Myofibril Stability Research Articles

Effect of ultrasound-assisted freezing combined with potassium alginate on the quality attributes and myofibril structure of large yellow croaker (Pseudosciaena crocea)

This research evaluated the effects of ultrasound-assisted freezing (UAF) at different power (160 W, 175 W, 190 W) combined with potassium alginate (PA, as the water-retaining agents) on the physicochemical quality, water-holding capacity (WHC), moisture migration and distribution, and myofibril structure of large yellow croaker. UAF-175 with PA as the freezing medium retained a better performance, such as color, texture, WHC, and water distribution. The pH and total volatile basic nitrogen (TVB-N) were the most desirable under the PA-UAF-175 treatment, as well as the flavor of the samples. In addition, it maintained the stability of myofibrils and reduced myofibril denaturation. Therefore, PA-UAF-175 treatment could be a possible freezing method as it maintained the quality properties and revealed better myofibril characteristics of large yellow croaker.

Effect of multi-frequency ultrasound assisted thawing on the quality of large yellow croaker (Larimichthys crocea)

The effects of mono-, dual- and tri-frequency ultrasound-assisted thawing (UAT) on the physicochemical quality, water-holding capacity, moisture migration and distribution and myofibrillary structure of frozen large yellow croaker (Larimichthys crocea) were detected. The results indicated that multifrequency UAT treatment significantly increased the thawing rate, maintained the stability of myofibrils and reduced the lipid oxidation. The multifrequency UAT samples had better water-holding capacity (higher water-holding capacity values, lower thawing loss and cooking loss) and physicochemical quality (higher hardness, springiness, resilience, chewiness and lower total volatile basic nitrogen (TVB-N) values, thiobarbituric acid reactive substances (TBARS) values), higher immobilized water content, and lower free water content. Therefore, the results provide a further understanding of the quality stability of frozen large yellow croaker treated by the multifrequency UAT.

Myofibril assembly and the roles of the ubiquitin proteasome system.

De novo assembly of myofibrils in vertebrate cross-striated muscles progresses in three distinct steps, first from a minisarcomeric alignment of several nonmuscle and muscle proteins in premyofibrils, followed by insertions of additional proteins and increased organization in nascent myofibrils, ending with mature contractile myofibrils. In a search for controls of the process of myofibril assembly, we discovered that the transition from nascent to mature myofibrils could be halted by inhibitors of three distinct functions of the ubiquitin proteasome system (UPS). First, inhibition of pathway to E3 Cullin ligases that ubiquitinate proteins led to an arrest of myofibrillogenesis at the nascent myofibril stage. Second, inhibition of p97 protein extractions of ubiquitinated proteins led to a similar arrest of myofibrillogenesis at the nascent myofibril stage. Third, inhibitors of proteolytic action by proteasomes also blocked nascent myofibrils from transitioning to mature myofibrils. In contrast, inhibitors of autophagy or lysosomes did not affect myofibrillogenesis. To probe for differences in the effects of UPS inhibitors during myofibrillogenesis, we analyzed by fluorescence recovery after photobleaching the exchange rates of two selected sarcomeric proteins (muscle myosin II heavy chains and light chains). In the presence of p97 and proteasomal inhibitors, the dynamics of each of these two myosin proteins decreased in the nascent myofibril stage, but were unaffected in the mature myofibril stage. The increased stability of myofibrils occurring in the transition from nascent to mature myofibril assembly indicates the importance of dynamics and selective destruction in the muscle myosin II proteins for the remodeling of nascent to mature myofibrils.

Reductions in ATPase activity, actin sliding velocity, and myofibril stability yield muscle dysfunction in Drosophila models of myosin-based Freeman-Sheldon syndrome.

Using Drosophila melanogaster, we created the first animal models for myosin-based Freeman–Sheldon syndrome (FSS), a dominant form of distal arthrogryposis defined by congenital facial and distal skeletal muscle contractures. Electron microscopy of homozygous mutant indirect flight muscles showed normal (Y583S) or altered (T178I, R672C) myofibril assembly followed by progressive disruption of the myofilament lattice. In contrast, all alleles permitted normal myofibril assembly in the heterozygous state but caused myofibrillar disruption during aging. The severity of myofibril defects in heterozygotes correlated with the level of flight impairment. Thus our Drosophila models mimic the human condition in that FSS mutations are dominant and display varied degrees of phenotypic severity. Molecular modeling indicates that the mutations disrupt communication between the nucleotide-binding site of myosin and its lever arm that drives force production. Each mutant myosin showed reduced in vitro actin sliding velocity, with the two more severe alleles significantly decreasing the catalytic efficiency of actin-activated ATP hydrolysis. The observed reductions in actin motility and catalytic efficiency may serve as the mechanistic basis of the progressive myofibrillar disarray observed in the Drosophila models as well as the prolonged contractile activity responsible for skeletal muscle contractures in FSS patients.

A Novel FLNC Frameshift and an OBSCN Variant in a Family with Distal Muscular Distrophy

Distal muscular dystrophies are a group of inherited primary muscle disorders showing progressive muscle atrophy and weakness preferentially in the hands and/or feet. We identified a novel frameshift mutation in the filamin C (FLNC c.5161delG) gene in two members of a French family affected by distal myopathy and in one healthy relative. Interestingly, the two affected members, but not the healthy relative, also carry a p.Arg4444Trpvariant in the gene coding for the giant protein obscurin(OBSCN). Both OBSCN and FLNC are Z-disk associated proteins. FLNC is an actin cross-linking protein, involved with Z-disk organization and muscle integrity. The FLNC c.5161delG mutation here described is one nucleotide away from a previously reported FLNC mutation (c.5160delC), identified in patients and in asymptomatic carriers of three families with distal muscular dystrophy, indicating a low penetrance of these FLNC frameshift mutations. Obscurin is a giant protein, which interacts with titin at both the M-band and the Z-disk and with muscle-specific ankyrins at the sarcoplasmic reticulum and the sarcolemma. The p.Arg4444Trpvariant we identified is localized within the OBSCN Ig59 that, together with Ig58, binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional studies indicated that the OBSCN p.Arg4444Trp variant results in a 10-fold decrease in binding to titin. Interestingly, in vitro and in vivo models show that loss of FLNC results in myofibril destabilization and that the decrease in the interaction between titin and obscurin is associated with cardiac and skeletal muscle diseases. On this line, we suggest that the combination of the OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation can cooperatively affect myofibril stability and increase the penetrance of muscular dystrophy.

A novel FLNC frameshift and an OBSCN variant in a family with distal muscular dystrophy.

A novel FLNC c.5161delG (p.Gly1722ValfsTer61) mutation was identified in two members of a French family affected by distal myopathy and in one healthy relative. This FLNC c.5161delG mutation is one nucleotide away from a previously reported FLNC mutation (c.5160delC) that was identified in patients and in asymptomatic carriers of three Bulgarian families with distal muscular dystrophy, indicating a low penetrance of the FLNC frameshift mutations. Given these similarities, we believe that the two FLNC mutations alone can be causative of distal myopathy without full penetrance. Moreover, comparative analysis of the clinical manifestations indicates that patients of the French family show an earlier onset and a complete segregation of the disease. As a possible explanation of this, the two French patients also carry a OBSCN c.13330C>T (p.Arg4444Trp) mutation. The p.Arg4444Trp variant is localized within the OBSCN Ig59 domain that, together with Ig58, binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional studies indicate that this OBSCN p.Arg4444Trp mutation decreases titin binding by ~15-fold. On this line, we suggest that the combination of the OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation, can cooperatively affect myofibril stability and increase the penetrance of muscular dystrophy in the French family.

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.

Pressure-induced changes of silver carp (Hypophthalmichthys molitrix) myofibrillar protein structure

Silver carp (Hypophthalmichthys molitrix) is one of the major species of freshwater fish in China. Modification of myofibrillar proteins induced by high pressure processing (HPP) has been investigated at pressures ranging from 200 to 500 MPa for 10 min at 20 °C. Influence of high pressure on conformational changes of myofibrils was studied by means of differential scanning calorimetry (DSC), surface hydrophobicity measurement, ultraviolet absorption spectra and second-derivative spectroscopy, intrinsic spectrofluorimetry, Raman spectroscopic analysis and circular dichroism (CD). DSC analyses revealed that the conformational stability of myofibrils is reduced to different extents when they are exposed to a pressure over 200 MPa. More sulfydryl groups as well as hydrophobic regions and amino acid residues which had ultraviolet absorbance had been found significantly after HPP (≥300 MPa). Changes in the Raman spectra were interpreted as the occurrence of secondary structural changes in pressure-induced myofibrillar proteins. CD analysis results demonstrated that α-helix was destroyed after processed at 400 MPa for 10 min. The results implicated that HPP can be used as a possible means of improving the functional properties for growing demand for healthier fish products.

Thin , a Trim32 ortholog, is essential for myofibril stability and is required for the integrity of the costamere in Drosophila

Myofibril stability is required for normal muscle function and maintenance. Mutations that disrupt myofibril stability result in individuals who develop progressive muscle wasting, or muscular dystrophy, and premature mortality. Here we present our investigations of the Drosophila l(2)thin [l(2)tn] mutant. The "thin" phenotype exhibits features of the human muscular disease phenotype in that tn mutant larvae show progressive muscular degeneration. Loss-of-function and rescue experiments determined that l(2)tn is allelic to the tn locus [previously annotated as both CG15105 and another b-box affiliate (abba)]. tn encodes a TRIM (tripartite motif) containing protein highly expressed in skeletal muscle and is orthologous to the human limb-girdle muscular dystrophy type 2H disease gene Trim32. Thin protein is localized at the Z-disk in muscle, but l(2)tn mutants showed no genetic interaction with mutants affecting the Z-line-associated protein muscle LIM protein 84B. l(2)tn, along with loss-of-function mutants generated for tn, showed no relative mislocalization of the Z-disk proteins α-Actinin and muscle LIM protein 84B. In contrast, tn mutants had significant disorganization of the costameric orthologs β-integrin, Spectrin, Talin, and Vinculin, and we present the initial description for the costamere, a key muscle stability complex, in Drosophila. Our studies demonstrate that myofibrils progressively unbundle in flies that lack Thin function through progressive costamere breakdown. Due to the high conservation of these structures in animals, we demonstrate a previously unknown role for TRIM32 proteins in myofibril stability.

Interaction Between the Relay Loop and the SH1-SH2 Helix Region in Drosophila Muscle Myosin is Essential for Normal Motor Function, Myofibril Stability and Muscle Contraction

Molecular modeling of Drosophila muscle myosin reveals that relay domain residue E499 interacts with SH1-SH2 residue R714 in a charge-dependent manner. To explore the significance of this interaction, we generated transgenic lines expressing myosin with a mutation in the relay loop domain (E499R) or the SH1-SH2 helix (R714E). Both mutations yield ∼75% reductions in CaATPase as well as basal or actin-activated MgATPase activity. Actin-sliding velocity of E499R is reduced by 65% and R714E shows no motility. Indirect flight muscles in late pupae of each mutant display disrupted myofibril assembly. Two-hour- and two-day-old adults have severely abnormal myofibril morphology and poor sarcomere organization, with no flight ability. Therefore, the putative interaction of the relay with SH1-SH2 is indispensable for normal motor function, myofibril stability and locomotion. We next constructed a putative compensatory mutant designed to restore function of the E499R or R714E mutant by generating a transgenic line that expresses both E499R and R714E. Interestingly, calcium, basal, and actin-stimulated ATPase values are restored to 65–70% of wild type and actin-sliding velocity is at 40%. The compensatory mutation suppresses the assembly defects in pupal muscle that are seen in both E499R and R714E and reduces disrupted myofibril morphology in two-hour old indirect flight muscles. However, the E499R-R714E mutant myosin was unable to maintain sarcomere organization in two-day-old flies or to restore flight ability. Overall, our results reveal that interaction of residues at the relay/SH1-SH2 helix interface is important for myosin and muscle function.

Mutating the Converter–Relay Interface of Drosophila Myosin Perturbs ATPase Activity, Actin Motility, Myofibril Stability and Flight Ability

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase ( V max) by ∼ 60% compared to wild-type myosin, but there is no change in apparent actin affinity ( K m). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by ∼ 15% or ∼ 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by ∼ 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional “cracking” of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle.

Converter Domain Residue R759 Interaction with Relay Loop Residue N509 in Drosophila Muscle Myosin is Critical for Motor Function, Myofibril Stability and Flight Ability

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of Drosophila melanogaster skeletal muscle myosin. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at the relay loop interaction site. The mutation depresses calcium, basal or actin-activated MgATPase values (Vmax) by ∼60% and actin sliding velocity ∼35% compared to wild-type myosin. Ultrastructure of two-day-old adult fibers shows cracking and frayed myofibrils with some disruption of the myofilament lattice which becomes more severe in one-week-old adults. Flight ability is reduced in two-day-old flies compared to controls and is absent in 1-week-old adults. Thus appropriate interaction between the relay loop and converter domain is essential for normal motor function, myofibril stability and locomotion. To examine the specificity of this interaction, we used a compensatory mutational approach to attempt to restore the function of the R759E mutant myosin. Our modeling indicates that relay loop residues N509 and D511 interact with converter domain residue R759. To verify our model, we generated two transgenic lines that express R759E and either the N509K or D511K mutations. Interestingly, calcium, basal, and actin stimulated ATPase values are restored to 70% and actin sliding velocity is restored to 90% in N509K/R759E but not in D511K/R759E. Structurally fibers from 2-day or one-week -old adults appear morphologically normal in N509K/R759E and their flight ability is like wild type. However, D511K/R759E myofibrils do not show any improvement compared to R759E and flight ability is worse than R759E. Overall, our results reveal the critical interaction between the converter domain with relay loop residues and their role in myosin motor function and myofibril assembly/stability.

An Integrative Analysis of the Muscle Myosin Motor Using Genetic and Transgenic Tools

We use Drosophila melanogaster to investigate the mechanisms by which myosin heavy chain isoforms contribute to muscle-specific cyto-architectures and contractile properties. We expressed various myosin isoforms in transgenic indirect flight muscles. Along with our collaborators, we examined 1) ATPase, in vitro actin sliding, step size and structure of the isolated myosin, 2) ultrastructure and mechanical properties of muscle fibers, and 3) locomotory abilities of the transgenic organisms. We found that isoform-specific differences in myosin cause relatively small structural variations in muscle assembly, but are critical to myofibril stability and function.

Essential role of obscurin in cardiac myofibrillogenesis and hypertrophic response: evidence from small interfering RNA-mediated gene silencing

Obscurin is a recently identified giant multidomain muscle protein (approximately 800 kDa) whose structural and regulatory functions remain to be defined. The goal of this study was to examine the effect of obscurin gene silencing induced by RNA interference on the dynamics of myofibrillogenesis and hypertrophic response to phenylephrine in cultured rat cardiomyocytes. We found that that the adenoviral transfection of short interfering RNA (siRNA) constructs targeting the first coding exon of obscurin sequence resulted in progressive depletion of cellular obscurin. Confocal microscopy demonstrated that downregulation of obscurin expression led to the impaired assembly of new myofibrillar clusters and considerable aberrations of the normal structure of the contractile apparatus. While the establishment of the initial periodic pattern of alpha-actinin localization remained mainly unaffected in siRNA-transfected cells, obscurin depletion did cause the defective lateral alignment of myofibrillar bundles, leading to their abnormal bifurcation, dispersal and multiple branching. Bending of immature myofibrils, apparently associated with the loss of their rigidity, a modified titin pattern, the absence of well-formed A-bands in newly formed contractile structures as documented by a diffuse localization of sarcomeric myosin labeling, and an occasional irregular periodicity of sarcomere spacing were typical of obscurin siRNA-treated cells. These results suggest that obscurin is indispensable for spatial positioning of contractile proteins and for the structural integration and stabilization of myofibrils, especially at the stage of myosin filament incorporation and A-band assembly. This demonstrates a vital role for obscurin in myofibrillogenesis and hypertrophic growth.

Comparison of Gel‐forming Properties of Silver Carp (Hypophthalmichthys molitrix) Surimi Prepared in Different Seasons

ABSTRACT: The gel‐forming properties of silver carp surimi made in different seasons were compared. Surimi prepared in winter and spring formed gel at 30°C, while autumn and summer surimi required a higher temperature of 40°C for gel formation. All surimi showed marked disintegration when incubated at 60°C. Ca2+‐ATPase inactivation rate of myofibrils prepared from 4 surimi samples showed that myofibrils in autumn and summer surimi were much more stable than those in winter and spring surimi by about 10°C. These results demonstrated a close relationship between the gel‐forming temperature of surimi and the thermal stability of myofibrils in surimi, namely that autumn and summer surimi containing stable myofibrils required higher temperature than winter and spring surimi for the gel formation.

Dynamics of obscurin localization during differentiation and remodeling of cardiac myocytes: obscurin as an integrator of myofibrillar structure.

Obscurin is a newly identified giant muscle protein whose functions remain to be elucidated. In this study we used high-resolution confocal microscopy to examine the dynamics of obscurin localization in cultures of rat cardiac myocytes during the assembly and disassembly of myofibrils. Double immunolabeling of neonatal and adult rat cells for obscurin and sarcomeric alpha-actinin, the major protein of Z-lines, demonstrated that, during myofibrillogenesis, obscurin is intensely incorporated into M-band areas of A-bands and, to a lesser extent, in Z-lines of newly formed sarcomeres. Presarcomeric structural precursors of myofibrils were intensely immunopositive for alpha-actinin and, unlike mature myofibrils, weakly immunopositive or immunonegative for obscurin. This indicates that most of the obscurin assembles in developing myofibrils after abundant incorporation of alpha-actinin and that massive integration of obscurin occurs at more advanced stages of sarcomere assembly. Immunoreactivity for obscurin in the middle of A-bands and in Z-lines of sarcomeres bridged the gaps between individual bundles of newly formed myofibrils, suggesting that this protein appears to be directly involved in their primary lateral connection and registered alignment into larger clusters. Close sarcomeric localization of obscurin and titin suggests that they may interact during myofibril assembly. Interestingly, the laterally aligned striated pattern of obscurin formed at a stage when desmin, traditionally considered as a molecular linker responsible for the lateral binding and stabilization of myofibrils at the Z-bands, was still diffusely localized. During the disassembly of the contractile system in adult myocytes, disappearance of the cross-striated pattern of obscurin preceded the disorganization of registered alignment and intense breakdown of myofibrils. The cross-striated pattern of desmin typical of terminally differentiated myocytes disappeared before or simultaneously with obscurin. During redifferentiation, as in neonatal myocytes, sarcomeric incorporation of obscurin closely followed that of alpha-actinin and occurred earlier than the striated arrangement of desmin intermediate filaments. The presence of obscurin in the Z-lines and its later assembly into the A/M-bands indicate that it may serve to stabilize and align sarcomeric structure when myosin filaments are incorporated. Our data suggest that obscurin, interacting with other muscle proteins and possibly with the sarcoplasmic reticulum, may have a role as a flexible structural integrator of myofibrils during assembly and adaptive remodeling of the contractile apparatus.

Determining structure/function relationships for sarcomeric myosin heavy chain by genetic and transgenic manipulation of Drosophila.

Drosophila melanogaster is an excellent system for examining the structure/function relationships of myosin. It yields insights into the roles of myosin in assembly and stability of myofibrils, in defining the mechanical properties of muscle fibers, and in dictating locomotory abilities. Drosophila has a single gene encoding muscle myosin heavy chain (MHC), with alternative RNA splicing resulting in stage- and tissue-specific isoform production. Localization of the alternative domains of Drosophila MHC on a three-dimensional molecular model suggests how they may determine functional differences between isoforms. We are testing these predictions directly by using biophysical and biochemical techniques to characterize myosin isolated from transgenic organisms. Null and missense mutations help define specific amino acid residues important in actin binding and ATP hydrolysis and the function of MHC in thick filament and myofibril assembly. Insights into the interaction of thick and thin filaments result from studying mutations in MHC that suppress ultrastructural defects induced by a troponin I mutation. Analysis of transgenic organisms expressing engineered versions of MHC shows that the native isoform of myosin is not critical for myofibril assembly but is essential for muscle function and maintenance of muscle integrity. We show that the C-terminus of MHC plays a pivotal role in the maintenance of muscle integrity. Transgenic studies using headless myosin reveal that the head is important for some, but not all, aspects of myofibril assembly. The integrative approach described here provides a multi-level understanding of the function of the myosin molecular motor.

Effects of postmortem storage on the ultrastructure of the endomysium and myofibrils in normal and callipyge longissimus.

These experiments were conducted to examine ultrastructural changes in longissimus from normal and callipyge lamb during 14 d of postmortem storage at 4 degrees C. Six crossbred ewe lambs (1/2 Dorset x 1/2 Romanov) were grain-fed and slaughtered at approximately 250 d of age. Leg conformation score was the basis for classifying carcasses into normal and callipyge. The normal and callipyge longissimus had mean Warner-Bratzler shear force of 2.8 (2.7, 2.4, and 3.4) and 9.0 (12.2, 6.9, and 7.9) kg, respectively, after 14 d of postmortem storage. The results of transmission electron microscopy demonstrated ultrastructural changes, including sarcolemma detachment, loss of myofibril lateral attachments, and I-band breaks in normal longissimus. Detachment of sarcolemma from myofibrils occurred in both phenotypes, but it was delayed by several days in callipyge longissimus. Thus, the sarcolemma detachment seems not to contribute significantly to postmortem tenderization. The endomysium of both phenotypes did not change with postmortem storage. In normal longissimus, the percentage of fractured I-bands increased from 0% at d 1 to 11% at d 3 (P<.05) and did not change between 3 and 14 d (15%) postmortem (P>.05). However, postmortem storage did not affect (0 to 3%) the frequency of the I-band breaks in the callipyge longissimus (P>.05). Therefore, the break in the I-band region in postmortem muscle is a change that is associated with postmortem tenderization. We conclude that the major factor responsible for the toughness of meat from callipyge longissimus is the postmortem stability of myofibrils.

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.

鶏筋原線維蛋白質の可溶化に及ぼすカルシウムイオンの影響

This study was conducted to determine the effect of calcium ions on the solubilization of chicken myofibrillar proteins that contribute to myofibril stability. Incubation of freshly prepared myofibrils in the solutions containing either 1mM CaCl2 or 1mM EGTA and 1mM (L-3-trans-Carboxyoxiran-2-Carbonyl)-L-L-Leu-Agmatin (E-64, an irreversible inhibitor of calpain) caused the increase in solubility of myofibrillar proteins with incubation time. In the presence of calcium ions, however, solubility at 6 days incubation was about three times greater than that in the absence of calcium ions. Gel electrophoresis indicated that 6 days' incubation under such conditions released large amounts of 45, 000, 36, 000, 34, 000 and 21, 000 dalton components and small amounts of 205, 000, 140, 000, 100, 000, 75, 000 and 55, 000 dalton components from myofibrils. Moreover, the addition of calcium ions caused the selective solubilization of several unknown components around 24, 000 and below 20, 000 daltons. It is unclear whether these unknown components are responsible for the weakening of myofibrillar structures closely related to meat tenderization.