Unloaded Shortening Velocity Research Articles

Transgenic Mouse α- and β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μM) is ~ 4-fold weaker than for β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

Hormone replacement therapy improves contractile function and myonuclear organization of single muscle fibres from postmenopausal monozygotic female twin pairs

Ageing is associated with a decline in muscle mass and strength leading to increased physical dependency in old age. Postmenopausal women experience a greater decline than men of similar age in parallel with the decrease in female sex steroid hormone production. We recruited six monozygous female twin pairs (55-59 years old) where only one twin pair was on hormone replacement therapy (HRT use = 7.8 ± 4.3 years) to investigate the association of HRT with the cytoplasmic volume supported by individual myonuclei (myonuclear domain (MND) size,) together with specific force at the single fibre level. HRT use was associated with a significantly smaller (∼27%; P < 0.05) mean MND size in muscle fibres expressing the type I but not the IIa myosin heavy chain (MyHC) isoform. In comparison to non-users, higher specific force was recorded in HRT users both in muscle fibres expressing type I (∼27%; P < 0.05) and type IIa (∼23%; P < 0.05) MyHC isoforms. These differences were fibre-type dependent, i.e. the higher specific force in fast-twitch muscle fibres was primarily caused by higher force per cross-bridge while slow-twitch fibres relied on both a higher number and force per cross-bridge. HRT use had no effect on fibre cross-sectional area (CSA), velocity of unloaded shortening (V0) and relative proportion of MyHC isoforms. In conclusion, HRT appears to have significant positive effects on both regulation of muscle contraction and myonuclei organization in postmenopausal women.

Factors Beyond Detachment Kinetics that Influence Unloaded Shortening Velocities of Muscle

Recent studies demonstrate the influence of actin-myosin attachment kinetics on in vitro actin filament sliding velocities (V), but it is unclear what factors determine whether velocities are detachment (d/τon) or attachment limited (Yengo et al. 2012. J Muscle Res Cell Motil.). We have developed a muscle model with molecular detail that suggests three key mechanisms by which attachment kinetics can influence unloaded shortening velocities. First, the saturation of myosin binding sites on actin by weakly bound myosin heads can result in attachment limited velocities that are slower than the detachment limit. Essentially, at high myosin densities the actin-activated ATPase activity (attachment limited) in the motility assay saturates before mechanical saturation (detachment limited). Second if actin-bound myosin heads that resist actin movement have a relatively low stiffness, unloaded shortening velocities can exceed the detachment limit. The non-linear compliance of positively and negatively strained myosin heads was demonstrated experimentally (Kaya and Higuchi. 2010. Science. 329: 686-9). Finally, our model shows that mechanically accelerated ADP release kinetics can result in velocities that exceed the detachment limit. These simulations accurately describe the observation of hypermotile velocities (Hooft et al. 2007. Biochemistry. 46:3513-20., Jackson and Baker. 2009. PCCP. 11:4808-14.). In summary, unloaded shortening velocities can be influenced by many factors other than detachment kinetics. Thus a mutation or perturbation that results in a change in V need not result from a change in only d or τon.

Contact between Cd4+ T Cells and Smooth Muscle Tissue Enhances Contractile Function

Smooth muscle (SM) hyper-contractility is central to the pathophysiology of several diseases, including asthma. Inflammatory cells and/or their products may alter the expression of contractile proteins, thereby enhancing SM mechanics. The goal of the current study was to characterize the maximum unloaded shortening velocity (Vmax), stress (force/cross sectional area), and contractile protein expression of Brown Norway (BN) rat airway SM in the presence of activated CD4+ T cells. T cells from OVA-sensitized BN rats were co-cultured with tracheal rings for 24h. The SM strips were then mounted in a mechanical testing apparatus, equilibrated and supramaximally stimulated with methacholine. A significant increase in Vmax (mean±S.E.) (0.15±0.01 l/s for control, 0.29±0.02 l/s for sensitized, p=0.0012) was observed whereas no significant changes in stress (84.5±13.9 kPa for control, 107.8±4.8 kPa for sensitized) were seen. Western blot analysis showed that levels of the (+)insert myosin heavy chain isoform and myosin light chain kinase (MLCK) were significantly increased. To determine whether contact between T cells and SM cells is required for this enhanced contractility, CD4+T cells were placed in the upper chamber of a transwell, separated by a membrane from the muscle strips placed below. The membrane had 0.4 μm pores that allowed the cytokines released from the CD4+T cells to reach the SM but prevented direct contact between cells. We found a significant increase in Vmax when the SM were in contact with the T cells (0.32±0.02 l/s) compared to the co-culture without contact (0.25±0.01 l/s) or compared to the SM alone (0.24±0.01 l/s; p=0.01). Thus, contact between SM and CD4+T cells is necessary for modulation of SM contractile properties and this may be mediated, at least in part, by increases in (+)insert myosin heavy chain and MLCK expression. Supported by: CIHR, NIH-RO1HL103405.

Evidence that Actin-Myosy Cycling in Muscle may not Pass through Rigor Configuration

In 1989, Sutoh, Tokunaga and Wakabayashi (J Mol Biol 206:357) prepared a monoclonal antibody (IgG) directed to junctional peptide between 50- and 20-kDa segments of myosin heavy chain, and showed that the antibody attaches around the tip of myosin heads facing actin filaments in muscle. using the same antibody, we successfuly position-mark the tip of myosin heads in synthetic myosin filaments kept in hydrated state in the gas environmental chamber, and succeeded in recording ATP-induced myosin head movement electron microscopically (Sugi et al. PNAS 105: 17396, 2008). We have examined the effect of this antibody on both ATP-dependent in vitro actin-myosin sliding and Ca-activated contraction of permealized rabbit psoas muscle fibers. Unexpectedly, the antibody (concentration, up to 2mg/ml) had no appreciable effect on the velocity profile of actin-myosin sliding and the contraction characteristics of muscle fibers, including magnitude of the maximum Ca-activated isometric force, the maximum unloaded shortening velocity, ans MgATPase activity during contraction. Since the junctional peptide is located between the two main actin- binding sites of myosin heads during formation of rigor linkages, attachment of massive antibodies to myosin heads completely covers their actin- binding sites. The possibility that the antibody does not diffuse into muscle fibers is excluded by the fact that other kinds of antibody diffuse into muscle fibers to inhibit Ca-activated force generation (e.g., Sugi et al. PNAS 89: 6134, 1992). Our work, therefore, suggests strongly that attachment-detachment cycle between actin and myosin in muscle may not pass through rigor configuration. It seems possible that so-called strong actin-myosin binding might be due to some electrostatic mechanism.

Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Rationale: Alterations to the cardiac regulatory light chain (RLC) phosphorylation state correlate with many types of cardiac disease including familial hypertrophic cardiomyopathies (FHC's) and heart failure (HF) post myocardial infarction (MI). However, little is known regarding the role of RLC phosphorylation in regulating cardiac contraction mechanics during shortening. Objective: Determine the mechanical effect of altered cRLC phosphorylation on the force-velocity characteristics of cardiac muscle. Methods and Results: SDS-PAGE electrophoresis determined the effects of heart failure on cRLC phosphorylation on human heart failure (HF) samples and a rat chronic-MI model. We report an increase in RLC phosphorylation in HF progression. Recombinant cRLC was cloned and expressed. Recombinant RLC was differentially phosphorylated in-vitro using cardiac myosin light chain kinase (cMLCK) and ZIP kinase. Recombinant RLC species were exchanged into permeabilised cardiac trabeculae to perform mechanical measurements during shortening (slack test and force-velocity experimentation protocols). We observed a decrease in peak power, velocity at peak power (VPP) and peak isometric force of contraction when reducing RLC phosphorylation. Conversely, enriching RLC phosphorylation increased peak power, VPP and peak maximal unloaded shortening velocity (VMAX) in comparison to control. Conclusions: Altering cRLC phosphorylation has a significant impact on the mechanical function of cardiac muscle and is dramatically altered during HF progression. These results highlight the importance of understanding thick filament regulation and specifically the ability of cRLC phosphorylation to regulate cardiac muscle function.

Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

Ca(2+) dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca(2+) binding in the NH(2)-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin's detailed dynamics, troponin's regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca(2+) slowed HDX up to 2 orders of magnitude within the NH(2)-lobe and the NH(2)-lobe-associated TnI switch helix, implying that Ca(2+) greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca(2+) and actin. Ca(2+)-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca(2+) destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca(2+)-mediated regulation: the coiled-coil's NH(2)-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca(2+)-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca(2+), including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca(2+) release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin's dynamic features.

Role of alpha-actinin-3 in contractile properties of human single muscle fibers: a case series study in paraplegics.

A common nonsense polymorphism in the ACTN3 gene results in the absence of α-actinin-3 in XX individuals. The wild type allele has been associated with power athlete status and an increased force output in numeral studies, though the mechanisms by which these effects occur are unclear. Recent findings in the Actn3−/− (KO) mouse suggest a shift towards ‘slow’ metabolic and contractile characteristics of fast muscle fibers lacking α-actinin-3. Skinned single fibers from the quadriceps muscle of three men with spinal cord injury (SCI) were tested regarding peak force, unloaded shortening velocity, force-velocity relationship, passive tension and calcium sensitivity. The SCI condition induces an ‘equal environment condition’ what makes these subjects ideal to study the role of α-actinin-3 on fiber type expression and single muscle fiber contractile properties. Genotyping for ACTN3 revealed that the three subjects were XX, RX and RR carriers, respectively. The XX carrier’s biopsy was the only one that presented type I fibers with a complete lack of type IIx fibers. Properties of hybrid type IIa/IIx fibers were compared between the three subjects. Absence of α-actinin-3 resulted in less stiff type IIa/IIx fibers. The heterozygote (RX) exhibited the highest fiber diameter (0.121±0.005 mm) and CSA (0.012±0.001 mm2) and, as a consequence, the highest peak force (2.11±0.14 mN). Normalized peak force was similar in all three subjects (P = 0.75). Unloaded shortening velocity was highest in R-allele carriers (P<0.001). No difference was found in calcium sensitivity. The preservation of type I fibers and the absence of type IIx fibers in the XX individual indicate a restricted transformation of the muscle fiber composition to type II fibers in response to long-term muscle disuse. Lack of α-actinin-3 may decrease unloaded shortening velocity and increase fiber elasticity.

Origin of Motion in the Human Ureter: Mechanics, Energetics and Kinetics of the Myosin Molecular Motors

Ureteral peristalsis is the result of coordinated mechanical motor performance of longitudinal and circular smooth muscle layer of the ureter wall. The main aim of this study was to characterize in smooth muscle of proximal segments of human ureter, the mechanical properties at level of muscle tissue and at level of myosin molecular motors. Ureteral samples were collected from 15 patients, who underwent nephrectomy for renal cancer. Smooth muscle strips longitudinally and circularly oriented from proximal segments of human ureter were used for the in vitro experiments. Mechanical indices including the maximum unloaded shortening velocity (Vmax), and the maximum isometric tension (P0) normalized per cross-sectional area, were determined in vitro determined in electrically evoked contractions of longitudinal and circular smooth muscle strips. Myosin cross-bridge (CB) number per mm2 (Ψ) the elementary force per single CB (Ψ) and kinetic parameters were calculated in muscle strips, using Huxley's equations adapted to nonsarcomeric muscles. Longitudinal smooth muscle strips exhibited a significantly (p<0.05) faster Vmax (63%) and a higher P0 (40%), if compared to circular strips. Moreover, longitudinal muscle strips showed a significantly higher unitary force (Ψ) per CB. However, no significant differences were observed in CB number, the attachment (f1) and the detachment (g2) rate constants between longitudinal and circular muscle strips. The main result obtained in the present work documents that the mechanical, energetic and unitary forces per CB of longitudinal layer of proximal ureter are better compared to the circular one; these preliminary findings suggested, unlike intestinal smooth muscle, a major role of longitudinal smooth muscle layer in the ureter peristalsis.

An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle.

A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ.With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4-30◦C) and substrate concentration. VF was 8.88 ± 0.26 μms⁻¹ at 30.6◦C and decreased to 1.60 ± 0.09 μms⁻¹ at 4.5◦C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40-50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5◦C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads.

Half-Sarcomere Mechanics and Energetics Indicate that Myosin Motors Slip Between Two Consecutive Actin Monomers during their Working Stroke

The coupling between chemical and mechanical steps of actomyosin ATPase cycle was studied in situ by using fast mechanical protocols in Ca2+-activated demembranated fibres from rabbit psoas under sarcomere length control (sarcomere length 2.4 μm, temperature 12°C). We determined the effects of the concentration of inorganic phosphate (Pi) on the force-velocity relation (T-V), on the stiffness-velocity relation (e-V) and on the isotonic velocity transient following a stepwise drop in force from the isometric plateau force (T0) (Piazzesi et al. J Physiol 545:145, 2002). With respect to control (no added Pi), the increase of [Pi] to 10 mM, i) reduced T0 by 50-60%, decreased the curvature of the T-V relation by 30% and increased the unloaded shortening velocity (V0) by 19%; ii) decreased the relative half-sarcomere stiffness at each shortening velocity by an extent that indicates that Pi has little effect on the force per attached myosin motor; iii) did not change the rate of early rapid shortening (phase 2) following the stepwise drop in force, while reduced its size and made the subsequent pause of shortening (phase 3) briefer. Steady state and transient mechanical responses and the known related energetics (Potma and Stienen J Physiol 496:1, 1996) are simulated with a kinetic-mechanical model of the actomyosin ATPase cycle that incorporates Huxley and Simmons mechanism of force generation. Muscle power and efficiency during isotonic shortening at high and intermediate loads can be predicted only if myosin motors at an intermediate stage of both the working stroke and product release can slip to the next Z-ward actin monomer. Supported by MIUR, Ministero della Salute and Ente Cassa di Risparmio di Firenze (Italy).

Recent insights into muscle fatigue at the cross-bridge level.

The depression in force and/or velocity associated with muscular fatigue can be the result of a failure at any level, from the initial events in the motor cortex of the brain to the formation of an actomyosin cross-bridge in the muscle cell. Since all the force and motion generated by muscle ultimately derives from the cyclical interaction of actin and myosin, researchers have focused heavily on the impact of the accumulation of intracellular metabolites [e.g., Pi, H+ and adenosine diphoshphate (ADP)] on the function these contractile proteins. At saturating Ca++ levels, elevated Pi appears to be the primary cause for the loss in maximal isometric force, while increased [H+] and possibly ADP act to slow unloaded shortening velocity in single muscle fibers, suggesting a causative role in muscular fatigue. However the precise mechanisms through which these metabolites might affect the individual function of the contractile proteins remain unclear because intact muscle is a highly complex structure. To simplify problem isolated actin and myosin have been studied in the in vitro motility assay and more recently the single molecule laser trap assay with the findings showing that both Pi and H+ alter single actomyosin function in unique ways. In addition to these new insights, we are also gaining important information about the roles played by the muscle regulatory proteins troponin (Tn) and tropomyosin (Tm) in the fatigue process. In vitro studies, suggest that both the acidosis and elevated levels of Pi can inhibit velocity and force at sub-saturating levels of Ca++ in the presence of Tn and Tm and that this inhibition can be greater than that observed in the absence of regulation. To understand the molecular basis of the role of regulatory proteins in the fatigue process researchers are taking advantage of modern molecular biological techniques to manipulate the structure and function of Tn/Tm. These efforts are beginning to reveal the relevant structures and how their functions might be altered during fatigue. Thus, it is a very exciting time to study muscle fatigue because the technological advances occurring in the fields of biophysics and molecular biology are providing researchers with the ability to directly test long held hypotheses and consequently reshaping our understanding of this age-old question.

Improved V̇O2 uptake kinetics and shift in muscle fiber type in high-altitude trekkers

The study investigated the effect of prolonged hypoxia on central [i.e., cardiovascular oxygen delivery (Q(a)O(2))] and peripheral (i.e., O(2) utilization) determinants of oxidative metabolism response during exercise in humans. To this aim, seven male mountaineers were examined before and immediately after the Himalayan Expedition Interamnia 8000-Manaslu 2008, lasting 43 days, among which, 23 days were above 5,000 m. The subjects showed a decrease in body weight (P < 0.05) and of power output during a Wingate Anaerobic test (P < 0.05) and an increase of thigh cross-sectional area (P < 0.05). Absolute maximal O(2) uptake (VO(2max)) did not change. The mean response time of VO(2) kinetics at the onset of step submaximal cycling exercise was reduced significantly from 53.8 s ± 10.9 to 39.8 s ± 10.9 (P < 0.05), whereas that of Q(a)O(2) was not. Analysis of single fibers dissected from vastus lateralis biopsies revealed that the expression of slow isoforms of both heavy and light myosin subunits increased, whereas that of fast isoforms decreased. Unloaded shortening velocity of fibers was decreased significantly. In summary, independent findings converge in indicating that adaptation to chronic hypoxia brings about a fast-to-slow transition of muscle fibers, resulting in a faster activation of the mitochondrial oxidative metabolism. These results indicate that a prolonged and active sojourn in hypoxia may induce muscular ultrastructural and functional changes similar to those observed after aerobic training.

Effects of myosin heavy chain (MHC) plasticity induced by HMGCoA‐reductase inhibition on skeletal muscle functions

The rate-limiting step of cholesterol biosynthetic pathway is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme reductase (HGMR), whose inhibitors, the statins, widely used in clinical practice to treat hypercholesterolemia, often cause myopathy, and rarely rhabdomyolysis. All studies to date are limited to the definition of statin-induced myotoxicity omitting to investigate whether and how HMGR inhibition influences muscle functions. To this end, 3-mo-old male rats (Rattus norvegicus) were treated for 3 wk with a daily intraperitoneal injection of simvastatin (1.5 mg/kg/d), and biochemical, morphological, mechanical, and functional analysis were performed on extensor digitorum longus (EDL) muscle. Our results show that EDL muscles from simvastatin-treated rats exhibited reduced HMGR activity; a 15% shift from the fastest myosin heavy-chain (MHC) isoform IIb to the slower IIa/x; and reduced power output and unloaded shortening velocity, by 41 and 23%, respectively, without any change in isometric force and endurance. Moreover, simvastatin-treated rats showed a decrease of maximum speed reached and the latency to fall off the rotaroad (∼-30%). These results indicate that the molecular mechanism of the impaired muscle function following statin treatment could be related to the plasticity of fast MHC isoform expression.

Muscle Protein Composition in Aerobically Trained Skeletal Muscle

Aerobic exercise training uniquely alters skeletal muscle fiber contractile function. However, it is unclear if these alterations are due to changes in the general muscle protein composition or specific contractile elements (actin and myosin). PURPOSE: To examine skeletal muscle protein composition in aerobically trained skeletal muscle as it relates to myofiber contractile function. METHODS: Skeletal muscle biopsy samples were obtained from the lateral head of the gastrocnemius of seven collegiate (RUN; 71±1 ml·kg-1·min-1) and eight recreational (REC; 46±2 ml·kg-1·min-1) runners. Muscle water content and protein composition were determined through freeze-drying, separation of protein fractions, SDS-PAGE, staining and densitometry. RESULTS: MHC I and MHC IIa myofibers from RUN exhibited a higher (p<0.05) unloaded shortening velocity and a trend (p=0.07) for a lower specific tension (P0/CSA) compared to REC. Muscle water content was lower (p<0.05) in RUN (73±1%) compared to REC (75±1%). Relative to muscle wet weight, total (RUN, 165±4; REC, 147±4 μg·mg-1) and myofibrillar (RUN, 106±6; REC, 82±3 μg·mg-1) protein fractions were greater (p<0.05) in RUN as compared to REC. However, this pattern was not apparent after correcting for muscle dry weight in the total (RUN, 612±1; REC, 652±27 μg·mg-1) and myofibrillar (RUN, 395±21; REC, 357±21 μg·mg-1) protein fractions. Myosin concentration was not different between REC (0.990.08 AU) and RUN (1.010.07 AU). Additionally, actin concentration was similar between REC (14±1 μg·mg-1 muscle wet wt) and RUN (17±2 μg·mg-1 muscle wet wt). CONCLUSION: These data suggest that muscle water content, total and myofibrillar protein content and the concentration of the main contractile proteins actin and myosin do not explain the robust differences in myofiber function between these groups of runners. Therefore, it is likely that the arrangement, rather than the composition of skeletal muscle proteins, is more relevant to understanding myofiber contractile function in aerobically trained human skeletal muscle. Supported by NIH Grant AG032127

Potential errors in fiber length measurements resulting from lever arm rotation during mechanical testing of muscle cells

In single muscle cell preparations fibers are often suspended between connectors, extending perpendicularly from a force transducer, and the lever arm of a torque motor. The fiber does not move along a horizontal plane when shortened or lengthened by lever arm rotation. An error from the true length (TL) is introduced if the expected length (EL) is calibrated along this horizontal optical plane. Lever arm length (LAL), initial fiber length (FLi), connector length (CL), and the magnitude of EL all contribute to this error. A mathematical model was used to determine the TL during shortening (0.96–0.80FLi) and lengthening (1.10–1.50FLi) at a constant LAL of 13.6mm. CL had the greatest impact on error. For FLi=2mm at the longest CL modeled (15mm), an expected shortening of 0.20FLi produced a true shortening of ∼0.17FLi, and an expected stretch to 1.50FLi resulted in a true stretch to almost 1.60FLi. Under these conditions, the true sarcomere length would be 4% and 6% longer than expected during shortening and lengthening, respectively. Because of their non-linear nature, length errors at long CL's may result in an over-estimation of unloaded shortening velocity during slack tests and a left-ward shift in the passive tension-fiber length relationship at long fiber lengths. Measurement errors decreased dramatically with shorter CL's, becoming negligible (<1%) at CL=3mm. We recommend that investigators keep CL as short as possible. Alternatively, we provide a method for adjusting the magnitude of the EL to yield a desired TL.

Increased Myosin Light Chain 3f Content Restores Age‐Induced Slowing of Single Skeletal Muscle Fiber Contraction

Aging is characterized by a progressive loss of muscle mass and impaired contractile functions (e.g. decline in force, velocity and power). Although these phenomena in aging muscle are well described, the underlying molecular mechanisms responsible for a decrease in contraction speed are not defined. Evidence suggests that the slowing of muscle contraction, as demonstrated by slower unloaded shortening velocity (Vo), is correlated with a decrease in relative myosin light chain 3f (MLC3f) content. In the current study, we first established recombinant adenovirus (rAd) gene transfer technology to increase MLC3f content in semimembranosus muscles of aged rats. We hypothesized that 1) age-induced decline in Vo would be associated with a decrease in relative MLC3f content and 2) increasing MLC3f content via rAd would restore Vo in myosin heavy chain (MHC) type II fibers. Vo (3.2±0.2 vs. 2.2±0.2 FL/s), %MLC3f (13.6±1.0 vs. 3.7±0.3%) and MLC3f/MLC2f ratio (0.28±0.04 vs. 0.10±0.01) decreased with age in type II single fibers. Increasing %MLC3f to 8.0% and MLC3f/MLC2f ratio to 0.20 via rAd provided significant protection against age-induced reduction in Vo without causing muscle damage or influencing single fiber diameter, force and MHC composition. To the best of our knowledge, these are the first data to demonstrate affirmative effects of MLC3f against slowing of contraction in aged skeletal muscle. RO1AG017768

Eccentric contraction-induced injury to type I, IIa, and IIa/IIx muscle fibers of elderly adults

Muscles of old laboratory rodents experience exaggerated force losses after eccentric contractile activity. We extended this line of inquiry to humans and investigated the influence of fiber myosin heavy chain (MHC) isoform content on the injury process. Skinned muscle fiber segments, prepared from vastus lateralis biopsies of elderly men and women (78 ± 2years, N = 8), were subjected to a standardized eccentric contraction (strain, 0.25 fiber length; velocity, 0.50 unloaded shortening velocity). Injury was assessed by evaluating pre- and post-eccentric peak Ca(2+)-activated force per fiber cross-sectional area (F (max)). Over 90% of the variability in post-eccentric F (max) could be explained by a multiple linear regression model consisting of an MHC-independent slope, where injury was directly related to pre-eccentric F (max), and MHC-dependent y-intercepts, where the susceptibility to injury could be described as type IIa/IIx fibers > type IIa fibers > type I fibers. We previously reported that fiber type susceptibility to the same standardized eccentric protocol was type IIa/IIx > type IIa = type I for vastus lateralis fibers of 25-year-old adults (Choi and Widrick, Am J Physiol Cell Physiol 299:C1409-C1417, 2010). Modeling combined data sets revealed significant age by fiber type interactions, with post-eccentric F (max) deficits greater for type IIa and type IIa/IIx fibers from elderly vs. young subjects at constant pre-eccentric F (max). We conclude that the resistance of the myofilament lattice to mechanical strain has deteriorated for type IIa and type IIa/IIx, but not for type I, vastus lateralis fibers of elderly adults.

The effect of skeletal myosin light chain kinase gene ablation on the fatigability of mouse fast muscle

Contraction-induced activation of a skeletal muscle specific Ca(2+) and calmodulin dependent myosin light chain kinase (skMLCK) catalyzes phosphorylation of the myosin regulatory light chain (RLC), a reaction that potentiates twitch force. The purpose of this study was to test the effect of skMLCK gene ablation on the fatigability of mouse extensor digitorum longus (EDL) muscle (in vitro at 25°C). Muscles were isolated from wildtype (WT, n = 10-12) and skeletal MLCK knockout (skMLCK KO, n = 10-12) mice and fatigued using a protocol consisting of 5 min of repeated tetanic stimulation (150 Hz for 1000 ms every 5 s). Both twitch (P(t)) and tetanic (P(o)) force as well as unloaded shortening velocity (V(o)) were assessed before, during and after fatiguing stimulation. Fatiguing stimulation increased RLC phosphorylation in WT but not skMLCK KO muscles (16 ± 0.01-0.63 ± 0.02 and 0.07 ± 0.02-0.08 ± 0.02 mol phos mol RLC, respectively). Although P(t) was potentiated above baseline in both WT and KO muscles, this increase was greater in WT than in KO muscles (to 1.37 ± 0.05 vs. 1.14 ± 0.02 of unpotentiated values, respectively). The difference in P(t) persisted until P(o) had been diminished to ~60% of baseline and thereafter P(t) declined to similar levels in both WT and KO muscles (to ~35% of initial). Overall, the time-course and decline in P(o) for WT and KO was similar (reduced to 0.20 ± 0.01 and 0.20 ± 0.01 of baseline, respectively) (P < 0.05). Initial values for V(o) were similar between WT and KO muscles and, moreover, the fatigue related decline in Vo was similar for both muscle genotypes (P < 0.05). Thus, our results demonstrate that skMLCK--catalyzed RLC phosphorylation augments isometric twitch force during moderate, but not severe, levels of fatigue.

Fiber Myosin Heavy Chain Polymorphism is Associated with a Heightened Susceptibility to Eccentric Damage

The purpose of this study was to model the relationship between fiber myosin heavy chain (MHC) isoform content and susceptibility to damage by a standardized eccentric contraction (+25% change in fiber length; 50% of unloaded shortening velocity, Vo). Damage was quantified as the pre- to post-eccentric change in Ca2+-activated force. Chemically skinned fibers from the soleus and EDL of C57BL/6 mice were studied in order to obtain MHC monomorphic (type I, IIa, IIx, IIb) and polymorphic (I/IIa, IIa/IIx, IIx/IIb) fibers. Fiber MHC isoform content was determined by SDS-PAGE. The mean (±SE) pre- to post-eccentric change in force was −5.2 ± 0.5 kN/m2 (n = 24) for type I fibers, −18.3 ± 1.5 (7) for I/IIa, −5.3 ± 0.7 (27) for IIa, −18.1 ± 2.3 (5) for IIa/IIx, −5.4 ± 0.6 (34) for IIx, −17.8 ± 1.1 (11) for IIx/IIb, and −5.7 ± 0.8 (24) for IIb. Multiple linear regression indicated that, independent of MHC content, greater pre-specific force was associated with a slightly greater post-force deficit (slope = 0.94). With pre-eccentric force held constant, no relationship was found between fiber Vo and post-eccentric force (p = 0.716) . In contrast, data were well described by a model using pre-eccentric force and MHC polymorphism as predictors (p < 0.001; r2 = 0.97): post-force of monomorphic fibers = 0.94(pre-force) + 2.04 kN/m2 post-force of polymorphic fibers = 0.94(pre-force) - 12.15 kN/m2 The significant difference (p < 0.001) in model y-intercepts reveals that, at similar pre-eccentric specific force, MHC polymorphism was associated with a 14 kN/m2 greater eccentric-induced reduction in force vs. monomorphic fibers. We conclude that MHC polymorphism is associated with a heightened sensitivity to high mechanical strain at the level of the myofilament lattice or cytoskeleton.