Muscle Fiber Stiffness Research Articles

Mechanical Properties of Single Muscle Fibers: Understanding Poor Muscle Quality in Older Adults with Diabetes.

BackgroundWhile aging causes muscle weakness, type 2 diabetes mellitus (T2DM) is also considered a high-risk factor for the induction of skeletal muscle weakness. Previous studies have reported increased collagen content in insulin-resistant skeletal muscles. Here, we studied the mechanical properties of aged skeletal muscle in patients with T2DM to investigate whether aged skeletal muscles with T2DM induce higher passive tension due to the abundance of extracellular matrix (ECM) inside or outside of the muscle fibers. MethodsSamples from the gluteus maximus muscles of older adults with diabetes (T2DM) and non-diabetic (non-DM) older adults who underwent elective orthopedic surgery were collected. Permeabilized single muscle fibers from these samples were used to identify their mechanical properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to quantify titin and fiber type distributions in these samples. ResultsWe confirmed a significant predominance of type I fiber ratio in both T2DM and non-DM aged muscles. While the average cross-sectional area and maximal active tension of the single fibers were smaller in the T2DM group than those in the non-DM group, the difference was not statistically significant. T2DM subjects showed significantly greater passive tension and lower titin-/ECM-based passive tension ratios than those in non-DM subjects, which indicated that more ECM but less titin contributed to the total passive tension. ConclusionBased on our findings, we concluded that T2DM may cause increased passive stiffness of single skeletal muscle fibers in older adults because of an excessive accumulation of ECM in and around single muscle fibers due to increased insulin resistance.

Age-related changes in human single muscle fibre passive elastic properties are sarcomere length dependent.

Studies have revealed an age-related decrease in the ability to produce force as well as an increase in the contractile stiffness and passive stress of single muscle fibres. However, further insight into age-related changes to the passive properties of human skeletal muscles is needed. The aim of this study was to characterize single muscle fibre passive properties from young and old males across a physiologic range of sarcomere lengths (SLs). Ten young ([YM]: mean age: 25.4years) and ten old ([OM]: mean age: 68.9years) males participated. Vastus lateralis muscles were biopsied and 182 fibres were tested and analyzed (90 YM and 92 OM) using a cumulative stretch-relaxation protocol. Passive mechanical characteristics of each fibre were obtained by fitting the integral of the logistic function to experimental stress-SL data. Muscle fibres from older individuals had significantly greater passive elastic moduli at short SL (1.9-2.65μm) compared with young (p<0.05). This resulted in a significantly larger passive stress for SLs between 2.1μm and 3.55μm for older individuals when compared with young (p<0.05). These results provide novel insight into the length dependency of changes in single muscle fibre passive mechanical properties with age.

History-dependence of muscle slack length following contraction and stretch in the human vastus lateralis.

In reduced muscle preparations, the slack length and passive stiffness of muscle fibres have been shown to be influenced by previous muscle contraction or stretch. In human muscles, such behaviours have been inferred from measures of muscle force, joint stiffness and reflex magnitudes and latencies. Using ultrasound imaging, we directly observed that isometric contraction of the vastus lateralis muscle at short lengths reduces the slack lengths of the muscle-tendon unit and muscle fascicles. The effect is apparent 60s after the contraction. These observations imply that muscle contraction at short lengths causes the formation of bonds which reduce the effective length of structures that generate passive tension in muscles. In reduced muscle preparations, stretch and muscle contraction change the properties of relaxed muscle fibres. In humans, effects of stretch and contraction on properties of relaxed muscles have been inferred from measurements of time taken to develop force, joint stiffness and reflex latencies. The current study used ultrasound imaging to directly observe the effects of stretch and contraction on muscle-tendon slack length and fascicle slack length of the human vastus lateralis muscle in vivo. The muscle was conditioned by (a) strong isometric contractions at long muscle-tendon lengths, (b) strong isometric contractions at short muscle-tendon lengths, (c) weak isometric contractions at long muscle-tendon lengths and (d) slow stretches. One minute after conditioning, ultrasound images were acquired from the relaxed muscle as it was slowly lengthened through its physiological range. The ultrasound image sequences were used to identify muscle-tendon slack angles and fascicle slack lengths. Contraction at short muscle-tendon lengths caused a mean 13.5degree (95% CI 11.8-15.0degree) shift in the muscle-tendon slack angle towards shorter muscle-tendon lengths, and a mean 5mm (95% CI 2-8mm) reduction in fascicle slack length, compared to the other conditions. A supplementary experiment showed the effect could be demonstrated if the muscle was conditioned by contraction at short lengths but not if the relaxed muscle was held at short lengths, confirming the role of muscle contraction. These observations imply that muscle contraction at short lengths causes the formation of bonds which reduce the effective length of structures that generate passive tension in muscles.

Robotic assessment of neuromuscular characteristics using musculoskeletal models: A pilot study

ObjectiveNon-invasive neuromuscular characterization aims to provide greater insight into the effectiveness of existing and emerging rehabilitation therapies by quantifying neuromuscular characteristics relating to force production, muscle viscoelasticity and voluntary neural activation. In this paper, we propose a novel approach to evaluate neuromuscular characteristics, such as muscle fiber stiffness and viscosity, by combining robotic and HD-sEMG measurements with computational musculoskeletal modeling. This pilot study investigates the efficacy of this approach on a healthy population and provides new insight on potential limitations of conventional musculoskeletal models for this application. MethodsSubject-specific neuromuscular characteristics of the biceps and triceps brachii were evaluated using robot-measured kinetics, kinematics and EMG activity as inputs to a musculoskeletal model. ResultsRepeatability experiments in five participants revealed large variability within each subjects evaluated characteristics, with almost all experiencing variation greater than 50% of full scale when repeating the same task. ConclusionThe use of robotics and HD-sEMG, in conjunction with musculoskeletal modeling, to quantify neuromuscular characteristics has been explored. Despite the ability to predict joint kinematics with relatively high accuracy, parameter characterization was inconsistent i.e. many parameter combinations gave rise to minimal kinematic error. Significance: The proposed technique is a novel approach for in vivo neuromuscular characterization and is a step towards the realization of objective in-home robot-assisted rehabilitation. Importantly, the results have confirmed the technical (robot and HD-sEMG) feasibility while highlighting the need to develop new musculoskeletal models and optimization techniques capable of achieving consistent results across a range of dynamic tasks.

Cardiomyopathy-related mutation (A30V) in mouse cardiac troponin T divergently alters the magnitude of stretch activation in α- and β-myosin heavy chain fibers.

The differential impact of α- and β-myosin heavy chain (MHC) on contractile dynamics causes a mutant cardiac troponin T (TnTA30V) to differently modulate cardiac contractile function. TnTA30V attenuated Ca2+-activated maximal tension and length-mediated cross-bridge recruitment against α-MHC but augmented these parameters against β-MHC, suggesting divergent contractile phenotypes.

Lecithin Prevents Cortical Cytoskeleton Reorganization in Rat Soleus Muscle Fibers under Short-Term Gravitational Disuse.

The aim of this study was to prevent the cortical cytoskeleton reorganization of rat soleus muscle fibers under short-term gravitational disuse. Once a day, we injected the right soleus muscle with 0.5 ml lecithin at a concentration of 200 mg/ml and the left soleus muscle with a diluted solution in an equal volume for 3 days prior to the experiment. To simulate microgravity conditions in rats, an anti-orthostatic suspension was used according to the Ilyin-Novikov method modified by Morey-Holton et al. for 6 hours. The following groups of soleus muscle tissues were examined: «C», «C+L», «HS», and «HS+L». The transversal stiffness of rat soleus muscle fibers after 6 hours of suspension did not differ from that of the control group for the corresponding legs; there were no differences between the groups without lecithin «C» and «HS» or between the groups with lecithin «C+L» and «HS+L». However, lecithin treatment for three days resulted in an increase in cell stiffness; in the «C+L» group, cell stiffness was significantly higher by 22.7% (p < 0.05) compared with that of group «C». The mRNA content of genes encoding beta- and gamma-actin and beta-tubulin did not significantly differ before and after suspension in the corresponding groups. However, there was a significant increase in the mRNA content of these genes after lecithin treatment: the beta-actin and gamma-actin mRNA content in group «C+L» increased by 200% compared with that of group «C», and beta-tubulin increased by 100% (as well as the mRNA content of tubulin-binding proteins Ckap5, Tcp1, Cct5 and Cct7). In addition, desmin mRNA content remained unchanged in all of the experimental groups. As a result of the lecithin injections, there was a redistribution of the mRNA content of genes encoding actin monomer- and filament-binding proteins in the direction of increasing actin polymerization and filament stability; the mRNA content of Arpc3 and Lcp1 increased by 3- and 5-fold, respectively, but the levels of Tmod1 and Svil decreased by 2- and 5-fold, respectively. However, gravitational disuse did not result in changes in the mRNA content of Arpc3, Tmod1, Svil or Lcp1. Anti-orthostatic suspension for 6 hours resulted in a decrease in the mRNA content of alpha-actinin-4 (Actn4) and alpha-actinin-1 (Actn1) in group «HS» compared with that of group «C» by 25% and 30%, respectively, as well as a decrease and increase in the ACTN4 protein content in the membrane and cytoplasmic fractions, respectively. Lecithin injection resulted in an increase in the Actn1 and Actn4 mRNA content in group «C+L» by 1.5-fold and more than 2-fold, respectively, compared with the levels in group «C». Moreover, in group «HS+L», the mRNA content did not change in these genes compared with the levels in group «C+L», and the ACTN4 protein content in the membrane and cytoplasmic fractions also remained unchanged. Thus, lecithin prevented the reduction of Actn1 and Actn4 mRNA and the migration of ACTN4 from the cortical cytoskeleton to the cytoplasm.

Stuck in gear: age-related loss of variable gearing in skeletal muscle.

Skeletal muscles power a broad diversity of animal movements, despite only being able to produce high forces over a limited range of velocities. Pennate muscles use a range of gear ratios, the ratio of muscle shortening velocity to fiber shortening velocity, to partially circumvent these force-velocity constraints. Muscles operate with a high gear ratio at low forces; fibers rotate to greater angles of pennation, enhancing velocity but compromising force. At higher forces, muscles operate with a lower gear ratio; fibers rotate little so limiting muscle shortening velocity, but helping to preserve force. This ability to shift gears is thought to be due to the interplay of contractile force and connective tissue constraints. In order to test this hypothesis, gear ratios were determined in the medial gastrocnemius muscles of both healthy young rats, and old rats where the interaction between contractile and connective tissue properties was assumed to be disrupted. Muscle fiber and aponeurosis stiffness increased with age (P<0.05) from 19.1±5.0 kPa and 188.5±24.2 MPa, respectively, in young rats to 39.1±4.2 kPa and 328.0±48.3 MPa in old rats, indicating a mechanical change in the interaction between contractile and connective tissues. Gear ratio decreased with increasing force in young (P<0.001) but not old (P=0.72) muscles, indicating that variable gearing is lost in old muscle. These findings support the hypothesis that variable gearing results from the interaction between contractile and connective tissues and suggest novel explanations for the decline in muscle performance with age.

The Muscle Mechanical Basis of Freeman-Sheldon Syndrome

We have generated the first animal models for Distal Arthrogryposis type 2A, commonly known as Freeman-Sheldon Syndrome (FSS). FSS is characterized by congenital contractures resulting from point mutations in skeletal muscle contractile proteins, yet how these mutations alter muscle contractile properties is not known. We created two myosin heavy chain transgenes containing point mutations Y583S and T178I, each known to cause dominant FSS. We expressed them in Drosophila muscles, isolated the indirect flight muscles from heterozygous mutant flies (mutation/+), and performed skinned muscle fiber mechanics. The most striking mechanical alteration was a dramatic increase in muscle stiffness. Y583S and T178I fibers’ elastic moduli increased 40% and 30%, respectively, under relaxed conditions and 70% and 46% when activated. The increased stiffness contributed to decreased work and power generation. Maximum power decreased by 45% and 62% because elevated stiffness increased work absorbed values during the lengthening portion of work-loop power measurements. A contributing factor to both decreased power and increased active stiffness was a reduction in rate for at least one strongly-bound cross-bridge cycle transition. This was inferred from 17% and 31% decreases in optimal frequency for power generation, and 27% and 39% decreases in apparent muscle rate constant 2πb. Another possible contributor to the stiffness changes might be muscle ultrastructure alterations. While we performed mechanics experiments on fibers from 2-hour-old flies, which showed normal muscle ultrastructure, disruption in myofibril hexagonal packing and morphology appeared at 2-days and increased with age. Impaired muscle function caused 29% and 63% decreases in flight ability for Y583S and T178I heterozygotes, respectively. We conclude that Freeman-Sheldon Syndrome likely results from highly elevated muscle fiber stiffness and decreased ability of the muscles to power limb movement. Supported by NIH grants R01 AR064274 to D.M.S. and R01 GM32443 to S.I.B.

Evidence for the Regulation of Actin-myosin Binding Strength by Lever Arm and Subfragment-2 Regions of Myosin Molecule in Contracting Skinned Muscle Fibers as Revealed by the Effect of Antibodies

It is generally believed that myosin heads (M) extending from myosin filaments form weak binding with actin filament (A) in the form of A-M-ADP-Pi, and strong binding with A in the form of A-M-ADP and A-M. During muscle contraction, M first attaches weakly to A in the form of M-ADP-Pi, and then release Pi to attach strongly with A in the form of A-MADP to exert power stroke producing myofilament sliding. The weak to strong binding transition between M and A is therefore essential for producing muscle contraction. After power stroke, associated with release of ADP, M detaches from A on binding with next ATP to form M-ADP-Pi. Thus, M repeats attachment-detachment cycle with A, coupled with ATP hydrolysis. Using antibodies to myosin lever arm domain (anti-LD antibody) and to myosin subfragment-2 region (anti-S-2 antibody), we have found that, if these antibodies attach to LD or S-2 regions, myosin heads no longer generate force, but still hydrolyse ATP at a normal rate without contributing to muscle fiber stiffness and internal resistance against fiber shortening. The puzzling effect of antibodies can be accounted for by assuming that in myosin molecules, with antibodies attached to their LD or S-2 region, (1) the strength of A-M binding is markedly reduced to be readily broken by small length perturbations or muscle fiber shortening, and (2) such myosin heads still repeat attachmentdetachment cycle coupled with ATP hydrolysis without passing through weak to strong A-M binding transition. Thus, the present work indicates functional influence of LD and S-2 regions on the A-M binding of myosin molecule, suggesting essential role of these regions in muscle contraction.

Passive stiffness of hindlimb muscles in anurans with distinct locomotor specializations

Anurans (frogs and toads) have been shown to have relatively compliant skeletal muscles. Using a meta-analysis of published data we have found that muscle stiffness is negatively correlated with joint range of motion when examined across mammalian, anuran and bird species. Given this trend across a broad phylogenetic sample, we examined whether the relationship held true within anurans. We identified four species that differ in preferred locomotor mode and hence joint range of motion (Lithobates catesbeianus, Rhinella marina, Xenopus laevis and Kassina senegalensis) and hypothesized that smaller in vivo angles (more flexed) at the knee and ankle joint would be associated with more compliant extensor muscles. We measured passive muscle tension during cyclical stretching (20%) around L0 (sarcomere lengths of 2.2μm) in fiber bundles extracted from cruralis and plantaris muscles. We found no relationship between muscle stiffness and range of motion for either muscle–joint complex. There were no differences in the passive properties of the cruralis muscle among the four species, but the plantaris muscles of the Xenopus and Kassina were significantly stiffer than those of the other two species. Our results suggest that in anurans the stiffness of muscle fibers is a relatively minor contributor to stiffness at the level of joints and that variation in other anatomical properties including muscle–tendon architecture and joint mechanics as well as active control likely contribute more significantly to range of motion during locomotion.

Collective effects of low cell pH and inorganic phosphate on force, velocity, stiffness, and power in rat slow and fast muscle fibers (1102.7)

Muscle fatigue is a loss of power resulting from a decline in both force and velocity. Intense exercise induces high rates of ATP hydrolysis and glycolysis, increasing inorganic phosphate (Pi) and H+. These factors are thought to induce fatigue by interfering with key steps in the cross bridge cycle, including ATP hydrolysis, ADP release, and rate and extent of cross‐bridge transition from the low‐ to high‐force state. Skinned fibers from rat soleus (slow) and gastrocnemius (fast) were isolated, and the combined effects of elevated H+ (pH 6.2) and Pi (30 mM) on force, velocity, stiffness, and power at low (15°C) and high (30°C) temperatures studied. At 30°C, pH 6.2 plus 30 mM Pi depressed force by 39% (158 to 97 kN/m2) in slow fibers and 42% (189 to 111 kN/m2) in fast fibers. Velocity was depressed by 17% (Vmax 4.23 to 3.56 Fl/s) in slow fibers and 44% (6.91 to 3.88 Fl/s) in fast fibers. The a/Po ratio was depressed in slow fibers but increased in fast fibers in pH 6.2, 30 mM Pi conditions, resulting in a greater power loss in slow (67%) versus fast (58%) fibers. Force‐stiffness plots normalized to rigor tension and stiffness show a higher number of low‐force cross bridges in fast versus slow fibers in pH 6.2, 30 mM Pi conditions. Our results show that the effects of elevated H+ and Pi are fiber‐type specific, and the larger decrease in force and velocity in type II fibers may in part be explained by a higher population of low‐force cross bridges.Grant Funding Source: Supported by a Marquette University Way Klinger Fellowship to RH Fitts

Thixotropy of Muscle Fibers Probed with Sinusoidal Oscillations

Mechanical properties of individual muscle fibers from rabbit psoas were probed by applying sinusoidal length changes at one end of the fiber and measuring the resulting force at the other end. At frequencies above 1 Hz, this oscillation resulted in a history-dependent reduction in muscle fiber stiffness in activated muscle fibers. Such a reduction in muscle stiffness in response to force is referred to as thixotropy. Perturbing the actin-myosin interaction by treatment with EDTA and blebbistatin caused this effect to disappear, suggesting that this thixotropy results from cross-bridge interactions. At frequencies less than 1 Hz, oscillation resulted in a history-dependent increase in muscle fiber stiffness in activated fibers. Disruption of actin-myosin interactions has no effect on this negative thixotropy, suggesting a different mechanism is at play.

Divergent effects of α- and β-myosin heavy chain isoforms on the N terminus of rat cardiac troponin T

Divergent effects of α– and β–myosin heavy chain (MHC) isoforms on contractile behavior arise mainly because of their impact on thin filament cooperativity. The N terminus of cardiac troponin T (cTnT) also modulates thin filament cooperativity. Our hypothesis is that the impact of the N terminus of cTnT on thin filament activation is modulated by a shift from α- to β-MHC isoform. We engineered two recombinant proteins by deleting residues 1–43 and 44–73 in rat cTnT (RcTnT): RcTnT1–43Δ and RcTnT44–73Δ, respectively. Dynamic and steady-state contractile parameters were measured at sarcomere length of 2.3 µm after reconstituting proteins into detergent-skinned muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rat hearts. α-MHC attenuated Ca2+-activated maximal tension (∼46%) in RcTnT1–43Δ fibers. In contrast, β-MHC decreased tension only by 19% in RcTnT1–43Δ fibers. Both α- and β-MHC did not affect tension in RcTnT44–73Δ fibers. The instantaneous muscle fiber stiffness measurements corroborated the divergent impact of α- and β-MHC on tension in RcTnT1–43Δ fibers. pCa50 (-log of [Ca2+]free required for half-maximal activation) decreased significantly by 0.13 pCa units in α-MHC + RcTnT1–43Δ fibers but remained unaltered in β-MHC + RcTnT1–43Δ fibers, demonstrating that β-MHC counteracted the attenuating effect of RcTnT1–43Δ on myofilament Ca2+ sensitivity. β-MHC did not alter the sudden stretch–mediated recruitment of new cross-bridges (ER) in RcTnT1–43Δ fibers, but α-MHC attenuated ER by 36% in RcTnT1–43Δ fibers. The divergent impact of α- and β-MHC on how the N terminus of cTnT modulates contractile dynamics has implications for heart disease; alterations in cTnT and MHC are known to occur via changes in isoform expression or mutations.

Loss of muscle strength during sepsis is in part regulated by glucocorticoids and is associated with reduced muscle fiber stiffness

Sepsis is associated with impaired muscle function but the role of glucocorticoids in sepsis-induced muscle weakness is not known. We tested the role of glucocorticoids in sepsis-induced muscle weakness by treating septic rats with the glucocorticoid receptor antagonist RU38486. In addition, normal rats were treated with dexamethasone to further examine the role of glucocorticoids in the regulation of muscle strength. Sepsis was induced in rats by cecal ligation and puncture, and muscle force generation (peak twitch and tetanic tension) was determined in lower extremity muscles. In other experiments, absolute and specific force as well as stiffness (reflecting the function of actomyosin cross bridges) were determined in isolated skinned muscle fibers from control and septic rats. Sepsis and treatment with dexamethasone resulted in reduced maximal twitch and tetanic force in intact isolated extensor digitorum longus muscles. The absolute and specific maximal force in isolated muscle fibers was reduced during sepsis together with decreased fiber stiffness. These effects of sepsis were blunted (but not abolished) by RU38486. The results suggest that muscle weakness during sepsis is at least in part regulated by glucocorticoids and reflects loss of contractility at the cellular (individual muscle fiber) level. In addition, the results suggest that reduced function of the cross bridges between actin and myosin (documented as reduced muscle fiber stiffness) may be involved in sepsis-induced muscle weakness. An increased understanding of mechanisms involved in loss of muscle strength will be important for the development of new treatment strategies in patients with this debilitating consequence of sepsis.

The N-Terminal Extension of Cardiac Troponin T Stabilizes the Blocked State of Cardiac Thin Filament

Cardiac troponin T (cTnT) is a key component of contractile regulatory proteins. cTnT is characterized by a ∼32 amino acid N-terminal extension (NTE), the function of which remains unknown. To understand its function, we generated a transgenic (TG) mouse line that expressed a recombinant chimeric cTnT in which the NTE of mouse cTnT was removed by replacing its 1–73 residues with the corresponding 1–41 residues of mouse fast skeletal TnT. Detergent-skinned papillary muscle fibers from non-TG (NTG) and TG mouse hearts were used to measure tension, ATPase activity, Ca2+ sensitivity (pCa50) of tension, rate of tension redevelopment, dynamic muscle fiber stiffness, and maximal fiber shortening velocity at sarcomere lengths (SLs) of 1.9 and 2.3 μm. Ca2+ sensitivity increased significantly in TG fibers at both short SL (pCa50 of 5.96 vs. 5.62 in NTG fibers) and long SL (pCa50 of 6.10 vs. 5.76 in NTG fibers). Maximal cross-bridge turnover and detachment kinetics were unaltered in TG fibers. Our data suggest that the NTE constrains cardiac thin filament activation such that the transition of the thin filament from the blocked to the closed state becomes less responsive to Ca2+. Our finding has implications regarding the effect of tissue- and disease-related changes in cTnT isoforms on cardiac muscle function.

Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics

Ala/Asp substitutions at Ser23/24 have been employed to investigate the functional impact of cardiac troponin I (cTnI) phosphorylation by protein kinase A (PKA). Some limitations of previous studies include the use of heterologous proteins and confounding effects arising from phosphorylation of cardiac myosin binding protein-C. Our goal was to probe the effects of cTnI phosphorylation using a homologous assay, so that altered function could be solely attributed to changes in cTnI. We reconstituted detergent-skinned rat cardiac papillary fibers with homologous rat cardiac troponin subunits to study the impact of Ala and Asp substitutions at Ser23/24 of rat cTnI (RcTnI S23A/24A and RcTnI S23D/24D). Both RcTnI S23A/24A and RcTnI S23D/24D showed a ∼36% decrease in Ca2+-activated maximal tension. Both RcTnI S23A/24A and RcTnI S23D/24D showed a ∼18% decrease in ATPase activity. Muscle fiber stiffness measurements suggested that the decrease in thin filament activation observed in RcTnI S23A/24A and RcTnI S23D/24D was due to a decrease in the number of strongly-bound crossbridges. Another major finding was that Ala and Asp substitutions in cTnI did not affect crossbridge detachment kinetics.

Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but its function is poorly understood, especially the role of the cardiac specific N-terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N-terminal extension was investigated with the Tg-Δ43 mouse model, whose myocardium expresses a truncated ELC. Low-angle X-ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing (≈ 1.5 nm) and no alterations in cross-bridge mass distribution in Tg-A57G mice compared to Tg-WT, expressing the full-length nonmutated ELC. The truncation mutation showed a 1.3-fold increase in I(1,1)/I(1,0), indicating a shift of cross-bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg-A57G muscle fibers compared to Tg-WT or Tg-Δ43. The equilibrium constant for the cross-bridge force generation step was smallest in Tg-Δ43. These results support an important role for the N-terminal ELC extension in prepositioning the cross-bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross-bridge properties and lead to pathological phenotypes.

Non-uniform distribution of strain during stretch of relaxed skeletal muscle fibers from rat soleus muscle.

Tension and regional average sarcomere length (L(s)) behavior were examined during repeated stretches of single, permeabilized, relaxed muscle fibers isolated from the soleus muscles of rats. We tested the hypothesis that during stretches of single permeabilized fibers, the global fiber strain is distributed non-uniformly along the length of a relaxed fiber in a repeatable pattern. Each fiber was subjected to eight constant-velocity stretch and release cycles with a strain of 32% and strain rate of 54% s(-1). Stretch-release cycles were separated by a 4.5 min interval. Throughout each stretch-release cycle, sarcomere lengths were measured using a laser diffraction technique in which 20 contiguous sectors along the entire length of a fiber segment were scanned within 2 ms. The results revealed that: (1) the imposed length change was not distributed uniformly along the fiber, (2) the first stretch-release cycle differed from subsequent cycles in passive tension and in the distribution of global fiber strain, and (3) a characteristic "signature" for the L(s) response emerged after cycle 3. The findings support the conclusions that longitudinal heterogeneity exists in the passive stiffness of individual muscle fibers and that preconditioning of fibers with stretch-release cycles produces a stable pattern of sarcomere strains.

Static Stiffness in Slow and Fast Mouse Muscle Fibers Expressing Different Titin Isoforms

We showed previously (Bagni et al., 2002) that most of the increase of muscle fiber stiffness during the early phases of a tetanic contraction is due to a non-crossbridge sarcomere component whose stiffness (called static stiffness) increases after stimulation with a time course very similar to the internal Ca2+ concentration. This led us to speculate that Ca2+ concentration, in addition to promote crossbridge formation, could also leads to a stiffening of a sarcomere structure, identified with the titin filament, either directly or through a titin-actin interaction leading to the observed sarcomere stiffness increase. According to this hypothesis, it is expected that static stiffness has different properties in muscles expressing titin with different mechanical properties. Therefore we compared the static stiffness values in soleus and EDL adult mouse muscles, which express titin isoforms with long and short PEVK segment, respectively. Considering that Ca2+ binding to E-rich motifs in the PEVK segment increases its bending rigidity, the higher proportion of these motifs in EDL compared to soleus is expected to lead to a greater static stiffness in EDL. Our results showed that in agreement with the titin hypothesis, the static stiffness measured in single fibers at 25°C was more than five times greater in EDL than in soleus and about two times greater than previously reported on FDB muscle. The static stiffness time course in EDL was about the same as in FDB but slightly faster than in soleus, and it became much faster at 35°C in both EDL and soleus similarly to tension time course. These results are in agreement with the idea that static stiffness depends on the increment of titin stiffness due to the interaction between Ca2+ and E-rich motifs in PEVK segment.

AHNAK1 and AHNAK2 are costameric proteins: AHNAK1 affects transverse skeletal muscle fiber stiffness

The AHNAK scaffold PDZ-protein family is implicated in various cellular processes including membrane repair; however, AHNAK function and subcellular localization in skeletal muscle are unclear. We used specific AHNAK1 and AHNAK2 antibodies to analyzed the detailed localization of both proteins in mouse skeletal muscle. Co-localization of AHNAK1 and AHNAK2 with vinculin clearly demonstrates that both proteins are components of the costameric network. In contrast, no AHNAK expression was detected in the T-tubule system. A laser wounding assay with AHNAK1-deficient fibers suggests that AHNAK1 is not involved in membrane repair. Using atomic force microscopy (AFM), we observed a significantly higher transverse stiffness of AHNAK1 −/− fibers. These findings suggest novel functions of AHNAK proteins in skeletal muscle.