Thin Myofilaments Research Articles

Troponin I and Its Clinical Significance

Troponin I is one of the regulatory proteins of the troponin complex (troponin I, troponin C and troponin T) found in both cardiac and skeletal muscle. The letter I stands for its inhibitory character as binding is prevented between actin and myosin in relaxed muscle. It plays a significant role in holding actin-tropomyosin complex in place by binding actin in thin myofilaments. Binding of calcium to troponin C causes certain conformational changes, leading to troponin I dislocation and thus exposing binding site of myosin on actin, which leads to muscle contraction. It is frequently used in the diagnosis of acute coronary syndrome and myocardial infarction, and also serves as a prognostic marker in their management (1). There are three distinct forms of troponin I with tissue-specific expression patterns: 1. Slow-twitch skeletal muscle isoform troponin I, 2. Fast-twitch skeletal muscle isoform troponin I and 3. Cardiac troponin I.

A multiscale finite element model of left ventricular mechanics incorporating baroreflex regulation

Cardiovascular function is regulated by a short-term hemodynamic baroreflex loop, which tries to maintain arterial pressure at a normal level. In this study, we present a new multiscale model of the cardiovascular system named MyoFE. This framework integrates a mechanistic model of contraction at the myosin level into a finite-element-based model of the left ventricle pumping blood through the systemic circulation. The model is coupled with a closed-loop feedback control of arterial pressure inspired by a baroreflex algorithm previously published by our team. The reflex loop mimics the afferent neuron pathway via a normalized signal derived from arterial pressure. The efferent pathway is represented by a kinetic model that simulates the net result of neural processing in the medulla and cell-level responses to autonomic drive. The baroreflex control algorithm modulates parameters such as heart rate and vascular tone of vessels in the lumped-parameter model of systemic circulation. In addition, it spatially modulates intracellular Ca2+ dynamics and molecular-level function of both the thick and the thin myofilaments in the left ventricle. Our study demonstrates that the baroreflex algorithm can maintain arterial pressure in the presence of perturbations such as acute cases of altered aortic resistance, mitral regurgitation, and myocardial infarction. The capabilities of this new multiscale model will be utilized in future research related to computational investigations of growth and remodeling.

A multiscale model of the cardiovascular system that regulates arterial pressure viaclosed loopbaroreflex control of chronotropism, cell-level contractility, and vascular tone.

Multiscale models of the cardiovascular system can provide new insights into physiological and pathological processes. PyMyoVent is a computer model that bridges from molecular- to organ-level function and which simulates a left ventricle pumping blood through the systemic circulation. Initial work with PyMyoVent focused on the end-systolic pressure volume relationship and ranked potential therapeutic strategies by their impact on contractility. This manuscript extends the PyMyoVent framework by adding closed-loop feedback control of arterial pressure. The control algorithm mimics important features of the physiological baroreflex and was developed as part of a long-term program that focuses on growth and biological remodeling. Inspired by the underlying biology, the reflex algorithm uses an afferent signal derived from arterial pressure to drive a kinetic model that mimics the net result of neural processing in the medulla and cell-level responses to autonomic drive. The kinetic model outputs control signals that are constrained between limits that represent maximum parasympathetic and maximum sympathetic drive and which modulate heart rate, intracellular Ca2+ dynamics, the molecular-level function of both the thick and the thin myofilaments, and vascular tone. Simulations show that the algorithm can regulate mean arterial pressure at user-defined setpoints as well as maintaining arterial pressure when challenged by changes in blood volume and/or valve resistance. The reflex also regulates arterial pressure when cell-level contractility is modulated to mimic the idealized impact of myotropes. These capabilities will be important for future work that uses computer modeling to investigate clinical conditions and treatments.

Zebrafish integrin a3b is required for cardiac contractility and cardiomyocyte proliferation

In cardiac muscle cells, heterodimeric integrin transmembrane receptors are known to serve as mechanotransducers, translating mechanical force to biochemical signaling. However, the roles of many individual integrins have still not been delineated. In this report, we demonstrate that Itga3b is localized to the sarcolemma of cardiomyocytes from 24 to 96 hpf. We further show that heterozygous and homozygous itga3b/bdf mutant embryos display a cardiomyopathy phenotype, with decreased cardiac contractility and reduced cardiomyocyte number. Correspondingly, proliferation of ventricular and atrial cardiomyoctyes and ventricular epicardial cells is decreased in itga3b mutant hearts. The contractile dysfunction of itga3b mutants can be attributed to cardiomyocyte sarcomeric disorganization, including thin myofilaments with blurred and shortened Z-discs. Together, our results reveal that Itga3b localizes to the myocardium sarcolemma, and it is required for cardiac contractility and cardiomyocyte proliferation.

The morphology of the elements of the myocardial contractile apparatus question and research prospects

Background. Over the past 50 years, specific methods for studying the ultrastructure of the heart have been rapidly developed. The complex interaction of various research methods makes it possible to more accurately form a representation of the spatial structure of the components of the myocardial contractile apparatus. Objective. To conduct a content analysis of the results of the study of the composition of the myocardial contractile apparatus. Conduct a broad analysis of literary references and form an understanding of the spatial structure of the components of the myocardial contractile apparatus in the prospect of research at different levels of cell organization. Methods. Processing of information sources was carried out by the method of complex meta-analysis of data analysis. Results. The morphological characteristics of the myocardial contractile apparatus include a number of broad profile elements. The system of composite elements of the contractile apparatus of cardiomyocytes is the most formed and developed in the structure of the cytoplasmic complex of organelles in the group of contractile cardiomyocytes. The complex of the contractile apparatus is represented by myofibrils, each of which consists of thousands of sarcomeres telophragm connected in series, containing actin (thin) and myosin (thick) myofilaments. The main methods for studying the contractile apparatus of the myocardium include how immunohistochemistry and transmission electron microscopy provide an understanding of the structure of components at various levels of organization of histoarchitectonics and ultrastructure of organelles.. The contractile apparatus of the myocardium includes species-specific organelles, which basically belong to a number of basic hardware systems of cardiomyocytes. Conclusion. Immunohistochemical methods should clearly show the localization of individual tipes of elements in the protein structure of the contractile apparatus of the myocardium, and therefore should include in the study methods the use of the following immunohistochemical markers that can show the configuration of thin and thick myofilaments. The results of analytical review and analysis of information sources on the characteristics of the components of the myofibrillar complex gives a choice of specific research methods and forms a more detailed understanding of the spatial organization of the morphology of the myocardial contractile apparatus.

Ultrastructural evidence for the participation of muscle cells in the formation of extracellular matrices in aporocotylid blood flukes (Digenea)

The muscle cells and extracellular matrices (ECMs) of two teleost-infecting blood flukes belonging to distinct evolutionary lineages of the Aporocotylidae (Digenea) were examined using Transmission Electron Microscopy. Four morphotypes of muscle cells were found in the freshwater species Sanguinicola sp., but were considered to be various developmental stages of a single cisternic type. In the marine species Aporocotyle simplex, three types of muscle cells were apparent, one of which is cisternic. The first ultrastructural evidence is presented for the exocytosis of the moderately dense contents of dilated cisternae of cisternic muscle cells into the extracellular space in both Sanguinicola sp. and A. simplex. The basal matrices of aporocotylids support various types of epithelia. In Sanguinicola sp., beneath the distal tegumental cytoplasm, there is a thin lamina densa, whereas the intestinal epithelium is supported by a lamina reticularis. In A. simplex, both a thin lamina densa and a thick lamina reticularis underlie the distal cytoplasm of the tegument and are present at the periphery of the ovary, but beneath the epithelial lining of the caeca and both genital and excretory ducts there is only a lamina reticularis. Significant variation in the development and amount of the ECM in marine and freshwater aporocotylids is described, since A. simplex has a much better developed ECM than occurs in Sanguinicola sp. Moreover, thin myofilaments of muscle fibres participate in the ECM formation in A. simplex and represent its dominant component. The presence of two mechanisms for ECM formation in A. simplex, as opposed to a single mechanism in Sanguinicola sp., may represent further evidence for the affiliation of these two taxa to divergent evolutionary lineages. The data presented are discussed in relation to available information on these structures in other neodermatan groups.

Discrete localization patterns of Arf6, and its activators EFA6A and BRAG2, and its effector PIP5kinaseγ on myofibrils of myotubes and plasma membranes of myoblasts in developing skeletal muscles of mice

Arf6 (ADP ribosylation factor 6), activated by Arf-GEF (guanine nucleoside exchange factor), is involved in the membrane trafficking and actin-remodeling which are critical for maintenance of cell organization and activity and for fusion of myoblasts to form myotubes/myofibers. EFA6A (exchange factor for Arf6 type A) and BRAG2 (brefeldin A-resistant Arf-GEF 2) represent members of discrete subfamilies of Arf-GEF, while PIP5Kγ (phosphatidylinositol4-phosphate5-kinase γ) produces PI 4,5-bisphosphate (PIP2) and it is target for Arf6. In the present study, immunoreactive bands for Arf6, EFA6A, BRAG2 and PIP5Kγ were detected in immunoblots of skeletal muscle homogenates of mice at E18D (embryonic day 18), while the bands for Arf6, EFA6A and PIP5Kγ were reduced in density and no significant bands for BRAG2 were discerned at P1D (postnatal 1 day). No immunoblot bands for any of the molecules were eventually detected in skeletal fibers of adult mice. Immunoreactivities for endogenous Arf6, EFA6A and PIP5Kγ were visualized using immuno-light microscopy localized as periodic striations running perpendicular to the longitudinal axes of skeletal muscle fibers of mice at E18D and P1D. All the striations were co-immunoreactive for β-actin in double immunofluorescence microscopy, and the immunoreactivities were confined to thin myofilaments at sarcomeric I-domains in immuno-electron microscopy. On the other hand, immunoreactivities for Arf6, BRAG2 and PIP5Kγ were conspicuous on plasmalemma of myoblasts at E14D, while immunoreactivity for EFA6A was already distinct in striations perpendicular to myofibrils in myotubes at E14D. The present findings suggest three possibilities: involvement of EFA6A-activated Arf6 together with PIP5Kγ in maturation of myofibrils, movement of Arf6 and PIP5Kγ from the plasmalemma of myoblasts to myofibrils of myotubes, and that of BRAG2 to the cytoplasm of myotubes; and further a function of EFA6A independent of the activation of Arf6 in immature myofibrils. In addition, the involvement of Arf6, BRAG2 and PIP5Kγ in the fusion of myoblasts into myotubes was supported by the present finding.

Myofilaments: Movers and Rulers of the Sarcomere.

Striated cardiac and skeletal muscles play very different roles in the body, but they are similar at the molecular level. In particular, contraction, regardless of the type of muscle, is a precise and complex process involving the integral protein myofilaments and their associated regulatory components. The smallest functional unit of muscle contraction is the sarcomere. Within the sarcomere can be found a sophisticated ensemble of proteins associated with the thick filaments (myosin, myosin binding protein-C, titin, and obscurin) and thin myofilaments (actin, troponin, tropomyosin, nebulin, and nebulette). These parallel thick and thin filaments slide across one another, pulling the two ends of the sarcomere together to regulate contraction. More specifically, the regulation of both timing and force of contraction is accomplished through an intricate network of intra- and interfilament interactions belonging to each myofilament. This review introduces the sarcomere proteins involved in striated muscle contraction and places greater emphasis on the more recently identified and less well-characterized myofilaments: cardiac myosin binding protein-C, titin, nebulin, and obscurin. © 2017 American Physiological Society. Compr Physiol 7:675-692, 2017.

Effect of the bendiocarb on the ultrastructure of rabbit skeletal muscle

Bendiocarb belongs to the group of carbamate insecticides that inhibit acetylcholinesterase. In agriculture, it is used to control a variety of insects, therefore it is important to examine every potential aspect of its toxicology. The aim of this study was to observe the effect of bendiocarb on the ultrastructure of the skeletal muscle in rabbits. Rabbits in all experimental groups received capsules of bendiocarb (96% Bendiocarb, Bayer, Germany) per os daily at a dose of 5 mg/kg body weight. Samples of skeletal muscles were collected on days 10 and 20. On day 10 of the experiment, muscle fibres were not affected consistently. The observed changes were moderate and focal. Electron microscopy revealed dilatation of sarcoplasmic reticulum, and myofilament disorganization. On day 20 of the experiment, the ultrastructural changes in muscle fibres were more intense and more frequent. The most important alteration was the disruption of the sarcomeres due to the lysis of both thick and thin myofilaments. However, in the unchanged regions of muscle fibres a prominent mitochondrial swelling was observed. Many mitochondria lacked cristae and thus appeared as large membrane-bound cytoplasmic vesicles. The results presented in this study indicate that bendiocarb affects the ultrastructure of skeletal muscles. The intensity of damage (dissolution of myofilaments and disruption of sarcomeres) was related to the duration of administration of bendiocarb.

Protein kinase C‐site phosphorylation of cardiac myosin binding protein‐C decreases cross‐bridge kinetics (1081.5)

Cardiac myosin binding protein‐C (cMyBP‐C) phosphorylation is necessary for the regulation of sarcomeric structure and myocardial contractility in vivo. Different from skeletal isoforms, cMyBP‐C has M‐domain phosphorylation motifs in which three serines, Ser273, 282 and 302, are known to be substrates for several kinases. Phosphorylation of these M‐domain sites regulates the interaction of cMyBP‐C with thick and thin myofilaments during muscle contraction. While protein kinase C (PKC) is known to phosphorylate Ser273 and Ser302, the precise functional consequences of PKC‐mediated cMyBP‐C phosphorylation are not fully characterized. Therefore, the objective of this study was to determine how PKC‐mediated phosphorylation at Ser273/302 might control the function of cMyBP‐C in the context of thick and thin myofilament regulation and sarcomere contraction. Using recombinant proteins and yeast‐2‐hybrid (Y2H) clones expressing Ser273‐Ser282‐Ser302 (cMyBP‐CWT), Ala273‐Asp282‐Ala302 (cMyBP‐CADA) or Asp273‐Ala282‐Asp302 (cMyBP‐CDAD), cosedimentation of C0C2 and Y2H of C1C2 assays with either cardiac myosin S2 region or sarcomeric actin revealed that PKC phosphorylation of Ser273/Ser302 in cMyBP‐C M‐domain is sufficient to maintain its interaction with myosin S2 region, but diminish its interaction with actin in vitro. Furthermore, mechanical studies using skinned ventricular fibers from cMyBP‐CDAD revealed that PKC phospho‐mimetic exhibits a significant decrease in active force, cross‐bridge kinetics and Mg2+‐ATPase activity and myofilament Ca2+ sensitivity in vitro, compared to the controls. Taken together, our studies demonstrated that two phosphorylatable (by PKC) serines in cMyBP‐C M‐domain enable the interaction between cMyBP‐C protein and myosin S2, but diminishes its interaction with actin, thus affecting the ability of this protein to regulate sarcomere contractility

Myopodin is an F-actin bundling protein with multiple independent actin-binding regions

The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric α-actinin, as well as its binding to actin, α-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of α-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.

Structural and ultrastructural changes of full‐cycle cultured Pacific bluefin tuna (Thunnus orientalis) muscle slices during chilled storage

This study examined the structural and ultrastructural changes of dorsal and ventral muscle tissues of full-cycle cultured Pacific bluefin tuna (PBT), Thunnus orientalis Temminck & Schlegel 1844, cut into slices simulating sashimi and placed in chilled storage for varying periods. Structural and ultrastructural changes were determined in order to understand the physical texture by breaking strength measurement. Progressive deterioration of myofibril structure was observed during chilled storage (4 °C) of PBT muscle slices over 5 days post mortem. Muscle degradation included detachment between myofibres, detachment of the plasmalemma, disruption of mitochondria, loss of Z-line density and alignment, cementation of myofibrils, loss of the hexagonal arrangement of thick versus thin myofilaments and migration of subsarcolemmal nuclei to intermyofibrillar spaces. Loss of myofibre-myofibre adhesion, detachment of the plasmalemma and disruption of other components did not lower the breaking strength of PBT muscle. This provides evidence that the muscle breaking strength of PBT is not only associated with the detachment of myofibres or detachment of the plasmalemma. Other factors that produce cement-like substances, such as cementation of the myofibrillar components and degradation of the sarcoplasmic reticulum, may also increase breaking strength.

Fine Architectonics and Protein Turnover Rate in Myofibrils of Glucocorticoid-Caused Myopathic Rats

Objective: The purpose of this study was to assess the relationship between the synthesis and degradation rate of thick and thin myofilaments, and changes in fine architectonics of myofibrils of glycolytic muscle fibers of glucocorticoid caused myopathic rats. Methods: Male Wistar rats receive dexamethasone to induce myopathy. Protein synthesis rate degradation and structure of myofibrils was measured. Results: Myofibrils from glycolytic muscle fibers of 16-week-old male rats of the Wistar Strain were used for analysis. Myofibrils of glucocorticoid caused myopathic rats were thinner than in the control group (1.10 ± 0.05 m2 and 2.56 ± 0.08 m2, respectively) and thick and thin myofilaments in myopathic muscle disappeared from 22.0 ± 2.7% of the myofibrils cross-section area. In the control group, myofilaments were absent from 1.8 ± 0.5%. In the myopathic group, a positive correlation was found between the area of myofibrils where myofilaments disappeared and the myofibrillar protein degradation rate (r=0.849; p

47‐YEAR‐OLD MAN WITH FRONTAL LOBE TUMOR

A 47 year-old man, who previously underwent epilepsy surgery in which silver clips had been used for hemostasis, presented with left extremity weakness. Investigation revealed a mass lesion of right frontal lobe near the place of previous surgery and where the silver clips resided. Intra-operative findings revealed a huge cere- bral tumor with poor blood supply and circumscribed by adjacent gliosis, with the silver clips located within the tumor parenchyma. Histologically, the tumor was composed of spindle-shaped cells with fusiform and tapered nuclei. The cells were positive for a-SMA, but negative for vimentin, pancytokeratin, EMA and CD34. Ultrastructurally, tumor cells had abundant dilated rER and thin myofilaments with local dense bodies, confirming the histo- logical diagnosis of Low-grade Myofibroblastic Sarcoma (LGM). LGM is a rare tumor with cerebral involvement being exception- ally unusual. Only one similar case has ever been described in the English-language literature. That our case was related to previ- ously placed silver clips in epilepsy surgery further adds to the novelty. Correspondence 232 Brain Pathology 21 (2011) 229–232 © 2011 The Authors; Brain Pathology © 2011 International Society of Neuropathology

Alterations in Caveolin Expression and Ultrastructure After Bladder Smooth Muscle Hypertrophy

Partial bladder outlet obstruction in male rabbits causes detrusor smooth muscle hypertrophy and voiding dysfunction similar to that observed in men with benign prostate hyperplasia. Using this model, we analyzed the protein expression and ultrastructure of caveolae and the intermediate size filament in detrusor smooth muscle following partial bladder outlet obstruction induced hypertrophy. Detrusor smooth muscle sections from bladder body were processed for immunofluorescence and electron microscopy. Western analysis was performed to determine the expression of caveolin isoform-1, 2 and 3, and intermediate size filament proteins. Detrusor smooth muscle cells from both normal and hypertrophied bladders contain orderly arrays of thick and thin myofilaments, interspersed with dense bodies. In addition, there was an increase in intermediate size filaments in the hypertrophic detrusor smooth muscle cells. The dense plaques in the inner membrane of hypertrophied detrusor smooth muscle were longer than those of the control. Detrusor smooth muscle from hypertrophied bladder revealed a decreased number of caveolae and a lack of their orderly distribution at the plasma membrane. Western blotting showed decreased expression of caveolin-1, 2 and 3 in hypertrophied detrusor smooth muscle. Caveolae serve as platforms for proteins and receptors that have a role in signal transduction. The decreased number of caveolae and caveolin protein expression in hypertrophied detrusor smooth muscle might contribute to alterations in signal transduction pathways that regulate the downstream effects of agonist induced contraction, including calcium sensitization, observed in obstructed bladder. In addition, the increased number of intermediate size filaments in the hypertrophied detrusor smooth muscle is likely to alter the cytoskeletal structure and affect the cellular transmission of passive and/or active force.

Surface and inner cell behaviour along skeletal muscle cell in vitro differentiation

During muscle tissue differentiation, in particular in the formation of myotubes from the myoblasts, plasma membrane changes its morpho-functional characteristics. In this study, muscle cell membrane behaviour has been studied along the differentiation of C2C12, a mouse myoblastic adherent cell line. Flat undifferentiated cells, cultured for 3–4 days in the differentiation medium, progressively become thick, long and multinucleated myotubes covered with microvilli. They lose stress fibers and adhesion to the underlying substrate evidentiating an actin redistribution, followed by the spatial organization of thick and thin myofilaments. Sarcomeres and myofibrils occasionally appear, even if a certain percentage of “myosacs” containing randomly oriented filaments can be identified all along the differentiation. M-cadherin, a molecule involved in cell–cell adhesion, also appears in the early differentiation stage, during myoblast fusion. Occasional focal contractions can also be observed in myotubes, which prompt an electrophysiological membrane analysis. When studied by means of patch clamp technique, resting membrane potential appears to undergo a transient depolarization, while input resistance increases until day 5 after differentiation induction, then successively decreases. Capacitance declines until day 5, later appearing enhanced. Moreover, with the induction of differentiation, the pattern of functional voltage-dependent ion channels changes. Therefore, during myogenesis, cell maturation is coupled with changes in cell membrane morphological features and functional characteristics.

The Effects of Sciatic Neurectomy on Rat Gastrocnemius Muscle

The effects of unilateral sciatic neurectomy on gastrocnemius muscle were studied in adult male rats. The morphological changes of both gastrocnemius muscles were observed by light and electron microscopy. Western blot analysis was performed to study the protein expression. Following the denervation, the affected muscle weights decreased significantly than normal. And the denervation led to a significant reduction in the area and diameter of muscle fibers on light microscopy. The affected muscle fibers showed the decreasing in size and the irregularity of myofibrilar arrangement on electron microscopy. On transverse view, the area of affected muscle fiber were smaller than normal. Their myofibrils were smaller and very irregular in size. The thin and thick myofilaments were not regular and partially lost. Mitochondria between myofibrils and subsarcolemmal area in affected muscle fibers were damaged and partially lost. The sacoplasmic reticulum and T-tubules were mostly lost and irregular. Some satellite cells were observed adjacent the muscle fiber, but they were inactive morphologically. On longitudinal view, most of myofibrils in affected muscle fibers were lost generally or partially although the their most sarcomeres were regularly arranged. Z line and myofilaments were lost partially and were partially irregularly arranged. The loss of thin myofilaments was more than that of thick myofilaments. Much debris of cell organelles were scattered among myofibrils. The expression of MyoD and myogenin were decrease significantly and the expression of p-mTOR and p-FOXO1 were decreased, too. On the other hand, MuRF1 was increased significantly. Taken together, the main effect of gastrocnemius muscle by sciatic neurectomy is the atrophy as a result of the loss of myofilaments and cell organelles through the decrease of protein synthesis and the increase of protein degradation.

Fibronexus in Low-Grade Myofibrosarcoma: A Case Report

Fibronexus (fibronexus junction) has been thought to be a characteristic ultrastructural feature of myofibroblasts, but it is controversial as to whether fibronexus is a characteristic of various myofibroblastic tumors. We report here a case of low-grade myofibrosarcoma with fibronexus arising in the right arm of an 80-year-old man. Histologically, the tumor was composed of relatively uniform and slender spindle cells arranged in fascicles. The nuclei with fusiform and tapered shapes were mildly hyperchromatic, but never exhibited pleomorphism. Mitotic figures were common, but no atypical mitosis was identified. At the tumor periphery, tumor cells had invaded into the surrounding skeletal muscle tissue. Tumor cells were positive diffusely for alpha-smooth muscle actin and less intensely for desmin, but were negative for h-caldesmon and S-100 protein. Ultrastructurally, tumor cells had well developed cytoplasmic organelles and varying amounts of peripheral or subplasmalemmal bundles of thin myofilaments with focal density. In addition, well formed, long fibronectin fibrils adjacent to the cell surface and fibronexus contacting intracellular myofilaments were easily identified. We believe that fibronexus is a useful ultrastructural feature for differentiating myofibrosarcoma from other myogenic sarcomas.

Abnormal recruitment of periendothelial cells to lymphatic capillaries in digestive organs of angiopoietin-2-deficient mice

The fine structure of lymphatic capillaries in the digestive organs of angiopoietin-2 (Ang2) knockout mice was studied by using both immunohistochemistry and electron microscopy. The genetic deletion of Ang2 yielded hypoplasia and disorganization of the lymphatic capillaries, with their shapes being irregular, and an aberrant recruitment of vascular periendothelial cells immunopositive for smooth muscle actin to the lymphatic capillaries. The abnormal lymphatic periendothelial cells were considered to be a type of pericyte for the lymphatic capillaries after the deletion of Ang2, because they were ultrastructurally characterized by abundant thin myofilaments in their cytoplasm and long cytoplasmic extensions similar to those shown by blood vascular pericytes. The genetic replacement of Ang2 with Ang1 rescued the defects, viz., the disorganization and disordered structure of the lymphatic capillaries. The present findings suggest that Ang2 serves the morphogenesis of lymphatic capillaries as an agonist for the receptor, Tie2, and that Ang1 can replace Ang2 in guiding lymphatic formation and development.

Molecular Determinants of Altered Ca 2+ Handling in Human Chronic Atrial Fibrillation

Abnormal Ca2+ handling may contribute to impaired atrial contractility and arrhythmogenesis in human chronic atrial fibrillation (cAF). Here, we assessed the phosphorylation levels of key proteins involved in altered Ca2+ handling and contractility in cAF patients. Total and phosphorylation levels of Ca2+-handling and myofilament proteins were analyzed by Western blotting in right atrial appendages of 49 patients in sinus rhythm and 52 cAF patients. We found a higher total activity of type 1 (PP1) and type 2A phosphatases in cAF, which was associated with inhomogeneous changes of protein phosphorylation in the cellular compartments, ie, lower protein kinase A (PKA) phosphorylation of myosin binding protein-C (Ser-282 site) at the thick myofilaments but preserved PKA phosphorylation of troponin I at the thin myofilaments and enhanced PKA (Ser-16 site) and Ca2+-calmodulin protein kinase (Thr-17 site) phosphorylation of phospholamban. PP1 activity at sarcoplasmic reticulum is controlled by inhibitor-1 (I-1), which blocks PP1 in its PKA-phosphorylated form only. In cAF, the ratio of Thr-35-phosphorylated to total I-1 was 10-fold higher, which suggests that the enhanced phosphorylation of phospholamban may result from a stronger PP1 inhibition by PKA-hyperphosphorylated (activated) I-1. Altered Ca2+ handling in cAF is associated with impaired phosphorylation of myosin binding protein-C, which may contribute to the contractile dysfunction after cardioversion. The hyperphosphorylation of phospholamban probably results from enhanced inhibition of sarcoplasmic PP1 by hyperphosphorylated I-1 and may reinforce the leakiness of ryanodine channels in cAF. Restoration of sarcoplasmic reticulum-associated PP1 function may represent a new therapeutic option for treatment of atrial fibrillation.