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Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of [Na.sup.+]/[H.sup.+] exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. [gamma]-Secretase inhibition with DAPT recovered apical brush border structure and functional [Na.sup.+]/[H.sup.+] exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID., Introduction Microvillus inclusion disease (MVID) is a rare congenital diarrhea and enteropathy (CoDE) caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B) (1-5). In general, patients with severe [...]

Myosin modulators are a novel class of pharmaceutical agents that are being developed to treat patients with a range of cardiomyopathies. The therapeutic goal of these drugs is to target cardiac myosins directly to modulate contractility and cardiac power output to alleviate symptoms that lead to heart failure and arrhythmias, without altering calcium signaling. In this Review, we discuss two classes of drugs that have been developed to either activate (omecamtiv mecarbil) or inhibit (mavacamten) cardiac contractility by binding to [beta]-cardiac myosin (MYH7). We discuss progress in understanding the mechanisms by which the drugs alter myosin mechanochemistry, and we provide an appraisal of the results from clinical trials of these drugs, with consideration for the importance of disease heterogeneity and genetic etiology for predicting treatment benefit., Myosin modulators are a novel class of pharmaceutical agents that were recently developed to treat patients with cardiomyopathies. Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are the two most common [...]

Emerging evidence indicates that the neuronal guidance molecule SLIT plays a role in tumor suppression, as SLIT- encoding genes are inactivated in several types of cancer, including lung cancer; however, it is not clear how SLIT functions in lung cancer. Here, our data show that SLIT inhibits cancer cell migration by activating RhoA and that myosin 9b (Myo9b) is a ROBO-interacting protein that suppresses RhoA activity in lung cancer cells. Structural analyses revealed that the RhoGAP domain of Myo9b contains a unique patch that specifically recognizes RhoA. We also determined that the ROBO intracellular domain interacts with the Myo9b RhoGAP domain and inhibits its activity; therefore, SLIT-dependent activation of RhoA is mediated by ROBO inhibition of Myo9b. In a murine model, compared with control lung cancer cells, SLIT-expressing cells had a decreased capacity for tumor formation and lung metastasis. Evaluation of human lung cancer and adjacent nontumor tissues revealed that Myo9b is upregulated in the cancer tissue. Moreover, elevated Myo9b expression was associated with lung cancer progression and poor prognosis. Together, our data identify Myo9b as a key player in lung cancer and as a ROBO-interacting protein in what is, to the best of our knowledge, a newly defined SLIT/ROBO/Myo9b/RhoA signaling pathway that restricts lung cancer progression and metastasis. Additionally, our work suggests that targeting the SLIT/ROBO/Myo9b/RhoA pathway has potential as a diagnostic and therapeutic strategy for lung cancer., Introduction As one of the most aggressive and highly metastatic malignancies, lung cancer ranks first in cancer-related deaths worldwide (1, 2). Metastasis is the leading cause of death among cancer [...]

We have isolated mesenchymal stem cells (MSCs) from tracheal aspirates of premature infants with respiratory distress. We examined the capacity of MSCs to differentiate into myofibroblasts, cells that participate in lung development, injury, and repair. Gene expression was measured by array, qPCR, immunoblot, and immunocytochemistry. Unstimulated MSCs expressed mRNAs encoding contractile (e.g., ACTA2, TAGLN), extra-cellular matrix (COL1A1 and ELN), and actin-binding (DBN1, PXN) proteins, consistent with a myofibroblast phenotype, although there was little translation into immunoreactive protein. Incubation in serum-free medium increased contractile protein (ACTA2, MYH11) gene expression. MSC-conditioned medium showed substantial levels of TGF-[beta]1, and treatment of serum-deprived cells with a type I activin receptor-like kinase inhibitor, SB-431542, attenuated the expression of genes encoding contractile and extracellular matrix proteins. Treatment of MSCs with TGF-[beta]1 further induced the expression of mRNAs encoding contractile (ACTA2, MYH11, TAGLN, DES) and extracellular matrix proteins (FN1, ELN, COL1A1, COL1A2), and increased the protein expression of [alpha]-smooth muscle actin, myosin heavy chain, and SM22. In contrast, human bone marrow-derived MSCs failed to undergo TGF-[beta]1-induced myofibroblastic differentiation. Finally, primary cells from tracheal aspirates behaved in an identical manner as later passage cells. We conclude that human neonatal lung MSCs demonstrate an mRNA expression pattern characteristic of myofibroblast progenitor cells. Autocrine production of TGF-[beta]1 further drives myofibroblastic differentiation, suggesting that, in the absence of other signals, fibrosis represents the 'default program' for neonatal lung MSC gene expression. These data are consistent with the notion that MSCs play a key role in neonatal lung injury and repair. alveolarization; bronchopulmonary dysplasia; myofibroblast; neonate doi: 10.1152/ajplung.00347.2009.

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice. inflammation; limb-girdle muscular dystrophy type 2B; Miyoshi myopathy; muscle injury doi: 10.1152/ajpcell.00122.2009

The Frank-Starling relationship of the heart yields increased stroke volume with greater end-diastolic volume, and this relationship is steeper after [beta]-adrenergic stimulation. The underlying basis for the Frank-Starling mechanism involves length-dependent changes in both [Ca.sup.2+] sensitivity of myofibrillar force and power output. In this study, we tested the hypothesis that PKA-induced phosphorylation of myofibrillar proteins would increase the length dependence of myofibrillar power output, which would provide a myofibrillar basis to, in part, explain the steeper Frank-Starling relations after [beta]-adrenergic stimulation. For these experiments, adult rat left ventricles were mechanically disrupted, permeabilized cardiac myocyte preparations were attached between a force transducer and position motor, and the length dependence of loaded shortening and power output were measured before and after treatment with PKA. PKA increased the phosphorylation of myosin binding protein C and cardiac troponin I, as assessed by autoradiography. In terms of myocyte mechanics, PKA decreased the [Ca.sup.2+] sensitivity of force and increased loaded shortening and power output at all relative loads when the myocyte preparations were at long sarcomere length (~2.30 [micro]m). PKA had less of an effect on loaded shortening and power output at short sarcomere length (~2.0 [micro]m). These changes resulted in a greater length dependence of myocyte power output after PKA treatment; peak normalized power output increased ~20% with length before PKA and ~40% after PKA. These results suggest that PKA-induced phosphorylation of myofibrillar proteins explains, in part, the steeper ventricular function curves (i.e., Frank-Starling relationship) after [beta]-adrenergic stimulation of the left ventricle. Frank-Starling; protein kinase A; myosin binding protein C; cardiac troponin I

Myosin storage myopathy (MSM) is a congenital myopathy characterized by the presence of subsarcolemmal inclusions of myosin in the majority of type I muscle fibers, and has been linked to 4 mutations in the slow/cardiac muscle myosin, [beta]-MyHC (MYH7). Although the majority of the >230 disease causing mutations in MYH7 are located in the globular head region of the molecule, those responsible for MSM are part of a subset of MYH7 mutations that are located in the [alpha]-helical coiled-coil tail. Mutations in the myosin head are thought to affect the ATPase and actin-binding properties of the molecule. To date, however, there are no reports of the molecular mechanism of pathogenesis for mutations in the rod region of muscle myosins. Here, we present analysis of 4 mutations responsible for MSM: L1793P, R1845W, E1886K, and H1901L. We show that each MSM mutation has a different molecular phenotype, suggesting that there are multiple mechanisms by which MSM can be caused. These mechanisms range from thermodynamic and functional irregularities of individual proteins (L1793P), to varying defects in the assembly and stability of filaments formed from the proteins (R1845W, E1886K, and H1901L). In addition to furthering our understanding of MSM, these observations provide the first insight into how mutations affect the rod region of muscle myosins, and provide a framework for future studies of disease-causing mutations in this region of the molecule.

Genetic selection for improved growth and overall meatiness has resulted in the occurrence of 2 major mutations in pigs, the Rendement Napole (RN) and Halothane (Hal) gene mutations. At the tissue level, these mutations influence energy metabolism in skeletal muscle and muscle fiber type composition, yet also influence total body composition. The RN mutation affects the adenosine monophosphate-activated protein kinase [gamma] subunit and results in increased glycogen deposition in the muscle, whereas the Hal mutation alters sarcoplasmic calcium release mechanisms and results in altered energy metabolism. From a meat quality standpoint, these mutations independently influence the extent and rate of muscle energy metabolism postmortem, respectively. Even though these mutations alter overall muscle energy metabolism and histochemically derived muscle fiber type independently, their effects have not been yet fully elucidated in respect to myosin heavy chain (MyHC) isoform content and those enzymes responsible for defining energetics of the tissue. Therefore, the objective of this study was to determine the collective effects of the RN and Hal genes on genes and gene products associated with different muscle fiber types in pig skeletal muscle. To overcome potential pitfalls associated with traditional muscle fiber typing, real-time PCR, gel electrophoresis, and Western blotting were used to evaluate MyHC composition and several energy-related gene expressions in muscles from wild-type, RN, Hal, and Hal-RN mutant pigs. The MyHC mRNA levels displayed sequential transitions from IIb to IIx and IIa in pigs bearing the RN mutation. In addition, our results showed MyHC protein isoform abundance is correlated with mRNA level supporting the hypothesis that MyHC genes are transcriptionally controlled. However, transcript abundance of genes involved in energy metabolism, including lactate dehydrogenase, citrate synthase, glycogen synthase, and peroxisome proliferator-activated receptor [alpha], was not different between genotypes. These data show that the RN and Hal gene mutations alter muscle fiber type composition and suggest that muscle fiber energy metabolism and speed of contraction, the 2 determinants of muscle fiber type, can be uncoupled. Key words: halothane, myosin heavy chain, porcine, Rendement Napole, skeletal muscle

The cytokinetic furrow arises from spatial and temporal regulation of cortical contractility. To test the role microtubules play in furrow specification, we studied myosin II activation in echinoderm zygotes by assessing serine l9-phosphorylated regulatory light chain (pRLC) localization after precisely timed drug treatments. Cortical pRLC was globally depressed before cytokinesis, then elevated only at the equator. We implicated cell cycle biochemistry (not microtubules) in pRLC depression, and differential microtubule stability in localizing the subsequent myosin activation. With no microtubules, pRLC accumulation occurred globally instead of equatorially, and loss of just dynamic microtubules increased equatorial pRLC recruitment. Nocodazole treatment revealed a population of stable astral microtubules that formed during anaphase; among these, those aimed toward the equator grew longer, and their tips coincided with cortical pRLC accumulation. Shrinking the mitotic apparatus with colchicine revealed pRLC suppression near dynamic microtubule arrays. We conclude that opposite effects of stable versus dynamic microtubules focuses myosin activation to the cell equator during cytokinesis.

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was [Ca.sup.2+] dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity. actin; blebbistatin; isometric tension; myosin light chain kinase; regulatory light chain phosphorylation; focal adhesions

Phosphorylation of the 20-kDa myosin regulatory light chains ([LC.sub.20]) plays a key role in the regulation of smooth muscle contraction. The level of [LC.sub.20] phosphorylation is governed by the relative activities of myosin light chain kinase and phosphatase pathways. The regulation of these two pathways differs in different smooth muscle types and in the actions of different vasoactive stimuli. Little is known concerning the regulation of [LC.sub.20] phosphorylation in the renal microcirculation. The available pharmacological probes are often nonspecific, and current techniques to directly measure [LC.sub.20] phosphorylation are not sensitive enough for quantification in small arterioles. We describe here a novel approach to address this important issue. Using SDS-PAGE with polyacrylamide-bound [Mn.sup.2+]-phosphate-binding tag and enhanced Western blot analysis, we were able to detect [LC.sub.20] phosphorylation using as little as 5 pg (250 amol) of isolated [LC.sub.20]. Phosphorylated and unphosphorylated [LC.sub.20] were detected in single isolated afferent arterioles, and [LC.sub.20] phosphorylation levels could be accurately quantified in pooled samples of three arterioles ( afferent arteriole; Phos-tag SDS-PAGE

Male but not female mice carrying a single R403Q missense allele for cardiac [alpha]-myosin heavy chain (M-[alpha].sup.MH[C.sup.R403Q/+] and F-[alpha]MH[C.sup.R403Q/+], respectively) develop significant hypertrophic cardiomyopathy (HCM) compared with male and female wild-type mice (M-[alpha]MH[C.sup.+/+] and F-[alpha]MH[C.sup.+/+], respectively) after Tilde 30 wk of age. We tested the hypothesis that myofilament mechanical performance differs between M-[alpha]MH [C.sup.403Q/+] and F-[alpha]MH[C.sup.R403Q/+ ] at younger ages (10-20 wk) and could account for sex differences in HCM development. The sensitivity of chemically skinned myocardial strips to [Ca.sup.2+] activation (p[Ca.sub.50]) was signifcantly (P < 0.05) enhanced in male mice independent of genotype (M-[alpha]MH[C.sup.R403Q/+] 5.70 [+ or -] 0.06, M-[alpha]MH[C.sup.+/+]: 5.63 [+ or -] + 0.05, F-[alpha]MH [C.sup.R403Q/+]: 5.57 [+ or -] 0.03, F-[alpha] MH[C.sup.+/+]: 5.54 [+ or -] 0.04) by two-way ANOVA, whereas maximum developed tension was significantly enhanced in [alpha]-MH[C.sup.R403Q/+] independent of sex (M-[alpha]MH[C.sup.R403Q/+]: 29.3 [+ or -] 2.3, M-[alpha]MH [C.sup.+/+]: 26.0 [+ or -] 1.4, F-[alpha]MH[C.sup.R403Q/+]: 30.2 [+ or -] 2.1, F-[alpha]MH[C.sup.+/+]: 26.2 [+ or -] 1.2 mN/[mm.sup.2]). The frequency of maximum work generated by sinusoidal length perturbation was significantly higher in [alpha]MH[C.sup.R403Q/+] mice than in sex-matched controls (M-[alpha]MH[CR403Q/+]: 2.26 [+ or -] 0.47, M-[alpha]MH[C.sup.+/+]: 1.29[+ or -] 0.18, F-[alpha]MH [C.sup.R403Q/+]: 3.21 [+ or -] 0.33, F-[alpha]MH[C.sup.+/+]: 2.52 [+ or -] 0.36 Hz). Unloaded shortening velocity was significantly enhanced in [alpha]MH[C.R403Q/+] and in female mice (M-[alpha]MH [C.sup.R403Q/+]: 2.26 [+ or -] 0.47, M-[alpha]MH[C.sup.+/+]: 1.29 [+ or -] 0.18, F-[alpha]MH[C.sup.R403Q/+]: 3.21 [+ or -] 0.33, F-[alpha]MH [C.sup.+/+]: 2.52 [+ or -] 0.36 muscle lengths/s), and normalized mechanical power, calculated from the tension-velocity relationship, was significantly enhanced in [alpha]MH[C.sup.R403Q/+] independent of sex (M[alpha]MH[C.sup.R403Q/+]: 60 [+ or -] 2 [10.sup.-3], M-[alpha]MH [C.sup.+/+]: 37 [+ or -] 3 [10.sup.- 3], F-[alpha]MH[C.sup.R403Q/+]: 57 [+ or -] 3 [10.sup.-3], F-[alpha]MH [C.sup.+/+] 25 [+ or -] 3 [10.sup.-3] muscle lengths/s x normalized tension). We did not find a statistically significant sex x mutation interaction for any measure of myofilament performance. Therefore, sarcomeric incorporation of the R403Q myosin similarly enhanced left ventricular myofilament mechanical performance in both male and female mice. The sex-dependent development of HCM due to the R403Q myosin may then be inhibited by female sex hormones, which may additionally underlie the observed sex differences for p[Ca.sub.50] and unloaded shortening velocity. hypertrophic cardiomyopathy; myosin heavy chain; isometric tension; calcium sensitivity; force-velocity

Fructose feeding has been shown to induce the cardiac [alpha]-myosin heavy chain (MHC) expression and protect the heart from ischemia- and reperfusion-mediated cell injury. This study was designed to investigate the mechanism involved in the effect of this sugar on MHC gene expression and cardiac protection. Adult mice were fed with a 6-propyl-2-thiouracil (PTU) diet or PTU combined with a fructose-rich diet. PTU treatment made animals hypothyroid and that resulted in total replacement of cardiac [alpha]-MHC with the [beta]-MHC isoform. Addition of fructose in the PTU diet led to reexpression of the [alpha]-MHC isoform to a significant level. Similar induction of [beta]-MHC expression was also seen when PTU diet was combined with resveratrol, an agonist of sirtuin (SIRT) 1 deacetylase. Analysis of heart lysate of these animals indicated that fructose feeding augmented the NAD-to-NADH ratio and the cardiac SIRT1 levels, thus suggesting a role of SIRT1 in fructose-mediated activation of [alpha]-MHC isoform. To analyze a direct effect of SIRT1 on MHC isoform expression, we generated transgenic mice expressing SIRTI in the heart. Treatment of these transgenic mice with PTU diet did not lead to disappearance of [alpha]-MHC, as it did in the nontransgenic animals. SIRT1 overexpression also activated the [alpha]-MHC gene promoter in transient transfection assays, thus confirming a role of SIRT1 in the induction of [alpha]-MHC expression. Fructose feeding also attenuated the MHC isoform shift and blocked the cardiac hypertrophy response associated with pressure overload, which was again associated with the induction of cardiac SIRT1 levels. These results demonstrate that fructose feeding protects the heart by induction of the SIRT1 deacetylase and highlight its role in the induction of [alpha]-MHC gene expression. sirtuins; resveratrol; cardiac hypertrophy

A shortening muscle is a machine that converts metabolic energy into mechanical work, but, when a muscle is stretched, it acts as a brake, generating a high resistive force at low metabolic cost. The braking action of muscle can be activated with remarkable speed, as when the leg extensor muscles rapidly decelerate the body at the end of a jump. Here we used time-resolved x-ray and mechanical measurements on isolated muscle cells to elucidate the molecular basis of muscle braking and its rapid control. We show that a stretch of only 5 nm between each overlapping set of myosin and actin filaments in a muscle sarcomere is sufficient to double the number of myosin motors attached to actin within a few milliseconds. Each myosin molecule has two motor domains, only one of which is attached to actin during shortening or activation at constant length. A stretch strains the attached motor domain, and we propose that combined steric and mechanical coupling between the two domains promotes attachment of the second motor domain. This mechanism allows skeletal muscle to resist external stretch without increasing the force per motor and provides an answer to the longstanding question of the functional role of the dimeric structure of muscle myosin. motor proteins | myosin II

Diverse forms of injury and stress evoke a hypertrophic growth response in adult cardiac myocytes, which is characterized by an increase in cell size, enhanced protein synthesis, assembly of sarcomeres, and reactivation of fetal genes, often culminating in heart failure and sudden death. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs that were regulated during cardiac hypertrophy and heart failure. We describe >12 miRNAs that are up- or down-regulated in cardiac tissue from mice in response to transverse aortic constriction or expression of activated calcineurin, stimuli that induce pathological cardiac remodeling. Many of these miRNAs were similarly regulated in failing human hearts. Forced overexpression of stress-inducible miRNAs was sufficient to induce hypertrophy in cultured cardiomyocytes. Similarly, cardiac overexpression of miR-195, which was up-regulated during cardiac hypertrophy, resulted in pathological cardiac growth and heart failure in transgenic mice. These findings reveal an important role for specific miRNAs in the control of hypertrophic growth and chamber remodeling of the heart in response to pathological signaling and point to miRNAs as potential therapeutic targets in heart disease. calcineurin | myosin heavy chain | thoracic aortic banding | cardiomyocytes

The FMR family of KH domain RNA-binding proteins is conserved from invertebrates to humans. In humans, inactivation of the X-linked FMR gene fragile X is the most common cause of mental retardation and leads to defects in neuronal architecture. While there are three FMR family members in humans, there is only a single gene, dfmr1, in flies. As in humans, inactivation of dfmr1 causes defects in neuronal architecture and in behavior, dfmr1 has other functions in the fly in addition to neurogenesis. Here we have analyzed its role during early embryonic development. We found that dfmr1 embryos display defects in the rapid nuclear division cycles that precede gastrulation in nuclear migration and in pole cell formation. While the aberrations in nuclear division are correlated with a deflect in the assembly of centromeric/centric heterochromatin, the defects in pole cell formation are associated with alterations in the actin--myosin cytoskeleton.

Byline: Helena Stibler (1), Lars Edstrom (1), Karsten Ahlbeck (2), Sten Remahl (3), Tor Ansved (1) Keywords: Critical illness myopathy; Electrophoresis; Morphology; Myosin/actin ratio; Neurophysiology Abstract: Objective To develop a rapid method to quantify myosin in muscle biopsy specimens from patients with critical illness myopathy (CIM). Design Percutaneous muscle biopsy specimens at different stages of CIM were examined by light microscopy and transmission electron microscopy (TEM) and by horizontal pore gradient SDS electrophoresis (SDS-PAGE). The myosin/actin ratio was calculated densitometrically. Neurophysiological examinations were performed at various times during the course of CIM. Setting All patients were treated in intensive care units at Karolinska Hospital. Patients and participants We studied 11 patients with CIM, 5 patients with axonal neuropathies, and 42 control individuals. Measurements and results The histopathological changes included in all cases muscle fiber atrophy, degeneration, regeneration, nuclear changes, and reduction in myofibrillar ATPase activity in both type I and II fibers. In severely affected muscles fiber type differentiation was lost. On TEM preferential loss of thick filaments was the dominant finding. In some patients changes were present only in parts of the specimen. The neurophysiological examinations indicated myopathy in five patients and combined myopathy and neuropathy in five and suggested neuropathy in one. The SDS-pore PAGE used showed a technical variation of 4--5%. Quantitative results were obtained within 1 day and night. The mean value of the myosin/actin ratio in controls was 1.37+-0.21 and in CIM patients 0.37+-0.17, without overlapping with the control values. Conclusions Considering the diagnostic difficulty using morphological and neurophysiological methods, especially in early stages of CIM, we suggest including SDS-pore PAGE to determine the myosin/actin ratio for rapid diagnosis of CIM. Author Affiliation: (1) Department of Neurology, Karolinska Hospital, Stockholm, Sweden (2) Department of Anestesiology, Karolinska Hospital, 17176, Stockholm, Sweden (3) Department of Neurophysiology, Karolinska Hospital, 17176, Stockholm, Sweden Article History: Received Date: 21/11/2002 Accepted Date: 27/05/2003 Online Date: 12/08/2003 Article note: This work was supported by grants from the Swedish Medical Research Council (nos. 07121, 03875) and the Karolinska Institute.

Daily administration of recombinant porcine somatotropin (rpST) has marked effects on growth performance of swine. Protein accretion rates are dramatically increased with rpST treatment. However, the effect of rpST on specific muscle proteins has not been addressed. The purpose of this study was to examine the effects of rpST on 1) the distribution pattern of isomyosins, 2) the total myosin heavy chain protein levels, and 3) myosin heavy chain mRNA abundance present in swine skeletal muscle. There were no differences in myosin isoform composition in the longissimus muscle resulting from porcine somatotropin treatment. There were also no differences in the myosin heavy chain protein concentration within the longissimus muscles. In accordance with the protein levels, myosin heavy chain mRNA expression was not altered with rpST treatment. This study shows that although the pig responds to rpST with increased protein accretion rates, the proportion of myosin per milligram of total protein remains constant. Myosin was not altered by type, concentration, or expression level. By combining these tools we are now able to examine a myosin on both a protein and message level throughout growth.