Skeletal muscle fiber hypercontraction induced by Bothrops asper myotoxic phospholipases A2 ex vivo does not involve a direct action on the contractile apparatus.

Myonecrosis is a frequent clinical manifestation of envenomings by Viperidae snakes, mainly caused by the toxic actions of secreted phospholipase A2 (sPLA2) enzymes and sPLA2-like homologs on skeletal muscle fibers. A hallmark of the necrotic process induced by these myotoxins is the rapid appearance of hypercontracted muscle fibers, attributed to the massive influx of Ca2+ resulting from cell membrane damage. However, the possibility of myotoxins having, in addition, a direct effect on the contractile machinery of skeletal muscle fibers when internalized has not been investigated. This question is here addressed by using an ex vivo model of single-skinned muscle fibers, which lack membranes but retain an intact contractile apparatus. Rabbit psoas skinned fibers were exposed to two types of myotoxins of Bothrops asper venom: Mt-I, a catalytically active Asp49 sPLA2 enzyme, and Mt-II, a Lys49 sPLA2-like protein devoid of phospholipolytic activity. Neither of these myotoxins affected the main parameters of force development in striated muscle sarcomeres of the skinned fibers. Moreover, no microscopical alterations were evidenced after their exposure to Mt-I or Mt-II. In contrast to the lack of effects on skinned muscle fibers, both myotoxins induced a strong hypercontraction in myotubes differentiated from murine C2C12 myoblasts, with drastic morphological alterations that reproduce those described in myonecrotic tissue in vivo. As neither Mt-I nor Mt-II showed direct effects upon the contractile apparatus of skinned fibers, it is concluded that the mechanism of hypercontraction triggered by both myotoxins in patients involves indirect effects, i.e., the large cytosolic Ca2+ increase after sarcolemma permeabilization.

Alterations of the skeletal muscle contractile apparatus in necrosis induced by myotoxic snake venom phospholipases A2: a mini-review.

Skeletal muscle necrosis is a common clinical manifestation of snakebite envenoming. The predominant myotoxic components in snake venoms are catalytically-active phospholipases A2 (PLA2) and PLA2 homologs devoid of enzymatic activity, which have been used as models to investigate various aspects of muscle degeneration. This review addresses the changes in the contractile apparatus of skeletal muscle induced by these toxins. Myotoxic components initially disrupt the integrity of sarcolemma, generating a calcium influx that causes various degenerative events, including hypercontraction of myofilaments. There is removal of specific sarcomeric proteins, owing to the hydrolytic action of muscle calpains and proteinases from invading inflammatory cells, causing an initial redistribution followed by widespread degradation of myofibrillar material. Experiments using skinned cardiomyocytes and skeletal muscle fibers show that these myotoxins do not directly affect the contractile apparatus, implying that hypercontraction is due to cytosolic calcium increase secondary to sarcolemmal damage. Such drastic hypercontraction may contribute to muscle damage by generating mechanical stress and further sarcolemmal damage.

Basal wall hypercontraction of Takotsubo cardiomyopathy in a patient who had been diagnosed with dilated cardiomyopathy: a case report.

BACKGROUND: Takotsubo cardiomyopathy is characterized by the basal hypercontractility and apical ballooning of the left ventriculum and T-wave inversion in the electrocardiogram. It has been suggested that Takotsubo cardiomyopathy might underlie the pathogenesis of persistent cardiac dysfunction; however, few reports are present demonstrating the advent of Takotsubo cardiomyopathy in patients with idiopathic cardiomyopathy. CASE PRESENTATION: A 64-year-old women was admitted due to dyspnea on effort and lower extremity edema. She had been diagnosed with idiopathic dilated cardiomyopathy 2.5 years before owing to the reduced left ventricular ejection fraction (24%), normal coronary artery, and interstitial fibrosis of the myocardial samples. On admission, her electrocardiogram showed giant negative T wave in II, III, aVF, and precordial leads. Echocardiography showed dyskinesis of the left ventricular apex and hypercontraction of the basal wall, which had not been observed in the previous examinations. Coronary angiography showed normal coronary arteries, and apical ballooning and basal hypercontractility was confirmed by left ventriculography. On day 15 of admission, contraction of apical wall was recovered, and basal hypercontraction was disappeared. CONCLUSION: The present case is the first report demonstrating appearance the transient basal wall hypercontraction along with the advent of Takotsubo cardiomyopathy in a patient diagnosed with dilated cardiomyopathy. Whether such findings are indicative of fair prognosis and have the utility of understanding the pathogenesis of dilated cardiomyopathy needs further investigation.

Eugenol and carvone as relaxants of arsenic and mercury hypercontracted rat trachea.

Exposure to arsenic and mercury is known to cause respiratory problems in both humans and animals. In this study, we elicit and compare maximum contraction caused by As(III) and Hg(II) when the pollutants are fully equilibrated with contractile machinery in resting mode. Hypercontraction of 27% and 69% was obtained following exposure of tracheal rings to 25 µM As(III) and 6 nM Hg(II) for 40 min, respectively. Co-incubation of tracheal rings with pollutants and verapamil, sodium nitroprusside or apocynin indicates that major contributors to As(III) and Hg(II) caused hypercontraction are reactive oxygen species (ROS) elevation and nitric oxide (NO) depletion. Changes in calcium influx have minor contribution in As(III) and Hg(II) caused increased contraction of tracheal tissues. Eugenol and carvone caused relaxation of 38% and 45% in pollutant unexposed rings, 56% and 49% in As(III)-exposed tracheal rings, and 54% and 47% in Hg(II)-exposed tracheal rings. Pathway delineation studies indicate that the major effect of eugenol originates from quenching of ROS whereas that of carvone originates from the blockage of extracellular calcium influx. Both molecules also show a minor stimulatory effect on NO generation. In line with their suggested mode of relaxation, eugenol is found to better ameliorate both As(III)- and Hg(II)-caused hypercontraction. Carvone, though a better relaxant than eugenol, comes out as poor ameliorator of both As(III)- and Hg(II)-caused hypercontraction, as the pathway on which it acts is not elevated following exposure to these pollutants.

Quercetin and resveratrol ameliorate nickel-mediated hypercontraction in isolated Wistar rat aorta.

PURPOSE: The ameliorative potential of quercetin and resveratrol on isolated endothelium-intact aortic rings incubated with nickel was examined. METHOD: The effect of varying concentrations of quercetin and resveratrol was investigated on isolated Wistar rat aortic rings using an organ bath system over vasoconstrictor phenylephrine (PE) at 1 µM. To delineate the mechanism of action, isolated aortic rings were pre-incubated with pharmacological modulators, such as verapamil 1 µM, apocynin 100 µM, indomethacin 100 µM or N-G-nitro-L-arginine methyl ester (L-NAME) 100 µM, separately, before incubation with 100 µM quercetin and 30 µM resveratrol. To assess the ameliorative and prophylactic potentials of quercetin and resveratrol, aortic rings were also incubated with quercetin or resveratrol for 40 min, followed by incubation with nickel for 40 min. RESULTS: At 100 µM, quercetin caused 29% inhibition of contraction, while resveratrol at 30 µM caused 55% inhibition of contraction in aortic rings compared with control. Aortic rings incubated with contractile modulators, such as verapamil, apocynin, indomethacin or N-G-nitro-L-arginine methyl ester (L-NAME), along with quercetin or resveratrol at their concentrations producing maximum relaxant effect, showed that both of these natural compounds exert their relaxant effect by inhibiting the generation of reactive oxygen species (ROS) from endothelial and smooth muscle cells, blocking voltage-gated calcium channels, and increasing the release of nitric oxide (NO). The mediation of hypercontraction by nickel is due to the increased ROS and the influx of calcium through voltage-dependent calcium channels. These natural compounds are shown to counter the nickel-induced effects, appearing as effective ameliorators. CONCLUSION: In this study, we found that quercetin and resveratrol act as ameliorators of nickel-mediated hypercontraction by decreasing ROS and enhancing NO release from endothelial cells.

Iliopsoas Hematomas in a Patient with Progressive Encephalomyelitis with Rigidity and Myoclonus.

Progressive encephalomyelitis with rigidity and myoclonus (PERM) is a rare and severe syndrome characterized by rigidity of the limb and truncal muscles, brainstem signs, myoclonus, and hyperekplexia. Iliopsoas hematoma is a serious complication of bleeding disorders that occurs most commonly in patients with hemophilia and also in association with anti-coagulant drug treatment. We herein present a case of PERM complicated with bilateral iliopsoas hematomas. His neurological symptoms improved after immunotherapy, and thereafter the iliopsoas hematomas disappeared. Neurologists should consider iliopsoas hematomas as a serious potential complication of PERM.

Role of calcium channels and endothelial factors in nickel induced aortic hypercontraction in Wistar rats.

AIM: To investigate the mechanism of nickel augmented phenylephrine (PE)-induced contraction in isolated segments of Wistar rat aorta. MATERIALS AND METHODS: Effect of varying concentrations of nickel on PE-induced contraction were investigated in isolated segments of Wistar rat aorta using an organ bath system. Aortic rings were pre-incubated with verapamil (1 µM and 20 µM), gadolinium, apocynin, indomethacin or N-G-nitro-L-arginine methyl ester (L-NAME) separately before incubation with nickel. RESULTS: Endothelium intact aortic rings incubated with 100 nM, 1 µM or 100 µM of nickel exhibited 80%, 43% and 28% increase in PE-induced contraction, respectively, while no such enhancing responses were observed in endothelium denuded aorta. Incubation of aortic rings with 1 µM and 20 µM verapamil suggested an involvement of influx of calcium through T-type calcium channels in smooth muscle cells, while aortic rings pre-incubated with gadolinium showed no role of store operated calcium channels in the nickel effect on PE-induced contractions. The enhancing effect of nickel on PE-induced contractions was inhibited by apocynin, indomethacin or L-NAME. CONCLUSION: Nickel has caused augmentation of PE-induced contractions as a result of the endothelial generation of reactive oxygen species (ROS) and cyclooxygenase 2 (COX2) dependent endothelium contracting factors (EDCFs), which increases the influx of extracellular calcium through T-type Ca2+ channels in smooth muscle cells.

Overexpression of miRNA-9 Generates Muscle Hypercontraction Through Translational Repression of Troponin-T in Drosophila melanogaster Indirect Flight Muscles.

MicroRNAs (miRNAs) are small noncoding endogenous RNAs, typically 21-23 nucleotides long, that regulate gene expression, usually post-transcriptionally, by binding to the 3'-UTR of target mRNA, thus blocking translation. The expression of several miRNAs is significantly altered during cardiac hypertrophy, myocardial ischemia, fibrosis, heart failure, and other cardiac myopathies. Recent studies have implicated miRNA-9 (miR-9) in myocardial hypertrophy. However, a detailed mechanism remains obscure. In this study, we have addressed the roles of miR-9 in muscle development and function using a genetically tractable model system, the indirect flight muscles (IFMs) of Drosophila melanogaster Bioinformatics analysis identified 135 potential miR-9a targets, of which 27 genes were associated with Drosophila muscle development. Troponin-T (TnT) was identified as major structural gene target of miR-9a. We show that flies overexpressing miR-9a in the IFMs have abnormal wing position and are flightless. These flies also exhibit a loss of muscle integrity and sarcomeric organization causing an abnormal muscle condition known as "hypercontraction." Additionally, miR-9a overexpression resulted in the reduction of TnT protein levels while transcript levels were unaffected. Furthermore, muscle abnormalities associated with miR-9a overexpression were completely rescued by overexpression of TnT transgenes which lacked the miR-9a binding site. These findings indicate that miR-9a interacts with the 3'-UTR of the TnT mRNA and downregulates the TnT protein levels by translational repression. The reduction in TnT levels leads to a cooperative downregulation of other thin filament structural proteins. Our findings have implications for understanding the cellular pathophysiology of cardiomyopathies associated with miR-9 overexpression.

A cis-regulatory mutation in troponin-I of Drosophila reveals the importance of proper stoichiometry of structural proteins during muscle assembly.

Rapid and high wing-beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations that have been instrumental in the identification of many proteins and residues that are important for muscle assembly, function, and disease. In this article, we present a molecular-genetic characterization of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). We show that fliH is a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms that are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. Our results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications for the identification of mutations and disease phenotypes in other species, including humans.