Skeletal Muscle Myosin Research Articles

Feed restriction delays developmental fast skeletal muscle myosin heavy chain isoforms in turkey poults selected for differential growth

Genetic selection has been very successful at significantly increasing BW and breast muscle proportion in commercial broiler and turkey strains. The mechanisms of breast muscle growth in poultry and the interactive effects of nutritional status and selection are not fully understood. The hypothesis underlying the current study is that feed restriction, simply as a vehicle for controlling early growth, would delay the temporal expression pattern of neonatal (nMyHC) and adult (aMyHC) fast skeletal muscle myosin heavy chain (MyHC) isoforms in the pectoralis major muscle of turkey poults. The poultry growth model used to evaluate this hypothesis consisted of a randombred control turkey line (RBC2) that represents commercial turkeys of the 1960s and a line developed from the RBC2 by selection for BW at 16 wk of age (F line). The F line has significantly heavier breast muscles than the RBC2 concomitant with increased BW, but the proportion of breast muscle relative to BW is similar. A quantitative indirect ELISA using fast skeletal MyHC isoform specific monoclonal antibodies revealed no significant line differences in the temporal expression of posthatch fast skeletal muscle MyHC in ad libitum fed poults. Feed restriction, however, altered the temporal expression patterns of nMyHC and aMyHC in both F line and RBC2 poults compared with the poults fed ad libitum.

Erratum to: Identification of functional differences between recombinant human α and β cardiac myosin motors.

The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding.

Temperature dependent measurements reveal similarities between muscle and non-muscle myosin motility

We examined the temperature dependence of muscle and non-muscle myosin (heavy meromyosin, HMM) with in vitro motility and actin-activated ATPase assays. Our results indicate that myosin V (MV) has a temperature dependence that is similar in both ATPase and motility assays. We demonstrate that skeletal muscle myosin (SK), smooth muscle myosin (SM), and non-muscle myosin IIA (NM) have different temperature dependence in ATPase compared to in vitro motility assays. In the class II myosins we examined (SK, SM, and NM) the rate-limiting step in ATPase assays is thought to be attachment to actin or phosphate release, while for in vitro motility assays it is controversial. In MV the rate-limiting step for both in vitro motility and ATPase assays is known to be ADP release. Consequently, in MV the temperature dependence of the ADP release rate constant is similar to the temperature dependence of in vitro motility. Interestingly, the temperature dependence of the ADP release rate constant of SM and NM was shifted toward the in vitro motility temperature dependence. Our results suggest that the rate-limiting step in SK, SM, and NM may shift from attachment-limited in solution to detachment limited in the in vitro motility assay. Internal strain within the myosin molecule or by neighboring myosin motors may slow ADP release which becomes rate-limiting in the in vitro motility assay. Within this small subset of myosins examined, the in vitro sliding velocity correlates reasonably well with actin-activated ATPase activity, which was suggested by the original study by Barany (J Gen Physiol 50:197-218, 1967).

Abstract 366: Engineered Human Cardiac Tissue from Skeletal Muscle Derived Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes

We have previously shown that rat skeletal muscle derived stem cells differentiate into an immature cardiomyocyte (CM) phenotype within a 3-dimensional collagen gel engineered cardiac tissue (ECT). Here, we investigated whether human skeletal muscle derived progenitor cells (skMDCs) can differentiate into a CM phenotype within ECT similar to rat skeletal muscle stem cells and compared the human skMDC-ECT properties with ECT from human induced pluripotent stem cell (iPSc) derived CMs. SkMDCs differentiated into a cardiac muscle phenotype within ECT and exhibited spontaneous beating activity as early as culture day 4 and maintained their activity for more than 2 weeks. SkMDC-ECTs stained positive for cardiac specific troponin-T and troponin-I, and were co-localized with fast skeletal muscle myosin heavy chain (sk-fMHC) with a striated muscle pattern similar to fetal myocardium. The iPS-CM-ECTs maintained spontaneous beating activity for more than 2 weeks from ECT construction. iPS-CM stained positive for both cardiac troponin-T and troponin-I, and were also co-localized with sk-fMHC while the striated expression pattern of sk-fMHC was lost similar to post-natal immature myocardium. Connexin-43 protein was expressed in both engineered tissue types, and the expression pattern was similar to immature myocardium. The skMDC-ECT significantly upregulated expression of cardiac-specific genes compared to conventional 2D culture. SkMDC-ECT displayed cardiac muscle like intracellular calcium ion transients. The contractile force measurements demonstrated functional properties of fetal type myocardium in both ECTs. Our results suggest that engineered human cardiac tissue from skeletal muscle progenitor cells mimics developing fetal myocardium while the engineered cardiac tissue from inducible pluripotent stem cell-derived cardiomyocytes mimics post-natal immature myocardium.

Mechanical and kinetic properties of β-cardiac/slow skeletal muscle myosin

We aimed to establish reference parameters to identify functional effects of familial hypertrophic cardiomyopathy-related point mutations in the β-cardiac/slow skeletal muscle myosin heavy chain (β-cardiac/MyHC-1). We determined mechanical and kinetic parameters of the β-cardiac/MyHC-1 using human soleus muscle fibers that express the same myosin heavy chain (MyHC-1) as ventricular myocardium (β-cardiac). The observed parameters are compared to previously reported data for rabbit psoas muscle fibers. We found all of the examined kinetic parameters to be slower in soleus fibers than in rabbit psoas muscle. Somewhat surprisingly, however, we also found that the stiffness of the β-cardiac/MyHC-1 head domain is more than 3-fold lower than the stiffness of the fast isoform of psoas fibers. Furthermore, and different from rabbit psoas muscle, in human soleus fibers both the occupancy of force-generating cross-bridge states as well as the elastic extension of force-generating heads increase with temperature. Thus, a myosin head in the force generating states makes an increasing contribution to force with temperature. We support some of our fiber data by data from in vitro motility and optical trapping assays. Initial findings with FHC-related point mutations in the converter imply that the differences in stiffness of the head domain between the slow and fast isoform may well be due to particular differences in the amino acid sequence of the converter. We show that the slower kinetics may be linked to a larger flexibility of the β-cardiac/MyHC-1 isoform compared to fast MyHC isoforms.

Allosteric communication in Dictyostelium myosin II

Myosin's affinities for nucleotides and actin are reciprocal. Actin-binding substantially reduces the affinity of ATP for myosin, but the effect of actin on myosin's ADP affinity is quite variable among myosin isoforms, serving as the principal mechanism for tuning the actomyosin system to specific physiological purposes. To understand the structural basis of this variable relationship between actin and ADP binding, we studied several constructs of the catalytic domain of Dictyostelium myosin II, varying their length (from the N-terminal origin) and cysteine content. The constructs varied considerably in their actin-activated ATPase activity and in the effect of actin on ADP affinity. Actin had no significant effect on ADP affinity for a single-cysteine catalytic domain construct, a double-cysteine construct partially restored the actin-dependence of ADP binding, and restoration of all native Cys restored it further, but full restoration of function (similar to that of skeletal muscle myosin II) was obtained only by adding all native Cys and an artificial lever arm extension. Pyrene-actin fluorescence confirmed these effects on ADP binding to actomyosin. We conclude that myosin's Cys content and lever arm both allosterically modulate the reciprocal affinities of myosin for ADP and actin, a key determinant of the biological functions of myosin isoforms.

Transgenic Expression and Purification of Myosin Isoforms Using the Drosophila melanogaster Indirect Flight Muscle System

We report a novel method for the generation of extremely pure transgenic myosin to carry out crystallization screening for x‐ray crystallography. We are optimizing a novel method for the use of genetically engineered Drosophila melanogaster to express histidine‐tagged recombinant muscle myosin isoforms. This method relies on several facts. First, that the Drosophila genome contains only one gene encoding all skeletal muscle myosin heavy chain isoforms. Second, that wild‐type or mutagenized versions of the gene can then be inserted into a specific location in the Drosophila genome. Third, that the Mhc10 fly line has endogenous myosin knocked out in the indirect flight muscles. Fourth, that the Actin88F promoter facilitates high‐level expression of accessible myosin in the thoracic indirect flight muscles. Finally, that cost of fruit fly colony propagation is relatively low. Using these advantages we devised a method for expressing and purifying two isoforms of recombinant histidine‐tagged myosin from an engineered fly strain. The myosin shows ATPase activity and is of sufficient purity and homogeneity for crystallization. This technique may prove useful for the expression and isolation of mutant myosins linked with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization. Supported by NIH R01 GM032443 to SIB.

Nitric oxide synthase inhibition prevents activity‐induced calcineurin–NFATc1 signalling and fast‐to‐slow skeletal muscle fibre type conversions

The calcineurin–NFAT (nuclear factor of activated T-cells) signalling pathway is involved in the regulation of activity-dependent skeletal muscle myosin heavy chain (MHC) isoform type expression. Emerging evidence indicates that nitric oxide (NO) may play a critical role in this regulatory pathway. Thus, the purpose of this study was to investigate the role of NO in activity-induced calcineurin–NFATc1 signalling leading to skeletal muscle faster-to-slower fibre type transformations in vivo. Endogenous NO production was blocked by administering L-NAME (0.75 mg ml(−1)) in drinking water throughout 0, 1, 2, 5 or 10 days of chronic low-frequency stimulation (CLFS; 10 Hz, 12 h day(−1)) of rat fast-twitch muscles (L+Stim; n = 30) and outcomes were compared with control rats receiving only CLFS (Stim; n = 30). Western blot and immunofluorescence analyses revealed that CLFS induced an increase in NFATc1 dephosphorylation and nuclear localisation, sustained by glycogen synthase kinase (GSK)-3β phosphorylation in Stim, which were all abolished in L+Stim. Moreover, real-time RT-PCR revealed that CLFS induced an increased expression of MHC-I, -IIa and -IId(x) mRNAs in Stim that was abolished in L+Stim. SDS-PAGE and immunohistochemical analyses revealed that CLFS induced faster-to-slower MHC protein and fibre type transformations, respectively, within the fast fibre population of both Stim and L+Stim groups. The final fast type IIA to slow type I transformation, however, was prevented in L+Stim. It is concluded that NO regulates activity-induced MHC-based faster-to-slower fibre type transformations at the transcriptional level via inhibitory GSK-3β-induced facilitation of calcineurin–NFATc1 nuclear accumulation in vivo, whereas transformations within the fast fibre population may also involve translational control mechanisms independent of NO signalling.

Myosin Isoform-Specificity of Contractile Elements Formed in Reconstituted Actomyosin Bundles

Forces generated by the contraction of actomyosin bundles underlie diverse physiological processes including muscle contraction, cell migration and cytokinesis. While actomyosin force transmission is well understood in the context of organized sarcomeres found in striated muscle, very little is known about the nature of actomyosin force transmission in non-sarcomeric bundles such as those found in smooth and non-muscle cells. We recently showed that contractile elements spontaneously arise during the contraction of disordered actomyosin bundles reconstituted solely from actin filaments and smooth muscle myosin. In turn, the contraction of disordered actomyosin bundles arises from a several contractile elements linked in series resulting in a bundle contraction velocity linearly proportional to bundle length and a length-independent contractile force. The physical properties of the contractile elements, including the rate of contraction, maximum contractile strain and the maximum force generated, depended on the relative quantities of actin and myosin. Here, we demonstrate that reconstituted actomyosin bundles formed with skeletal muscle myosin form qualitatively similar contractile elements with similar dependencies on myosin concentration. However, the physical properties of the contractile units formed with skeletal muscle myosin dramatically differ from those formed with smooth muscle myosin, in a fashion consistent with differences in the myosin mechanochemistry and thick filament geometry of smooth and skeletal muscle myosin. Contractile elements formed by skeletal muscle myosin contract significantly faster, generate more force and can shorten to greater extents than those formed with smooth muscle myosin. Our results demonstrate that both smooth and skeletal muscle myosin isoforms can facilitate the spontaneous assembly of contractile elements within disordered actomyosin bundles and provide insight into the mechanisms underlying contraction in non-sarcomeric actomyosin bundles.

A Method for the Transgenic Expression and Purification of Skeletal Muscle Myosin II Isoforms using Drosophila Melanogaster

There are many biologically interesting myosin isoforms that are expressed at levels too low for direct purification from in vivo sources. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We are optimizing a novel method for the use of Drosophila melanogaster that has been genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method relies on several facts. First, that the Drosophila genome contains only one gene encoding all skeletal muscle myosin isoforms. Second, that this DNA can be manipulated with molecular biology techniques and then be inserted into a specific location in the Drosophila genome. Third, that the Mhc10 fly line has endogenous myosin knocked out in the indirect flight muscles. Fourth, that the Actin88F promoter facilitates high-level expression of accessible myosin in the thoracic indirect flight muscles. Finally, that cost of fruit fly colony propagation is relatively low. Employing these advantages, we have created a system for the production of extremely pure skeletal muscle myosin. We demonstrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. This myosin shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. Our technique may prove useful for the expression and isolation of mutant myosins linked with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.

Differential Impact of Temperature and Magnesium on Myosin V and Myosin II

We examined the impact of temperature and free magnesium concentration on monomeric FlAsH labeled myosin V (MV FlAsH), dimeric myosin V (MV HMM), and dimeric fast skeletal muscle myosin II (SK HMM) using ATPase and motility assays. Our results indicate that MV HMM and SK HMM both have a linear dependence on temperature that is similar in both ATPase and motility assays. However, MV FlAsH contains a different temperature dependence in ATPase and motility assays suggesting its short lever arm may impart a high strain dependence in the motility assay. MV HMM and MV FlAsH are inhibited by high concentrations of magnesium in both ATPase and motility assays. The rate-limiting step in myosin V is known to be ADP release, which we demonstrate correlates well with the magnesium and temperature dependence of ATPase and motility assays. Interestingly, SK HMM exhibits magnesium inhibition in ATPase assays, but only a slight decrease is observed in the motility assay. In SK HMM the rate-limiting step in ATPase assays is thought to be attachment to actin or phosphate release, while in motility assays it is controversial. Our results indicate that SK HMM is better described by an attachment limited model in the motility assay. Magnesium may reduce the duty ratio of SK HMM which alters ATPase activity but not velocity in the motility assay. Future experiments will determine if magnesium alters the actin binding and/or the product release steps in myosin V and skeletal muscle myosin. Myosin V contains a tyrosine (residue 439) in the switch II region, which is an alanine at the corresponding position in myosin II, suggesting this residue may play a key role in differentially altering magnesium coordination in the active site of myosins.

Instability of the S1/S2 Hinge is not Affected by the Myosin Filament

The S1/S2 hinge of myosin is thought to be unstable in some species of myosin such as myosin VI and certain scallop muscle myosin. To examine the stability of the S1/S2 hinge of rabbit skeletal muscle myosin, gravitational force spectrometry was used to measure the separation of myosin heads bound in rigor to actin immobilized on glass microspheres. The molecular lengths of this separation were measured at different levels of applied force up to the limit that the rigor bond would sustain of about 10 pN in these experiments. The molecular lengths were found to increase linearly with the applied force up to lengths exceeding 100 nm which requires a partial unraveling of the S2 coiled coil in the single myosin molecule. Free-fall force spectrometry on such single myosin molecules confirmed that the coiled coil could reversibly unravel and refold at forces less than 13 pN. In order to test whether thick filament formation would affect this instability under force, synthetic cofilaments of proteolytically isolated myosin rod and intact myosin were prepared at different ratios of myosin to rod up to ratios where it is expected that only one intact myosin would be present per myosin rod cofilament. The force-distance relationships for myosin in the cofilament were not substantially different from those of single myosin molecules. These data indicate the possibility that vertebrate skeletal muscle myosin may also utilize S1/S2 hinge unraveling to provide flexibility during crossbridge formation so that a productive binding orientation is achieved. The reversibility of the unraveling of the coiled coil might contribute to the mechanism of motility under some circumstances as has been proposed for non-muscle myosin VI.

Myosin-Induced Detachment Causes Differences Between Ensemble and Single Molecule Myosin Kinetics

Single molecule measurements of mechanochemistry have greatly increased our understanding of muscle contraction. However, since trillions of myosin molecules work together in muscle, extrapolation to in vivo function requires additional understanding of how motors behave in an ensemble. Early findings suggested that myosin behaves similarly at both the single molecule and ensemble levels; but more recent experiments suggest otherwise. Using a combination of simulation and theory, we show that the force-dependence of ADP release causes myosin-myosin interactions that make ensemble myosin behavior differ fundamentally from single myosin behavior.We use solution data to estimate the parameters of a simple 4-state kinetic model for actomyosin interaction. For smooth muscle myosin, we add the measured force-dependence of ADP release to this model. Simulations of the model successfully predict the results of four experiments: 1. single molecule measurements of step size and strong binding liftetime; 2. In vitro motility measurements of actin speed as a function of [ATP]; 3. In vitro motility measurements of actin speed at low myosin density as a function of actin filament length; and 4. laser trap measurements of velocity as a function of force for small myosin ensembles. For skeletal muscle myosin, we use a subset of these data to estimate the force-dependence of ADP release and successfully predict the remaining data. The model is therefore consistent with both single molecule and ensemble data.In the model, myosin binding to actin accelerates the detachment of previously bound myosin. This myosin-induced detachment causes strong binding lifetime to depend on the number of myosin molecules interacting with actin. Counter-intuitively, this result implies that even when actin speed is “detachment limited” (meaning speed equals myosin's step size times the ADP release rate), increasing the attachment rate can increase speed.

Phosphorylation of Cardiac Myosin Binding Protein-C Acts as On/Off Switch to Modulate cMyBP-C's Inhibition of Actomyosin Power Generation in the Load-Clamp Laser Trap Assay

Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains- C0-C10, binds myosin LMM via its C-terminus, while its N-terminus binds both myosin S2 and actin. These N-terminal interactions can be attenuated by phosphorylation of serines within the C1-C2 linker. In vivo, cMyBP-C exists in a range of phosphorylation states, which may alter its regulation of actomyosin. Therefore, N-terminal fragments (C0-C3) of cMyBP-C were bacterially expressed with one or more phosphorylatable serines (S273, S282, S302) mutated to either alanines to mimic dephosphorylation or aspartic acids as phosphomimetics. Using C0-C3 fragments that were effectively mono-, di- or tri-phosphorylated, we characterized how the extent of cMyBP-C phosphorylation modulated the the force:velocity relationship generated by a small ensemble (∼8 motors) of skeletal muscle myosin in the load-clamped laser trap assay. Mono-phosphorylated C0-C3 inhibited unloaded velocity by 70% as did unphosphorylated C0-C3, while di- and tri-phosphorylated constructs were indistinguishable and less inhibitory (30%). Maximal force was unaffected by the extent of phosphorylation. Thus, the un- and mono-phosphorylated C0-C3 constructs depressed the force-velocity relationship, which was well fitted by a modified Hill equation containing an assumed viscous load due to C0-C3 binding to actin (Weith et al., 2011). In addition, both un- and mono- phosphorylated C0-C3 reduced peak power by ∼40%, while neither di- nor tri-phosphorylated C0-C3 had any effect on peak power. cMyBP-C phosphorylation appears to modulate cMyBP-C's inhibition of actomyosin power generation in an “on/off” manner.

Metallomics investigations on potential binding partners of methylmercury in tuna fish muscle tissue using complementary mass spectrometric techniques

In this study, the binding behaviour of methylmercury (MeHg(+)) towards proteins is investigated. Free sulfhydryl groups in cysteine residues are known to be the most likely binding partners, due to the high affinity of mercury to sulphur. However, detailed knowledge about discrete binding sites in living organisms has been so far scarce. A metallomics approach using different methods like size-exclusion chromatography (SEC) and liquid chromatography (LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) as well as complementary mass spectrometric techniques (electrospray ionisation-tandem mass spectrometry, ESI-MS/MS) are combined to sequence and identify possible target proteins or peptides after enzymatic digestion. Potential targets for MeHg(+) in tuna fish muscle tissue are investigated using the certified reference material CRM464 as a model tissue. Different extraction procedures appropriate for the extraction of proteins are evaluated for their efficiency using isotope dilution analysis for the determination of total Hg in the extracts. Due to the high chemical stability of the mercury-sulphur bond, the bioconjugate can be quantitatively extracted with a combination of tris(hydroxymethyl)aminomethane (TRIS) and sodium dodecyl sulphate (SDS). Using different separation techniques such as SEC and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) it can be shown that major binding occurs to a high-molecular weight protein (M(w) > 200 kDa). A potential target protein, skeletal muscle myosin heavy chain, could be identified after tryptic digestion and capillary LC-ESI-MS/MS.

Formation of Myosin-Photochromic ATP Analogue-Phosphate Analogue Ternary Complexes that Transit Reversibly Among Different ATPase Transient States by Light Irradiation

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Previously, we have cross-linked reactive cysteine SH1 and SH2 of skeletal muscle myosin subfragment-1(S1) with the sulfhydryl-reactive bifunctional azobenzene derivative, azobenzene-dimaleimide (ABDM) and succeeded to induce lever arm swinging reversibly by photo-irradiation. The results suggested that it might be possible to control or drive motor proteins by light irradiation. In the present study, we have employed photochromic ATP analogues that change its structure reversibly by light irradiation in order to photo-regulate function of myosin. Phenylazobenzoyl-iminoethyl-Tri-Phosphate (PABITP) have been designed and synthesized. PABITP was hydrolyzed by skeletal muscle myosin subfragment-1 (S1) and induced dissociation of acto-myosin. Formations of S1-PABIDP-Pi analogues (AlF4-, BeFn) ternary complexes that may mimic different transient states in ATPase cycle were examined. The formations of the ternary complexes were monitored by competing with fluorescent ADP analogue Mant-ADP. Trans PABITP formed the ternary complexes with AlF4- and BeFn. Interestingly, formation of S1-PABIDP-BeFn was saturated at 50%. It is known that there are two species of BeFn, which have different number of fluorine. One of them forms M∗•ATP state and the other forms M∗∗•ADP•Pi state. As S1-ADP-AlF4- ternary complex mimic only M∗∗•ADP•Pi state. Therefore, trans PABIDP forms predominantly ternary complexes mimics M∗∗•ADP•Pi with BeFn and AlF4-. The photo-isomerization to cis PABIDP by UV irradiation decreased the stability of the ternary complexes. The results suggest that trans PABITP may mimic transient state of M∗∗ADP-Pi state and isomerization to cis PABITP induced transition to other state in ATPase cycle.

Myofibrillar myopathy caused by a mutation in the motor domain of mouse MyHC IIb

Ariel is a mouse mutant that suffers from skeletal muscle myofibrillar degeneration due to the rapid accumulation of large intracellular protein aggregates. This fulminant disease is caused by an ENU-induced recessive mutation resulting in an L342Q change within the motor domain of the skeletal muscle myosin protein MYH4 (MyHC IIb). Although normal at birth, homozygous mice develop hindlimb paralysis from Day 13, consistent with the timing of the switch from developmental to adult myosin isoforms in mice. The mutated myosin (MYH4(L342Q)) is an aggregate-prone protein. Notwithstanding the speed of the process, biochemical analysis of purified aggregates showed the presence of proteins typically found in human myofibrillar myopathies, suggesting that the genesis of ariel aggregates follows a pathogenic pathway shared with other conformational protein diseases of skeletal muscle. In contrast, heterozygous mice are overtly and histologically indistinguishable from control mice. MYH4(L342Q) is present in muscles from heterozygous mice at only 7% of the levels of the wild-type protein, resulting in a small but significant increase in force production in isolated single fibres and indicating that elimination of the mutant protein in heterozygotes prevents the pathological changes observed in homozygotes. Recapitulation of the L342Q change in the functional equivalent of mouse MYH4 in human muscles, MYH1, results in a more aggregate-prone protein.

Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system

Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.

Impact of static magnetic fields on human myoblast cell cultures

Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle α1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal muscle tissue engineering approaches.