Ultrastructure of cardiac muscle of Trochosa terricola Thor., Pardosa amentata Clerck,P. pullata Clerck,and Pisaura mirabilis Clerck (Araneae: Lycosidae,Pisauridae)

Summary The ultrastructure of the cardiac muscle in some araneae has been investigated. The sarcolemma invaginates at the Z band and may extend into the cells through several myofibrils. Numerous T-tubules are given off both from sarcolemmal invaginations and also directly from the cell surface. In passing through the Z band, the luminal diameter of T-tubules greatly increases. Dyadic and a few triadic couplings are found mainly at the A-I level. Peripheral couplings were not seen.The ruthenium red solutions employed were prepared as described by Myklebust (1975), but the fixative contained 2% sucrose. The hearts were fixed for 3 1/2h in the ice cold solution, washed in buffer and post-fixed for 1 1/2h in a ruthenium red/OsO₄ solution. Dehydration and embedding was performed as described above. Sections were cut on a Reichert Om U2 microtome, stained with lead citrate (Reynolds, 1963) and examined in a Philips 300 electron microscope at 80 kV.Semithin sections for light microscopy were stained with toluidine blue (Mercer and Birbeck, 1972).I am much obliged to cand. real Erling Hauge for identifying the specimens used in the investigation proportion of relaxed sarcomeres. The fixed pieces were washed in a buffered sucrose solution and post-fixed in a 1% OsO₄ solution buffered with sodium cacodylate for 1 h. Following washes in a compatible buffer and in distilled water, the tissue was stained en bloc for l h in a 2% aqueous uranyl acetate solution. The tissue was dehydrated through cold acetone and embedded in Epon 812.     

Contribution of myosin rod protein to the structural organization of adult and embryonic muscles in Drosophila

Myosin rod protein (MRP) is a naturally occurring 155 kDa protein in Drosophila that includes the myosin heavy chain (MHC) rod domain, but contains a unique 77 amino acid residue N-terminal region that replaces the motor and light chain-binding domains of S1. MRP is a major component of myofilaments in certain direct flight muscles (DFMs) and it is present in other somatic, cardiac and visceral muscles in adults, larvae and embryos, where it is coexpressed and polymerized into thick filaments along with MHC. DFM49 has a relatively high content of MRP, and is characterized by an unusually disordered myofibrillar ultrastructure, which has been attributed to lack of cross-bridges in the filament regions containing MRP. Here, we characterize in detail the structural organization of myofibrils in adult and embryonic Drosophila muscles containing various MRP/MHC ratios and in embryos carrying a null mutation for the single MHC gene. We examined MRP in embryonic body wall and intestinal muscles as well as in DFMs with consistent findings. In DFMs numbers 49, 53 and 55, MRP is expressed at a high level relative to MHC and is associated with disorder in the positioning of thin filaments relative to thick filaments in the areas of overlap. Embryos that express MRP in the absence of MHC form thick filaments that participate in the assembly of sarcomeres, suggesting that myofibrillogenesis does not depend on strong myosin-actin interactions. Further, although thick filaments are not well ordered, the relative positioning of thin filaments is fairly regular in MRP-only containing sarcomeres, confirming the hypothesis that the observed disorder in MRP/MHC containing wild-type muscles is due to the combined action between the functional behavior of MRP and MHC myosin heads. Our findings support the conclusion that MRP has an active function to modulate the contractile activity of muscles in which it is expressed.     

The membrane systems of the cardiac muscle cell of Meganychtiphanes norvegica (M. Sars), euphauciacea,crustacea

Summary The membrane systems of cardiac muscle cells of the euphausiacean Meganychtiphanes norvegica are described. Transverse tubules are found both at the Z-band level (T_z-tubules) and at the H-band level (T_h-tubules). Within the sarcomere narrow longitudinal tubules branch off from the T_z-tubules. At the H-band level these tubules expand forming flattened cisternae in dyadic and triadic couplings with the sarcoplasmic reticulum (SR). Adjacent myofibrils are separated by a well developed SR. Modifications of the SR are seen at the H-band level where junctional cisternae are formed.     

D-Titin: a giant protein with dual roles in chromosomes and muscles

Previously, we reported that chromosomes contain a giant filamentous protein, which we identified as titin, a component of muscle sarcomeres. Here, we report the sequence of the entire titin gene in Drosophila melanogaster, D-Titin, and show that it encodes a two-megadalton protein with significant colinear homology to the NH(2)-terminal half of vertebrate titin. Mutations in D-Titin cause chromosome undercondensation, chromosome breakage, loss of diploidy, and premature sister chromatid separation. Additionally, D-Titin mutants have defects in myoblast fusion and muscle organization. The phenotypes of the D-Titin mutants suggest parallel roles for titin in both muscle and chromosome structure and elasticity, and provide new insight into chromosome structure.     

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.     

Residual force enhancement in myofibrils and sarcomeres

Residual force enhancement has been observed following active stretch of skeletal muscles and single fibres. However, there has been intense debate whether force enhancement is a sarcomeric property, or is associated with sarcomere length instability and the associated development of non-uniformities. Here, we studied force enhancement for the first time in isolated myofibrils (n=18) that, owing to the strict in series arrangement, allowed for evaluation of this property in individual sarcomeres (n=79). We found consistent force enhancement following stretch in all myofibrils and each sarcomere, and forces in the enhanced state typically exceeded the isometric forces on the plateau of the force-length relationship. Measurements were made on the plateau and the descending limb of the force-length relationship and revealed gross sarcomere length non-uniformities prior to and following active myofibril stretching, but in contrast to previous accounts, revealed that sarcomere lengths were perfectly stable under these experimental conditions. We conclude that force enhancement is a sarcomeric property that does not depend on sarcomere length instability, that force enhancement varies greatly for different sarcomeres within the same myofibril and that sarcomeres with vastly different amounts of actin-myosin overlap produce the same isometric steady-state forces. This last finding was not explained by differences in the amount of contractile proteins within sarcomeres, vastly different passive properties of individual sarcomeres or (half-) sarcomere length instabilities, suggesting that the basic mechanical properties of muscles, such as force enhancement, force depression and creep, which have traditionally been associated with sarcomere instabilities and the corresponding dynamic redistribution of sarcomere lengths, are not caused by such instabilities, but rather seem to be inherent properties of the mechanisms of contraction.     

History dependence of force production in submaximal stimulated rat medial gastrocnemius muscle

Studies on maximal stimulated muscle have shown that history dependence is an integral part of muscle force production. The purpose of this study was to evaluate the history dependent effects in submaximal stimulated muscle. For this purpose, lengthening and shortening experiments were performed on the medial gastrocnemius muscle of the rat. The length dependence of lengthening induced force enhancement and shortening induced force depression was studied in maximal (80 Hz) and submaximal (30 Hz) stimulated muscles. The most important results of this study are (a) submaximal stimulation reduces the maximal amplitude of the history effects and (b) lengthening induced force enhancement and shortening induced force deficit are affected differently by submaximal stimulation. The implication of these results for the underlying mechanism and functional relevance of the history dependent effects is discussed.     

粘虫蛾飞行肌的发育：超微结构特征分析

应用电子显微镜对正常条件下饲养的0-16日龄粘虫Mythimna separata (Walker)雌蛾飞行肌超微结构的研究结果表明：肌原纤维直径。线粒体和横管的体积分量均随蛾龄的增加而增加,到7日龄达到最大值以后又随蛾龄的增加而下降;但是,肌节长度则随蛾龄的增加而缩短,到7日龄达到最短后又随蛾龄的增加而延长,从而使整条发育曲线呈“V”字形;肌原纤维和肌质网体积分量变化不大或无规律可寻;二位体在发育初期和未期的比例较高,而三位体在4和7口龄比例较高;肌丝排列从0～7日龄均是有序的,肌动蛋白丝(细丝)和肌球蛋白丝(粗丝)的数量比为3：1,粗丝的数量变化也不大,每根肌原纤维约有600根。但从10日龄开始肌丝排列出现紊乱,细丝全部消失,粗丝降解、数量减少了30%,从而使肌原纤维留下一片片的空白。根据这些结果,把7日龄前的飞行肌发育过程视为生长过程,而把10日龄后的视作降解过程。最后对粘虫蛾飞行肌与卵巢发育及其飞行能力变化的关系进行了讨论。