Electron paramagnetic resonance reveals age-related myosin structural changes in rat skeletal muscle fibers

We tested the hypothesisthat low specific tension (force/cross-sectional area) in skeletalmuscle from aged animals results from structural changes in myosin thatoccur with aging. Permeabilized semimembranosus fibers from young adultand aged rats were spin labeled site specifically at myosin SH1(Cys-707). Electron paramagnetic resonance (EPR) was then used toresolve and quantify the structural states of the myosin head todetermine the fraction of myosin heads in the strong-binding (forcegenerating) structural state during maximal isometric contraction.Fibers from aged rats generated 27 ± 0.8% less specific tensionthan fibers from younger rats (P < 0.001). EPRspectral analyses showed that, during contraction, 31.6 ± 2.1%of myosin heads were in the strong-binding structural state in fibersfrom young adult animals but only 22.1 ± 1.3% of myosin heads infibers from aged animals were in that state (P = 0.004). Biochemical assays indicated that the age-related change inmyosin structure could be due to protein oxidation, as indicated by adecrease in the number of free cysteine residues. We conclude thatmyosin structural changes can provide a molecular explanation forage-related decline in skeletal muscle force generation.

     

Muscle activity and aging affect myosin structural distribution and force generation in rat fibers.

The purpose of this study was to determine whether increased muscle activity could reverse myosin structural alterations that occur in aged rat muscle and whether those alterations could be induced in young rat muscle by decreased activity. Semimembranosus muscle activity was increased by electrical stimulation (200-ms trains, 154 Hz, 5 V) through a nerve cuff on the tibial branch of the ischiatic nerve. The protocol consisted of 5 sets of 6-10 maximal isometric contractions performed twice per week for 4 or 8-10 wk. Decreased muscle activity was induced by denervation of the semimembranosus muscle for 2 or 4 wk. Semimembranosus fibers were then studied for Ca(2+)-activated force generation. Fibers were also spin labeled on the myosin catalytic domain and studied using electron paramagnetic resonance (EPR) spectroscopy to assess myosin structural distribution. Increased muscle activity for 4 and 8-10 wk in approximately 32-mo-old rats resulted in -16 and +4% changes in specific tension, respectively (P < 0.01). EPR spectra showed that the fraction of myosin heads in the strong-binding structural state during contraction was reduced at 4 wk (0.241 +/- 0.020 vs. 0.269 +/- 0.018, P = 0.046) but returned to normal by 8-10 wk (P = 0.67). Decreased muscle activity for 2 and 4 wk in approximately 9-mo-old rats resulted in 23 and 34% reductions, respectively, in specific tension; EPR spectra showed 16 and 35% decreases in strong-binding myosin (P < 0.01). These data support the hypothesis that changes in muscle activity affect muscle strength, at least in part through alterations in myosin structure and function.     

Unilateral lower limb suspension does not mimic bed rest or spaceflight effects on human muscle fiber function.

We used Ca2+-activated skinned muscle fibers to test the hypothesis that unilateral lower leg suspension (ULLS) alters cross-bridge mechanisms of muscle contraction. Soleus and gastrocnemius biopsies were obtained from eight subjects before ULLS, immediately after 12 days of ULLS (post-0 h), and after 6 h of reambulation (post-6 h). Post-0 h soleus fibers expressing type I myosin heavy chain (MHC) showed significant reductions in diameter, absolute and specific peak Ca2+-activated force, unloaded shortening velocity, and absolute and normalized peak power. Fibers obtained from the gastrocnemius were less affected by ULLS, particularly fibers expressing fast MHC isoforms. Post-6 h soleus fibers produced less absolute and specific peak force than did post-0 h fibers, suggesting that reambulation after ULLS induced cell damage. Like bed rest and spaceflight, ULLS primarily affects soleus over gastrocnemius fibers. However, in contrast to these other models, slow soleus fibers obtained after ULLS showed a decrease in unloaded shortening velocity and a greater reduction in specific force.     

Force generation,but not myosin ATPase activity,declines with age in rat muscle fibers

We tested the hypothesis thatage-associated decline in muscle function is related to a change inmyosin ATPase activity. Single, glycerinated semimembranosus fibersfrom young (8-12 mo) and aged (32-37 mo) Fischer 344 × Brown Norway male rats were analyzed simultaneously for force andmyosin ATPase activity over a range of Ca²⁺ concentrations.Maximal force generation was ~20% lower in fibers from aged animals(P = 0.02), but myosin ATPase activity was not different between fibers from young and aged rats: 686 ± 46 (n = 30) and 697 ± 46 µM/s (n = 33) (P = 0.89). The apparent rate constant for thedissociation of strong-binding myosin from actin was calculated to be~30% greater in fibers from aged animals (P = 0.03),indicating that the lower force produced by fibers from aged animals isdue to a greater flux of myosin heads from the strong-binding state tothe weak-binding state during contraction. This is in agreement withour previous electron paramagnetic resonance experiments that showed areduced fraction of myosin heads in the strong-binding state during amaximal isometric contraction in fibers from older rats.

     

Myosin subunits and contractile properties of single fibers from hypokinetic rat muscles

The effects of prolonged hypokinesia on the contractile properties and myosin isozymes of single fibers from the synergistic fast-twitch plantaris (PL) and slow-twitch soleus (SOL) skeletal muscles of adult rats were studied after 28 days of hindlimb suspension. There was a 31% increase in the mean maximal velocity of unloaded shortening (Vmax) among fibers from SOL with no change in the mean Vmax of fibers from PL after suspension. The myosin heavy and light chain (MHC and MLC) composition of bundles and the MHC composition of single fibers from control and suspended muscles were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase in the relative amount of fast-type MHC's in hypokinetic SOL and a smaller increase in the amount of fast-type MHC's in the PL. Relatively minor changes occurred in the MLC's during hypokinesia. As Vmax increased among individual fibers from control and suspended muscles, the relative amount of fast-type MHC's increased. The results demonstrate that the myosin isozyme composition of skeletal muscle, especially the heavy chains, is altered during hypokinesia, and this finding provides an explanation for changes in Vmax of rat single muscle fibers under the same conditions.     

Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers

To understand the molecular mechanism of oxidation-induced inhibition of muscle contractility, we have studied the effects of hydrogen peroxide on permeabilized rabbit psoas muscle fibers, focusing on changes in myosin purified from these fibers. Oxidation by 5 mM peroxide decreased fiber contractility (isometric force and shortening velocity) without significant changes in the enzymatic activity of myofibrils and isolated myosin. The inhibitory effects were reversed by treating fibers with dithiothreitol. Oxidation by 50 mM peroxide had a more pronounced and irreversible inhibitory effect on fiber contractility and also affected enzymatic activity of myofibrils, myosin, and actomyosin. Peroxide treatment also affected regulation of contractility, resulting in fiber activation in the absence of calcium. Electron paramagnetic resonance of spin-labeled myosin in muscle fibers showed that oxidation increased the fraction of myosin heads in the strong-binding structural state under relaxing conditions (low calcium) but had no effect under activating conditions (high calcium). This change in the distribution of structural states of myosin provides a plausible explanation for the observed changes in both contractile and regulatory functions. Mass spectroscopy analysis showed that 50 mM but not 5 mM peroxide induced oxidative modifications in both isoforms of the essential light chains and in the heavy chain of myosin subfragment 1 by targeting multiple methionine residues. We conclude that 1) inhibition of muscle fiber contractility via oxidation of myosin occurs at high but not low concentrations of peroxide and 2) the inhibitory effects of oxidation suggest a critical and previously unknown role of methionines in myosin function.     

Age-related changes in contractile properties of single skeletal fibers from the soleus muscle.

Peak absolute force, specific tension (peak absolute force per cross-sectional area), cross-sectional area, maximal unloaded shortening velocity (Vo; determined by the slack test), and myosin heavy chain (MHC) isoform compositions were determined in 124 single skeletal fibers from the soleus muscle of 12-, 24-, 30-, 36-, and 37-mo-old Fischer 344 Brown Norway F1 Hybrid rats. All fibers expressed the type I MHC isoform. The mean Vo remained unchanged from 12 to 24 mo but did decrease significantly from the 24- to 30-mo time period (from 1.71 +/- 0.13 to 0.85 +/- 0.09 fiber lengths/s). Fiber cross-sectional area remained constant until 36 mo of age, at which time there was a 20% decrease from the values at 12 mo of age (from 5,558 +/- 232 to 4,339 +/- 280 micrometer2). A significant decrease in peak absolute force of single fibers occurred between 12 and 24 mo of age (from 51 +/- 2 x 10(-5) to 35 +/- 2 x 10(-5) N) and then remained constant until 36 mo, when another 43% decrease occurred. Like peak absolute force, the specific tension decreased significantly between 12 and 24 mo by 20%, and another 32% decline was observed at 37 mo. Thus, by 24 mo, there was a dissociation between the loss of fiber cross-sectional area and force. The results suggest time-specific changes of the contractile properties with aging that are independent of each other. Underlying mechanisms responsible for the time-dependent and contractile property-specific changes are unknown. Age-related changes in the molecular dynamics of myosin may be the underlying mechanism for altered force production. The presence of more than one beta/slow MHC isoform may be the mechanism for the altered Vo with age.     

Aging, muscle fiber type, and contractile function in sprint-trained athletes.

Biopsy samples were taken from the vastus lateralis of 18- to 84-yr-old male sprinters (n = 91). Fiber-type distribution, cross-sectional area, and myosin heavy chain (MHC) isoform content were identified using ATPase histochemistry and SDS-PAGE. Specific tension and maximum shortening velocity (V(o)) were determined in 144 single skinned fibers from younger (18-33 yr, n = 8) and older (53-77 yr, n = 9) runners. Force-time characteristics of the knee extensors were determined by using isometric contraction. The cross-sectional area of type I fibers was unchanged with age, whereas that of type II fibers was reduced (P < 0.001). With age there was an increased MHC I (P < 0.01) and reduced MHC IIx isoform content (P < 0.05) but no differences in MHC IIa. Specific tension of type I and IIa MHC fibers did not differ between younger and older subjects. V(o) of fibers expressing type I MHC was lower (P < 0.05) in older than in younger subjects, but there was no difference in V(o) of type IIa MHC fibers. An aging-related decline of maximal isometric force (P < 0.001) and normalized rate of force development (P < 0.05) of knee extensors was observed. Normalized rate of force development was positively associated with MHC II (P < 0.05). The sprint-trained athletes experienced the typical aging-related reduction in the size of fast fibers, a shift toward a slower MHC isoform profile, and a lower V(o) of type I MHC fibers, which played a role in the decline in explosive force production. However, the muscle characteristics were preserved at a high level in the oldest runners, underlining the favorable impact of sprint exercise on aging muscle.     

Effect of hindlimb suspension on the functional properties of slow and fast soleus fibers from three strains of mice.

Cross-sectional area (CSA), peak Ca2+-activated force (Po), fiber specific force (Po/CSA), and unloaded shortening velocity (Vo) were measured in slow-twitch [containing type I myosin heavy chain (MHC)] and fast-twitch (containing type II MHC) chemically skinned soleus muscle fiber segments obtained from three strains of weight-bearing and 7-day hindlimb-suspended (HS) mice. HS reduced soleus slow MHC content (from approximately 50 to approximately 33%) in CBA/J and ICR strains without affecting slow MHC content in C57BL/6 mice ( approximately 20% of total MHC). Two-way ANOVA revealed HS-induced reductions in CSA, Po, and Po/CSA of slow and fast fibers from all strains. Fiber Vo was elevated post-HS, but not consistently across strains. No MHC x HS treatment interactions were observed for any variable for C57BL/6 and CBA/J mice, and the two significant interactions found for the ICR strain (CSA, Po) appeared related to inherent pre-HS differences in slow vs. fast fiber CSA. In the mouse HS models studied here, fiber atrophy and contractile dysfunction were partially dependent on animal strain and generally independent of fiber MHC isoform content.     

The mechanism of muscle contraction

Knowledge of the mechanism of contraction has been obtained from studies of the interaction of actin and myosin in solution, from an elucidation of the structure of muscle fibers, and from measurements of the mechanics and energetics of fiber contraction. Many of the states and the transition rates between them have been established for the hydrolysis of ATP by actin and myosin subfragments in solution. A major goal is to now understand how the kinetics of this interaction are altered when it occurs in the organized array of the myofibril. Early work on the structure of muscle suggested that changes in the orientation of myosin cross-bridges were responsible for the generation of force. More recently, fluorescent and paramagnetic probes attached to the cross-bridges have suggested that at least some domains of the cross-bridges do not change orientation during force generation. A number of properties of active cross-bridges have been defined by measurements of steady state contractions of fibers and by the transients which follow step changes in fiber length or tension. Taken together these studies have provided firm evidence that force is generated by a cyclic interaction in which a myosin cross-bridge attaches to actin, exerts force through a "powerstroke" of 12 nm, and is then released by the binding of ATP. The mechanism of this interaction at the molecular level remains unknown.     

Invited Review: plasticity and energetic demands of contraction in skeletal and cardiac muscle.

Numerous studies have explored the energetic properties of skeletal and cardiac muscle fibers. In this mini-review, we specifically explore the interactions between actin and myosin during cross-bridge cycling and provide a conceptual framework for the chemomechanical transduction that drives muscle fiber energetic demands. Because the myosin heavy chain (MHC) is the site of ATP hydrolysis and actin binding, we focus on the mechanical and energetic properties of different MHC isoforms. Based on the conceptual framework that is provided, we discuss possible sites where muscle remodeling may impact the energetic demands of contraction in skeletal and cardiac muscle.     

Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of isolated skeletal muscle fibers

This study examines the myosin isozyme heterogeneity (in terms of both alkali light chains and myosin heavy chains) among skeletal muscle fibers of the rabbit and correlates these isozyme differences with the differences in a contractile property, the velocity of unloaded shortening, of the fibers. The mean velocities of unloaded shortening (pCa 4.3; 12 degrees C) were as follows: psoas IIb fibers, 2.07 fiber lengths/s (n = 25); tibialis anterior (IIb) fibers, 1.63 fiber lengths/s (n = 18); vastus intermedius IIa fibers, 0.98 fiber lengths/s (n = 15); fibers (IIa) from chronically stimulated tibialis anterior, 0.86 fiber lengths/s (n = 16). Peptide maps of the myosins showed that the myosin heavy chains of the two groups of IIb fibers were indistinguishable from each other, but different from the heavy chains of the IIa fibers. However, the difference in maximal shortening velocity of the two groups of IIb fibers was correlated with a difference in the alkali light chain ratio deduced from the intensity ratio of myosin isoforms separated by gel electrophoresis under nondenaturing conditions. The vastus intermedius (IIa) and chronically stimulated tibialis anterior (IIa) fibers were indistinguishable in terms of either velocities of unloaded shortening or myosin isozyme contents. Soleus fibers contained only slow-twitch myosin. Thus, among fibers that contained a variety of myosin isozymes, differences in shortening velocities were correlated with the alkali light chain ratio, myosin heavy chain type, or a combination of both.     

Structural features of cross-bridges in isometrically contracting skeletal muscle

Two-dimensional x-ray diffraction was used to investigate structural features of cross-bridges that generate force in isometrically contracting skeletal muscle. Diffraction patterns were recorded from arrays of single, chemically skinned rabbit psoas muscle fibers during isometric force generation, under relaxation, and in rigor. In isometric contraction, a rather prominent intensification of the actin layer lines at 5.9 and 5.1 nm and of the first actin layer line at 37 nm was found compared with those under relaxing conditions. Surprisingly, during isometric contraction, the intensity profile of the 5.9-nm actin layer line was shifted toward the meridian, but the resulting intensity profile was different from that observed in rigor. We particularly addressed the question whether the differences seen between rigor and active contraction might be due to a rigor-like configuration of both myosin heads in the absence of nucleotide (rigor), whereas during active contraction only one head of each myosin molecule is in a rigor-like configuration and the second head is weakly bound. To investigate this question, we created different mixtures of weak binding myosin heads and rigor-like actomyosin complexes by titrating MgATPgammaS at saturating [Ca2+] into arrays of single muscle fibers. The resulting diffraction patterns were different in several respects from patterns recorded under isometric contraction, particularly in the intensity distribution along the 5.9-nm actin layer line. This result indicates that cross-bridges present during isometric force generation are not simply a mixture of weakly bound and single-headed rigor-like complexes but are rather distinctly different from the rigor-like cross-bridge. Experiments with myosin-S1 and truncated S1 (motor domain) support the idea that for a force generating cross-bridge, disorder due to elastic distortion might involve a larger part of the myosin head than for a nucleotide free, rigor cross-bridge.     

Orientation of intermediate nucleotide states of indane dione spin-labeled myosin heads in muscle fibers.

We have used electron paramagnetic resonance to study the orientation of myosin heads in the presence of nucleotides and nucleotide analogs, to induce equilibrium states that mimic intermediates in the actomyosin ATPase cycle. We obtained electron paramagnetic resonance spectra of an indane dione spin label (InVSL) bound to Cys 707 (SH1) of the myosin head, in skinned rabbit psoas muscle fibers. This probe is rigidly immobilized on the catalytic domain of the head, and the principal axis of the probe is aligned nearly parallel to the fiber axis in rigor (no nucleotide), making it directly sensitive to axial rotation of the head. On ADP addition, all of the heads remained strongly bound to actin, but the spectral hyperfine splitting increased by 0.55 +/- 0.02 G, corresponding to a small but significant axial rotation of 7 degrees. Adenosine 5'-(adenylylim-idodiphosphate) (AMPPNP) or pyrophosphate reduced the actomyosin affinity and introduced a highly disordered population of heads similar to that observed in relaxation. For the remaining oriented population, pyrophosphate induced no significant change relative to rigor, but AMPPNP induced a slight but probably significant rotation (2.2 degrees +/- 1.6 degrees), in the direction opposite that induced by ADP. Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) relaxed the muscle fiber, completely dissociated the heads from actin, and produced disorder similar to that in relaxation by ATP. ATP gamma S plus Ca induced a weak-binding state with most of the actin-bound heads disordered. Vanadate had negligible effect in the presence of ADP, but in isometric contraction vanadate substantially reduced both force and the fraction of oriented heads. These results are consistent with a model in which myosin heads are disordered early in the power stroke (weak-binding states) and rigidly oriented later in the power stroke (strong-binding states), whereas transitions among the strong-binding states induce only slight changes in the axial orientation of the catalytic domain.     

Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle

The effects of isoproterenol on isometric force, unloaded shortening velocity, and myosin phosphorylation were examined in thin muscle bundles (0.1-0.2 mm diam) dissected from lamb tracheal smooth muscle. Methacholine (10(-6) M) induced rapid increases in isometric force and in phosphorylation of the 20,000-Da myosin light chain. Myosin phosphorylation remained elevated during steady-state maintenance of isometric force. The shortening velocity peaked at 15 s after stimulation with methacholine and then declined to approximately 45% of the maximal value by 3 min. Isoproterenol pretreatment inhibited methacholine-stimulated myosin light chain phosphorylation, shortening velocity, and force during the early stages of force generation. However, the inhibitory effect of isoproterenol on force and myosin phosphorylation is proportionally greater than that on shortening velocity. Isoproterenol pretreatment also caused a rightward non-parallel shift in the methacholine dose-response curves for both isometric tension and myosin light chain phosphorylation. These data demonstrate that isoproterenol attenuates the contractile properties of airway smooth muscles by affecting the rate and extent of myosin light chain phosphorylation, perhaps through a mechanism that involves the synergistic interaction of myosin light chain kinase phosphorylation and Ca2+ metabolism.     

Myosin heavy chain composition of single cells from avian slow skeletal muscle is strongly correlated with velocity of shortening during development

We have determined the myosin heavy chain (MHC) composition (using a sensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system) and the maximal velocity of shortening (Vmax) of single cells from neonatal and adult chicken anterior latissimus dorsi (ALD) muscles. In addition, the MHC, myosin light chain, and regulatory protein (i.e., troponin and tropomyosin subunits) compositions of bundles of ALD fibers were determined at late embryonic, neonatal, and adult ages. At young ages, there are two MHCs in ALD muscle, SM1 and SM2, with SM1 decreasing in relative amount with increasing age, as shown previously by others. The mean Vmax of single fibers also decreases from neonatal to adult ages. A strong quantitative correlation is demonstrated between the specific MHC composition and Vmax among individual cells of the ALD muscle at several ages. Since virtually no changes occur in the regulatory protein and myosin light chain compositions of the ALD muscle between late embryonic and adult ages, it appears that the MHC composition of an individual cell in this muscle is the primary determinant of the maximal shortening velocity. These results are the first to illustrate the functional significance of the developmental transition in myosin heavy chain composition of an avian slow skeletal muscle, consistent with our previous findings on mammalian muscle.     

Structural changes in isometrically contracting insect flight muscle trapped following a mechanical perturbation

The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.5% changes in length (either a stretch or a release) within 2 ms to isometrically contracting insect flight muscle (IFM) fibers followed after 5-6 ms by rapid freezing against a liquid helium cooled copper mirror. ET of freeze-substituted fibers, embedded and thin-sectioned, provides 3-D cross-bridge images, sorted by multivariate data analysis into ~40 classes, distinct in average structure, population size and lattice distribution. Individual actin subunits are resolved facilitating quasi-atomic modeling of each class average to determine its binding strength (weak or strong) to actin. ~98% of strong-binding acto-myosin attachments present after a length perturbation are confined to "target zones" of only two actin subunits located exactly midway between successive troponin complexes along each long-pitch helical repeat of actin. Significant changes in the types, distribution and structure of actin-myosin attachments occurred in a manner consistent with the mechanical transients. Most dramatic is near disappearance, after either length perturbation, of a class of weak-binding cross-bridges, attached within the target zone, that are highly likely to be precursors of strong-binding cross-bridges. These weak-binding cross-bridges were originally observed in isometrically contracting IFM. Their disappearance following a quick stretch or release can be explained by a recent kinetic model for muscle contraction, as behaviour consistent with their identification as precursors of strong-binding cross-bridges. The results provide a detailed model for contraction in IFM that may be applicable to contraction in other types of muscle.     

Effect of denervation on ATP consumption rate of diaphragm muscle fibers.

Denervation (DNV) of rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) content in fibers expressing MHC(2X) isoform but not in fibers expressing MHC(slow) and MHC(2A). Since MHC is the site of ATP hydrolysis during muscle contraction, we hypothesized that ATP consumption rate during maximum isometric activation (ATP(iso)) is reduced following unilateral DIAm DNV and that this effect is most pronounced in fibers expressing MHC(2X). In single-type-identified, permeabilized DIAm fibers, ATP(iso) was measured using NADH-linked fluorometry. The maximum velocity of the actomyosin ATPase reaction (V(max) ATPase) was determined using quantitative histochemistry. The effect of DNV on maximum unloaded shortening velocity (V(o)) and cross-bridge cycling rate [estimated from the rate constant for force redevelopment (k(TR)) following quick release and restretch] was also examined. Two weeks after DNV, ATP(iso) was significantly reduced in fibers expressing MHC(2X), but unaffected in fibers expressing MHC(slow) and MHC(2A). This effect of DNV on fibers expressing MHC(2X) persisted even after normalization for DNV-induced reduction in MHC content. With DNV, V(o) and k(TR) were slowed in fibers expressing MHC(2X), consistent with the effect on ATP(iso). The difference between V(max) ATPase and ATP(iso) reflects reserve capacity for ATP consumption, which was reduced across all fibers following DNV; however, this effect was most pronounced in fibers expressing MHC(2X). DNV-induced reductions in ATP(iso) and V(max) ATPase of fibers expressing MHC(2X) reflect the underlying decrease in MHC content, while reduction in ATP(iso) also reflects a slowing of cross-bridge cycling rate.     

Myosin essential light chain in health and disease

The essential light chain of myosin (ELC) is known to be important for structural stability of the alpha-helical lever arm domain of the myosin head, but its function in striated muscle contraction is poorly understood. Two ELC isoforms are expressed in fast skeletal muscle, a long isoform and its NH(2)-terminal approximately 40 amino acid shorter counterpart, whereas only the long ELC is observed in the heart. Biochemical and structural studies revealed that the NH(2)-terminus of the long ELC can make direct contacts with actin, but the effects of the ELC on the affinity of myosin for actin, ATPase, force, and the kinetics of force generating myosin cross-bridges are inconclusive. Myosin containing the long ELC has been shown to have slower cross-bridge kinetics than myosin with the short isoform. A difference was also reported among myosins with long isoforms. Increased shortening velocity was observed in atrial compared with ventricular muscle fibers. The common findings suggest that ELC provides the fine tuning of the myosin motor function, which is regulated in an isoform and tissue-dependent manner. The functional importance of the ELC is further implicated by the discovery of ELC mutations associated with Familial Hypertrophic Cardiomyopathy. The pathological phenotypes vary in severity, but more notably, almost all ELC mutations result in sudden cardiac death at a young age. This review summarizes the functional roles of striated muscle ELC in normal healthy muscle and in disease. Transgenic animal models and phenotypic characterization of ELC-mediated remodeling of the heart are also discussed.     

Reptilian skeletal muscle: contractile properties of identified, single fast-twitch and slow fibers from the lizard Dipsosaurus dorsalis

Contractile properties and innervation patterns were determined in identified single fibers from the iliofibularis muscle of the desert iguana, Dipsosaurus dorsalis. Single fibers from both the red and white regions of the iliofibularis muscle were dissected along their length under oil and a portion was mounted on transducers for determination of maximum isometric tension (Po) and unloaded shortening velocity (Vmax) using the slack test method. Fibers were chemically skinned and activated by high Ca++. The remaining portion of the muscle fiber was mounted on a glass slide and histochemically treated to demonstrate myosin ATPase activity. Fibers studied functionally could therefore be classified as fast or slow according to their myosin ATPase activity, and they could also be classified metabolically according to the region of the muscle from which they were dissected. Fast-twitch glycolytic (FG) fibers from the white region and fast-twitch oxidative, glycolytic (FOG) and slow fibers from the red region had shortening velocities at 25 degrees C of 7.5, 4.4, and 1.5 l X s-1, respectively. Po did not differ in the three fiber types, averaging 279 kN X m-2. In a second experiment, 10 microns sections were examined every 30 microns through the proximal-most 7.5 mm of the iliofibularis muscle for motor endplates. Sections were stained to demonstrate regions of acetylcholinesterase activity. Fibers with visible endplates were classified in serial sections by histochemical treatment for myosin ATPase and succinic dehydrogenase. All slow fibers examined (n = 22) exhibited multiple endplates, averaging one every 725 microns.(ABSTRACT TRUNCATED AT 250 WORDS)