Nonproductive Splicing Prevents Expression of MYH7b Protein in the Mammalian Heart.

Background Although the roles of alpha-myosin heavy chain (α-MyHC) and beta-myosin heavy chain (ß-MyHC) proteins in cardiac contractility have long been appreciated, the biological contribution of another closely related sarcomeric myosin family member, MYH7b (myosin heavy chain 7b), has become a matter of debate. In mammals, MYH7b mRNA is transcribed but undergoes non-productive alternative splicing that prevents protein expression in a tissue-specific manner, including in the heart. However, several studies have recently linked MYH7b variants to different cardiomyopathies or have reported MYH7b protein expression in mammalian hearts. Methods and Results By analyzing mammalian cardiac transcriptome and proteome data, we show that the vast majority of MYH7b RNA is subject to exon skipping and cannot be translated into a functional myosin molecule. Notably, we discovered a lag in the removal of introns flanking the alternatively spliced exon, which could retain the non-coding RNA in the nucleus. This process could play a significant role in controlling MYH7b expression as well as the activity of other cardiac genes. Consistent with the negligible level of full-length protein coding mRNA, no MYH7b protein expression was detected in adult mouse, rat, and human hearts by Western blot analysis. Furthermore, proteome surveys including quantitative mass spectrometry analyses revealed only traces of cardiac MYH7b protein and even then, only in a subset of individual samples. Conclusions The comprehensive analysis presented here suggests that previous studies showing cardiac MYH7b protein expression were likely attributable to antibody cross-reactivity. More importantly, our data predict that the MYH7b disease-associated variants may operate through the alternately spliced RNA itself.

SOX9 is dispensable for the initiation of epigenetic remodeling and the activation of marker genes at the onset of chondrogenesis.

SOX9 controls cell lineage fate and differentiation in major biological processes. It is known as a potent transcriptional activator of differentiation-specific genes, but its earliest targets and its contribution to priming chromatin for gene activation remain unknown. Here, we address this knowledge gap using chondrogenesis as a model system. By profiling the whole transcriptome and the whole epigenome of wild-type and Sox9-deficient mouse embryo limb buds, we uncover multiple structural and regulatory genes, including Fam101a, Myh14, Sema3c and Sema3d, as specific markers of precartilaginous condensation, and we provide evidence of their direct transactivation by SOX9. Intriguingly, we find that SOX9 helps remove epigenetic signatures of transcriptional repression and establish active-promoter and active-enhancer marks at precartilage- and cartilage-specific loci, but is not absolutely required to initiate these changes and activate transcription. Altogether, these findings widen our current knowledge of SOX9 targets in early chondrogenesis and call for new studies to identify the pioneer and transactivating factors that act upstream of or along with SOX9 to prompt chromatin remodeling and specific gene activation at the onset of chondrogenesis and other processes.

Cytosolic proteome profiling of monocytes for male osteoporosis.

In male Caucasians with discordant hip bone mineral density (BMD), we applied the subcellular separation and proteome profiling to investigate the monocytic cytosol. Three BMD-associated proteins (ALDOA, MYH14, and Rap1B) were identified based on multiple omics evidence, and they may influence the pathogenic mechanisms of osteoporosis by regulating the activities of monocytes. INTRODUCTION: Osteoporosis is a serious public health problem, leading to significant mortality not only in aging females but also in males. Peripheral blood monocytes (PBMs) play important roles in bone metabolism by acting as precursors of osteoclasts and producing cytokines important for osteoclast development. The first cytosolic sub-proteome profiling analysis was performed in male PBMs to identify differentially expressed proteins (DEPs) that are associated with BMDs and risk of osteoporosis. METHODS: Here, we conducted a comparative proteomics analysis in PBMs from Caucasian male subjects with discordant hip BMD (29 low BMD vs. 30 high BMD). To decrease the proteome complexity and expand the coverage range of the cellular proteome, we separated the PBM proteome into several subcellular compartments and focused on the cytosolic fractions, which are involved in a wide range of fundamental biochemical processes. RESULTS: Of the total of 3796 detected cytosolic proteins, we identified 16 significant (P < 0.05) and an additional 22 suggestive (P < 0.1) DEPs between samples with low vs. high hip BMDs. Some of the genes for DEPs, including ALDOA, MYH14, and Rap1B, showed an association with BMD in multiple omics studies (proteomic, transcriptomic, and genomic). Further bioinformatics analysis revealed the enrichment of DEPs in functional terms for monocyte proliferation, differentiation, and migration. CONCLUSIONS: The combination strategy of subcellular separation and proteome profiling allows an in-depth and refined investigation into the composition and functions of cytosolic proteome, which may shed light on the monocyte-mediated pathogenic mechanisms of osteoporosis.

Coupling of mitochondrial function and skeletal muscle fiber type by a miR-499/Fnip1/AMPK circuit.

Upon adaption of skeletal muscle to physiological and pathophysiological stimuli, muscle fiber type and mitochondrial function are coordinately regulated. Recent studies have identified pathways involved in control of contractile proteins of oxidative-type fibers. However, the mechanism for coupling of mitochondrial function to the muscle contractile machinery during fiber type transition remains unknown. Here, we show that the expression of the genes encoding type I myosins, Myh7/Myh7b and their intronic miR-208b/miR-499, parallels mitochondrial function during fiber type transitions. Using in vivo approaches in mice, we found that miR-499 drives a PGC-1α-dependent mitochondrial oxidative metabolism program to match shifts in slow-twitch muscle fiber composition. Mechanistically, miR-499 directly targets Fnip1, an AMP-activated protein kinase (AMPK)-interacting protein that negatively regulates AMPK, a known activator of PGC-1α. Inhibition of Fnip1 reactivated AMPK/PGC-1α signaling and mitochondrial function in myocytes. Restoration of the expression of miR-499 in the mdx mouse model of Duchenne muscular dystrophy (DMD) reduced the severity of DMD Thus, we have identified a miR-499/Fnip1/AMPK circuit that can serve as a mechanism to couple muscle fiber type and mitochondrial function.

Genetic Dissection of Cardiac Remodeling in an Isoproterenol-Induced Heart Failure Mouse Model.

We aimed to understand the genetic control of cardiac remodeling using an isoproterenol-induced heart failure model in mice, which allowed control of confounding factors in an experimental setting. We characterized the changes in cardiac structure and function in response to chronic isoproterenol infusion using echocardiography in a panel of 104 inbred mouse strains. We showed that cardiac structure and function, whether under normal or stress conditions, has a strong genetic component, with heritability estimates of left ventricular mass between 61% and 81%. Association analyses of cardiac remodeling traits, corrected for population structure, body size and heart rate, revealed 17 genome-wide significant loci, including several loci containing previously implicated genes. Cardiac tissue gene expression profiling, expression quantitative trait loci, expression-phenotype correlation, and coding sequence variation analyses were performed to prioritize candidate genes and to generate hypotheses for downstream mechanistic studies. Using this approach, we have validated a novel gene, Myh14, as a negative regulator of ISO-induced left ventricular mass hypertrophy in an in vivo mouse model and demonstrated the up-regulation of immediate early gene Myc, fetal gene Nppb, and fibrosis gene Lgals3 in ISO-treated Myh14 deficient hearts compared to controls.

Akt2/ZEB2 may be a biomarker for exfoliant cells in ascitic fluid in advanced grades of serous ovarian carcinoma.

Ovarian cancers present a mild clinical course when diagnosed early but an aggressive pathway when diagnosed in the peri- and postmenopausal periods. However, the predictability of tumor progression is stochastic and is difficult to predict. In the present study, we hypothesized to examine the key pathways that are dysregulated to promote epithelial-mesenchymal transition in serous ovarian carcinoma. Examination of these steps would help to identify ascitic fluid with cells poised for metastasis or otherwise. We focused on examining the Akt2 expression, mainly because of its report as being overamplified in the aggressive variants of ovarian cancer, as well as TGFbeta-sensitivity of Akt2 that forms the key basis for metastasis initiation of most kinds of carcinoma. We obtained primary ovarian carcinoma samples as well as ascitic fluid and distantly metastatic ovarian carcinoma to examine the expression of Akt2. The results of the study demonstrated that in malignant exfoliated ovarian cancer cells, Smad4 expression was tremendously increased in the nuclei, suggesting nuclear translocation of Smad, which thereafter may have activated ZEB2, and thereafter genomically affected the expression of E-cadherin, myosin II, and vimentin, key components for initiating and sustaining metastasis. All of these may have been stimulated by increased cellular expression of Akt2 in metastatic variants of the serous ovarian carcinoma. The reliance on Akt2 and TGF beta signaling may also potentiate the case for Akt inhibitors or small molecule inhibitors of TGFbeta signaling like doxycycline as adjunct chemotherapy in serous ovarian carcinoma, especially the metastatic variants.

Analysis of cell cycle shortening and developmental interruption in Dictyostelium discoideum allC RNAi mutants.

Dictyostelium discoideum allC RNAi mutant cells are motile and aggregate together, but do not undergo further morphological development. The relatively quick growth rate of allC RNAi mutants compared to wild-type D. discoideum results in a shortened mutant cell cycle. However, at present, little is known about the mechanism underlying this phenomenon. Here, we used semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR, two-dimensional gel electrophoresis, and mass spectrometry/mass spectrometry to elucidate the phenomenon. We found significant downregulation of myosin II heavy chain, D. discoideum calcium-dependent cell adhesion molecule-1 (DdCAD-1) mRNA, DdCAD-1 protein, D. discoideum mRNA for 14-3-3 and 14-3-3 protein, and type A von Willebrand factor domain-containing protein mRNA in allC RNAi mutants. The results suggest that downregulation of the myosin II heavy chain could be one of key factors causing the developmental interruption and that downregulation of the 14-3-3 protein and the type A von Willebrand factor domain-containing protein mRNA plays an important role in shortening the cell cycle of allC RNAi mutants.

RCB20, an experimental benzimidazole derivative, affects tubulin expression and induces gross anatomical changes in Taenia crassiceps cysticerci.

Helminth ß-tubulins are the targets of benzimidazole (BZM) carbamate compounds. The specificity of the interactions between such compounds and their in vivo targets depends on the presence of specific amino acid residues in the target molecules. To discover new and effective anthelmintic drugs, we used a medicinal chemistry approach to synthesize a series of BZM derivatives that exploited the BZM moiety as a template. We have previously found that one compound, 2-(trifluoromethyl)-1H-benzimidazole (RCB20), has better in vitro and in vivo activity than albendazole sulfoxide (ABZSO). In the present study, the effect of RCB20 and ABZSO treatment on expression of Taenia crassiceps cysticerci cytoskeletal proteins such as actin, myosin II, and tubulin isoforms was examined. The effects of RCB20 and ABZSO after 11 days treatment of the parasites was evaluated by light, confocal, and electron microscopy, and by immunochemistry and immunohistochemistry. The RCB20-induced effects were more rapid than the ABZSO-induced effects on the parasites. In the RCB20-treated parasites, we observed gross-structural damage at the whole parasite level, particularly in the inner tissues and flame cells. Changes in the expression patterns of the cytoskeletal proteins, as assessed by immunohistochemistry and immunoblotting, revealed that the most important drug-induced effect on the parasites was a reduction in the expression level of tyrosinated α-tubulins. Our research findings suggest that RCB20 treatment affected posttranslational modification of parasite α-tubulin molecules, which involved removal of the α-tubulin carboxy-terminal tyrosine.

Myh7b/miR-499 gene expression is transcriptionally regulated by MRFs and Eos.

The sarcomeric myosin gene, Myh7b, encodes an intronic microRNA, miR-499, which regulates cardiac and skeletal muscle biology, yet little is known about its transcriptional regulation. To identify the transcription factors involved in regulating Myh7b/miR-499 gene expression, we have mapped the transcriptional start sites and identified an upstream 6.2 kb region of the mouse Myh7b gene whose activity mimics the expression pattern of the endogenous Myh7b gene both in vitro and in vivo. Through promoter deletion analysis, we have mapped a distal E-box element and a proximal Ikaros site that are essential for Myh7b promoter activity in muscle cells. We show that the myogenic regulatory factors, MyoD, Myf5 and Myogenin, bind to the E-box, while a lymphoid transcription factor, Ikaros 4 (Eos), binds to the Ikaros motif. Further, we show that through physical interaction, MyoD and Eos form an active transcriptional complex on the chromatin to regulate the expression of the endogenous Myh7b/miR-499 gene in muscle cells. We also provide the first evidence that Eos can regulate expression of additional myosin genes (Myosin 1 and ß-Myosin) via the miR-499/Sox6 pathway. Therefore, our results indicate a novel role for Eos in the regulation of the myofiber gene program.

Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules.

Neurons in the central nervous system (CNS) fail to regenerate axons after injuries due to the diminished intrinsic axon growth capacity of mature neurons and the hostile extrinsic environment composed of a milieu of inhibitory factors. Recent studies revealed that targeting a particular group of extracellular inhibitory factors is insufficient to trigger long-distance axon regeneration. Instead of antagonizing the growing list of impediments, tackling a common target that mediates axon growth inhibition offers an alternative strategy to promote axon regeneration. Neuronal growth cone, the machinery that derives axon extension, is the final converging target of most, if not all, growth impediments in the CNS. In this study, we aim to promote axon growth by directly targeting the growth cone. Here we report that pharmacological inhibition or genetic silencing of nonmuscle myosin II (NMII) markedly accelerates axon growth over permissive and nonpermissive substrates, including major CNS inhibitors such as chondroitin sulfate proteoglycans and myelin-associated inhibitors. We find that NMII inhibition leads to the reorganization of both actin and microtubules (MTs) in the growth cone, resulting in MT reorganization that allows rapid axon extension over inhibitory substrates. In addition to enhancing axon extension, we show that local blockade of NMII activity in axons is sufficient to trigger axons to grow across the permissive-inhibitory border. Together, our study proposes NMII and growth cone cytoskeletal components as effective targets for promoting axon regeneration.

Dupuytren's fibroblast contractility by sphingosine-1-phosphate is mediated through non-muscle myosin II.

PURPOSE: Previous studies suggest that Dupuytren's disease is caused by fibroblast and myofibroblast contractility within Dupuytren's nodules; however, the stimulus for cell contractility is unknown. Sphingosine-1-phosphate (S1P) is a serum-derived lysophospholipid mediator that enhances cell contractility by activating the S1P receptor, S1P(2). It is hypothesized that S1P stimulates Dupuytren's fibroblast contractility through S1P(2) activation of non-muscle myosin II (NMMII). This investigation examined the role of S1P and NMMII activation in Dupuytren's disease progression and suggests potential targets for treatment. METHODS: We enmeshed Dupuytren's fibroblasts into fibroblast-populated collagen lattices (FPCLs) and assayed S1P-stimulated FPCL contraction in the presence of the S1P(2) receptor inhibitor JTE-013, the Rho kinase inhibitor Y-27632, the myosin light chain kinase inhibitor ML-7, and the NMMII inhibitor blebbistatin. Tissues from Dupuytren's fascia (n = 10) and normal palmar fascia (n = 10) were immunostained for NMMIIA and NMMIIB. RESULTS: Sphingosine-1-phosphate stimulated FPCL contraction in a dose-dependent manner. Inhibition of S1P(2) and NMMII prevented S1P-stimulated FPCL contraction. Rho kinase and myosin light chain kinase inhibited both S1P and control FPCL contraction. Dupuytren's nodule fibroblasts robustly expressed NMMIIA and NMMIIB, compared with quiescent-appearing cords and normal palmar fascia. CONCLUSIONS: Sphingosine-1-phosphate promotes Dupuytren's fibroblast contractility through S1P(2), which stimulates activation of NMMII. NMMII isoforms are ubiquitously expressed throughout Dupuytren's nodules, which suggests that nodule fibroblasts are primed to respond to S1P stimulation to cause contracture formation. S1P-promoted activation of NMMII may be a target for disease treatment.

The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells.

PURPOSE: Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility. METHODS: BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. Ca(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated Ca(2+) responses. RESULTS: Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the Ca(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the thrombin effect on paracrine and gap junctional IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [Ca(2+)](i) transients in response to thrombin or ATP. CONCLUSIONS: Blebbistatin prevents the inhibitory effects of thrombin on intercellular Ca(2+) wave propagation. The findings demonstrate that myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.

Co-expression of myosin II regulatory light chain and the NMDAR1 subunit in neonatal and adult mouse brain.

Movement of glutamate receptors in neurons likely involves direct and indirect association of receptor subunits with microtubule- and actin-based motor proteins. We have previously shown that myosin II regulatory light chain (RLC) binds directly to subunits of the NMDA-type glutamate receptor (NR), suggesting that NMDA receptors are closely associated with a myosin II motor complex. Using a polyclonal antibody predicted to recognize all RLC isoforms previously described in rodent brain, we report the expression of RLC and the NR1 subunit in cortex, hippocampus and cerebellum of postnatal day 0 (P0) and adult mouse. Although myosin RLC was not exclusively localized with NR1 by immunohistochemistry, co-staining was striking in the neuronal soma of deep cortical neurons and Purkinje neurons of the cerebellum which showed a punctate, perinuclear pattern of immunoreactivity. These neuronal populations were identified using a monoclonal antibody directed against a nuclear-specific, transcriptional repressor, chicken ovalbumin upstream promoter-transcription factor (COUP-TF)-interacting protein 2 (CTIP2). Co-expression of NR1 and a myosin II motor was validated using an isoform specific anti-nonmuscle myosin II-B heavy chain (NMHC II-B) antibody. Our findings support the idea that there is regional heterogeneity in the molecular composition of the NMDA receptor-associated cytoskeleton, and suggest that NR subunits may be associated with an actin-based, myosin II-B motor within the endomembrane system of some neuronal populations. Differential staining patterns observed with light and heavy chain antibodies, however, suggest that there is also heterogeneity in the composition of myosin II complexes in brain.

Alpha-smooth muscle actin expression enhances cell traction force.

Using an established corneal stromal cell differentiation model, we manipulated alpha-smooth muscle actin (alpha-SMA) protein expression levels in fibroblasts by treating them with TGF-beta1, bFGF, TGF-beta type I receptor inhibitor (SB-431542), and siRNA against alpha-SMA. The corresponding cell traction forces (CTFs) were determined by cell traction force microscopy. With all these treatments, we found that alpha-SMA is not required for CTF induction, but its expression upregulates CTF. This upregulation involves the modification of stress fibers but does not appear to relate to non-muscle myosin II expression or beta-actin expression. Moreover, there exists a linear relationship between alpha-SMA protein expression level and CTF magnitude. Finally, CTFs were found to vary among a population of myofibroblasts, suggesting that alpha-SMA protein expression levels of individual cells also vary.

Multicellular rosette formation links planar cell polarity to tissue morphogenesis.

Elongation of the body axis is accompanied by the assembly of a polarized cytoarchitecture that provides the basis for directional cell behavior. We find that planar polarity in the Drosophila embryo is established through a sequential enrichment of actin-myosin cables and adherens junction proteins in complementary surface domains. F-actin accumulation at AP interfaces represents the first break in planar symmetry and occurs independently of proper junctional protein distribution at DV interfaces. Polarized cells engage in a novel program of locally coordinated behavior to generate multicellular rosette structures that form and resolve in a directional fashion. Actin-myosin structures align across multiple cells during rosette formation, and adherens junction proteins assemble in a stepwise fashion during rosette resolution. Patterning genes essential for axis elongation selectively affect the frequency and directionality of rosette formation. We propose that the generation of higher-order rosette structures links local cell interactions to global tissue reorganization during morphogenesis.

Isoenergetic dietary protein restriction decreases myosin heavy chain IIx fraction and myosin heavy chain production in humans.

The synthesis of muscle protein is restrained during dietary protein restriction. This is widely understood to vary quantitatively with the degree of nutritional deprivation, but there has been little discussion of qualitative changes in muscle protein deriving from dietary protein restriction. We studied 14 healthy subjects in a 2-sample study. Subjects were randomly assigned to a diet providing an ample, American-style protein intake (1.67 g. kg fat-free mass(-1). d(-1)) or a diet approximating the mean minimum adult protein requirement (0.71 g. kg fat-free mass(-1). d(-1)). We found that consumption of an isoenergetic diet at the mean adult minimum protein requirement for 4 wk produced an 81% lower fractional synthesis rate of myosin heavy chain (MHC) proteins in vastus lateralis muscle than did consumption of an ample protein diet (P = 0.05). Protein deprivation altered the skeletal muscle myosin composition such that the proportion of the total myosin content represented by fast-twitch MHC IIx was 51% lower than with ample intake (P = 0.013). The steady state content of MHC IIx messenger RNA (mRNA) did not differ in subjects consuming the minimum requirement of protein, suggesting that the reduced proportion of MHC IIx arises from posttranscriptional events. A 68% lower rate of 3-methylhistidine excretion with protein restriction (P < 0.01) suggests that myofibrillar protein degradation was lower. We conclude that dietary amino acid scarcity produces a change in myosin isoform distribution via posttranscriptional mechanisms. The relative contribution of inhibited myosin synthesis and inhibited degradation to the altered myosin isoform composition remains unknown. This has implications for the mechanisms by which amino acids govern muscle protein composition in vivo, and further exploration is required.

Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretion.

The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine beta-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca2+ and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks.

Induction of nonmuscle myosin heavy chain II-C by butyrate in RAW 264.7 mouse macrophages.

RAW 264.7 macrophages express nonmuscle myosin heavy chain II-A as the only significant nonmuscle myosin heavy chain isoform, with expression of nonmuscle myosin heavy chain II-B and II-C low or absent. Treatment of the cells with sodium butyrate, an inhibitor of histone deacetylase, led to the dose-dependent induction of nonmuscle myosin heavy chain II-C. Trichostatin A, another inhibitor of histone deacetylase, also induced nonmuscle myosin heavy chain II-C. Induction of nonmuscle myosin heavy chain II-C in response to these histone deacetylase inhibitors was attenuated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. Bacterial lipopolysaccharide alone had no effect on basal nonmuscle myosin heavy chain II-C expression, but attenuated butyrate-mediated induction of nonmuscle myosin heavy chain II-C. The effects of lipopolysaccharide were mimicked by the nitric oxide donors sodium nitroprusside and spermine NONOate, suggesting a role for nitric oxide in the lipopolysaccharide-mediated down-regulation of nonmuscle myosin heavy chain II-C induction. This was supported by experiments with the inducible nitric-oxide synthase inhibitor 1400W, which partially blocked the lipopolysaccharide-mediated attenuation of nonmuscle myosin heavy chain induction. 8-Bromo-cGMP had no effect on nonmuscle myosin heavy chain induction, consistent with a cGMP-independent mechanism for nitric oxide-mediated inhibition of nonmuscle myosin heavy chain II-C induction.

Altered expression of skeletal muscle myosin isoforms in cancer cachexia.

Cachexia is commonly seen in cancer and is characterized by severe muscle wasting, but little is known about the effect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression of myosin isoforms toward increased fast (type II) isoform expression. We injected mice with murine C-26 adenocarcinoma cells, a tumor cell line that has been shown to cause muscle wasting. Mice were killed 21 days after tumor injection, and hindlimb muscles were removed. Myosin heavy chain (MHC) and myosin light chain (MLC) content was determined in muscle homogenates by SDS-PAGE. Body weight was significantly lower in tumor-bearing (T) mice. There was a significant decrease in muscle mass in all three muscles tested compared with control, with the largest decrease occurring in the soleus. Although no type IIb MHC was detected in the soleus samples from control mice, type IIb comprised 19% of the total MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. These data are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.