Average Sarcomere Length Research Articles

Stretch-shortening cycles protect against the age-related loss of power generation in rat single muscle fibres

Aging is associated with impaired strength and power during isometric and shortening contractions, however, during lengthening (i.e., eccentric) contractions, strength is maintained. During daily movements, muscles undergo stretch-shortening cycles (SSCs). It is unclear whether the age-related maintenance of eccentric strength offsets age-related impairments in power generation during SSCs owing to the utilization of elastic energy or other cross-bridge based mechanisms. Here we investigated how aging influences SSC performance at the single muscle fibre level and whether performing active lengthening prior to shortening protects against age-related impairments in power generation. Single muscle fibres from the psoas major of young (∼8 months; n = 31 fibres) and old (∼32 months; n = 41 fibres) male F344BN rats were dissected and chemically permeabilized. Fibres were mounted between a force transducer and length controller and maximally activated (pCa 4.5). For SSCs, fibres were lengthened from average sarcomere lengths of 2.5 to 3.0 μm and immediately shortened back to 2.5 μm at both fast and slow (0.15 and 0.60 Lo/s) lengthening and shortening speeds. The magnitude of the SSC effect was calculated by comparing work and power during shortening to an active shortening contraction not preceded by active lengthening. Absolute isometric force was ∼37 % lower in old compared to young rat single muscle fibres, however, when normalized to cross-sectional area (CSA), there was no longer a significant difference in isometric force between age groups, meanwhile there was an ∼50 % reduction in absolute power in old as compared with young. We demonstrated that SSCs significantly increased power production (75–110 %) in both young and old fibres when shortening occurred at a fast speed and provided protection against power-loss with aging. Therefore, in older adults during everyday movements, power is likely ‘protected’ in part due to the stretch-shortening cycle as compared with isolated shortening contractions.

Gaining new understanding of sarcomere length non-uniformities in skeletal muscles.

Sarcomere lengths are non-uniform on all structural levels of mammalian skeletal muscle. These non-uniformities have been associated with a variety of mechanical properties, including residual force enhancement and depression, creep, increased force capacity, and extension of the plateau of the force-length relationship. However, the nature of sarcomere length non-uniformities has not been explored systematically. The purpose of this study was to determine the properties of sarcomere length non-uniformities in active and passive muscle. Single myofibrils of rabbit psoas (n = 20; with 412 individual sarcomeres) were subjected to three activation/deactivation cycles and individual sarcomere lengths were measured at 4 passive and 3 active points during the activation/deactivation cycles. The myofibrils were divided into three groups based on their initial average sarcomere lengths: short, intermediate, and long average sarcomere lengths of 2.7, 3.2, and 3.6µm. The primary results were that sarcomere length non-uniformities did not occur randomly but were governed by some structural and/or contractile properties of the sarcomeres and that sarcomere length non-uniformities increased when myofibrils went from the passive to the active state. We propose that the mechanisms that govern the systematic sarcomere lengths non-uniformities observed in active and passive myofibrils may be associated with the variable number of contractile proteins and the variable number and the adjustable stiffness of titin filaments in individual sarcomeres.

Impact of stretch on sarcomere length variability in isolated fully relaxed rat cardiac myocytes.

The contractility of cardiac muscle is greatly affected by preload via the Frank-Starling mechanism (FSM). It is based on preload-dependent activation of sarcomeres-the elementary contractile units in muscle cells. Recent findings show a natural variability in sarcomere length (SL) in resting cardiomyocytes that, moreover, is altered in an actively contracting myocyte. SL variability may contribute to the FSM, but it remains unresolved whether the change in the SL variability is regulated by activation process per se or simply by changes in cell stretch, i.e., average SL. To separate the roles of activation and SL, we characterized SL variability in isolated, fully relaxed rat ventricular cardiomyocytes (n = 12) subjected to a longitudinal stretch with the carbon fiber (CF) technique. Each cell was tested in three states: without CF attachment (control, no preload), with CF attachment without stretch, and with CF attachment and ~ 10% stretch of initial SL. The cells were imaged by transmitted light microscopy to retrieve and analyze individual SL and SL variability off-line by multiple quantitative measures such as coefficient of variation or median absolute deviation. We found that CF attachment without stretch did not affect the extent of SL variability nor average SL. In stretched myocytes, the averaged SL significantly increased, while the SL variability remained unchanged. This result clearly indicates that the non-uniformity of individual SL is not sensitive to the average SL itself in fully relaxed myocytes. We conclude that SL variability per se does not contribute to the FSM in the heart.

Absence of Bcl11b in murine heart leads to mitochondrial dysfunction and cardiomyocyte death

B cell lymphoma 11b (Bcl11b/Ctip2) is a zinc finger transcriptional factor involved in a number of physiological processes, including cardiac hypertrophy. In humans, genome-wide association studies show that a common genetic variation in the 3’ region of Bcl11b gene is associated with higher risk of aortic stiffness and increased rate of cardiovascular disease. In mice, Bcl11b invalidation specifically in cardiomyocytes leads to cardiac malfunction, fibrosis augmentation and cell death. Our specific aims are: To investigate the signalling pathways related to cardiac cell death and to assess the sarcomere structure of cardiomyocytes in the absence of Bcl11b. The experiments were conducted using cardiac specific Bcl11b knock-out mice induced by tamoxifen administration 3 and 7 days after the invalidation, cardiac tissues were collected to perform RNA sequencing and subsequently to analyse related molecular pathways using bioinformatic tools. In parallel, cardiomyocytes were purified and isolated by conducting Langendorff perfusion technique to have a better insight into the impact of Bcl11b. A simplified mathematical model, Fourier transform analysis was used to assess sarcomeric length following Bcl11b invalidation in cardiomyocytes. Based on the RNA sequencing results, Bcl11b invalidation has an impact on the regulation of over 2000 genes. Among them, genes related to cell death and mitochondrial functions are the most affected regarding the enrichment scores. Interestingly, a considerable number of genes fall into the “Cuproptosis” pathway, a newly identified, non-apoptotic cell death mechanism. The oxygen consumption of mitochondria was also measured using the Seahorse assay. Bcl11b deletion leads to the decrease in basal level of mitochondrial respiration with no effect on the maximum respiratory capacity. Finally, adult cardiomyocytes in the mutant groups showed a moderate hypertrophy (15% bigger than the control group). However, Fourier transform analysis exhibited stability in the average sarcomere length of these cells. The obtained data suggest that cardiac specific invalidation of Bcl11b induces mitochondrial dysfunction and possibly cell death through the “Cuproptosis” pathway.

Characterizing residual and passive force enhancements in cardiac myofibrils

Residual force enhancement (RFE), an increase in isometric force after active stretching of a muscle compared with the purely isometric force at the corresponding length, has been consistently observed throughout the structural hierarchy of skeletal muscle. Similar to RFE, passive force enhancement (PFE) is also observable in skeletal muscle and is defined as an increase in passive force when a muscle is deactivated after it has been actively stretched compared with the passive force following deactivation of a purely isometric contraction. These history-dependent properties have been investigated abundantly in skeletal muscle, but their presence in cardiac muscle remains unresolved and controversial. The purpose of this study was to investigate whether RFE and PFE exist in cardiac myofibrils and whether the magnitudes of RFE and PFE increase with increasing stretch magnitudes. Cardiac myofibrils were prepared from the left ventricles of New Zealand White rabbits, and the history-dependent properties were tested at three different final average sarcomere lengths (n = 8 for each), 1.8, 2, and 2.2 μm, while the stretch magnitude was kept at 0.2 μm/sarcomere. The same experiment was repeated with a final average sarcomere length of 2.2 μm and a stretching magnitude of 0.4 μm/sarcomere (n = 8). All 32 cardiac myofibrils exhibited increased forces after active stretching compared with the corresponding purely isometric reference conditions (p < 0.05). Furthermore, the magnitude of RFE was greater when myofibrils were stretched by 0.4 compared with 0.2 μm/sarcomere (p < 0.05). We conclude that, like in skeletal muscle, RFE and PFE are properties of cardiac myofibrils and are dependent on stretch magnitude.

Unique morphological architecture of the hamstring muscles and its functional relevance revealed by analysis of isolated muscle specimens and quantification of structural parameters.

The structural and functional differences of individual hamstrings have not been sufficiently evaluated. This study aimed to clarify the morphological architecture of the hamstrings including the superficial tendons in detail using isolated muscle specimens, together with quantification of structural parameters of the muscle. Sixteen lower limbs of human cadavers were used in this study. The semimembranosus (SM), semitendinosus (ST), biceps femoris long head (BFlh), and biceps femoris short head (BFsh) were dissected from cadavers to prepare isolated muscle specimens. Structural parameters, including muscle volume, muscle length, fiber length, sarcomere length, pennation angle, and physiological cross-sectional area (PCSA) were measured. In addition, the proximal and distal attachment areas of the muscle fibers were measured, and the proximal/distal area ratio was calculated. The SM, ST, and BFlh were spindle-shaped with the superficial origin and insertion tendons on the muscle surface, and the BFsh was quadrate with direct attachment to the skeleton and BFlh tendon. The muscle architecture was pennate in the four muscles. The four hamstrings possessed either of two types of structural parameters, one with shorter fiber length and larger PCSA, as in the SM and BFlh, and the other with longer fiber length and smaller PCSA, as in the ST and BFsh. Sarcomere length was unique in each of the four hamstrings, and thus the fiber length was suitably normalized using the average sarcomere length for each, instead of uniform length of 2.7μm. The proximal/distal area ratio was even in the SM, large in the ST, and small in the BFsh and BFlh. This study clarified that the superficial origin and insertion tendons are critical determinants of the unique internal structure and structural parameters representing the functional properties of the hamstring muscles.

Do actin-myosin cross-bridges exhibit a viscoelastic behaviour?

The S2 domain of myosin is the segment that links the myosin head (S1 domain) to the thick filament backbone and elongates to allow S1 to execute the power stroke. It has been suggested that S2 exhibits non-viscous behaviour, implying no lag in reaching steady-state force following power stroke elongation. However, the elongation step in the cross-bridge cycle occurs extremely fast, and the cross-bridge detaches rapidly following it without allowing perceptible changes in force. If a muscle is transferred to a rigor solution following activation, all cross-bridges become stuck in a post power stroke state, which allows for investigation of their viscoelastic behaviour. The objective of this study was to investigate the viscoelastic behaviour of cross-bridges by examining their force following transfer to a rigor solution. Fibres (n = 8) were activated at an average sarcomere length (SL) of 3.0 µm, and then transferred into a rigor solution for 90 s. Since active stretch has the potential to influence the behaviour of cross-bridges, fibres were activated at a SL of 2.4 µm, actively stretched to a SL of 3.0 µm, and at steady-state transferred to a rigor solution for 90 s. Active force and stiffness were compared before and after incubation in rigor, and between purely isomeric and active stretch contractions. Although stiffness, and thus the presumed proportion of attached cross-bridges, increased when fibres were transferred to rigor, force at steady-state decreased by 16.5 +/- 4.0% and 10.0 +/- 1.3%, in the purely isometric and active stretch conditions, respectively. These results suggest that cross-bridges exhibit a viscoelastic behaviour, where their force decreases while they remain attached in a constant conformation. The difference in force decrease between the purely isometric and active stretch conditions suggests that active stretch modulates cross-bridge properties. Further research is warranted to investigate the cross-bridge nanostructure before and after rigor incubation.

Force loss induced by inhibiting cross-bridge cycling is mitigated in eccentric contraction.

We examined whether the force loss induced by 2,3-butanedione monoxime affects isometric and eccentric forces differently. Single skinned muscle fibers were activated at an average sarcomere length of 2.4 μm and then stretched to 3.0 μm. This trial was performed with and without 2,3-butanedione monoxime to calculate the magnitude of force loss attained at several time points: pre-stretch phase at 2.4 μm, eccentric phase, end of eccentric contraction, and post-stretch phase at 3.0 μm. The magnitude of force loss was significantly larger in the pre-stretch phase than at the other time points. Further, the mitigated force loss in the eccentric contraction was more prominent in the long condition than in the short condition. We suggest that the eccentric force is relatively preserved compared with the reference isometric force (pre-stretch) when cross-bridge cycling is inhibited, possibly because of the contribution of the elastic force produced by titin.

Residual force enhancement is reduced in permeabilized fiber bundles from mdm muscles.

Residual force enhancement (RFE) is the increase in steady-state force after active stretch relative to the force during isometric contraction at the same final length. The muscular dystrophy with myositis (mdm) mutation in mice, characterized by a small deletion in N2A titin, has been proposed to prevent N2A titin-actin interactions so that active mdm muscles are more compliant than wild type (WT). This decrease in active muscle stiffness is associated with reduced RFE. We investigated RFE in permeabilized soleus (SOL) and extensor digitorum longus (EDL) fiber bundles from WT and mdm mice. On each fiber bundle, we performed active and passive stretches from an average sarcomere length of 2.6-3.0 µm at a slow rate of 0.04 µms-1, as well as isometric contractions at the initial and final lengths. One-way ANOVA showed that SOL and EDL fiber bundles from mdm mice exhibited significantly lower RFE than WT mice (P<0.0001). This result is consistent with previous observations in single myofibrils and intact muscles. However, it contradicts the results from a previous study that appeared to show that compensatory mechanisms could restore titin force enhancement in single fibers from mdm psoas. We suggest that RFE measured previously in mdm single fibers was an artifact of the high variability in passive tension found in degenerating fibers, which begins after ∼24 days of age. The results are consistent with the hypothesis that RFE is reduced in mdm skeletal muscles owing to impaired Ca2+-dependent titin-actin interactions resulting from the small deletion in N2A titin.

Fast stretching of skeletal muscle fibres abolishes residual force enhancement.

The steady-state isometric force of a muscle after active stretching is greater than the steady-state force for a purely isometric contraction at the same length and activation level. The mechanisms underlying this property, termed residual force enhancement (rFE), remain unknown. When myofibrils are actively stretched while cross-bridge cycling is inhibited, rFE is substantially reduced, suggesting that cross-bridge cycling is essential to produce rFE. Our purpose was to further investigate the role of cross-bridge cycling in rFE by investigating whether fast stretching that causes cross-bridge slipping is associated with a loss of rFE. Skinned fibre bundles from rabbit psoas muscles were stretched slowly (0.08 µms-1) or rapidly (800 µms-1) while activated, from an average sarcomere length of 2.4 to 3.2 µm. Force was enhanced by 38±4% (mean±s.e.m) after the slow stretches but was not enhanced after the fast stretches, suggesting that proper cross-bridge cycling is required to produce rFE.

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.

Residual force enhancement is attenuated for quick stretch conditions

It is generally accepted that the magnitude of residual force enhancement is not affected by stretching velocity. However, we recently found that when the stretching velocity was too large, the magnitude of residual force enhancement was attenuated or vanished in cat soleus muscle in situ. Therefore, the purpose of this study was to extend the knowledge by conducting very quick stretches in skinned rabbit psoas and soleus muscle fibers. Skinned psoas (N = 17) or soleus (N = 13) muscle fibers were activated isometrically at an average sarcomere length of 2.7 μm, and then, actively stretched to an average sarcomere length of 3.0 μm in 0.5 or 500 ms, followed by an isometric contraction at that length. In addition, a purely isometric reference contraction was conducted at an average sarcomere length of 3.0 μm to calculate the magnitude of residual force enhancement for both stretch velocities. The magnitude of residual force enhancement was significantly different between 0.5 ms stretch (101.5 ± 5.6% for psoas, and 101.5 ± 2.3% for soleus) and 500 ms stretch (106.2 ± 5.9% for psoas, and 106.8 ± 5.1% for soleus) while the magnitude of residual force enhancement was not significantly different between muscles. In line with our previous finding, the magnitude of residual force enhancement can vary when the stretch velocity is very large. This finding can contribute to clarify the key mechanism for inducing residual force enhancement and to explain contradicting results obtained in previous studies.

Residual force enhancement is reduced in permeabilized fiber bundles from mdm muscles

Residual Force enhancement (RFE) is defined as the increase in steady state force after active stretch compared with force after isometric contraction at the same final length. Recent studies have demonstrated a pivotal role for titin in RFE. In myofibrils stretched beyond overlap of thick and thin filaments, Ca2+ activated myofibrils had a higher titin-based force and a steeper increase with force with stretch than passively stretched myofibrils at the same average sarcomere length. These results suggest higher titin-based stiffness in active myofibrils, perhaps due to N2A titin-actin interactions in active wild type (WT) muscle. Mdm mice carry a small deletion in titin that includes 53 amino acids in Ig83 in the N2A region. It has been proposed that the mdm mutation prevents N2A titin-actin interactions so that active mdm muscles are more compliant than WT muscles. This decrease in active muscle stiffness should be associated with a reduction in RFE. Based on this rationale, we investigated RFE in permeabilized soleus (SOL) and extensor digitorum longus (EDL) fiber bundles from 20-30 day old mdm mice (n=5), harvested at a young age to diminish the effects of degeneration and regeneration, and 30-60 day old WT mice (n=5). Contractile stress and RFE of EDL (mdm n=19 and WT n=20) and SOL fibers (WT n=19 and mdm n=16) was analyzed using JMP and Graph pad (PRISM) software. Statistics included Shapiro-Wilk (normality), Levene's and Bartlett (homogeneity of variances), Welch's one-way ANOVA (unequal variances), Dunnett's T3 (multiple comparisons), and T-tests with Welch's correction (unpaired). To investigate differences in RFE between genotypes, we performed isometric contractions, as well as active and passive stretch from an average sarcomere length of 2.6 - 3.0 µm on each fiber. This stretch amplitude, within the physiological range, resulted in no observable breakage or slipping of fibers during experiments. We predicted that mdm fiber bundles would show reduced RFE compared to WT fibers. Contractile stress when activated at 2.6 µm was significantly greater in WT EDL (Welch's one-way ANOVA; P<0.05) bundle and SOL (Welch's one-way ANOVA; P<0.05) compared to fiber bundles. Our results showed that mdm muscles exhibit significantly lower RFE than WT for both SOL (Welch's one-way ANOVA; P<0.0001) and EDL (Welch's one-way ANOVA; P<0.0001). This result is consistent with previous observations in single myofibrils and intact muscles. The data support the hypothesis that titin contributes to RFE. We suggest that RFE is reduced in mdm muscles due to impaired Ca2+ dependent titin-actin interactions, resulting from the small deletion in titin that includes 53 amino acids in Ig83 in the N2A region.

Energy Cost of Force Production After a Stretch-Shortening Cycle in Skinned Muscle Fibers: Does Muscle Efficiency Increase?

Muscle force is enhanced during shortening when shortening is preceded by an active stretch. This phenomenon is known as the stretch-shortening cycle (SSC) effect. For some stretch-shortening conditions this increase in force during shortening is maintained following SSCs when compared to the force following a pure shortening contraction. It has been suggested that the residual force enhancement property of muscles, which comes into play during the stretch phase of SSCs may contribute to the force increase after SSCs. Knowing that residual force enhancement is associated with a substantial reduction in metabolic energy per unit of force, it seems reasonable to assume that the metabolic energy cost per unit of force is also reduced following a SSC. The purpose of this study was to determine the energy cost per unit of force at steady-state following SSCs and compare it to the corresponding energy cost following pure shortening contractions of identical speed and magnitude. We hypothesized that the energy cost per unit of muscle force is reduced following SSCs compared to the pure shortening contractions. For the SSC tests, rabbit psoas fibers (n = 12) were set at an average sarcomere length (SL) of 2.4 μm, activated, actively stretched to a SL of 3.2 μm, and shortened to a SL of 2.6 or 3.0 μm. For the pure shortening contractions, the same fibers were activated at a SL of 3.2 μm and actively shortened to a SL of 2.6 or 3.0 μm. The amount of ATP consumed was measured over a 40 s steady-state total isometric force following either the SSCs or the pure active shortening contractions. Fiber stiffness was determined in an additional set of 12 fibers, at steady-state for both experimental conditions. Total force, ATP consumption, and stiffness were greater following SSCs compared to the pure shortening contractions, but ATP consumption per unit of force was the same between conditions. These results suggest that the increase in total force observed following SSCs was achieved with an increase in the proportion of attached cross-bridges and titin stiffness. We conclude that muscle efficiency is not enhanced at steady-state following SSCs.

Modifiability of residual force depression in single muscle fibers following uphill and downhill training in rats.

Following active muscle shortening, steady‐state isometric force is less than a purely isometric contraction at the same muscle length and level of activation; this is known as residual force depression (rFD). It is unknown whether rFD at the single muscle fiber level can be modified via training. Here we investigated whether rFD in single muscle fibers is modifiable through downhill and uphill running in the extensor digitorum longus (EDL) and soleus (SOL) muscles in rats. Rats were run uphill or downhill 5 days/week for 4 weeks. After muscles were dissected and chemically permeabilized, single fibers were tied between a length controller and force transducer, transferred to an activating solution, with ATP and pCa of 4.2 for mechanical testing. rFD was quantified after active fiber shortening from an average sarcomere length (SL) of 3.1–2.5 µm at a relative speed of 0.15 fiber lengths/s (slow) and 0.6 fiber lengths/s (fast). rFD was calculated as the difference in force (normalized to cross‐sectional area) during the isometric steady‐state phase following active shortening and the purely isometric contraction. In addition to rFD, mechanical work of shortening and stiffness depression were also calculated. rFD was present for both the EDL (6–15%) and SOL (1–2%) muscles, with no effect of training. rFD was greater for the EDL than SOL which closely corresponded to the greater stiffness depression in the EDL, indicating a greater inhibition of cross‐bridge attachments. These results indicate that while rFD was observed, training did not appear to alter this intrinsic history‐dependent property of single muscle fibers.

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.

Differences in stretch-shortening cycle and residual force enhancement between muscles

It has been suggested that cross bridge kinetics and residual force enhancement (RFE) affect force in the stretch-shortening cycle (SSC). Because cross bridge kinetics and titin isoforms, which are thought to be related to RFE, differ between muscles, the SSC effect may be also muscle-dependent. Thus, we compared the SSC effect between psoas and soleus muscles, which have a distinct fiber type distribution and different titin isoforms. Four tests (SSC, SSC control, RFE, RFE control) were conducted using isolated, skinned fibers of psoas and soleus. In the SSC tests, fibers were activated at an average sarcomere length of 2.4 μm, stretched to 3.0 μm, and shortened to 2.4 μm. In the SSC control tests, fibers were activated at an average sarcomere length of 3.0 μm and then shortened to 2.4 μm. The relative increase in mechanical work obtained during shortening between tests was defined as the SSC effect. In the RFE tests, fibers were activated at an average sarcomere length of 2.4 μm and then stretched to 3.0 μm, while the RFE control tests consisted of an isometric contraction at 3.0 μm. The difference in steady-state isometric force between tests was defined as RFE. The SSC effect was greater in soleus than in psoas, while the RFE was the same for both muscles. Since the SSC effect was greater in soleus, while the RFE was the same, the observed greater SSC effect is probably not directly caused by RFE, but may be related to differences in cross bridge kinetics.

Residual force enhancement and force depression in human single muscle fibres

Residual force depression (rFD) and residual force enhancement (rFE) are intrinsic contractile properties of muscle. rFD is characterized as a decrease in steady-state isometric force following active shortening compared with a purely isometric contraction at the same muscle length and level of activation. By contrast, isometric force is increased following active lengthening compared to a reference isometric contraction at the same muscle length and level of activation; this is termed rFE. To date, there have been no investigations of rFD and rFE in human muscle fibres, therefore the purpose of this study was to determine whether rFD and rFE occur at the single muscle fibre level in humans. rFD and rFE were investigated in maximally activated single muscle fibres biopsied from the vastus lateralis of healthy adults. To induce rFD, fibres were activated and shortened from an average sarcomere length (SL) of 3.2–2.6 μm. Reference isometric contractions were performed at an average SL of 2.6 μm. To induce rFE, fibres were actively lengthened from an average SL of 2.6–3.2 μm and a reference isometric contraction was performed at an average SL of 3.2 μm. Isometric steady-state force was lower following active shortening (p < 0.05), and higher following active lengthening (p < 0.05), as compared to the reference isometric contractions. We demonstrated rFD and rFE in human single fibres which is consistent with previous animal models. The non-responder phenomenon often reported in rFE studies involving voluntary contractions at the whole human level was not observed at the single fibre level.

On sarcomere length stability during isometric contractions before and after active stretching.

Sarcomere length (SL) instability and SL non-uniformity have been used to explain fundamental properties of skeletal muscles, such as creep, force depression following active muscle shortening and residual force enhancement following active stretching of muscles. Regarding residual force enhancement, it has been argued that active muscle stretching causes SL instability, thereby increasing SL non-uniformity. However, we recently showed that SL non-uniformity is not increased by active muscle stretching, but it remains unclear if SL stability is affected by active stretching. Here, we used single myofibrils of rabbit psoas muscle and measured SL non-uniformity and SL instability during isometric contractions and for isometric contractions following active stretching at average SLs corresponding to the descending limb of the force-length relationship. We defined isometric contractions as contractions during which mean SL remained constant. SL instability was quantified by the rate of change of individual SLs over the course of steady-state isometric force and SL non-uniformity was defined as deviations of SLs from the mean SL at an instant of time. We found that whereas the mean SL remained constant during isometric contraction, by definition, individual SLs did not. SLs were more stable in the force-enhanced, isometric state following active stretching compared with the isometric reference state. We also found that SL instability was not correlated with the rate of change of SL non-uniformity. Also, SL non-uniformity was not different in the isometric and the post-stretch isometric contractions. We conclude that since SL is more stable but similarly non-uniform in the force-enhanced compared with the corresponding isometric reference contraction, it appears unlikely that either SL instability or SL non-uniformity contribute to the residual force enhancement property of skeletal muscle.