Both d- and l-specific Lactate Dehydrogenases Co-exist in Individual Cephalopods

Both d-lactate-specific and l-lactate-specific lactate dehydrogenases coexist in individual cephalopods, contrary to the commonly held view that invertebrate species may contain one or other, but not both. We describe the tissue distribution of these lactate dehydrogenases and of octopine dehydrogenase, the major pyruvate reductase activity in cephalopods, in three species: common squid (Loligo vulgaris), cuttlefish (Sepia officinalis) and lesser octopus (Eledone cirrhosa). The l-specific lactate dehydrogenase of squid is shown to be a dimer of 36,000 dalton subunits.     

Comparative morphology of changeable skin papillae in octopus and cuttlefish

A major component of cephalopod adaptive camouflage behavior has rarely been studied: their ability to change the three‐dimensionality of their skin by morphing their malleable dermal papillae. Recent work has established that simple, conical papillae in cuttlefish (Sepia officinalis) function as muscular hydrostats; that is, the muscles that extend a papilla also provide its structural support. We used brightfield and scanning electron microscopy to investigate and compare the functional morphology of nine types of papillae of different shapes, sizes and complexity in six species: S. officinalis small dorsal papillae, Octopus vulgaris small dorsal and ventral eye papillae, Macrotritopus defilippi dorsal eye papillae, Abdopus aculeatus major mantle papillae, O. bimaculoides arm, minor mantle, and dorsal eye papillae, and S. apama face ridge papillae. Most papillae have two sets of muscles responsible for extension: circular dermal erector muscles arranged in a concentric pattern to lift the papilla away from the body surface and horizontal dermal erector muscles to pull the papilla's perimeter toward its core and determine shape. A third set of muscles, retractors, appears to be responsible for pulling a papilla's apex down toward the body surface while stretching out its base. Connective tissue infiltrated with mucopolysaccharides assists with structural support. S. apama face ridge papillae are different: the contraction of erector muscles perpendicular to the ridge causes overlying tissues to buckle. In this case, mucopolysaccharide‐rich connective tissue provides structural support. These six species possess changeable papillae that are diverse in size and shape, yet with one exception they share somewhat similar functional morphologies. Future research on papilla morphology, biomechanics and neural control in the many unexamined species of octopus and cuttlefish may uncover new principles of actuation in soft, flexible tissue. J. Morphol. 275:371–390, 2014. © 2013 Wiley Periodicals, Inc.     

Ozz-E3 Ubiquitin Ligase Targets Sarcomeric Embryonic Myosin Heavy Chain during Muscle Development

Muscle contractile proteins are expressed as a series of developmental isoforms that are in constant dynamic remodeling during embryogenesis, but how obsolete molecules are recognized and removed is not known. Ozz is a developmentally regulated protein that functions as the adaptor component of a RING-type ubiquitin ligase complex specific to striated muscle. Ozz^−/− mutants exhibit defects in myofibrillogenesis and myofiber differentiation. Here we show that Ozz targets the rod portion of embryonic myosin heavy chain and preferentially recognizes the sarcomeric rather than the soluble pool of myosin. We present evidence that Ozz binding to the embryonic myosin isoform within sarcomeric thick filaments marks it for ubiquitination and proteolytic degradation, allowing its replacement with neonatal or adult isoforms. This unique function positions Ozz within a system that facilitates sarcomeric myosin remodeling during muscle maturation and regeneration. Our findings identify Ozz-E3 as the ubiquitin ligase complex that interacts with and regulates myosin within its fully assembled cytoskeletal structure.     

Differentiation of slow and fast fibers in tentacles of Sepia officinalis (Mollusca)

The tentacles of Sepia officinalis are cylindrical muscular structures that can be quickly everted and elongated to capture prey. The combination of both velocity and extensive elongation of the tentacles is due to the presence of both cross-striated and helical muscles. The complex organization and differentiation of different fibers has been studied in cuttlefish extracted from egg gel coats at different developmental stages, and in completely developed animals. Tentacle muscles start to differentiate centrifugally from the area close to the axial nervous system, where two types of myocytes can be recognized. These populations of myocytes, which may be distinguished morphologically and which express different myosin isoforms, give rise to fast and slow muscles. The presence in molluscs of slow and fast muscles arising from different populations of myocytes, as in vertebrate muscle development, could be considered as an example of evolutionary conservation.     

Cuttlefish skin papilla morphology suggests a muscular hydrostatic function for rapid changeability

Coleoid cephalopods adaptively change their body patterns (color, contrast, locomotion, posture, and texture) for camouflage and signaling. Benthic octopuses and cuttlefish possess the capability, unique in the animal kingdom, to dramatically and quickly change their skin from smooth and flat to rugose and three‐dimensional. The organs responsible for this physical change are the skin papillae, whose biomechanics have not been investigated. In this study, small dorsal papillae from cuttlefish (Sepia officinalis) were preserved in their retracted or extended state, and examined with a variety of histological techniques including brightfield, confocal, and scanning electron microscopy. Analyses revealed that papillae are composed of an extensive network of dermal erector muscles, some of which are arranged in concentric rings while others extend across each papilla's diameter. Like cephalopod arms, tentacles, and suckers, skin papillae appear to function as muscular hydrostats. The collective action of dermal erector muscles provides both movement and structural support in the absence of rigid supporting elements. Specifically, concentric circular dermal erector muscles near the papilla's base contract and push the overlying tissue upward and away from the mantle surface, while horizontally arranged dermal erector muscles pull the papilla's perimeter toward its center and determine its shape. Each papilla has a white tip, which is produced by structural light reflectors (leucophores and iridophores) that lie between the papilla's muscular core and the skin layer that contains the pigmented chromatophores. In extended papillae, the connective tissue layer appeared thinner above the papilla's apex than in surrounding areas. This result suggests that papilla extension might create tension in the overlying connective tissue and chromatophore layers, storing energy for elastic retraction. Numerous, thin subepidermal muscles form a meshwork between the chromatophore layer and the epidermis and putatively provide active papillary retraction. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

Enhancement of hybrid-fiber types in rat soleus muscle after clenbuterol administration during hindlimb unloading

Our objective was to determine the effects of a clenbuterol (CB) treatment orally administered (2 mg per kg) to rats submitted to 14 days of hindlimb unloading (HU). The morphological and the contractile properties as well as the myosin heavy chain isoforms contained in each fiber type were determined in whole soleus muscles. As classically described after HU, a decrease in muscle wet weight and in body mass associated with a loss of muscular force, an evolution of the contractile parameters towards those of a fast muscle type, and the emergence of fast myosin heavy chain isoforms were observed. The CB treatment in the HU rats helped reduce the decrease in 1) muscle and body weights, 2) force and 3) the proportion of slow fibers, without preventing the emergence of fast myosin isoforms. Clenbuterol induced a complex remodelling of the muscle typing promoting the combination of both slow and fast myosin isoforms within one fiber. To conclude, our data demonstrate that CB administration partially counteracts the effects produced by HU, and they allow us to anticipate advances in the treatment of muscular atrophy.     

A finite element simulation scheme for biological muscular hydrostats

An explicit finite element scheme is developed for biological muscular hydrostats such as squid tentacles, octopus arms and elephant trunks. The scheme is implemented by embedding muscle fibers in finite elements. In any given element, the fiber orientation can be assigned arbitrarily and multiple muscle directions can be simulated. The mechanical stress in each muscle fiber is the sum of active and passive parts. The active stress is taken to be a function of activation state, muscle fiber shortening velocity and fiber strain; while the passive stress depends only on the strain. This scheme is tested by simulating extension of a squid tentacle during prey capture; our numerical predictions are in close correspondence with existing experimental results. It is shown that the present finite element scheme can successfully simulate more complex behaviors such as torsion of a squid tentacle and the bending behavior of octopus arms or elephant trunks.     

Myosin II isoforms in smooth muscle: heterogeneity and function

Both smooth muscle (SM) and nonmuscle class II myosin molecules are expressed in SM tissues comprising hollow organ systems. Individual SM cells may express one or more of multiple myosin II isoforms that differ in myosin heavy chain (MHC) and myosin light chain (MLC) subunits. Although much has been learned, the expression profiles, organization within contractile filaments, localization within cells, and precise roles in various contractile functions of these different myosin molecules are still not well understood. However, data supporting unique physiological roles for certain isoforms continues to build. Isoform differences located in the S1 head region of the MHC can alter actin binding and rates of ATP hydrolysis. Differences located in the MHC tail can alter the formation, stability, and size of the myosin thick filament. In these distinct ways, both head and tail isoform differences can alter force generation and muscle shortening velocities. The MLCs that are associated with the lever arm of the S1 head can affect the flexibility and range of motion of this domain and possibly the motion of the S2 and motor domains. Phosphorylation of MLC(20) has been associated with conformational changes in the S1 and/or S2 fragments regulating enzymatic activity of the entire myosin molecule. A challenge for the future will be delineation of the physiological significance of the heterogeneous expression of these isoforms in developmental, tissue-specific, and species-specific patterns and or the intra- and intercellular heterogeneity of myosin isoform expression in SM cells of a given organ.     

The expression and functional activities of smooth muscle myosin and non‐muscle myosin isoforms in rat prostate

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non‐muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM‐A/B) and myosin light chain 17 isoforms (LC_17a/b), and isoform ratios were determined via competitive RT‐PCR. SM MHC and NM MHC isoforms (NMMHC‐A, NMMHC‐B and NMMHC‐C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC_17a and SM1 but with relatively equal expression of SM‐A/SM‐B at the mRNA level. Meanwhile, isoforms of NMMHC‐A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC‐A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre‐contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.     

Changes in the human muscle force-velocity relationship in response to resistance training and subsequent detraining.

Previous studies show that cessation of resistance training, commonly known as "detraining," is associated with strength loss, decreased neural drive, and muscular atrophy. Detraining may also increase the expression of fast muscle myosin heavy chain (MHC) isoforms. The present study examined the effect of detraining subsequent to resistance training on contractile performance during slow-to-medium velocity isokinetic muscle contraction vs. performance of maximal velocity "unloaded" limb movement (i.e., no external loading of the limb). Maximal knee extensor strength was measured in an isokinetic dynamometer at 30 and 240 degrees/s, and performance of maximal velocity limb movement was measured with a goniometer during maximal unloaded knee extension. Muscle cross-sectional area was determined with MRI. Electromyographic signals were measured in the quadriceps and hamstring muscles. Twitch contractions were evoked in the passive vastus lateralis muscle. MHC isoform composition was determined with SDS-PAGE. Isokinetic muscle strength increased 18% (P < 0.01) and 10% (P < 0.05) at slow and medium velocities, respectively, along with gains in muscle cross-sectional area and increased electromyogram in response to 3 mo of resistance training. After 3 mo of detraining these gains were lost, whereas in contrast maximal unloaded knee extension velocity and power increased 14% (P < 0.05) and 44% (P < 0.05), respectively. Additionally, faster muscle twitch contractile properties along with an increased and decreased amount of MHC type II and MHC type I isoforms, respectively, were observed. In conclusion, detraining subsequent to resistance training increases maximal unloaded movement speed and power in previously untrained subjects. A phenotypic shift toward faster muscle MHC isoforms (I --> IIA --> IIX) and faster electrically evoked muscle contractile properties in response to detraining may explain the present results.     

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O‐GlcNAc modified. Moreover, it was demonstrated that O‐GlcNAc moieties involved in contractile protein interactions could modulate Ca²⁺ activation parameters of contraction. In order to better understand how O‐GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O‐GlcNAc modification in purified contractile protein homogenates. Using an MS‐based method that relies on mild β‐elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O‐GlcNAc site in the subdomain four of actin and four O‐GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O‐GlcNAc sites might modulate the actin–tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein–protein interactions but could also modulate the contractile properties of skeletal muscle.     

Actin gene family evolution and the phylogeny of coleoid cephalopods (Mollusca: Cephalopoda)

Phylogenetic analysis conducted on a 784-bp fragment of 82 actin gene sequences of 44 coleoid cephalopod taxa, along with results obtained from genomic Southern blot analysis, confirmed the presence of at least three distinct actin loci in coleoids. Actin isoforms were characteri zed through phylogenetic analysis of representative cephalopod sequences from each of the three isoforms, along with translated actin cDNA sequences from a diverse array of metazoan taxa downloaded from GenBank. One of the three isoforms found in cephalopods was closely related to actin sequences expressed in the muscular tissues of other molluscs. A second isoform was most similar to cytoplasmic-specific actin amino acid sequences. The muscle type actins of molluscs were found to be distinct from those of arthropods, suggesting at least two independent derivations of muscle actins in the protostome lineage, although statistical support for this conclusion was lacking. Parsimony and maximum-likelihood analyses of two of the isoforms from which >30 orthologous coleoid sequences had been obtained (one of the cytoplasmic actins and the muscle actin) supported the monophyly of several higher-level coleoid taxa. These included the superorders Octopodiformes and Decapodiformes, the order Octopoda, the octopod suborder Incirrata, and the teuthoid suborder Myopsida. The monophyly of several taxonomic groups within the Decapodiformes was not supported, including the orders Teuthoidea and Sepioidea and the teuthoid suborder Oegopsida. Parametric bootstrap analysis conducted on the simulated cytoplasmic actin data set provided statistical support to reject the monophyly of the Sepioidea. Although parametric bootstrap analysis of the muscle actin isoform did not reject sepioid monophyly at the 5% level, the results (rejection at P: = 0.068) were certainly suggestive of sepioid nonmonophyly.     

Sarcomeric Gene Expression and Contractility in Myofibroblasts

Myofibroblasts are unusual cells that share morphological and functional features of muscle and nonmuscle cells. Such cells are thought to control liver blood flow and kidney glomerular filtration rate by having unique contractile properties. To determine how these cells achieve their contractile properties and their resemblance to muscle cells, we have characterized two myofibroblast cell lines. Here, we demonstrate that myofibroblast cell lines from kidney mesangial cells (BHK) and liver stellate cells activate extensive programs of muscle gene expression including a wide variety of muscle structural proteins. In BHK cells, six different striated myosin heavy chain isoforms and many thin filament proteins, including troponin T and tropomyosin are expressed. Liver stellate cells express a limited subset of the muscle thick filament proteins expressed in BHK cells. Although these cells are mitotically active and do not morphologically differentiate into myotubes, we show that MyoD and myogenin are expressed and functional in both cell types. Finally, these cells contract in response to endothelin-1 (ET-1); and we show that ET-1 treatment increases the expression of sarcomeric myosin.     

Myosin light chain kinase and the role of myosin light chain phosphorylation in skeletal muscle

Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca²⁺/calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca²⁺ binding to calmodulin forming a (Ca²⁺)₄•calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca²⁺ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca²⁺/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca²⁺-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.     

Changes in muscle proteomics in the course of the Caudwell Research Expedition to Mt. Everest

This study employed differential proteomic and immunoassay techniques to elucidate the biochemical mechanisms utilized by human muscle (vastus lateralis) in response to high altitude hypoxia exposure. Two groups of subjects, participating in a medical research expedition (A, n = 5, 19d at 5300 m altitude; B, n = 6, 66d up to 8848 m) underwent a ≈ 30% drop of muscular creatine kinase and of glycolytic enzymes abundance. Protein abundance of most enzymes of the tricarboxylic acid cycle and oxidative phosphorylation was reduced both in A and, particularly, in B. Restriction of α‐ketoglutarate toward succinyl‐CoA resulted in increased prolyl hydroxylase 2 and glutamine synthetase. Both A and B were characterized by a reduction of elongation factor 2alpha, controlling protein translation, and by an increase of heat shock cognate 71 kDa protein involved in chaperone‐mediated autophagy. Increased protein levels of catalase and biliverdin reductase occurred in A alongside a decrement of voltage‐dependent anion channels 1 and 2 and of myosin‐binding protein C, suggesting damage to the sarcomeric structures. This study suggests that during acclimatization to hypobaric hypoxia the muscle behaves as a producer of substrates activating a metabolic reprogramming able to support anaplerotically the tricarboxylic acid cycle, to control protein translation, to prevent energy expenditure and to activate chaperone‐mediated autophagy.     

503unc,a small and muscle‐specific zebrafish promoter

The muscle‐specific UNC‐45b assists in the folding of sarcomeric myosin. Analysis of the zebrafish unc‐45b upstream region revealed that unc‐45b promoter fragments reliably drive GFP expression after germline transmission. The muscle‐specific 503‐bp minimal promoter 503unc was identified to drive gene expression in the zebrafish musculature. In transgenic Tg(?503unc:GFP) zebrafish, GFP fluorescence was detected in the adaxial cells, their slow fiber descendants, and the fast muscle. At later stages, robust GFP fluorescence is eminent in the cardiac, cranial, fin, and trunk muscle, thereby recapitulating the unc‐45b expression pattern. We propose that the 503unc promoter is a small and muscle‐specific promoter that drives robust gene expression throughout the zebrafish musculature, making it a valuable tool for the exploration of zebrafish muscle. genesis 51:443–447. © 2013 Wiley Periodicals, Inc.     

The functional morphology of the musculature of squid (Loliginidae) arms and tentacles

The arms and tentacles of squid (Family Loliginidae: Sepioteuthis sepioidea (Blainville), Loligo pealei (LeSueur), Loligo plei (Blainville), Loliguncula brevis (Blainville)) do not possess the hardened skeletal elements or fluid-filled cavities that typically provide skeletal support in other animals. Instead, these appendages are made up almost entirely of muscle. It is suggested here that the musculature serves as both the effector of movement and as the skeletal support system itself. High-speed movie recordings were used to observe prey capture by loliginid squid. Extension of the tentacles (1 pair) during prey capture is probably brought about by contraction of transverse muscle fibers and circular muscle fibers. Contraction of longitudinal muscle fibers causes retraction of the tentacles. Torsion of the tentacles during extension may be the result of contraction of muscle fibers arranged in a helical array. The inextensible but manipulative arms (4 pairs) may utilize a transverse muscle mass to resist the longitudinal compression caused by contraction of the longitudinal muscles which bend the arms. A composite connective tissue/muscle helical fiber array may twist the arms.     

Muscle development in squid: Ultrastructural differentiation of a specialized muscle fiber type

The ultrastructural differentiation of two muscle fiber types of the squid Sepioteuthis lessoniana was correlated with development of prey-capture behavior. Transmission electron microscopy was used to document the differentiation of the fast-contracting cross-striated muscle cells of the tentacles and the obliquely striated muscle cells of the arms of specimens sampled at one week intervals from hatching to 5 weeks. By using high-speed video recordings, the ultrastructural differentiation was correlated with changes in prey-capture behavior that occur during development and growth. The ultrastructural analysis focused on the muscle cells of the transverse muscle of the tentacles and the transverse muscle of the arms. For the first 2 weeks after hatching, the tentacle transverse muscle fibers do not show the adult ultrastructure and are indistinguishable from the obliquely striated fibers of the transverse muscle of the arms. Transverse striation of the tentacle muscle cells appears at approximately three weeks and adult ultrastructure is present by 4–5 weeks after hatching. The high-speed video recordings show correlated behavioral changes. During the first 2–3 weeks after hatching, the animals use a different prey-capture mode from the adults; they jet forward and capture the prey with splayed arms and tentacles rather than employing the rapid tentacular strike. © 1996 Wiley-Liss, Inc.