CaMKII activation in early diabetic hearts induces altered sarcoplasmic reticulum-mitochondria signaling.

Prediabetic myocardium, induced by fructose-rich diet (FRD), is prone to increased sarcoplasmic reticulum (SR)-Ca2+ leak and arrhythmias due to increased activity of the Ca2+/calmodulin protein kinase II (CaMKII). However, little is known about the role of SR-mitochondria microdomains, mitochondrial structure, and mitochondrial metabolisms. To address this knowledge gap we measured SR-mitochondrial proximity, intracellular Ca2+, and mitochondrial metabolism in wild type (WT) and AC3-I transgenic mice, with myocardial-targeted CaMKII inhibition, fed with control diet (CD) or with FRD. Confocal images showed significantly increased spontaneous Ca2+ release events in FRD vs. CD WT cardiomyocytes. [3H]-Ryanodine binding assay revealed higher [3H]Ry binding in FRD than CD WT hearts. O2 consumption at State 4 and hydrogen peroxide (H2O2) production rate were increased, while respiratory control rate (RCR) and Ca2+ retention capacity (CRC) were decreased in FRD vs. CD WT isolated mitochondria. Transmission Electron Microscopy (TEM) images showed increased proximity at the SR-mitochondria microdomains, associated with increased tethering proteins, Mfn2, Grp75, and VDAC in FRD vs. CD WT. Mitochondria diameter was decrease and roundness and density were increased in FRD vs. CD WT specimens. The fission protein, Drp1 was significantly increased while the fusion protein, Opa1 was unchanged in FRD vs. CD WT hearts. These differences were prevented in AC3-I mice. We conclude that SR-mitochondria microdomains are subject to CaMKII-dependent remodeling, involving SR-Ca2+ leak and mitochondria fission, in prediabetic mice induced by FRD. We speculate that CaMKII hyperactivity induces SR-Ca2+ leak by RyR2 activation which in turn increases mitochondria Ca2+ content due to the enhanced SR-mitochondria tethering, decreasing CRC.

Relative lipid oxidation associates directly with mitochondrial fusion phenotype and mitochondria-sarcoplasmic reticulum interactions in human skeletal muscle.

Disturbances in skeletal muscle lipid oxidation might induce ectopic fat deposition and lipotoxicity. Nevertheless, the cellular mechanisms that regulate skeletal muscle lipid oxidation have not been fully determined. We aimed to determine whether there was an association between relative whole body lipid oxidation and mitochondrial size or mitochondria-sarcoplasmic reticulum interactions in the skeletal muscle. Twelve healthy men were included [mean (standard deviation), 24.7 (1.5) yr old, 24.4 (2.6) kg/m2]. The respiratory quotient (RQ) was used to estimate relative lipid oxidation at rest and during exercise (50% maximal oxygen consumption, 600 kcal expended). A skeletal muscle biopsy was obtained from the vastus lateralis at rest. Transmission electron microscopy was used to determine mitochondrial size and mitochondria-sarcoplasmic reticulum interactions (≤50 nm of distance between organelles). Protein levels of fusion/fission regulators were measured in skeletal muscle by Western blot. Resting RQ and exercise RQ associated inversely with intermyofibrillar mitochondrial size (r = -0.66 and r = -0.60, respectively, P < 0.05). Resting RQ also associated inversely with the percentage of intermyofibrillar mitochondria-sarcoplasmic reticulum interactions (r = -0.62, P = 0.03). Finally, intermyofibrillar mitochondrial size associated inversely with lipid droplet density (r = -0.66, P = 0.01) but directly with mitochondria fusion-to-fission ratio (r = 0.61, P = 0.03). Our results show that whole body lipid oxidation is associated with skeletal muscle intermyofibrillar mitochondrial size, fusion phenotype, and mitochondria-sarcoplasmic-reticulum interactions in nondiabetic humans.

Muscle function decline and mitochondria changes in middle age precede sarcopenia in mice.

Sarcopenia is the degenerative loss of muscle mass and strength with aging. Although a role of mitochondrial metabolism in muscle function and in the development of many diseases has been described, the role of mitochondrial topology and dynamics in the process of muscle aging is not fully understood. This work shows a time line of changes in both mitochondrial distribution and skeletal muscle function during mice lifespan. We isolated muscle fibers from flexor digitorum brevis of mice of different ages. A fusion-like phenotype of mitochondria, together with a change in orientation perpendicular to the fiber axis was evident in the Adult group compared to Juvenile and Older groups. Moreover, an increase in the contact area between sarcoplasmic reticulum and mitochondria was evident in the same group. Together with the morphological changes, mitochondrial Ca2+ resting levels were reduced at age 10-14 months and significantly increased in the Older group. This was consistent with a reduced number of mitochondria-to-jSR pairs in the Older group compared to the Juvenile. Our results support the idea of several age-dependent changes in mitochondria that are accentuated in midlife prior to a complete sarcopenic phenotype.

El Diazepam altera la ultraestructura del atrio fetal de ratón / Diazepam alters the ultrastructure of fetal atrium in rat

El diazepam (DZ) es un tranquilizante menor sintético, utilizado en pacientes con trastornos psicológicos y psiquiátricos. Es sedante, miorrelajante, anticonvulsionante y antipsicótico. El DZ atraviesa la barrera placentaria humana y la del ratón. Mujeres jóvenes que son adictas al fármaco, si se embarazan y continúan utilizándolo, sobre todo durante el primer trimestre, exponen a sus hijos a presentar alteraciones psicomotoras. El propósito de este trabajo fue investigar si el DZ administrado durante la gestación,induce alteraciones ultraestructurales del miocardio fetal de ratón. El grupo (DZ) de hembras gestantes deratón de la cepa CD-1 fue tratado con dosis únicas diarias de 1,0 mg/kg/pc/sc del día 6 al 17 y un grupo (C)que recibió solución salina. El día 18 las hembras de ambos grupos se anestesiaron, los fetos se perfundieron por vía intracardiaca con paraformaldehído al 1 % y glutaraldehido al 2,5 %, se les extrajo el corazón, se disecó el atrio, se fijó en OsO4 al 1 % y se incluyó en resina epóxica. Los cortes finos se contrastaron conacetato de uranilo y citrato de plomo y se observaron en un microscopio electrónico de transmisión. En los miocitos de los fetos del grupo DZ las sarcómeras del miocardio compacto tenían menor longitud que las del grupo C. Se observaron zonas con miofibrillas desorganizadas. El retículo sarcoplásmico de algunos miocitos presentaba cisternas distendidas y fragmentadas, mitocondrias alteradas y se observaron abundantes polirribosomas. Los cambios podrían deberse al efecto del DZ sobre la síntesis de actina y miosina pesada y sobre los organelos citoplásmicos, mediados por receptores benzodiazepínicos periféricos presentes en la membrana externa de las mitocondrias y asociados a canales de calcio dependientes de voltaje. Las alteraciones ultraestructurales del miocardio atrial de fetos de ratones expuestos in utero a DZ podrían tener efectos posnatales.

Diazepam (DZ) is a syntheticminor tranquilizer, used in patients with psychologicaland psychiatric disorders. It is a relaxing sedative,anticonvulsant and antipsychotic. DZ crosses thehuman placental barrier in mouse. Young women who are addicted to the drug, if they become pregnantand continue to use it, particularly during the firsttrimester, expose their children to psychomotor disorders. The purpose of this study was to investigate whether DZ administered during pregnancy induces ultrastructural alterations of fetal mouse myocardium.The group (DZ) of pregnant female mice of the CD-1strain was treated with a single daily dose of 1.0 mg/ kg / pc / sc of day 6 to 17 and a group (C) that received saline solution. On day 18 females of bothgroups were anesthetized, the fetuses were perfusedby intracardiac route with 1 % paraformaldehyde and 2.5 % glutaraldehyde, the heart was removed, theatrium was dissected, fixed in 1 % OsO4, it wasimmersed in epoxy resin. The fine sections werecontrasted with uranyl acetate and lead citrate and observed in a transmission electron microscope. Inthe myocytes of the fetuses of the DZ group, the sarcomers of the compact myocardium were shorter than those of the C group. Areas with disorganized myofibrils were observed. The sarcoplasmic reticulumof some myocytes had distended and fragmented 996cisterns, altered mitochondria, and abundant polyribosomes were observed. The changes may bedue to the effect of DZ on the synthesis of actin and heavy myosin and on cytoplasmic organelles mediatedby peripheral benzodiazepine receptors present onthe outer membrane of the mitochondria and associated with voltage-dependent calcium channels.Ultrastructural alterations of the atrial myocardium of fetuses of mice exposed to DZ in utero may have postnatal effects.

Mastoparan effects in skeletal muscle damage: An ultrastructural view until now concealed.

Animal venoms have been valuable sources for development of new drugs and important tools to understand cellular functioning in health and disease. The venom of Polybia paulista, a neotropical social wasp belonging to the subfamily Polistinae, has been sampled by headspace solid phase microextraction and analyzed by gas chromatography-mass spectrometry. Recent study has shown that mastoparan, a major basic peptide isolated from the venom, reproduces the myotoxic effect of the whole venom. In this study, Polybia-MPII mastoparan was synthesized and studies using transmission electron microscopy were carried out in mice tibial anterior muscle to identify the subcellular targets of its myotoxic action. The effects were followed at 3 and 24 h, 3, 7, and 21 days after mastoparan (0.25 mug/muL) intramuscular injection. The peptide caused disruption of the sarcolemma and collapse of myofibril arrangement in myofibers. As a consequence, fibers presented heteromorphic amorphous masses of agglutinated myofilaments very often intermingled with denuded sarcoplasmic areas sometimes only surrounded by a persistent basal lamina. To a lesser extent, a number of fibers apparently did not present sarcolemma rupture but instead appeared with multiple small vacuoles. The results showed that sarcolemma, sarcoplasmic reticulum (SR), and mitochondria were the main targets for mastoparan. In addition, a number of fibers showed apoptotic-like nuclei suggesting that the peptide causes death both by necrosis and apoptosis. This study presents a hitherto unexplored view of the effects of mastoparan in skeletal muscle and contributes to discuss how the known pharmacology of the peptide is reflected in the sarcolemma, SR, mitochondria, and nucleus of muscle fibers, apparently its subcellular targets.

Alterations in the ultrastructure of cardiac autonomic nervous system triggered by crotoxin from rattlesnake (Crotalus durissus cumanensis) venom.

This study explored the toxic effects of crotoxin isolated from Crotalus durissus cumanensis venom on the ultrastructure of mice cardiac autonomic nervous system. Mice were intravenously injected with saline (control group) and crotoxin diluted in saline venom (study group) at a dose of 0.107 mg/kg mouse body weight. Samples from the inter-ventricular septum were prepared for electron microscopy after 6 h (G1), 12 h (G2), 24 h (G3) and 48 h (G4). The G1 group showed some cardiomyocyte with pleomorphic mitochondria. Capillary swollen walls, nerve cholinergic endings with depleted acetylcholine vesicles in their interior and other depletions were observed. A space completely lacking in contractile elements was noticed. The G2 group demonstrated a myelinic figure, a subsarcolemic region with few myofibrils and nervous cholinergic terminal with scarce vacuoles in their interior. The G3 group demonstrated a structure with a depleted axonic terminal, mitochondrias varying in size and enhanced electron density. In addition, muscular fibers with myofibrillar structure disorganization, a depleted nervous structure surrounded by a Schwann cell along with an abundance of natriuretic peptides, were seen. An amyelinic terminal with depleted Schwann cell and with scarce vesicles was also observed. Finally, axonic lysis with autophagic vacuoles in their interior and condensed mitochondria was observed in the G4 group. This work describes the first report of ultrastructural damage caused by crotoxin on mice cardiac autonomic nervous system.

A transverse tubule NADPH oxidase activity stimulates calcium release from isolated triads via ryanodine receptor type 1 S -glutathionylation.

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.

Cross-talk between the sarcoplasmic reticulum and the mitochondrial calcium handling systems may play an important role in the regulation of contraction in anococcygeus smooth muscle.

Mitochondrial Ca(2+) and its relation with the contraction induced by phenylephrine was investigated. In normal Ca(2+), carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone (FCCP) and oligomycin produced contraction similar to that promoted by phenylephrine. Phenylephrine-induced contraction was reduced by FCCP+oligomycin. In Ca(2+)-free, FCCP+oligomycin did not induce contraction. Response to FCCP+oligomycin was reduced upon Ca(2+) repletion and this response was lower than that to phenylephrine. Ca(2+) concentration was increased by FCCP+oligomycin. Since a profuse net of sarcoplasmic reticulum encloses mitochondria, a cross-talk between the two organelles may play an important role in the phenylephrine-induced contraction in presence of Ca(2+) encountered in both sarcoplasmic reticulum and extracellular medium of anococcygeus cells.

Protective role of antioxidant vitamin E and catechin on idarubicin-induced cardiotoxicity in rats.

Idarubicin is an anthracycline antibiotic extensively used in acute leukemia. In the present study we investigated whether vitamin E and catechin can reduce the toxic effects of idarubicin. Vitamin E (200 IU kg(-1) week(-1)), catechin (200 mg kg(-1) week(-1)), idarubicin (5 mg kg(-1) week(-1)), idarubicin + vitamin E (200 IU kg(-1) week(-1)), and idarubicin + catechin (200 mg kg(-1) week(-1)) combinations were given to male Sprague-Dawley rats weighing 210 to 230 g (N = 6/group). Idarubicin-treated animals exhibited a decrease in body and heart weight, a decrease in myocardial contractility, and changes in ECG parameters (P<0.01). Catechin + idarubicin- and vitamin E + idarubicin-treated groups exhibited similar alterations, but changes were attenuated in comparison to those in cardiac muscle of idarubicin-treated rats (P<0.05). Superoxide dismutase and catalase activity was reduced in the idarubicin-treated group (P<0.05). Glutathione peroxidase levels were decreased in the idarubicin-treated group (P<0.05) and reached maximum concentrations in the catechin- and catechin + idarubicin-treated groups compared to control (P<0.01). Malondialdehyde activity was decreased in the catechin + idarubicin-treated groups compared to control and increased in the other groups, reaching maximum concentrations in the vitamin E-treated group (P<0.01). In electron microscopy studies, swelling of the mitochondria and dilatation of the sarcoplasmic reticulum of myocytes were observed in the idarubicin-treated groups. In groups that were given idarubicin + vitamin E and idarubicin + catechin, the only morphological change was a weak dilatation of the sarcoplasmic reticulum. We conclude that catechin and vitamin E significantly reduce idarubicin-induced cardiotoxicity in rats.

Potential definition of the time of death from autolytic myocardial cells: a morphometric study.

Morphometric methods and transmission electron microscopy were used to quantify modifications occurring in the mitochondria of dog myocardium during the first four hours of autolysis. Myocardial fragments were obtained from the outer free wall of the left ventricle, during anesthesia (control-zero) and at 15, 45, 120, and 240 min after cardiac arrest, maintaining the heart "in situ" at 22 degrees C. During the 240 min of autolysis, the main parameters evaluated showed: (a) a decrease in the number of mitochondria from 0.31 to 0.12 per micron 3 of cytoplasm. The decrease over the first 45 min reached 50% of the initial value; (b) an increase in mitochondrial volume, three times greater after the first 45 min (from 0.92 to 2.68 micron 3) and four times greater after 240 min (from 0.92 to 3.79 micron 3); (c) an increase in mitochondrial outer membrane surface area from 5.51 to 12.54 micron 2; (d) an increase in the surface area of individual mitochondria inner membrane and cristae from 27.60 to 56.96 micron 2. The progressive nature of the alterations and the difference in the numerically expressed values allow correlation with the time of somatic death. The authors emphasize the need for further studies in order to complement the present study.

Ultrastructural aspects of the retractor ocular bulbi muscle in the opossum (Didelphis albiventris).

In the present study the different types of muscle fibers of the retractor ocular bulbi muscle of the South American opossum were classified according to their ultrastructural characteristics. The tridimensional characteristics of the neuromuscular junctions present in this muscle were also demonstrated by scanning electron microscopy (SEM). Five adult opossums, three males and two females, were perfused with fixative solution through the left ventricle and their right retractor ocular bulbi muscle was prepared for the ultrastructural study of muscle fibers. The contralateral muscle was used for the study of neuromuscular junctions by SEM. Three types of fibers were detected, denoted 1r, 2r and 3r. Only simple neuromuscular junctions of the plate type were visualized by SEM.

Sarcoplasmic lipase and non-specific esterase inhibition in myofibers of rats intoxicated with the organophosphate isofenphos.

The expression of sarcoplasmic esterases, lipases as well as the lipid content in the myofibers of the diaphragm of rats intoxicated with the organophosphate isofenphos was studied. Lipid accumulation was documented at light, electron microsopic and by morphometric studies. The distribution of these lipid droplets was irregular and abundant in myofibers with numerous mitochondria (predominantly oxidative fibers). Histochemical inhibition of sarcoplasmic esterases and lipases was observed in the intoxicated animals. This sarcoplasmic inhibition of esterases occurs roughly in parallel to the inhibition of plasma cholinesterase activity. The inhibition of sarcoplasmic lipases may explain, at least partially, the accumulation of lipids. This inhibition probably makes difficult the use of lipids as fuel, especially in the oxidative fibers. In contrast to the small amount of muscle necrosis, (1.30+/-0.745), metabolic muscle impairment was intense and extensive, i.e., decreased activities of esterases and lipases in the sarcoplasm, that should contribute to muscle weakness. Therefore, because segmental necrosis was most prominent in oxidative fibers (and these fibers use lipids as the principal fuel and contain the greater amount of lipases in the sarcoplasm), it is possible that inhibition of activity of lipases is responsible for the segmental necrosis. Although the exact role of these metabolic changes is not known, it is possible that they contribute not only to the induction and evolution of muscle cell necrosis but also to the muscle weakness and clinical impairment of animals and humans in the acute intoxication by these compounds.

Functional evidence of a transmembrane channel within the Ca2+ transport ATPase of sarcoplasmic reticulum.

Ca2+ efflux can be studied conveniently following dilution of sarcoplasmic reticulum (SR) vesicles preloaded with 45Ca2+ by active transport. The rates of efflux are highly dependent on ATPase substrates and cofactors (Pi, Mg2+, Ca2+ and ADP) in the efflux medium. On the other hand, phenothiazines stimulate efflux through a passive permeability channel with no coupled catalytic events. Efflux activation by manipulation of catalytically active ATPase ligands, as well as by the catalytically inactive phenothiazines, can be prevented by thapsigargin, which is a highly specific inhibitor of the Ca(2+)-ATPase. This demonstrates that the passive channel activated by phenothiazines is an integral part of the ATPase, and can operate either uncoupled or coupled to catalytic events.

Thermodynamics of Ca2+ transport through sarcoplasmic reticulum membranes during the transient-state of simulated reactions.

The kinetics of a chemical model of Ca2+ transport and coupled ATPase activity in sarcoplasmic reticulum membranes were solved for the transient-state of simulated reactions, using a numerical integration procedure. The simulation conditions reproduced in vitro experiments using either fragmented membranes or vesicles with Ca2+ accumulating ability. The results yielded the concentrations of all the ligands and intermediates of the enzymatic cycle as a function of the reaction time. These results were applied to calculations of several thermodynamic variables: (1) the step by step profile of the standard free energy change of the cycle. (2) The step by profile of the actual free energy change of the cycle, and its evolution with the reaction time. (3) The separate contributions of ATP hydrolysis and Ca2+ transport to the overall free energy change with the reaction. (4) The dependence of the velocity of the free energy change with the reaction time. (5) The efficiency of the transport system, and its change with the reaction time. (6) The separate contributions of the Ca2+ gradient and some enzymatic intermediates as free energy stores. The main findings are: (1) the step by step diagrams of the free energy change calculated from the results of the kinetic analysis better describe the thermodynamic profile of the cycle than previously reported diagrams of the standard free energy and basic free energy changes. The relative contribution of each partial step to the driving force of the whole reactions, as well as their changes upon the advancement of the reactions, are derived from the diagrams. (2) Free energy yielded by ATP hydrolysis is stored by the system, not only as a Ca2+ gradient, but also as enzymatic intermediates of the reaction. The progressive increase of both free energy pools upon the advancement of the reaction is quantitated.

Ultrastructural characteristics of different stages of human chagasic myocarditis.

Myocardial damage was analyzed in the different stages of Chagas' disease. Myocardial biopsies from chagasic patients, whose clinical histories were initially unknown to the examiner, were examined and evaluated by electron microscopy using a table in which 244 characteristics were considered. When the ultrastructural results were associated with their respective clinical reports, it was found that Chagas' disease stages showed significant myocardium damage between stages IA and II: IA (normal EKG and cineventriculogram), 13.0%; IB (early left ventricular damage with normal EKG), 14.5%; II (advanced left ventricular damage, abnormal EKG), 25.5%; stage III (congestive heart failure) showed a decrement to 22.5% if compared with stage II. The acute stage of the illness was characterized by the presence of the parasites within the myocytes which were surrounded by inflammatory cell infiltrate. During Chagas' disease evolution the most affected organelle was the sarcoplasmic reticulum.

Cardiac sarcoplasmic reticulum characteristics in hypertrophic hearts from spontaneously hypertensive rats.

Sarcoplasmic reticulum (SR) from left ventricles of rats that developed spontaneous hypertension was studied in vitro. Similar increases of left ventricular mass were found when grouping the animals into mild and severe hypertensives (average systolic arterial pressure of 168 +/- 4 and 202 +/- 6 mmHg, respectively). The amount of SR protein (mg/g of left ventricle) was higher when obtained from hypertrophic ventricles of both hypertensive groups than from ventricles of the control group. The result agreed with the enhanced Ca2+ uptake exhibited by left ventricular homogenates of the hypertensive groups. Consequently, Ca2+ uptake in SR microsomes isolated per gram of left ventricle (nmol Ca2+/g muscle) was 51.62 +/- 10.06 and 64.99 +/- 12.84 in mildly and severely hypertensive groups vs. 17.37 +/- 5.79 in the control group (P less than 0.05). The SR microsomes obtained from ventricles of hypertensive rats showed an enhanced Ca2+ activated ATPase activity that was not accompanied by increased Ca2+ uptake at saturating calcium concentrations, but by increased affinity for calcium (K'app. of 1.09 +/- 0.28 and 2.67 +/- 0.16 microM in SR microsomes of hypertrophic and control ventricles respectively; P less than 0.05). The rates of calcium loss, measured in SR vesicles passively loaded with 45Ca, were similar when assayed in SR obtained from ventricles of both hypertensive and normal rats. These results enable us to suggest that in hearts of rats presenting spontaneous hypertension, the function of the SR system could account for a normal handling of cytosolic calcium. They might support the absence of mechanical alterations described in hearts of young rats of the SHR strain.

Aspectos ultra-estruturais das tríades no músculo deltóide de pacientes com a doença de Basedow-Graves / Ultrastructural aspects of the triads in the deltoid muscle of patients with Basedow-Graves' disease

Analisa-se a ultra-estrutura das tríades no músculo deltóide de pacientes com a doença de Basedow-Graves, comparando-a com a de indivíduos normais. Detectaram evidentes dilataçöes em alguns túbulos e cisternas que constituem as referidas tríades, nos músculos de pacientes com a doença