Unloaded Shortening Research Articles

Calcium-activated force of human muscle fibers following a standardized eccentric contraction

Peak Ca(2+)-activated specific force (force/fiber cross-sectional area) of human chemically skinned vastus lateralis muscle fiber segments was determined before and after a fixed-end contraction or an eccentric contraction of standardized magnitude (+0.25 optimal fiber length) and velocity (0.50 unloaded shortening velocity). Fiber myosin heavy chain (MHC) isoform content was assayed by SDS-PAGE. Posteccentric force deficit, a marker of damage, was similar for type I and IIa fibers but threefold greater for type IIa/IIx hybrid fibers. A fixed-end contraction had no significant effect on force. Multiple linear regression revealed that posteccentric force was explained by a model consisting of a fiber type-independent and a fiber type-specific component (r(2) = 0.91). Preeccentric specific force was directly associated with a greater posteccentric force deficit. When preeccentric force was held constant, type I and IIa fibers showed identical susceptibility to damage, while type IIa/IIx fibers showed a significantly greater force loss. This heightened sensitivity to damage was directly related to the amount of type IIx MHC in the hybrid fiber. Our model reveals a fiber-type sensitivity of the myofilament lattice or cytoskeleton to mechanical strain that can be described as follows: type IIa/IIx > type IIa = type I. If these properties extend to fibers in vivo, then alterations in the number of type IIa/IIx fibers may modify a muscle's susceptibility to eccentric damage.

Atrial contractile protein content and function are preserved in patients with coronary artery disease and atrial fibrillation

Atrial fibrillation (AF) causes atrial contractile dysfunction. The focus of this study was to determine whether the contractile deficit of human AF is the result of altered contractile protein abundance and/or function. Atrial tissue from patients with chronic AF undergoing open-heart surgery was compared with the tissue from patients in normal sinus rhythm (NSR). Myosin isoform composition and content were determined. Intact native thin filament and cardiac myosin contractile protein performance were independently assessed in an in-vitro motility assay. Myosin isoform expression and total myosin content were not different between AF and NSR. Calcium-activated native thin filament function was similar between AF and NSR as measured by calcium sensitivity and maximal activation. Myosin isolated from the atria of AF and NSR groups showed similar unloaded shortening speeds and isometric force generation. Unlike human ventricular dysfunction where contractile protein function is directly affected, the contractile deficit of AF is not the result of alterations in myosin content or contractile protein function.

Changes in the force-velocity relationship of fatigued muscle: implications for power production and possible causes.

Slowing of the contractile properties of skeletal muscle is one of the characteristic features of fatigue. First studied as a slowing of relaxation from an isometric contraction, it has become apparent that this slowing is indicative of functional changes in muscle responsible for a major loss of power with all its functional repercussions. There are three factors contributing to the loss of power in mammalian muscle at physiological temperatures, a decrease in isometric force, which mainly indicates a reduction in the number of active cross bridges, a slowing of the maximum velocity of unloaded shortening and an increased curvature of the force-velocity relationship. This latter change is a major cause of loss of power but is poorly understood. It is probably associated with an increase in the proportion of cross bridges in the low force state but there are no clear candidates for the metabolic changes that are responsible for this shift in cross bridge states. The possibility is discussed that the reduction in activating calcium that occurs with metabolically depleted muscle, alters the distribution of cross bridge states, affecting both shortening velocity and curvature.

Contractile properties of muscle fibers from the deep and superficial digital flexors of horses

Equine digital flexor muscles have independent tendons but a nearly identical mechanical relationship to the main joint they act upon. Yet these muscles have remarkable diversity in architecture, ranging from long, unipennate fibers ("short" compartment of DDF) to very short, multipennate fibers (SDF). To investigate the functional relevance of the form of the digital flexor muscles, fiber contractile properties were analyzed in the context of architecture differences and in vivo function during locomotion. Myosin heavy chain (MHC) isoform fiber type was studied, and in vitro motility assays were used to measure actin filament sliding velocity (V(f)). Skinned fiber contractile properties [isometric tension (P(0)/CSA), velocity of unloaded shortening (V(US)), and force-Ca(2+) relationships] at both 10 and 30°C were characterized. Contractile properties were correlated with MHC isoform and their respective V(f). The DDF contained a higher percentage of MHC-2A fibers with myosin (heavy meromyosin) and V(f) that was twofold faster than SDF. At 30°C, P(0)/CSA was higher for DDF (103.5 ± 8.75 mN/mm(2)) than SDF fibers (81.8 ± 7.71 mN/mm(2)). Similarly, V(US) (pCa 5, 30°C) was faster for DDF (2.43 ± 0.53 FL/s) than SDF fibers (1.20 ± 0.22 FL/s). Active isometric tension increased with increasing Ca(2+) concentration, with maximal Ca(2+) activation at pCa 5 at each temperature in fibers from each muscle. In general, the collective properties of DDF and SDF were consistent with fiber MHC isoform composition, muscle architecture, and the respective functional roles of the two muscles in locomotion.

Relationship Bewteen Viscoelastic Stress Relaxation and Passive Stiffness in Single Muscle Fibers of Human Skeletal Muscles

Human performance requires muscles to store energy when deformed or stretched and to recoil after being stretched. The association of viscoelastic and elastic properties with stiffness or extensibility still remains unclear in human single muscle fibers. PURPOSE: To evaluate the viscoelastic stress relaxation and passive stiffness of human single skeletal muscle fibers and to find the relationship between stress relaxation and stiffness. METHODS: Chemically skinned single muscle fibers (n=107) were prepared from biopsy specimens obtained from vastus lateralis muscles of 6 healthy volunteers (3 females and 3 males; mean age: 24.3±4.1 yr). Slack tests were used to determine specific force (maximal force/cross-sectional area, CSA) and unloaded shortening velocity. Stress relaxation and passive force were measured at pCa 9.0 using a stepwise stretch with 0.2 μm increments of muscle length from 2.4 to 4.2 μm. Peak responses to each ramp stretch and steady state force at the end of stress relaxation (after 1 min) were measured to obtain passive tension (steady state force/CSA), passive stiffness (slope of length-tension curve), peak steady-state force ratio (PSSFR, peak force/steady state force) and viscoelastic absorption (tension decayxt1/2). The myosin heavy chain isoform of each fiber was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: Specific force and unloaded shortening velocity were not significantly different between females and males for type I (n=29 for females and 20 for males) and IIa (n= 15 and 18) fibers. There were no sex and fiber type differences in passive tension and PSSFR. Passive stiffness and viscoelastic absorption were greater in type IIa, but not type I fibers of males vs. females. PSSFR at maximum stretch was 5.61±1.99 suggesting 82.2% loss of peak response during stress relaxation. PSSFR was negatively correlated with stiffness (r = -0.369, p = 0.001) but not passive tension. Viscoelastic absorption was correlated with stiffness (r = 0.432, p < 0.001). CONCLUSION: We found a sex difference in viscoelastic properties and stiffness only for type IIa fibers. PSSFR indicating relative force loss during stress relaxation can be useful to assess viscoelastic and elastic properties along with passive stiffness.

Enhancement of shortening velocity, power, and acto‐myosin crossbridge (CB) kinetics following long‐term treatment with propionyl‐L‐carnitine, coenzyme Q10, and omega‐3 fatty acids in BIO TO‐2 cardiomyopathic Syrian hamsters papillary muscle

Impaired functions of myocardial muscle cells in human and animals, is a primary defect associated with idiopathic dilated cardiomyopathy (DCM). The pathophysiological mechanisms implicated in the DCM are yet to be clarified and an effective therapy is still not available. The BIO TO-2 cardiomyopathic Syrian Hamsters (CMSHs) represent an animal model of idiopathic DCM. The aim of this study was to investigate the effect of long-term treatment (2 months) with propionyl-L-carnitine (PLC), coenzyme Q(10), omega-3 fatty acids and a combination of these three agents (formulation HS12607) on mechanical properties and acto-myosin crossbridges (CBs) kinetics of left ventricular (LV) papillary muscle from control and treated 10 month old BIO TO-2 CMSHs. Isometric and isotonic contractile properties of isolated papillary muscle from control and treated CMSHs were investigated, and acto-myosin CB number, force and kinetics were calculated using Huxley's equations. Mechanical parameter values were higher in treated than in control hamsters, particularly when substances were administered together in a coformulation (HS12607). Compared to control, HS12607-treated papillary muscles showed a significant increase of maximum peak isometric tension (P(o)) (30.06 +/- 4.91 vs. 19.74 +/- 5.00 mN/mm(2)), maximum extent of muscle shortening (0.13 +/- 0.03 vs. 0.07 +/- 0.02 L/L(max)), maximum unloaded shortening velocity (1.18 +/- 0.24 vs. 0.53 +/- 0.13 L/L(max) s(-1)) and maximum peak of power output (5.52 +/- 1.61 vs. 1.58 +/- 0.83). The curvature of the hyperbolic force-velocity relationships did not differ between control and treated hamsters. When compared to controls, acto-myosin CB number increased in treated hamsters [(6.67 +/- 1.91) 10(10)/mm(2) vs. (3.55 +/- 2.08) 10(10)/mm(2)], whereas the unitary force of single CB was similar in control and treated animals. The peak value of the rate constant for CB attachment (f(1)) and detachment (g(2)) was higher in treated animals when compared to control. (93.87 +/- 25.82 vs.47.28 +/- 10.88 s(-1) and 214.40 +/- 44.64 vs. 95.56 +/- 23.49 s(-1), respectively). In conclusion, the present study illustrates that supplementation with PLC, CoQ(10) and omega-3 fatty acids improved motor parameters, energetic, and CB kinetics of BIO TO-2 CMSH papillary muscle indicating that these naturally occurring substances may be a valid adjuvant to conventional therapy in DCM.

Force deficits of human type I, IIa, and IIa/IIx muscle fibers following a single in vitro eccentric contraction

Fast muscles from small laboratory animals show greater force deficits after eccentric contractions than slow muscles. How this applies to humans is unclear as humans do not express the same fast myosin heavy chain (MHC) isoforms as these animal models. To investigate relationships between eccentric damage and fiber type in human muscle, we measured peak Ca2+ activated force (15°C) of chemically skinned fiber segments (vastus lateralis, 6 subjects, age = 24 ± 2) before and after a single eccentric contraction (25% strain, 50% of pre‐eccentric unloaded shortening velocity). Fiber type was determined by gel electrophoresis. Fibers showing visible post‐eccentric myofibrillar tearing (N = 8) were excluded from analysis. At baseline, type IIa (125 ± 6 kN/m2; n = 28) and IIa/IIx (133 ± 7 kN/m2; n = 12) fibers produced greater Ca2+ activated force than type I fibers (111 ± 3 kN/m2; n = 38). A significant fiber type × time interaction revealed similar force deficits for type I (11 ± 1 kN/m2 or 10 ± 1% of pre force) and IIa (14 ± 2 kN/m2, 11 ± 1%) fibers but a 3‐fold greater deficit for IIa/IIx fibers (40 ± 5 kN/m2, 30 ± 3%). Control fibers subjected to a single isometric contraction showed force deficits of only 2 ± 1 kN/m2 (1 ± 1 %) with no difference between fiber types. These results indicate that human fast hybrid fibers (IIa/IIx) are more sensitive to a standardized in vitro eccentric contraction then either type I or type IIa fibers.

The ontogeny of muscle structure and locomotory function in the long-finned squidDoryteuthis pealeii

Understanding the extent to which changes in muscle form and function underlie ontogenetic changes in locomotory behaviors and performance is important in understanding the evolution of musculoskeletal systems and also the ecology of different life stages. We explored ontogenetic changes in the structure, myosin heavy chain (MHC) expression and contractile properties of the circular muscles that provide power for jet locomotion in the long-finned squid Doryteuthis pealeii. The circular muscle fibers of newly hatched paralarvae had different sizes, shapes, thick filament lengths, thin:thick filament ratio, myofilament organization and sarcoplasmic reticulum (SR) distribution than those of adults. Viewed in cross section, most circular muscle cells were roughly triangular or ovoid in shape with a core of mitochondria; however, numerous muscle cells with crescent or other unusual cross-sectional shapes and muscle cells with unequal distributions of mitochondria were present in the paralarvae. The frequency of these muscle cells relative to 'normal' circular muscle cells ranged from 1:6 to 1:10 among the 19 paralarvae we surveyed. The thick filaments of the two types of circular fibers, superficial mitochondria-rich (SMR) and central mitochondria-poor (CMP), differed slightly in length among paralarvae with thick filament lengths of 0.83+/-0.15 microm and 0.71+/-0.1 microm for the SMR and CMP fibers, respectively (P 0.05; ANOVA). During ontogeny the thick filament lengths of both the CMP and SMR fibers increased significantly to 1.78+/-0.27 microm and 3.12+/-0.56 microm, respectively, in adults (P<0.0001 for both comparisons; ANOVA with Tukey's highly significant difference post hoc tests). When sectioned parallel to their long axes, the SMR and CMP fibers of both paralarvae and adults exhibited the myofilament arrangements typical of obliquely striated muscle cells but the angle of obliquity of the dense bodies was 22.8+/-2.4 deg. and 4.6+/-0.87 deg. for paralarvae and adults, respectively. There were also differences in the distribution of the anastomosing network of SR. In paralarvae, the outer and central zones of SR were well developed but the intramyoplasmic zone was greatly reduced in some cells or was scattered non-uniformly across the myoplasm. Whereas in adults the intramyoplasmic SR region was composed primarily of flattened tubules, it was composed primarily of rounded vesicles or tubules when present in the paralarvae. The ontogenetic differences in circular muscle structure were correlated with significant differences in their contractile properties. In brief tetanus at 20 degrees C, the mean unloaded shortening velocity of the paralarval circular muscle preparations was 9.1 L(0) s(-1) (where L(0) was the preparation length that generated the peak isometric stress), nearly twice that measured in other studies for the CMP fibers of adults. The mean peak isometric stress was 119+/-15 mN mm(-2) physiological cross section, nearly half that measured for the CMP fibers of adults. Reverse transcriptase-polymerase chain reaction analysis of paralarval and adult mantle samples revealed very similar expression patterns of the two known isoforms of squid MHC. The ontogenetic differences in the structure and physiology of the circular muscles may result in more rapid mantle movements during locomotion. This prediction is consistent with jet pulse durations observed in other studies, with shorter jet pulses providing hydrodynamic advantages for paralarvae.

The Isotonic Velocity Transient Following a Sudden Rise in Force Imposed on the Muscle Sarcomere During Unloaded Shortening Reveals a Rate Limiting Step in Detached Myosin Motors

Energy balance studies indicate that ATP splitting by myosin motors during rapid shortening of skeletal muscle is not sufficient to account for the energy (mostly heat) output (Rall et al., J Gen Physiol 68(1), 13, 1976; Homsher et al., J Physiol 321, 423, 1981). We investigated the kinetic step of the myosin ATP-ase cycle related to this phenomenon in single muscle fibers from Rana esculenta (∼2.15 μm sarcomere length, 4°C), by recording the isotonic velocity transient following a force step from zero to the isometric tetanic value (T0). Once the isometric tetanus had developed, the force was first clamped to zero for a range of times from 10 ms to 18 ms (during which the fiber shortened at the maximum velocity by 30 nm hs−1 to 50 nm hs−1) and then raised again to T0 in a stepwise manner (∼120 μs). The elastic lengthening induced by the force step was followed by a transient isotonic lengthening, the size of which ranged from 40 to 60 nm hs−1 depending on the size of the preceding shortening. The lengthening velocity was larger for larger shortening size and progressively decreased to approach the isometric condition with a half-time of 2-3 ms. Similarly, the half-sarcomere stiffness recovered the isometric value e0 from the unloaded shortening value of 0.4 e0 with an exponential time course with τ ∼3 ms. We conclude that during rapid shortening a ∼3 ms-transition between detached states of the myosin motor, likely related to the ATP hydrolysis, becomes rate limiting. Accumulation of motors in the state preceding the hydrolysis step can account for the unexplained energy during rapid shortening. Supported by MIUR (Italy).

Temperature Dependence of MgATP and MgADP Affinity of Fast and Slow Rat Myosin Isoforms: An in Vitro Motility Assay Approach

It has been suggested that the rate of ADP release from acto-myosin and the rate of acto-myosin dissociation by ATP may play different roles to define unloaded shortening velocity of slow and fast myosins and tha their role might change with temperature (Nytray et al. 2006; Iorga et al. 2007). In this study, the in vitro motility assay (IVMA) approach was used to study the effect of MgATP and MgADP on actin sliding velocity (Vf) on slow and fast skeletal myosin isoforms at different temperatures. The velocity of actin filaments sliding on pure slow (myosin 1) and pure fast (myosin 2B) myosin isoforms from the rat was determined in a range of [MgATP] (0.01-2mM) and in the presence or absence of 2mM MgADP. Experiments were performed at 20, 25 and 35 °C. The rate constants of ADP release and ATP binding to acto-myosin were calculated. The inhibition of Vf by MgADP was greater in slow then fast isoforms and the rate constant of ADP release was higher in fast than slow isoform. The results suggest that, in slow isoforms, the maximum velocity could be limited by the rate of ADP dissociation from actomyosin. The ADP inhibitory effect decreased and the rate constant of ADP release increased in each isoform with temperature accounting for the increase in Vf. No differences were found between isoforms and among temperatures in the rate constant of ATP binding. The possibility that the rate of acto-myosin dissociation induced by ATP could play a role in defining Vf in fast isoforms will be discussed.

The Effects of Head-Head Interactions on Myosin-Based Actin Sliding Velocities

Myosin generates force with its weak-to-strong actin binding transition and senses force through a strain-dependent step closely associated with ADP release. We have developed both analytical and computational models of the cooperative interplay between these force-generating and force-sensing biochemical transitions. These models make several novel predictions for unloaded shortening muscle, such as rate constants, k-D, for ADP release that are [ADP]- and [ATP]-dependent. The model also predicts that the acceleration of k-D is associated with a dissipation of interhead strain. To test these model predictions, we use an in vitro motility assay to determine the effects of [ADP] and [ATP] on actin sliding velocities, V, and to determine changes in interhead strain by measuring the rate of actin filament breaking. Our results show a non-hyperbolic nucleotide-dependence of V and a nucleotide-dependence of the rate of actin filament breaking that are both consistent with our cooperative model.

Correction of Error in Fiber Length Due to Lever Arm Rotation During Mechanical Tests of Single Muscle Fibers

The single muscle fiber preparation is a popular technique to assess mechanical properties at the cellular level and how these properties change with aging, disease, exercise, etc. Fibers are usually attached to wires extending perpendicularly from the lever arm of a high speed motor and force transducer that are then mounted to the stage of a microscope. Because of lever arm rotation, the fiber does not move along the optical plane, introducing a length measurement error if length is measured along the optical plane. The purpose of this study was to 1) calculate the error of lever arm movement and 2) provide a correction equation. The error was calculated assuming 2 mm fiber length at up to 50% displacement, because this is a commonly used stretch or slack magnitude (∼15% slacking with unloaded shortening velocity, ∼30% lengthening response to eccentric contraction, ∼50% stretch with passive tension). The range of error was hyperbolically enlarged as either lever arm length or optical plane stretch magnitude increased, and this error was exacerbated with the increase of both factors. For example, with a 5 mm connector attached to the lever arm, the relative error [100×(actual displacement-target displacement)/target displacement] was 0.59%, 1.36%, and 1.78% with 10, 30, and 50% of stretch or slacking, respectively. However, with a 10 mm connector, the relative error was 2.38%, 5.66%, and 7.64%, and in the case of a 15 mm connector, the relative error increased dramatically up to 5.36%, 12.69%, and 17.10%. We suggest the following correction equation to eliminate potential errors; q=b+f−r|mT+1|/(m2T2+m2+T2+1)1/2, where q = observed fiber length, b=connector length, f=fiber length, h=lever arm length, r=(b2+h2)1/2, m=-h/(b+f), T=[p2-(d-r)1/2/(d2+r2-p2)]1/2, p=target fiber length, and d=[(b+f)2+h2]1/2.

Aldosterone improves contractile function of adult rat ventricular cardiomyocytes in a non-acute way: potential relationship to the calcium paradox of aldosteronism

Heart failure is accompanied by electrolyte disturbance including reduced calcium and sodium in the extracellular milieu but increased calcium within cells, a phenomenon called "calcium paradox". Aldosteronism is considered as part of this disorder. Aldosterone antagonism is known to reduce cardiac mortality on top of standard therapies such as antagonism of the renin-angiotensin-system. However, the effect of aldosterone on cardiac function under basal conditions and conditions more closely related to those seen in heart failure remains elusive. In order to address this question the function of isolated cardiomyocytes was determined as unloaded cell shortening. Cardiomyocytes were isolated from adult rat hearts and cultured for 24 h in the presence of aldosterone. Thereafter, cell shortening was determined in cells that were electrically paced (0.5-2.0 Hz). The effect of aldosterone on cell shortening was investigated under basal and maximal inotropic stimulation, preincubation with angiotensin II and myocytes from spontaneously hypertensive rats. The composition of the culture medium was modified according to the extracellular milieu found in patients with end-stage heart failure. Aldosterone increased cell shortening in a frequency-dependent way under basal conditions and conditions of low calcium. It potentiated the effect of beta-adrenoceptor stimulation, increased the formation of oxygen radicals, and increased diastolic and systolic calcium. In conclusion, chronic exposure to aldosterone improves the function of cardiomyocytes under basal conditions and electrolyte disturbances that mimic the situation found in heart failure patients.

Variation in the determinants of power of chemically skinned human muscle fibres

We have explored the extent to which the maximal velocity of unloaded shortening (V(max)), the force generated per unit cross-sectional area (P(0)) and the curvature of the force-velocity relationship (a/P(0) in the Hill equation) contribute to differences in peak power of chemically skinned single fibres from the quadriceps muscle of healthy young male subjects. The analysis was restricted to type I and IIA fibres that contained a single type of myosin heavy chain on electrophoretic separation. Force-velocity relationships were determined from isotonic contractions of maximally activated fibres at 15 degrees C. Mean (+/- s.d.) peak powers were 1.99 +/- 0.72 watts per litre (W L(-1)) for type I fibres and 6.92 +/- 2.41 W L(-1), for type IIA fibres. The most notable feature, however, was the very large, sevenfold, range of power outputs within a single fibre type. This wide range was a consequence of variations in each of the three components determining power: P(0), V(max) and a/P(0). Within a single fibre type, P(0) varied threefold, and V(max) and a/P(0) two- to threefold. There were no obvious relationships between P(0) and V(max) or between P(0) and a/P(0). However, there was a suggestion of an inverse relationship between a/P(0) and V(max), the effect being to reduce, somewhat, the impact of differences in V(max) on peak power. In searching for the causes of variation in peak power of fibres of the same type, it appears likely that there are two factors, one that affects P(0) and another that leads to variation in both V(max) and a/P(0).

Nebulin plays a direct role in promoting strong actin-myosin interactions.

The role of the actin filament-associated protein nebulin on mechanical and kinetic properties of the actomyosin motor was investigated in skeletal muscle of wild-type (wt) and nebulin-deficient (nebulin(-)(/)(-)) mice that were 1 d old, an age at which sarcomeric structure is still well preserved. In Ca2+-activated skinned fibers from psoas muscle, we determined the Ca2+ dependence of isometric force and stiffness, the rate of force redevelopment after unloaded shortening (k(TR)), the power during isotonic shortening, and the unloaded shortening velocity (V(0)). Our results show a 65% reduction in isometric force in nebulin(-)(/)(-) fibers at saturating [Ca2+], whereas neither thin-filament length nor the Ca2+ sensitivity of the contractile system is affected. Stiffness measurements indicate that the reduction in isometric force is due to a reduction in the number of actin-attached myosin motors, whereas the force of the motor is unchanged. Furthermore, in nebulin(-)(/)(-) fibers, k(TR) is decreased by 57%, V(0) is increased by 63%, and the maximum power is decreased by 80%. These results indicate that, in the absence of nebulin, the attachment probability of the myosin motors to actin is decreased, revealing a direct role for nebulin in promoting strong actomyosin interactions responsible for force and power production.

Causes of fatigue in slow-twitch rat skeletal muscle during dynamic activity

Skeletal muscle fatigue is most often studied in vitro at room temperature and is classically defined as a decline in maximum force production or power output, exclusively linked to repeated isometric contractions. However, most muscles shorten during normal use, and we propose that both the functional correlate of fatigue, as well as the fatigue mechanism, will be different during dynamic contractions compared with static contractions. Under isoflurane anesthesia, fatigue was induced in rat soleus muscles in situ by isotonic shortening contractions at 37 degrees C. Muscles were stimulated repeatedly for 1 s at 30 Hz every 2 s for a total of 15 min. The muscles were allowed to shorten isotonically against a load corresponding to one-third of maximal isometric force. Maximal unloaded shortening velocity (V(0)), maximum force production (F(max)), and isometric relaxation rate (-dF/dt) was reduced after 100 s but returned to almost initial values at the end of the stimulation protocol. Likewise, ATP and creatine phosphate (CrP) were reduced after 100 s, but the level of CrP was partially restored to initial values after 15 min. The rate of isometric force development, the velocity of shortening, and isotonic shortening were also reduced at 100 s, but in striking contrast, did not recover during the remainder of the stimulation protocol. The regulatory myosin light chain (MLC2s) was dephosphorylated after 100 s and did not recover. Although metabolic changes may account for the changes of F(max), -dF/dt, and V(0), dephosphorylation of MLC2s may be involved in the fatigue seen as sustained slower contraction velocities and decreased muscle shortening.

The cardiac troponin C mutation Leu29Gln found in a patient with hypertrophic cardiomyopathy does not alter contractile parameters in skinned murine myocardium

The present study investigates the effects of the first mutation of troponin C (hcTnCL29Q) found in a patient with hypertrophic cardiomyopathy (HCM) on force–pCa relations and the interplay with phosphorylation of sarcomeric PKA substrates. In triton-skinned murine cardiac fibers, the endogenous mcTnC was extracted and the fibers were subsequently reconstituted with recombinant wild-type and mutant hcTnC. Force–pCa relations of preparations containing hcTnCL29Q or hcTnCWT were similar. Incubation of fibers reconstituted with the recombinant proteins with phosphatase to dephosphorylate sarcomeric PKA substrates induced an increase in Ca2+ sensitivity, slightly more pronounced (0.04 pCa units) in hcTnCL29Q-containing fibers. Incubation of the dephosphorylated fibers with PKA induced significant rightward shifts of force–pCa relations of similar magnitude with both, hcTnCL29Q and hcTnCWT. No significant effects of hcTnCL29Q on the velocity of unloaded shortening were observed. In conclusion, no major differences in contractile parameters of preparations containing hcTnCL29Q compared to hcTnCWT were observed. Therefore, it appears unlikely that hcTnCL29Q induces the development of HCM by affecting the regulation of Ca2+-activated force and interference with PKA-mediated modulation of the Ca2+ sensitivity of contraction.

Force And Unloaded Shortening Velocity During Fatigue In Skeletal Myosin Light Chain Kinase Deficient Muscles

The myosin regulatory light chain (R-LC) of type II fibers is phosphorylatable. Contraction-induced activation of a skeletal muscle specific Ca2+ - calmodulin-dependent myosin light chain kinase (MLCK) catalyzes the phosphorylation reaction, an event that may modulate myosin motor behavior. The influence of this molecular mechanism on the contractile performance of fatigued fast twitch skeletal muscle remains unclear, however. PURPOSE: To determine the effect of skeletal muscle MLCK gene ablation on the fatigability of force and velocity in mouse extensor digitorum longus (EDL) muscles. METHODS: Muscles from wildtype (WT) and skeletal MLCK knockout (KO) mice (n=10) were studied in vitro at 25° C during a fatigue protocol consisting of 5 minutes of repetitive high frequency stimulation (150 Hz for 1000 ms every 5 sec). Isometric twitch (Pt) and tetanic (Po) forces and unloaded shortening velocity (Vo) were assessed throughout the fatigue protocol. RESULTS: Pt was increased to a greater extent in WT than in KO muscles (36.8 ± 5.9% vs. 14.9 ± 2.7%, p < 0.05) during the initial stages of stimulation when Po was depressed less than 40% in both muscle types; thereafter, Pt declined to similar levels in both WT and KO muscles (to ∼ 35% of initial levels). On the other hand, values for Vo were greater for WT than for KO muscles (10.3 ± 0.90 vs. 8.4 ± 0.36 fiber lengths/s) during the final stages of stimulation, when Po was reduced ∼ 90% in both muscle types. CONCLUSION: These results suggest that MLCK gene ablation selectively impairs low frequency force during moderate fatigue and impairs unloaded shortening velocity during severe fatigue of fast twitch skeletal muscle. Supported by NSERC.

Orthovanadate Slows Kinetics Of The Acto-Myosin Interaction In Skinned Muscle Fibers By Competition Between Myosin-ADP-Pi and Myosin-ADP-Vi Cross-Bridges For Actin Sites

Orthovanadate (Vi) is a phosphate analog that has been used to study the relationship between biochemical reactions and structural and mechanical transitions in the cross-bridge cycle, with great success. In solution, Vi binds to myosin with high affinity, forming a stable myosin-ADP-Vi complex. In permeabilized muscle fibers, Vi binds to an actin-myosin-ADP state during cross-bridge cycling, and suppresses isometric tension, isometric stiffness and unloaded shortening velocity. We investigated the effects of Vi on the mechanics and kinetics of the actin-myosin interaction in skinned fibres from rabbit psoas (sarcomere length 2.4 μm, temperature 12 °C). After correction for myofilament compliance, the observed changes in stiffness indicate that the reduction in T0 isdue to a proportional reduction in the number of myosin cross-bridges attached to actin (Caremani et al., Biophys J 480a/2289-Pos, 2007). The effect of [Pi] (range 0-15 mM added [Pi]) on T0 and the rate constant of force development following a period of unloaded shortening (kD) in the presence of Vi (0.1 mM) suggest that Vi acts as a competitive inhibitor of Pi for the myosin-ADP state (Caremani et al., Biophys J 128a/621-Pos, 2008). Simulations show that the effects of Vi on force, stiffness and rate of force redevelopment, as well as on the actomyosin ATPase (Wilson et al, Biophys J 68:216-226,1995) and velocity of unloaded shortening(Chase et al, J Physiol 460:231-246, 1993), can be explained with a straightforward modification of the kinetic scheme of Dantzig et al, J Physiol 451:247-278, 1992 to include myosin-ADP-Vi heads competing with myosin-ADP- Pi heads for actin sites. Supported by NIH (R01 AR049033.03) and MiUR (Italy).

The Molecular Effects of Skeletal Muscle Fatigue on Myosin Mechanics and Kinetics

Skeletal muscle, during periods of exertion, experiences several different fatigue based changes in contraction including reductions in force, velocity, power output, and energy usage. The physiological bases of fatigue induced changes in contractility stem from many different factors including changes in neuronal activity, excitation-contraction coupling, and actomyosin contraction. The direct changes in the actomyosin contractile complex have previously been shown to be the result of alterations in the levels of metabolites, oxidative damage, and an increase in myosin regulatory light chain phosphorylation. Here, we measured the direct molecular effects of fatigue-like conditions on actomyosin velocity and force generation using the in vitro motility assay. We examined how changes in ATP, ADP, Pi, and pH affect the ability of the myosin to translocate actin. We show that fatigue-induced increases in metabolites decrease unloaded shortening velocity, with ADP and pH contributing the most to the observed changes in velocity. We also examined whether myosin regulatory light chain phosphorylation alters the sensitivity of the myosin to fatigue-like conditions. We show that under fatigue-like conditions, phosphorylation of the myosin regulatory light chian enhances force production and reduces actin sliding velocity, similar to the effects of phosphorylation under fatigue-like conditions observed in both muscle fiber and in vitro motility studies. Furthermore, we found that force production by dephosphorylated myosin is very sensitive to fatigue-like conditions whereas force production by phosphorylated myosin is rather fatigue insensitive. These results suggest that phosphorylation of the myosin regulatory light chain in skeletal muscle may serve as a protective mechanism against fatigue. Supported by NIH-HL077280, AHA-0435434T (J.R.M.), AHA-0815704D (to M.J.G.), and NIH-HL071778 (D.S-C.).