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Preserving morphological features that are important for cell function and structure is a critical parameter for in vitro experiments with rat cardiomyocytes. Lentiviral vectors are commonly used as gene transfer tool because of its high flexibility, efficiency to deliver expression cassettes and versatility of transducing quiescent cells. The tropism of the recombinant viral particle can be determined depending on the virus envelope, which shows a specific binding to cell surface receptors on the target cell. The combination of promoter arrangement and viral envelope must be optimized to achieve a greater transduction efficiency and a higher transgene expression. In this study we explored the optimization of promoters and heterologous envelopes to transduce primary culture of neonatal rat ventricular myocytes. Our results suggest a robust expression driven by the cytomegalovirus promoter, and high efficiency transduction mediated by VSV-G envelope with no apparent compromising ultrastructural features of genetically modified cells.

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The myotendinous junction (MTJ) is the muscle-tendon interface and constitutes an integrated mechanical unit to force transmission. Joint immobilization promotes muscle atrophy via disuse, while physical exercise can be used as an adaptative stimulus. In this study, we aimed to investigate the components of the MTJ and their adaptations and the associated elements triggered with aquatic training after joint immobilization. Forty-four male Wistar rats were divided into sedentary (SD), aquatic training (AT), immobilization (IM), and immobilization/aquatic training (IMAT) groups. The samples were processed to measure fiber area, nuclear fractal dimension, MTJ nuclear density, identification of telocytes, sarcomeres, and MTJ perimeter length. In the AT group, the maintenance of ultrastructure and elements in the MTJ region were observed; the IM group presented muscle atrophy effects with reduced MTJ perimeter; the IMAT group demonstrated that aquatic training after joint immobilization promotes benefits in the muscle fiber area and fractal dimension, in the MTJ region shows longer sarcomeres and MTJ perimeter. We identified the presence of telocytes in the MTJ region in all experimental groups. We concluded that aquatic training is an effective rehabilitation method after joint immobilization due to reduced muscle atrophy and regeneration effects on MTJ in rats.

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Knowing the ultrastructure of skeletal muscle is critical to understand how it works under normal situation and the disorders caused by extreme or pathological conditions. Sarcomere is the basic structural unit of striated muscle tissue. An important element of sarcomere architecture are the intermediate filaments, including the desmin protein. Desmin protein contributes to maintenance of cell integrity, efficient transmission of force and mechanochemical signaling within the myocyte. Because of this, desmin protein has constantly been a focus of research that investigates its alterations associated to damage and muscle atrophy under different conditions. The purpose of the following literature review is to describe the basic concepts of muscle ultrastructure, emphasizing the desmin protein role under conditions of muscle disuse atrophy and aging.

Conocer la ultraestructura del músculo esquelético es crítico para entender cómo trabaja bajo situaciones normales y en desórdenes causados por condiciones extremas o patológicas. La sarcómera es la unidad de estructura básica del tejido muscular estriado. Elementos importantes en la arquitectura de la sarcómera son los filamentos intermedios, incluyendo la proteína desmina. La proteína desmina contribuye en mantener la integridad celular, la transmisión eficiente de fuerza y la señalización mecanoquímica dentro del miocito. Debido a lo anterior, la proteína desmina ha sido constante foco de investigación en trabajos que estudian sus alteraciones asociadas a daño y atrofia muscular bajo diferentes condiciones. El propósito de la siguiente revisión de la literatura es describir los conceptos básicos de la ultraestructura muscular, enfatizando en el rol de la proteína desmina bajo condiciones de atrofia muscular por desuso y envejecimiento.

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Remobilization of a previously immobilized rat right hindlimb in the ankle plantar-flexion-shortened position by free movement alone or associated with intermittent passive stretching was assessed by analysis of gait variables and dorsiflexion range of motion. The variables were related with the expression of extracellular matrix proteins and the addition of serial sarcomeres. Sixty-four female Wistar rats were divided into 8 groups: immobilized, free for 10 days, immobilized/stretched/free for 1, 3 or 10 days, immobilized/free for 1, 3 or 10 days. Gait variables, range of motion, serial sarcomeres number, localization and staining intensity of fibronectin, and expressions of types I and III collagen were analyzed. The hypokinesia changed the functional variables of gait, reduced the dorsiflexion range of motion (ROM), increased the number of fibers with intracellular fibronectin/total number of fibers (FIF/TNF), and decreased the expression of the type I collagen. After three days, morphological changes were exacerbated and the number of serial sarcomeres was increased in both groups, immobilized/stretched/free and immobilized/free. Functional impairment, ROM restriction and increased FIF/TNF were also observed. Despite the above described alterations, 10 days of stretching program increased the effectiveness of remobilization leading to recovery of the abnormalities observed in the muscle.

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The cardiac lethal mutation in Mexican axolotl (Ambystoma mexicanum) results in a lack of contractions in the ventricle of mutant embryos. Previous studies have demonstrated that tropomyosin, a component of thin filaments, is greatly reduced in mutant hearts lacking myofibril organization. Confocal microscopy was used to examine the structure and comparative amount of tropomyosin at heartbeat initiation and at a later stage. The formation of functional sarcomeres coincided with contractions in normal hearts at stage 35. A-bands and I-bands were formed at stage 35 and did not change at stage 39. The widening of Z-bodies into z-lines was the main developmental difference between stage 35 and 39 normal hearts. Relative to normal hearts, a reduction of sarcomeric protein levels in mutant hearts at stage 35 was found, and a greater reduction occurred at later stages. The lower level of tropomyosin limited the areas where organized myofibrils formed in the mutant. The areas that had tropomyosin staining also had staining for alpha-actinin and myosin. Early myofibrils formed in these areas but the A-bands and I-bands were shorter than normal. At a later stage in the mutant, A-bands and I-bands remained shorter and importantly the Z-bodies also did not form wider z-lines.

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We investigated whether veratrine (5 microl, 10 ng/kg) injected into the mouse extensor digitorum longus (EDL) (fast-twitch) and soleus (SOL) (slow-twitch) muscles provokes distinctive ultrastructural disturbances 15, 30 and 60 min later. The mitochondria in SOL were affected earlier (within 15 min) than in EDL. Swelling of the sarcoplasmic reticulum terminal cisternae was more marked in EDL than in SOL and caused distortion of sarcomeres so that fragmentation of myofilaments was more pronounced in EDL. Hypercontracted sarcomeres were seen mainly in SOL and veratrine caused infoldings of the sarcolemma only in this muscle. In both muscles, the T-tubules remained unaffected and by 60 min after veratrine most of the above alterations had reverted to normal. Pretreatment with tetrodotoxin prevented the alterations induced by veratrine. This suggests that most of the alterations resulted from the enhanced influx of Na+ into muscle fibers. These results emphasize the importance of considering the type of muscle when studying the action of myotoxic agents.

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The effects of veratrine have been investigated in mammalian, amphibian, and crustacean muscle, but not in fish. In this work, the action of veratrine was studied in the lateral muscle of the freshwater teleost Oreochromis niloticus after intramuscular injection. Histoenzymological typing and electron microscopy of muscle fibers before and 15, 30, and 60 min after veratrine injection (10 ng/kg fish) were used to indirectly assess the morphological changes and the oxidative and m-ATPase activities. In some cases, muscles were pretreated with tetrodotoxin to determine whether the ultrastructural changes were the result of Na(+) channel activation by veratrine. Veratrine altered the metabolism of fibers mainly after 30 min. Oxidative fibers showed decreased NADH-TR activity, whereas that of glycolytic and oxidative-glycolytic type fibers increased. There was no change in the m-ATPase activity of the three fiber types, except at 60 min postveratrine, when a novel fiber type, which showed no reversal after acidic and alkaline preincubations, appeared. Ultrastructural damage involved sarcomeres, myofibrils, and mitochondria, but the T-tubules remained intact. Pretreatment with tetrodotoxin (1 ng/ml) prevented the ultrastructural changes caused by veratrine. These results show that in fish skeletal muscle veratrine produces some effects that are not seen in mammalian muscle.

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The degenerative process of the myofibers of the diaphragm of rats intoxicated with the organophosphate isofenphos, a compound that inhibits esterases, was studied at different intervals of intoxication. Early disorganization of the intermyofibrillar network and of the myofilaments, as well as dilatation of organelles, were observed by use of transmission electron microscopy. These changes precede macrophage invasion of the muscle fibers. Early expression of ubiquitin was observed in segments of muscle fibers by immunohistochemistry. Bands of polyubiquitin complexes in muscle homogenates were observed by immunoblotting. These bands disappeared in later stages of intoxication. A 42.5-kDa band corresponds to actin, as observed by immunoblotting using antisarcometric actin. This indicates relatively large amounts of polyubiquitin complex associated with sarcomeric actin in muscle fibers in early stages of intoxication. Based on these results it seems that actin is an important target in organophosphate-induced myofiber degradation and that the degradation of this protein-by the polyubiquitin pathway-may play an important role in the early disorganization of the sarcomere, as observed by electron microscopy. A possible role of the ubiquitin proteolytic pathway is that of trying to eliminate proteins modified in the early phases of muscle fiber degeneration, which is a necessary step for regeneration of the posterior segmental muscle.

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In a wide variety of cellular settings, from organelle transport to muscle contraction, Ca2+ binding to members of the EF hand family of proteins controls the interaction between actin and different myosins that are responsible for generating movement. In vertebrate skeletal and cardiac muscle the Ca(2+)-binding protein troponin C (TnC) is one subunit of the ternary troponin complex which, through its association with actin and tropomyosin on the thin filament, inhibits the actomyosin interaction at submicromolar Ca2+ concentrations and stimulates the interaction at micromolar Ca2+ concentrations. Because TnC does not interact directly with actin or tropomyosin, the Ca(2+)-binding signal must be transmitted to the thin filament via the other two troponin subunits: troponin I (TnI), the inhibitory subunit, and troponin T (TnT), the tropomyosin-binding subunit. Thus, the troponin complex is a Ca(2+)-sensitive molecular switch and the structures of and interactions between its components have been of great interest for many years. Although the crystal structure of TnC has been known for almost a decade, the molecular structures of TnI and TnT are not known and therefore convincing models of the organization of the troponin complex and the Ca(2+)-induced changes in its structure have not been forthcoming. Recent advances on a wide variety of fronts including 1) the bacterial expression and characterization of mutants of TnC, TnI, and TnT; 2) cross-linking and fluorescence studies; and 3) the determination of the crystal and nuclear magnetic resonance structures of synthetic and recombinant troponin fragments and complexes between EF hand proteins and their target peptides have provided new insights into the nature of the interactions between troponin subunits. This review discusses these recent advances with the aim of critically assessing molecular models of the nature of the Ca(2+)-induced structural transition in troponin.

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Seventeen patients with coronary disease submitted to myocardial revascularization were studied. Ten patients had a hypertrophied ventricle, and 7 had normal ventricular mass. Myocardial biopsies were obtained before ischemia and at the time of reperfusion and were assessed for: volume fraction of fibrous tissue, myocyte diameter, morphometric mitochondrial studies and ultrastructural changes. The volume fraction of fibrous tissue in patients with hypertrophied ventricle was 1.9 +/- 0.04, and in patients with normal ventricular mass was 0.9 +/- 0.01 (p less than 0.05). The diameter of the myocyte was 23 +/- 0.3 microns and 18 +/- 1.2 microns for patients with hypertrophied and normal ventricular mass, respectively (p less than 0.01). The value of volumetric density for pre-ischemia samples in patients with a hypertrophied ventricle was 23 +/- 2.2 and in patients with normal ventricular mass was 35 +/- 2.7 (p less than 0.02). Grades 3 and 4 of damaged mitochondria were significantly increased in reperfusion samples from patients with a hypertrophied ventricle compared to pre-ischemia samples. Collagen growth was increased in hypertrophied hearts which were also more sensitive to the ischemia/reperfusion mechanism.

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