Cross-talk between glycogen synthase kinase 3ß (GSK3ß) and p38MAPK regulates myocyte enhancer factor 2 (MEF2) activity in skeletal and cardiac muscle.

Characterizing the signaling network that controls MEF2 transcription factors is crucial for understanding skeletal and cardiac muscle gene expression. Glycogen synthase kinase 3ß (GSK3ß) regulates MEF2 activity indirectly through reciprocal regulation of p38MAPK. Cross-talk between GSK3ß and p38MAPK regulates MEF2 activity in skeletal and cardiac muscle. Understanding cross-talk in the signaling network converging at MEF2 control has therapeutic implications in cardiac and skeletal muscle pathology. Glycogen synthase kinase 3ß (GSK3ß) is a known regulator of striated muscle gene expression suppressing both myogenesis and cardiomyocyte hypertrophy. Since myocyte enhancer factor 2 (MEF2) proteins are key transcriptional regulators in both systems, we assessed whether MEF2 is a target for GSK3ß. Pharmacological inhibition of GSK3ß resulted in enhanced MEF2A/D expression and transcriptional activity in skeletal myoblasts and cardiac myocytes. Even though in silico analysis revealed GSK3ß consensus (S/T)XXX(S/T) sites on MEF2A, a subsequent in vitro kinase assay revealed that MEF2A is only a weak substrate. However, we did observe a posttranslational modification in MEF2A in skeletal myoblasts treated with a GSK3ß inhibitor which coincided with increased p38MAPK phosphorylation, a potent MEF2A activator, indicating that GSK3ß inhibition may de-repress p38MAPK. Heart specific excision of GSK3ß in mice also resulted in up-regulation of p38MAPK activity. Interestingly, upon pharmacological p38MAPK inhibition (SB203580), GSK3ß inhibition loses its effect on MEF2 transcriptional activity suggesting potent cross-talk between the two pathways. Thus we have documented that cross-talk between p38MAPK and GSK3ß signaling converges on MEF2 activity having potential consequences for therapeutic modulation of cardiac and skeletal muscle gene expression.

hMEF2C gene encodes skeletal muscle- and brain-specific transcription factors.

The myocyte enhancer-binding factor 2 (MEF2) site is an essential element of many muscle-specific enhancers and promoters that binds nuclear proteins from muscle and brain. Recently, we have cloned a family of MEF2 transcription factors produced by two genes that, at the mRNA level, are broadly expressed and produce tissue-specific isoforms by posttranscriptional processes (Y.-T. Yu, R. E. Breitbart, L. B. Smoot, Y. Lee, V. Mahdavi, and B. Nadal-Ginard, Genes Dev. 6:1783-1798, 1992). Here, we report the isolation and functional characterization of cDNA clones encoding four MEF2 factors derived from a separate gene that we have named hMEF2C. In contrast to those of the previously reported genes, the transcripts of the hMEF2C gene are restricted to skeletal muscle and brain. One of the alternate exons is exclusively present in brain transcripts. The products of this gene have DNA-binding and trans-activating activities indistinguishable from those of the previously reported MEF2 factors. The hMEF2C gene is induced late during myogenic differentiation, and its expression is limited to a subset of cortical neurons. The potential targets for this transcription factor in a subset of neurons are not known at this time. The strict tissue-specific pattern of expression of hMEF2C in comparison with the more ubiquitous expression of other MEF2 genes suggests a different mode of regulation and a potentially important role of hMEF2C factors in myogenesis and neurogenesis.

18F-labeled resin microspheres as surrogates for 90Y resin microspheres used in the treatment of hepatic tumors: a radiolabeling and PET validation study.

(90)Y-labeled resin microspheres (SIR-Spheres) are currently used to treat patients with primary and metastatic solid liver tumors. This treatment is typically palliative since patients have exhausted all other standard treatment options. Improving the quality of life and extending patient survival are typical benchmarks for tracking patient response. However, the current method for predicting microsphere biodistributions with (99m)Tc-labeled macroaggregated albumin (MAA) does not correlate well with patient response. This work presents the development of a new (18)F-labeled resin microsphere to serve as a surrogate for the treatment microsphere and to employ the superior resolution and sensitivity of positron emission tomography (PET). The (18)F microsphere biodistributions were determined in a rabbit using PET imaging and histological review. The PET-based uptake ratio was shown to agree with the histological findings to better than 3%. In addition, the radiolabeling process was shown to be rapid, efficient and relatively stable in vivo.

Heart Failure and MEF2 Transcriptome Dynamics in Response to ß-Blockers.

Myocyte Enhancer Factor 2 (MEF2) mediates cardiac remodelling in heart failure (HF) and is also a target of ß-adrenergic signalling, a front-line treatment for HF. We identified global gene transcription networks involved in HF with and without ß-blocker treatment. Experimental HF by transverse aortic constriction (TAC) in a MEF2 "sensor" mouse model (6 weeks) was followed by four weeks of ß-blockade with Atenolol (AT) or Solvent (Sol) treatment. Transcriptome analysis (RNA-seq) from left ventricular RNA samples and MEF2A depleted cardiomyocytes was performed. AT treatment resulted in an overall improvement in cardiac function of TAC mice and repression of MEF2 activity. RNA-seq identified 65 differentially expressed genes (DEGs) due to TAC treatment with enriched GO clusters including the inflammatory system, cell migration and apoptosis. These genes were mapped against DEGs in cardiomyocytes in which MEF2A expression was suppressed. Of the 65 TAC mediated DEGs, AT reversed the expression of 28 mRNAs. Rarres2 was identified as a novel MEF2 target gene that is upregulated with TAC in vivo and isoproterenol treatment in vitro which may have implications in cardiomyocyte apoptosis and hypertrophy. These studies identify a cohort of genes with vast potential for disease diagnosis and therapeutic intervention in heart failure.

Smad proteins function as co-modulators for MEF2 transcriptional regulatory proteins.

An emerging theme in transforming growth factor-ss (TGF-ss) signalling is the association of the Smad proteins with diverse groups of transcriptional regulatory proteins. Several Smad cofactors have been identified to date but the diversity of TGF-ss effects on gene transcription suggests that interactions with other co-regulators must occur. In these studies we addressed the possible interaction of Smad proteins with the myocyte enhancer-binding factor 2 (MEF2) transcriptional regulators. Our studies indicate that Smad2 and 4 (Smad2/4) complexes cooperate with MEF2 regulatory proteins in a GAL4-based one-hybrid reporter gene assay. We have also observed in vivo interactions between Smad2 and MEF2A using co-immunoprecipitation assays. This interaction is confirmed by glutathione S:-transferase pull-down analysis. Immunofluorescence studies in C2C12 myotubes show that Smad2 and MEF2A co-localise in the nucleus of multinuclear myotubes during differentiation. Interestingly, phospho-acceptor site mutations of MEF2 that render it unresponsive to p38 MAP kinase signalling abrogate the cooperativity with the Smads suggesting that p38 MAP Kinase-catalysed phosphorylation of MEF2 is a prerequisite for the Smad-MEF2 interaction. Thus, the association between Smad2 and MEF2A may subserve a physical link between TGF-ss signalling and a diverse array of genes controlled by the MEF2 cis element.

Multiple reaction monitoring as a method for identifying protein posttranslational modifications.

The activity of many transcriptional regulators is significantly altered by posttranslational modifications of specific sites. For example, the activity of the muscle-restricted transcription factor family myocyte enhancer factor 2 (MEF2) is tightly controlled by phosphorylation. This modification is responsible for either an increase or a decrease in transcriptional activity, depending on the specific amino acid residues that are phosphorylated by signal-dependent kinases. Although mass spectrometry-based methods, such as precursor ion and neutral loss scans, are extremely useful for identifying unknown phosphopeptides from a complex mixture, they do not take advantage of any prior knowledge about the protein being investigated. Quite often a significant amount of information is available. This may include the primary sequence, type of phosphorylation (serine/threonine vs. tyrosine), or predicted phosphoacceptor sites (consensus peptide that is targeted by a kinase). This information can be used to predict precursor and fragment ion m/z values for a multiple reaction monitoring (MRM) experiment. By using these highly sensitive MRM experiments to trigger dependent product ion scans on a hybrid quadrupole linear ion-trap instrument, we were able to identify low levels of phosphorylation of MEF2A (a member of the MEF2 family), and alpha-casein. This method of monitoring protein phosphorylation at specific phosphoacceptor sites may prove useful in understanding the physiological regulation of protein function.

Persistence of glucose metabolism after exercise in trained and untrained soleus muscle.

We tested the hypothesis that increments in glucose metabolism in muscles from trained animals are caused by training adaptations in skeletal muscle and not by the residual effects of the last training session. The effects of a single bout of exercise on glucose metabolism (glycolysis and glycogenesis) were compared, against appropriate controls, in untrained (experiment 1) and trained (experiment 2) rat soleus muscles immediately (t = 0) and 3, 6, 24, 48, and 96 h after a standardized bout of exercise. [3H]Glucose incorporation into glycogen and glycolysis was measured in vitro in the absence and presence of insulin (0.1 and 10 nM). Experiment 1: A single bout of exercise provoked an increase in glycogenesis in the exercised, untrained muscles compared with the nonexercised, untrained muscles (0-96 h; P = 0.006). Glycolysis was not altered (0-96 h; P > 0.05). Experiment 2: In the exercised trained soleus, rates of glycolysis were greater than in the exercised, untrained soleus, at insulin concentrations of 0.1 nM (0-96 h; P = 0.005) and 10 nM (0-96 h; P = 0.01), but not in the absence of insulin (0-96 h; P > 0.05). No differences were observed in the rates of glycogenesis (0-96 h; P > 0.05). Therefore, acute exercise provokes increments in glycogenesis, whereas training increases glycolysis, in the presence of insulin, for some time after exercise. We speculate that insulin-dependent increments in glycolysis in trained muscles are a consequence of increased glucose transport caused by a greater pool of insulin-translocatable, intracellular glucose transporters.

Pro-survival function of MEF2 in cardiomyocytes is enhanced by ß-blockers.

ß1-Adrenergic receptor (ß1-AR) stimulation increases apoptosis in cardiomyocytes through activation of cAMP/protein kinase A (PKA) signaling. The myocyte enhancer factor 2 (MEF2) proteins function as important regulators of myocardial gene expression. Previously, we reported that PKA signaling directly represses MEF2 activity. We determined whether (a) MEF2 has a pro-survival function in cardiomyocytes, and (b) whether ß-adrenergic/PKA signaling modulates MEF2 function in cardiomyocytes. Initially, we observed that siRNA-mediated gene silencing of MEF2 induces cardiomyocyte apoptosis as indicated by flow cytometry. ß1-AR activation by isoproterenol represses MEF2 activity and promotes apoptosis in cultured neonatal cardiomyocytes. Importantly, ß1-AR mediated apoptosis was abrogated in cardiomyocytes expressing a PKA-resistant form of MEF2D (S121/190A). We also observed that a ß1-blocker, Atenolol, antagonizes isoproterenol-induced apoptosis while concomitantly enhancing MEF2 transcriptional activity. ß-AR stimulation modulated MEF2 cellular localization in cardiomyocytes and this effect was reversed by ß-blocker treatment. Furthermore, Kruppel-like factor 6, a MEF2 target gene in the heart, functions as a downstream pro-survival factor in cardiomyocytes. Collectively, these data indicate that (a) MEF2 has an important pro-survival role in cardiomyocytes, and (b) ß-adrenergic signaling antagonizes the pro-survival function of MEF2 in cardiomyocytes and ß-blockers promote it. These observations have important clinical implications that may contribute to novel strategies for preventing cardiomyocyte apoptosis associated with heart pathology.

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.

Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

Pneumatoceles causing respiratory compromise. Treatment by percutaneous decompression.

Pulmonary pneumatoceles are uncommon but generally benign, thin-walled parenchymal air collections arising in association with acute pneumonia. Rarely, they may attain such size as to severely affect respiration. We describe the percutaneous placement of multiple drains in a patient with pneumonia due to atypical measles and large pneumatoceles. Decompression via tubes resulted in improved ventilation and acceleration of recovery.

The influence of ambient and viewbox light upon visual detection of low-contrast targets in a radiograph.

Thirty-one observers (five radiologists, five radiology residents, and 21 nonradiologists) examined low-contrast images of 13 polyvinylchloride cylinders. Image density ranged from 1.91 to 2.05 OD units with a background density of 2.09 OD units. The experiment tested visual performance under various clinically relevant lighting conditions by asking the observers to locate a groove in each cylinder. Use of low ambient light and restricting light from surrounding veiwboxes significantly improved performance (P less than 0.0001). In general, the less the amount of extraneous light, the higher was the detection rate. Some observers did significantly better than others. Reduced scattering of light within the eye is suggested as an explanation for their increased performance.

Thrombolysis versus surgery as the initial management for native artery occlusion: efficacy, safety, and cost.

BACKGROUND: A controversy has evolved as to which therapy, thrombolysis or thromboembolectomy, represents the optimal initial treatment for acute native artery occlusion. METHODS: Forty-eight cases of acute class I or II limb ischemia caused by native artery occlusion were retrospectively analyzed between 1988 and 1993. Nineteen of the patients were initially treated with thrombolysis (group 1), and 29 underwent thromboembolectomy (group 2). RESULTS: Initial clinical improvement was seen in 11 (57.9%) of 19 extremities in group 1, with complete clot resolution in 21%, partial lysis in 47.4%, and no angiographic improvement in 31.6%. Significantly superior results were achieved in group 2; 28 (97%) of 29 limbs showed clinical improvement after initial surgical therapy (p = 0.001). Limb salvage was 88.2% in group 1 and 96.6% in group 2 (p = 0.5). Adjunctive procedures for limb salvage were necessary in 10 (52.6%) of 19 limbs in group 1 compared with only five (17.2%) of 29 limbs in group 2 (p = 0.013). Perioperative mortality was 10.5% and 10.3% (p = 1.0), whereas major postoperative complications occurred in 63.2% and 37% of patients in groups 1 and 2, respectively (p = 0.14). Hospital and professional patient charges were analyzed for the 12 most recent patients from each group. Total mean charges per patient were higher in group 1 ($45,171) than in group 2 ($24,898) (p = 0.046). CONCLUSIONS: Patients initially treated surgically achieved better immediate clinical results with significant cost savings and without significant differences in morbidity, mortality, or limb salvage compared with patients treated initially by thrombolysis.

Portal hypertension: surgical management in the 1990s.

BACKGROUND: Although liver transplantation offers definitive treatment for portal hypertension with end-stage liver failure, surgical portosystemic shunts avoid the risks of transplantation and immunosuppressive therapy, and transjugular intrahepatic portosystemic shunt (TIPS) creates a portosystemic shunt with minimal operative risk. The appropriate applications of these modalities are discussed. METHODS: All adults undergoing primary liver transplantation alone (PLT, n = 265), PLT after TIPS (n = 34), PLT after surgical shunts (n = 12), surgical shunt alone (n = 13), TIPS alone (n = 35), or surgical shunt after PLT (n = 5) served as the basis of this study. RESULTS: In contrast to surgical shunts before PLT, TIPS before PLT increased the 1-year graft survival. Surgical shunts alone were done in 18 patients with normal or near normal liver function with 100% survival. TIPS alone offered effective symptomatic relief to most patients, all of whom were judged not to be surgical candidates. CONCLUSIONS: TIPS, surgical shunts, and liver transplantation each have a logical role in management of portal hypertension. Surgical candidates with Child's B or C liver failure should be treated with liver transplantation, and TIPS offers effective treatment for nonsurgical candidates. Surgical shunts can be performed with excellent results in patients with Child's A liver disease. Portal vein occlusion with normal liver function can be successfully treated with surgical shunts.

Adrenal hormones enhance glycogenolysis in nonexercising muscle during exercise.

The purpose of this study was to investigate whether epinephrine exerts an effect on glycogen metabolism in nonexercising (Non-Ex) as well as in exercising (Ex) skeletal muscle. Rats ran (15 m/min; 8% grade) on their forelimbs while their hindlimbs (Non-Ex) were suspended above the treadmill. Electromyographic records confirmed the lack of significant contractile activity in muscles during suspension. Plasma epinephrine levels were manipulated in three experimental groups (n = 20 for each group): adrenalectomized (ADX), intact adrenals (IA), and IA + epinephrine injection (+Ep). Another group of rats performed normal exercise on all four limbs (15 m/min; 8% grade). Muscle glycogen levels were measured in selected hindlimb muscles at t = 0 and after 90 min exercise (15 m/min; 8% grade) or suspended rest. In the absence of epinephrine (ADX), no glycogen loss was found (P greater than 0.05) in Non-Ex muscles during the exercise period. In the IA group (epinephrine levels elevated sixfold above basal at t = 90 min), glycogen levels in the nonexercising soleus, plantaris, and red and white gastrocnemius were significantly (P less than 0.05) depleted to 62 +/- 6, 67 +/- 6, 58 +/- 5, and 67 +/- 9% of control values, respectively. Similar decrements occurred in these muscles when exercise was performed on all four limbs (P greater than 0.05). We conclude that glycogenolysis occurs in nonexercising skeletal muscle independent of contractile activity, probably due to the effect of epinephrine. Furthermore, the present data strongly suggest that glycogen depletion patterns in muscles during exercise cannot be used as an index of motor unit recruitment.

The effect of phosphate, sodium chloride, sodium nitrite, storage temperature and pH on the growth of enteropathogenic Escherichia coli in a laboratory medium.

The effect of eight phosphates in combination with sodium chloride/nitrite mixtures on growth of mixed strains of enteropathogenic Escherichia coli over a period of 10 weeks at three pH values (5.6, 6.2, 6.8) and at seven temperatures ranging from 10 degrees C to 35 degrees C is reported. All eight phosphates inhibited growth to varying degrees in at least some of the conditions investigated. Instances of inhibition increased with concentration of sodium chloride/nitrite and were more frequent at lower temperatures and pH values.

Ureteral injury during laparoscopy-assisted anterior lumbar fusion.

STUDY DESIGN: A case report of ureteral injury as a complication incurred during a laparoscopy-assisted lumbar fusion. OBJECTIVE: To alert orthopedic surgeons to this injury, which may occur during such surgery. SUMMARY OF BACKGROUND DATA: Laparoscopy-assisted lumbar fusion is a minimally invasive surgical technique to accomplish lumbar fusion with excellent patient satisfaction, reduced hospital stay, and decreased rehabilitative time. METHOD AND RESULTS: A case report is presented detailing ureteral injury as a complication of laparoscopy-assisted lumbar fusion and the subsequent treatment of the injury. CONCLUSION: Laparoscopy-assisted lumbar fusion is a new, less invasive technique with excellent patient satisfaction; however, ureteral injury may occur, and the surgeon should keep this in mind if a postoperative fluid collection occurs in the pelvis.

Masked high-frequency bone-conduction audiometry: test reliability.

The present study examines the reliability of masked high-frequency bone-conduction threshold measurements in 95 normal-hearing subjects. High-frequency pure-tone air-and bone-conduction thresholds were measured with a dedicated laboratory high-frequency auditory evaluation system using matched, modified Koss Pro/4X Plus earphones, and the Pracitronic KH 70/5 bone vibrator. A 400-Hz wide band masking noise centered at the frequency of the test tone was used to mask the nontest ear. Monaural masked bone-conduction threshold measurements were obtained at the ipsilateral mastoid of the ear with better high-frequency hearing. Two measurements were performed in each session, and each subject participated in two sessions. In several comparisons for test-retest consistency, high-frequency bone-conduction threshold measurements were as repeatable as air-conduction thresholds of identical frequency, or bone-conduction thresholds for frequencies of 4 kHz and less. High-frequency bone-conduction threshold measurement appears to be a sufficiently reliable tool for diagnosis of auditory disorders.

Physical and behavioral aspects of middle ear disease in school children.

Middle ear disease is today's primary aural health problem in school-age children. Until recently, pediatric health researchers were aware only of the medical consequences that middle ear disease had no children. Except in a few cases of chronic otitis media, the effects of middle ear disease were considered transitory. However, current research on middle ear disease suggests permanent changes in hearing sensitivity, reduced development of the auditory neural network and developmental delays in speech, language and cognitive skills dependent on hearing. This article presents a review of recent studies on the physical and behavioral aspects of middle ear disease and its sequelae in school children.

Immittance screening for aural problems in school children.

The predominant aural health problem in school-age children is middle ear inflammation with effusion. The conventional hearing screening method has ineffectively detected half of those school children with middle ear problems. Acoustic immittance measurements, recently added to the audiologist's battery of tests, effectively and economically screens middle ear effusion. Unfortunately, acoustic immittance does not screen hearing sensitivity as does the conventional pure-tone sweep check. This article describes the principles of acoustic immittance and compares it with pure-tone audiometry; discusses current immittance screening practices; and outlines how acoustic immittance can be included in a school hearing conservation program.

Glycogen synthase kinase 3ß represses MYOGENIN function in alveolar rhabdomyosarcoma.

MYOGENIN is a member of the muscle regulatory factor family that orchestrates an obligatory step in myogenesis, the terminal differentiation of skeletal muscle cells. A paradoxical feature of alveolar rhabdomyosarcoma (ARMS), a prevalent soft tissue sarcoma in children arising from cells with a myogenic phenotype, is the inability of these cells to undergo terminal differentiation despite the expression of MYOGENIN. The chimeric PAX3-FOXO1 fusion protein which results from a chromosomal translocation in ARMS has been implicated in blocking cell cycle arrest, preventing myogenesis from occurring. We report here that PAX3-FOXO1 enhances glycogen synthase kinase 3ß (GSK3ß) activity which in turn represses MYOGENIN activity. MYOGENIN is a GSK3ß substrate in vitro on the basis of in vitro kinase assays and MYOGENIN is phosphorylated in ARMS-derived RH30 cells. Constitutively active GSK3ß(S9A) increased the level of a phosphorylated form of MYOGENIN on the basis of western blot analysis and this effect was reversed by neutralization of the single consensus GSK3ß phosphoacceptor site by mutation (S160/164A). Congruently, GSK3ß inhibited the trans-activation of an E-box reporter gene by wild-type MYOGENIN, but not MYOGENIN with the S160/164A mutations. Functionally, GSK3ß repressed muscle creatine kinase (MCK) promoter activity, an effect which was reversed by the S160/164A mutated MYOGENIN. Importantly, GSK3ß inhibition or exogenous expression of the S160/164A mutated MYOGENIN in ARMS reduced the anchorage independent growth of RH30 cells in colony-formation assays. Thus, sustained GSK3ß activity represses a critical regulatory step in the myogenic cascade, contributing to the undifferentiated, proliferative phenotype in alveolar rhabdomyosarcoma (ARMS).