological signs of atrophy and degeneration of tenotomized rabbit muscles, muscle fiber death of and growth of connective tissue were eliminated on day 11 of exposure by simultaneous Achilles tenotomy. The effect of colchicine application on the tenotomized muscle was almost the same as denervation (10 mM solution was applicated at the sciatic nerve for 10 min), but in the case of colchicine the degenerative changes were more pronounced. Denervation of the tenotomized muscle or colchicine application maintained the fast fiber cross-sectional area at the level of intact control. Sciatic nerve denervation or colchicine application prevented the rise in soleus muscle TrkB and TrkС expression and altered some neurotrophin expression. Denervation with tenotomy decreased TrkB mRNA expression by 3.4 times (p < 0.05), TrkC — by 3.2 times (p < 0.05), NT3-by 3.4 times (p < 0.05) compared with the tentotomized contralateral limb, colchicine with tenotomy — by 3.6 and 2.9 times respectively (p < 0.05).

Influence of residual force enhancement and elongation of attached cross-bridges on stretch-shortening cycle in skinned muscle fibers.

Increased muscle force during stretch‐shortening cycles (SSCs) has been widely examined. However, the mechanisms causing increased muscle force in SSCs remain unknown. The purpose of this study was to determine the influence of residual force enhancement and elongation of attached cross‐bridges on the work enhancement in SSCs. For the Control condition, skinned rabbit soleus fibers were elongated passively from an average sarcomere length of 2.4 to 3.0 μm, activated and then actively shortened to 2.4 μm. For the Transition condition, fibers were elongated actively from an average sarcomere length of 2.4 to 3.0 μm. Two seconds after the end of active lengthening, fibers were actively shortened to 2.4 μm. In the SSC condition, fibers were lengthened actively from an average sarcomere length of 2.4 to 3.0 μm, and then immediately shortened actively to 2.4 μm. Increased muscle force in the SSCs was quantified by the increase in mechanical work during active shortening compared to the mechanical work measured during the purely active shortening contractions. Work enhancement was significantly greater in the SSC compared to the Transition conditions. This difference was associated with the pause given between the active lengthening and shortening phase in the Transition test, which likely resulted in a reduction of the average elongation of the attached cross‐bridges caused by active stretching. Since some work enhancement was still observed in the Transition condition, another factor, for example the stretch‐induced residual force enhancement, must also have contributed to the work enhancement in SSCs.

Hypertrophic Cardiomyopathy Cardiac Troponin C Mutations Differentially Affect Slow Skeletal and Cardiac Muscle Regulation.

Mutations in TNNC1—the gene encoding cardiac troponin C (cTnC)—that have been associated with hypertrophic cardiomyopathy (HCM) and cardiac dysfunction may also affect Ca2+-regulation and function of slow skeletal muscle since the same gene is expressed in both cardiac and slow skeletal muscle. Therefore, we reconstituted rabbit soleus fibers and bovine masseter myofibrils with mutant cTnCs (A8V, C84Y, E134D, and D145E) associated with HCM to investigate their effects on contractile force and ATPase rates, respectively. Previously, we showed that these HCM cTnC mutants, except for E134D, increased the Ca2+ sensitivity of force development in cardiac preparations. In the current study, an increase in Ca2+ sensitivity of isometric force was only observed for the C84Y mutant when reconstituted in soleus fibers. Incorporation of cTnC C84Y in bovine masseter myofibrils reduced the ATPase activity at saturating [Ca2+], whereas, incorporation of cTnC D145E increased the ATPase activity at inhibiting and saturating [Ca2+]. We also tested whether reconstitution of cardiac fibers with troponin complexes containing the cTnC mutants and slow skeletal troponin I (ssTnI) could emulate the slow skeletal functional phenotype. Reconstitution of cardiac fibers with troponin complexes containing ssTnI attenuated the Ca2+ sensitization of isometric force when cTnC A8V and D145E were present; however, it was enhanced for C84Y. In summary, although the A8V and D145E mutants are present in both muscle types, their functional phenotype is more prominent in cardiac muscle than in slow skeletal muscle, which has implications for the protein-protein interactions within the troponin complex. The C84Y mutant warrants further investigation since it drastically alters the properties of both muscle types and may account for the earlier clinical onset in the proband.

Effects of fiber type on force depression after active shortening in skeletal muscle

The aim of this study was to investigate force depression in Type I and Type II muscle fibers. Experiments were performed using skinned fibers from rabbit soleus and psoas muscles. Force depression was quantified after active fiber shortening from an average sarcomere length (SL) of 3.2µm to an average SL of 2.6µm at an absolute speed of 0.115fiber length/s and at a relative speed corresponding to 17% of the unloaded shortening velocity (V0) in each type of fibers. Force decay and mechanical work during shortening were also compared between fiber types. After mechanical testing, each fiber was subjected to myosin heavy chain (MHC) analysis in order to confirm its type (Type I expressing MHC I, and Type II expressing MHC IId). Type II fibers showed greater steady-state force depression after active shortening at a speed of 0.115fiber length/s than Type I fibers (14.5±1.5% versus 7.8±1.7%). Moreover, at this absolute shortening speed, Type I fibers showed a significantly greater rate of force decay during shortening and produced less mechanical work than Type II fibers. When active shortening was performed at the same relative speed (17% V0), the difference in force depression between fiber types was abolished. These results suggest that no intrinsic differences were at the origin of the disparate force depressions observed in Type I and Type II fibers when actively shortened at the same absolute speed, but rather their distinct force–velocity relationships.

An Active Role for the Z-Band during Contraction and Relaxation

In striated muscle, the Z-band transmits force between contracting sarcomeres; however, it is not merely a static anchor for actin filaments cross-linked by alpha-actinin. Shimamoto observed a coordinated yielding of adjacent sarcomeres in response to stretch, and its disruption by an alpha-actinin antibody [1], indicating an active Z-band function and communication between adjacent sarcomeres. We hypothesized the inter-sarcomeric communication must involve a previously observed but poorly understood, reversible transition between two Z-band structures descriptively called small-square and basketweave [2], implying that alpha-actinin antibody must shift the equilibrium between the two states. Using EM, we imaged Z-bands of chemically-skinned relaxed rabbit soleus fibers and found that alpha-actinin antibody induces complete conversion to the basketweave form of the Z-band, whereas control fibers without antibody show the expected small square form. Since the small-square form was previously associated with relaxed fibers and basketweave with contracted fibers [2], our results suggest that the basketweave Z-band may store elastic energy, which can then favor or drive sarcomere relaxation when the Z-band reverts to the small-square structure. By locking the Z-band in the basketweave form, the antibody prevents the relaxation and concomitant yielding of adjacent sarcomeres previously observed [1]. In parallel mechanics experiments, we found antibody treatment did not interfere with maximal calcium-activated isometric contraction and relaxation; however isometric force and stretch-activation force during sub-maximal activation by ADP were both reduced after antibody treatment. Thus, the alteration of Z-band form by antibody only affects the muscle's dynamic response to stretch during partial activation.1. Shimamoto, Y, et al. Inter-sarcomere coordination in muscle revealed through individual sarcomere response to quick stretch. PNAS, 2009. 106(29):11954-9.2. Goldstein, MA, et al. The Z-band lattice in skeletal muscle before, during and after tetanic contraction. J Muscle Res, 1986. 7(6):527-36.