Changes In Myosin Light Chain Research Articles

Abnormalities of the Duo/Ras-Related C3 Botulinum Toxin Substrate 1/p21-Activated Kinase 1 Pathway Drive Myosin Light Chain Phosphorylation in Frontal Cortex in Schizophrenia

Recent studies on GTPases have suggested that reduced Duo and cell division cycle 42 (Cdc42) transcript expression is involved in dendritic spine loss in schizophrenia. In murine models, Duo and Cdc42 phosphorylate p21-activated kinase 1 (PAK1), which modifies the activity of regulatory myosin light chain (MLC) and cofilin by altering their phosphorylation. Therefore, we hypothesized that in schizophrenia abnormal Duo and Cdc42 expression result in changes in MLC and/or cofilin phosphorylation, which might alter actin cytoskeleton dynamics underlying dendritic spine maintenance. We performed Western blot protein expression analysis in postmortem brains from patients diagnosed with schizophrenia and a comparison group. We focused our studies in the anterior cingulate cortex (ACC; n = 33 comparison group; n = 36 schizophrenia) and dorsolateral prefrontal cortex (DLPFC; n = 29 comparison group; n = 35 schizophrenia). In both ACC and DLPFC, we found a reduction of Duo expression and PAK1 phosphorylation in schizophrenia. Cdc42 protein expression was decreased in ACC but not in DLPFC. In ACC, we observed decreased PAK1 phosphorylation and increased MLC phosphorylation (pMLC), whereas in DLPFC pMLC remained unchanged. These data suggest a novel mechanism that might underlie dendritic spine loss in schizophrenia. The increase in pMLC seen in ACC might be associated with dendritic spine shrinkage. The lack of an effect on pMLC in DLPFC suggests that in schizophrenia PAK1 downstream pathways are differentially affected in these cortical areas.

RhoA and NF-κB are involved in lipopolysaccharide-induced brain microvascular cell line hyperpermeability

The aim of this study was to investigate the signaling mechanisms surrounding changes in tight junction (TJ) and the permeability of brain microvascular cell lines induced by lipopolysaccharide (LPS). To confirm that LPS induces endothelial barrier hyperpermeability by disrupting tight junction, Bend.3 cells were exposed to LPS, and changes in endothelial permeability (transendothelial electrical resistance (TEER) assay), F-actin dynamics (Rhodamine-Phalloidin staining) and tight junction protein expression (western blot or immunofluorescence) were monitored. Moreover, to ensure that both RhoA and NF-κB participated in the regulatory mechanisms, Bend.3 cells were transfected with n19RhoA and DNMu-IκBα plasmids, and the above experiments were repeated. To clarify the relationship between RhoA and NF-κB in the process, the activities of NF-κB (via luciferase reporter assays) and RhoA (via pull-down assays) were detected in transfected and untreated Bend.3 cells. Lastly, to investigate whether RhoA and NF-κB regulate MLC phosphorylation, we measured changes in myosin light chain (MLC) phosphorylation in untreated and transfected Bend.3 cells by western blot. LPS caused RhoA and NF-κB activation, MLC phosphorylation, F-actin rearrangement, tight junction disruption and barrier dysfunction. These effects were suppressed by inhibitors of RhoA or NF-κB; inhibiting RhoA was more efficient. Inactivating RhoA prohibited LPS-induced NF-κB activation, but the inverse was not true. LPS induces brain microvascular endothelial barrier hyperpermeability by disrupting TJs, in part through RhoA and NF-κB activation, in which RhoA is the positive upstream regulator for NF-κB.

Double Phosphorylation of the Myosin Regulatory Light Chain during Rigor Mortis of Bovine Longissimus Muscle

To investigate changes in myosin light chains (MyLCs) during postmortem aging of the bovine longissimus muscle, we performed two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results of fluorescent differential gel electrophoresis showed that two spots of the myosin regulatory light chain (MyLC2) at pI values of 4.6 and 4.7 shifted toward those at pI values of 4.5 and 4.6, respectively, by 24 h postmortem when rigor mortis was completed. Meanwhile, the MyLC1 and MyLC3 spots did not change during the 14 days postmortem. Phosphoprotein-specific staining of the gels demonstrated that the MyLC2 proteins at pI values of 4.5 and 4.6 were phosphorylated. Furthermore, possible N-terminal region peptides containing one and two phosphoserine residues were detected in each mass spectrum of the MyLC2 spots at pI values of 4.5 and 4.6, respectively. These results demonstrated that MyLC2 became doubly phosphorylated during rigor formation of the bovine longissimus, suggesting involvement of the MyLC2 phosphorylation in the progress of beef rigor mortis. Bovine; myosin regulatory light chain (RLC, MyLC2); phosphorylation; rigor mortis; skeletal muscle.

Effects of Cholesterol-Lowering Statins on the Aqueous Humor Outflow Pathway

To investigate the effects of cholesterol-lowering statin drugs on trabecular meshwork cellular properties and aqueous humor outflow. Primary cell cultures of porcine trabecular meshwork (PTM) and ciliary body (PCB) were treated with either lovastatin or compactin, to determine the effects of statins on cell shape, actin cytoskeletal organization, and cell-extracellular matrix interactions (focal adhesions) by immunofluorescence staining. Changes in myosin light-chain (MLC) phosphorylation were evaluated by Western blot analysis. Changes in Rho GTPase content of membrane fractions from lovastatin-treated PTM cells were assessed by Western blot analysis. A constant-flow, organ-culture perfusion system was used to measure the effects of statins on aqueous humor outflow facility in the anterior segments of porcine eyes. PTM and PCB cells treated with lovastatin or compactin exhibited dramatic changes in cell shape and cytoskeletal organization within 24 hours, consisting of cell rounding, actin depolymerization, and decreased focal adhesions. These effects were found to be reversible on supplementation with geranylgeranyl pyrophosphate. Both lovastatin and compactin decreased MLC phosphorylation in PTM and PCB cells. PTM cells treated with lovastatin exhibited marked decreases in membrane-bound Rho GTPase. In addition, perfusion of organ-cultured porcine eye anterior segments with 100 microM lovastatin for 96 hours caused a significant increase in aqueous humor outflow facility (110%) compared with control eyes, in a reversible manner. This study demonstrates that the statin drugs lovastatin and compactin induce changes in cell shape and actin cytoskeletal organization and decrease MLC phosphorylation in PTM and PCB cells, all of which are events that are likely to lead to cellular and tissue relaxation. In addition, these effects of the statins appear to be mediated by inhibition of isoprenylation of the small GTP-binding proteins such as Rho GTPase. An important finding is that statins exert an ocular hypotensive response in an organ-culture perfusion model, indicating the potential for this class of drugs in glaucoma therapy.

The effect of autografting on the myosin composition in skeletal muscle fibers.

The purpose of the study was to investigate the changes in myosin heavy chain (MHC) and myosin light chain (MLC) isoforms following autotransplantation of extensor digitorum longus muscles. Muscles were grafted in "standard" and "nerve-intact" conditions. MHC and MLC isoforms were analyzed by sodium dodecyl sulphate gel electrophoresis. Changes in MHC isoforms 10, 30, and 60 days after grafting were similar in the "standard" and the "nerve-intact" grafts. In contrast to MHC, changes in MLC were different in the 10th day groups, but the same in the 30th day groups. Sixty days after grafting the content of MLC isoforms was the same as the control muscles. These data indicate that transient loss of functional innervation, even for a short time, has permanent effect on the composition of MHC but not MLC isoforms in regenerating skeletal muscle fibers.

Myosin light-chain phosphorylation in diabetic cardiomyopathy in rats

The regulatory myosin light chain (MLC) is phosphorylated in cardiac muscle by Ca 2+/calmodulin-dependent MLC kinase (MLCK) and is considered to play a modulatory role in the activation of myofibrillar adenosine triphosphatase (ATPase) and the process of force generation. Since the depression in cardiac contractile function in chronic diabetes is associated with a decrease in myofibrillar ATPase activity, we investigated changes in MLC phosphorylation in diabetic heart. Rats Were made diabetic by injecting streptozotocin (65 mg/kg intravenously); and the hearts were removed 8 weeks later; some 6-week diabetic animals were injected with insulin (3 U/d) for 2 weeks. Changes in the relative MLC and MLCK protein contents were measured by electrophoresis and immunoblot assay, whereas phosphorylated and unphosphorylated MLCs were separated on 10% acrylamide/urea gel and identified by Western blot. MLC and MLCK contents were decreased markedly (40% to 45%) and MLC phosphorylation was decreased significantly (30% to 45%) in the diabetic rat heart homogenate in comparison to control values. The changes in MLC and MLCK content in diabetic heart were partially reversible, whereas changes in MLC phosphorylation were normalized upon treatment with insulin. These results suggest that decreased protein contents of MLC and MLCK and phosPhorylation of MLC may contribute to the depression of cardiac myofibrillar ATPase activity and heart dysfunction in diabetic cardiomyopathy.

Postoperative changes in myosin light chain and comparison with postoperative clinical variables in patients who have undergone valve surgery.

We measured the levels of creatine kinase-MB (CK-MB) and myosin light chain (MLCI) in 86 patients after double valve replacement to investigate whether measurement of the postoperative changes in myosin could be useful in patients undergoing valve surgery. In particular, we compared the postoperative levels of CK-MB and MLCI with the occurrence of postoperative low cardiac output syndrome (LOS). In both groups of patients (group I: without LOS, group II: with LOS), the peak CK-MB level was observed on postoperative day 1, and the peak myosin level on postoperative day 5. No significant differences were found in the postoperative levels of CK-MB between the two groups. On the other hand, a significant difference in the levels of MLCI on postoperative day 5 was observed (p < 0.05). Our results also showed that the peak level of CK-MB significantly correlated with the cardiopulmonary bypass time (r = 0.50), while the peak level of MLCI was significantly correlated with the postoperative cardiac index (r = -0.45). Our retrospective findings suggest that postoperative measurement of MLCI may be useful for estimating the postoperative myocardial impairment after valve surgery.

Changes in serum myosin light chain I following aortocoronary bypass operations.

The changes in myosin light chain I (MLC I) following aortocoronary bypass were studied in 31 patients and the curves of these changes were classified into three different patterns. A peak level of 14.7 +/- 1.54 ng/ml was seen in seven patients on postoperative day (POD) 2 which decreased suddenly to less than twice the normal value by POD 7 (group 1). Another 19 patients showed a peak level of 20.7 +/- 16.5 ng/ml on POD 5, which decreased slowly and was still high even by POD 7 (group 2). The remaining five patients developed only a slight increase in the MLC I level after the operation, with a peak value of 5.5 +/- 0.8 ng/ml (group 3). Creatine kinase myocardial band and glutamic oxaloacetic transaminase also remained low in this group. No correlation existed between the peak value of MLC I and the aortic cross-clamping time, or between the peak value of MLC I and the cardiopulmonary bypass time. Furthermore, the difference in cardiac output before and after the operation did not significantly differ among the three groups. These findings indicate that the measurement of MLC I is useful for diagnosing perioperative myocardial damage and may also be useful in the study of myocardial protection.

Heart and Hypertension

The manifestations of cardiac involvement in hypertension include: (1) the development of hypertensive heart disease characterized by left ventricular hypertrophy (LVH), and (2) the consequences of coronary atherosclerosis, as angina pectoris, myocardial infarction, and sudden cardiac death. Whereas the former is directly related to increased blood pressure, the latter are sequelae of atherosclerosis per se, and hypertension acts only as a risk factor in this regard. This can partially explain why antihypertensive treatment is effective in diminishing the incidence of congestive heart failure, which is the final consequence of LVH, but is not very effective in preventing coronary complications. It is generally accepted about LVH that increased arterial pressure is the major stimulus to cardiac hypertrophy in hypertension; however, there are a lot of both quantitative and qualitative events suggesting that other factors beside blood pressure levels can modulate the development of LVH, in particular neurohumoral influences. From a morphological point of view, hypertrophy of the cardiac muscle is defined as an increase in the size of existing myocardial fibers. In most experimental models, myocardial hypertrophy is associated with myosin isoenzymatic changes, consisting in a shift from the faster migrating isoenzyme V1 to V3, a form that migrates more slowly. However these changes do not occur in all animal species and particularly in humans. In the hypertrophied human ventricle, a decreased ATPase activity of myofibrils was observed, probably related to changes in myosin light chains. Presently the changes in ATPase activity and in ventricular contractility do not still have a clear molecular basis in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

高強度・持久トレーニングによるラット骨格筋タンパクの変化

Effects of long-term with high intensity endurance training upon histochemically assessed myofibrillar actomyosin ATPase, myosin composition and creatine kinase content were analyzed in muscles from rats which were trained to run up to 240min/day at 40m/min. Following 16 weeks of the training, in M. soleus, proportions of slow-twitch oxidative (S0) fibers and slow type myosin light chains were increased; no changes in myosin light chain were significantly observed. Furthermore, a decrease in creatine kinase was found in this muscle, indicating that SO fibers might have acquired slower properties. On the other hand, in M. extensor digitorum longus, there was only a transformation between fast-twitch fiber subgroups, with a decrease in LC3f by which fast myosin constructed was characterized by the highest myofibrillar ATPase activities. These data suggest that fiber type transformation may be brought about not only from fast-twitch glycolytic (FG) fibers to fast-twitch oxdative glycolytic (FOG) fibers but also from FOG fibers to SO fibers by long-term with high intensity endurance training.

&lt;b&gt;Effect of denervation on phosphorylation of the myosin light chain in rat extensor digitorum longus &lt;/b&gt;&lt;b&gt;muscles &lt;/b&gt;

To test the hypothesis that myosin light chain phosphorylation in the rat extensor digitorum longus muscle is under neural control, we studied changes in the extent of phosphorylation of the myosin light chain following sciatic nerve denervation using two-dimensional gel electrophoresis. The phosphorylated form of myosin light chain 2 was rapidly decreased and disappeared 3 days after denervation. No other changes in myosin light chains were observed. The results suggest that phosphorylation of the myosin light chain in fast skeletal muscle is under positive control of fast-type motor-neuron activity.

Correlation between protein phenotype and gene expression in adult rabbit fast twitch muscles undergoing a fast to slow fiber transformation in response to electrical stimulation [formula omitted

In this report we have defined three distinct stages of the fast to slow transformation of muscle in terms of the myosin isoenzyme pattern in a non-denaturing gel system. In phase I a rearrangement of fast isoenzymes with no increase in slow isoforms took place. Phase II is characterised by a complex pattern of fast and slow isoenzymes and of isoenzymes of intermediate mobility. Phase III shows full conversion to the slow isoform with residual traces of fast and intermediate components. Changes in myosin light chains, as revealed by two dimensional gel analysis, showed good correlation with their corresponding mRNAs as determined by translation of extracted total RNA in a nuclease treated reticulocyte lysate cell-free system. This suggests that in the fast to slow transformation gene expression is regulated at the level of transcription.

Changes in myosin light chains in the rat soleus after thyroidectomy

The occurrence of physiological and structural changes in skeletal muscle in hypothyroidism is well-documented...

Salt-induced conformational changes in skeletal myosin light chains, troponin-C, and parvalbumin.

Evidence for salt-induced changes in myosin light chains [dissociated by treatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)], troponin-C (TnC), and parvalbumin was obtained from chymotryptic digestion, circular dichroism, fluorescence, and difference absorption studies. High salt (0.6 M NaCl) protects the DTNB light chain from proteolysis, increases its alpha-helical content, and quenches the tryptophan fluorescence. These effects are similar to the changes induced by Ca2+ but smaller in magnitude. TnC is affected by monovalent cations in a similar manner. Changes in the alpha-helical content resemble the effect of Ca2+. The enhancement of tyrosine fluorescence reflects conformational changes in the Ca2+-Mg2+ binding sites. The increase in the fluorescence of dansylaziridine-labeled TnC suggests perturbation of Ca2+-specific sites by salt. Cancellation of this effect by Mg2+ binding to the high-affinity sites is indicative of site-site interactions. In Whiting parvalbumin, salt-induced a perturbation of tryptophan absorption similar in nature to the Ca2+ effect.