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.

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.

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.

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).

Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells.

Activator protein-1 (AP-1) is a ubiquitous transcription factor that paradoxically also has some tissue-specific functions. In skeletal muscle cells, we document that the AP-1 subunit, Fra-2, is expressed in the resident stem cells (Pax7-positive satellite cells) and also in the analogous undifferentiated 'reserve' cell population in myogenic cultures, but not in differentiated myofiber nuclei. Silencing of Fra-2 expression enhances the expression of differentiation markers such as muscle creatine kinase and myosin heavy chain, indicating a possible role of Fra-2 in undifferentiated myogenic progenitor cells. We observed that Fra-2 is a target of cytokine-mediated extracellular signal-regulated kinase-1/2 signaling in cultured muscle cells, and extensive mass spectrometry and mutational analysis identified S320 and T322 as regulators of Fra-2 protein stability. Interestingly, Fra-2 S320 phosphorylation occurs transiently in activated satellite cells and is extinguished in myogenin-positive differentiating cells. Thus, cytokine-mediated Fra-2 expression and stabilization is linked to regulation of myogenic progenitor cells having implications for the molecular regulation of adult muscle stem cells and skeletal muscle regeneration.

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.

Direct interaction between myocyte enhancer factor 2 (MEF2) and protein phosphatase 1alpha represses MEF2-dependent gene expression.

The myocyte enhancer factor 2 (MEF2) transcription factors play important roles in neuronal, cardiac, and skeletal muscle tissues. MEF2 serves as a nuclear sensor, integrating signals from several signaling cascades through protein-protein interactions with kinases, chromatin remodeling factors, and other transcriptional regulators. Here, we report a novel interaction between the catalytic subunit of protein phosphatase 1alpha (PP1alpha) and MEF2. Interaction occurs within the nucleus, and binding of PP1alpha to MEF2 potently represses MEF2-dependent transcription. The interaction utilizes uncharacterized domains in both PP1alpha and MEF2, and PP1alpha phosphatase activity is not obligatory for MEF2 repression. Moreover, a MEF2-PP1alpha regulatory complex leads to nuclear retention and recruitment of histone deacetylase 4 to MEF2 transcription complexes. PP1alpha-mediated repression of MEF2 overrides the positive influence of calcineurin signaling, suggesting PP1alpha exerts a dominant level of control over MEF2 function. Indeed, PP1alpha-mediated repression of MEF2 function interferes with the prosurvival effect of MEF2 in primary hippocampal neurons. The PP1alpha-MEF2 interaction constitutes a potent locus of control for MEF2-dependent gene expression, having potentially important implications for neuronal cell survival, cardiac remodeling in disease, and terminal differentiation of vascular, cardiac, and skeletal muscle.

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.

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.

Effect of MR angiography on the diagnosis and treatment of patients with suspected renovascular disease.

PURPOSE: Although the diagnostic accuracy of renal magnetic resonance (MR) angiography is established, its effect on referring physicians is unknown. The authors prospectively measured the effect of MR angiography results on referring physicians' diagnosis and treatment (plans) of patients with suspected renovascular disease. MATERIALS AND METHODS: Referring physicians prospectively completed questionnaires before and after MR angiography was performed during evaluation of their patients with suspected renovascular disease. The questionnaires asked them to estimate the probability (0%-100%) of their most likely diagnosis before and after receiving the imaging information. They were also asked for their anticipated and final treatment plans. The authors calculated the mean gain in diagnostic percentage confidence and the proportion of patients with changed initial diagnoses or anticipated management. A paired t-test was used to assess significance of the gains in diagnostic percentage confidence. RESULTS: Physicians prospectively completed pre- and post-MR-angiography questionnaires for 30 patients. MR angiography improved mean diagnostic certainty by 35% (P < .0001). MR angiography changed physicians' initial diagnoses in 12 patients (40%). Anticipated treatment plans were changed in 20 patients (67%). Invasive procedures were avoided in eight patients (27%). CONCLUSION: MR angiography has a substantial effect on the diagnostic and therapeutic decision-making of physicians managing patients with suspected renovascular disease.

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.

Cell signaling and the regulation of muscle-specific gene expression by myocyte enhancer-binding factor 2.

Skeletal muscle is an extremely adaptable tissue. Underlying the biochemical adaptation invoked by changes in activity or during development are dramatic alterations in gene expression. Recent advances in our understanding of the molecular machinery that regulates gene expression in muscle is allowing insight into the pathways that control muscle growth and differentiation. We review developments concerning how cellular signaling pathways induce genetic reprogramming in skeletal muscle.

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.

Peripheral vascular disease and renal transplant artery stenosis: a reappraisal of transplant renovascular disease.

BACKGROUND: Renal transplant artery stenosis (RTAS) continues to be a problematic, but potentially correctable, cause of post-transplant hypertension and graft dysfunction. Older transplant recipients, prone to peripheral vascular disease (PVD), may have pseudoRTAS with PVD involving their iliac system. METHODS: We retrospectively analyzed 819 patients who underwent kidney transplantation between 1993 and 1997 to determine the contribution of pseudoRTAS to renal transplant renovascular disease. Univariate analyses were performed for donor and recipient variables, including age, weight, gender, race, renal disease, cholesterol and creatinine values, human leukocyte antigen (HLA) matching, cytomegalovirus (CMV) infection, and immunosuppressive medications. Significant variables were then analyzed by a Cox proportional hazards model. RESULTS: Ninety-two patients (11.2%) underwent renal transplant arteriogram (Agram) or magnetic resonance angiography (MRA) for suspected RTAS. RTAS or pseudoRTAS, defined as one or more hemodynamically significant lesions in the transplant artery or iliac system, was evident in 44 patients (5.4%). Variables significantly associated with RTAS by univariate analysis were weight at the time of transplant (p = 0.0258), male gender (p = 0.034), discharge serum creatinine > 2 mg/dL (p = 0.0041), and donor age (p = 0.0062). Variables significantly associated with pseudoRTAS by univariate analysis were weight at the time of transplant (p = 0.0285), recipient age (p = 0.0049), insulin-dependent diabetes mellitus (IDDM; p = 0.0042), panel reactive antibody (PRA) at transplant (p = 0.018), and body mass index (p = 0.04). Weight at transplant and donor age remained significantly associated with an increased risk for RTAS in a multivariate stepwise Cox proportional hazards model. IDDM, transplant PRA, weight at transplant, and donor age were significantly associated with an increased risk for pseudoRTAS in a multivariate stepwise Cox proportional hazards model. Importantly, both RTAS and pseudoRTAS were associated with poorer graft survival (p < 0.007 for each). CONCLUSIONS: Renal transplant renovascular disease encompasses pre-existing PVD acting as pseudoRTAS, as well as classical RTAS. Efforts to identify and correct renal transplant renovascular disease of either nature are important, given its negative impact on graft survival.

Post-translational control of the MEF2A transcriptional regulatory protein.

Myocyte enhancer factor 2 (MEF2) transcriptional regulatory proteins are key regulators of muscle-specific gene expression and also play a general role in the cellular response to growth factors, cytokines and environmental stressors. To identify signaling pathway components that might mediate these events, the potential role of MAP kinase and PKC signaling in the modulation of MEF2A phosphorylation and transcriptional activity were therefore studied. In transient transfection reporter assays, activated p38 MAP kinase potently increased MEF2A trans -activating potential, PKC[delta] and [epsiv] isotypes enhanced MEF2A transactivation to a lesser extent, while the ERK1/2 and JNK/SAPK pathways were without effect. A GAL4-based assay system showed that p38 MAP kinase and PKC[delta] target the MEF2A transactivation domain. We also observed an increase in p38 MAP kinase activity in congruence with the increase in MEF2A expression in differentiating primary muscle cells. COS cells overexpressing MEF2A alone or with one of the kinases were metabolically labeled with [32P]orthophosphate and MEF2A was immunoprecipitated using specific anti-MEF2A antibodies. MEF2A from cells co-transfected with activated p38 MAP kinase showed a decreased electrophoretic mobility due to phosphorylation. Subsequent phosphopeptide mapping and phosphoamino acid analysis indicated the appearance of several phoshopeptides due to p38 MAP kinase activation of MEF2A which were due to phosphorylation on serine and threonine residues. These studies position MEF2A as a nuclear target for the p38 MAP kinase signaling pathway.

Interaction of myocyte enhancer factor 2 (MEF2) with a mitogen-activated protein kinase, ERK5/BMK1.

Myocyte enhancer factor 2 (MEF2) has been implicated in the complex hierarchical regulation of muscle-specific gene expression and differentiation. While the MyoD family members are able to initiate the skeletal muscle differentiation program, whether MEF2 is sufficient in directing skeletal muscle differentiation is still controversial. Furthermore, how MEF2 transactivates its target genes is not fully understood. It has been suggested that the interactions of MEF2 with other factors modify its transcriptional activity. Therefore, the identification of MEF2-interacting factors may be important in understanding the mechanism by which MEF2 activates its target genes. In this study, a mitogen-activated protein kinase (MAP kinase), ERK5/BMK1 was found to interact with MEF2 in a yeast two hybrid screen. The interaction was confirmed by a glutathione S -transferase-pull down assay and a co-immunoprecipitation study indicating that endogenous ERK5 and MEF2 interact with each other in vivo . The interacting domain of MEF2 was mapped to the N-terminus which contains the highly conserved MADS and MEF2 domains. Functionally, ERK5/BMK1 was able to phosphorylate MEF2 in vitro . Furthermore, when cotransfected with ERK5/BMK1, the transactivation capacity of MEF2 was enhanced. These results suggest that the functions of MEF2 could be regulated through ERK5/BMK1.