Adult mouse intrinsic laryngeal muscles express high levels of the myogenic regulatory factor, MYF-5

The intrinsic laryngeal muscles display unique structural and functional characteristics that distinguish them from the skeletal muscle of the trunk and limbs. These features include relatively small muscle fibers, super-fast contraction speed, and fatigue resistance. The molecular basis of tissue-specific functions and other characteristics is differential gene expression. Accordingly, we have investigated the molecular basis of the functional specialization of the intrinsic laryngeal muscles by examining the expression of two key genes in the larynx, known to be important for skeletal muscle development and function: (a) the muscle regulatory factor, Myf-5, and (b) the superfast-contracting myosin heavy chain (EO-MyHC). We have found that the adult thyroarytenoid muscles express much higher levels of both Myf-5 and EO-MyHC messenger ribonucleic acid (mRNA), compared to lower hindlimb skeletal muscle where Myf-5 mRNA levels are very low and EO-MyHC is not detectable. These findings suggest that the unique functional characteristics of the intrinsic laryngeal muscles may be based in laryngeal muscle-specific gene expression directed by a unique combination of muscle regulatory factors. Such laryngeal muscle-specific genes may allow the future development of new treatments for laryngeal muscle dysfunction.

Altering in vivo fate of heart-infiltrating progenitors from pro-fibrotic into F4/80+macrophages prevents progression of myocarditis into inflammatory dilated cardiomyopathy

Rationale: Experimental autoimmune myocarditis (EAM) mirrors important pathogenic aspects of inflammatory cardiomyopathy, a common cause of heart failure. In EAM, TGF-β-dependent conversion of heart-infiltrating prominin-1+ progenitors into myofibroblasts is critical for development of fibrosis and the end-stage heart failure phenotype. Therapeutic strategies modulating the in vivo fate of prominin-1+ progenitors might therefore prevent TGF-β-mediated cardiac fibrosis and pathological remodelling. Methods and Results: EAM was induced in BALB/c mice using alpha-myosin heavy chain (aMyHC) peptide/complete Freund's adjuvant (CFA) immunization. Prominin-1+ cells were isolated from the inflamed hearts at day 21 after immunization, expanded and treated with Macrophage Colony-Stimulating Factor (M-CSF) or Transforming Growth Factor-beta (TGF-β). Herein, we demonstrated that M-CSF turns, ex vivo and in the EAM, heart-infiltrating prominin-1+ progenitors into immunosuppressive F4/80/CD11b/CD16/32/NOS2-expressing, nitric oxide producing and E.coli bacteria phygocyting macrophages, and protect further TGF-β-stimulated differentiation into pathogenic myofibroblasts. Systemic M-CSF treatment during myocarditis completely prevented post-inflammatory fibrosis, T cell relapse and left ventricular dysfunction. Mechanistically, M-CSF-induced macrophage differentiation from prominin-1+ progenitors critically required nitric oxide synthase 2. Accordingly, M-CSF treatment failed to reduce myocardial fibrosis development in Nos2-/- mice. Conclusions: Altering the in vivo fate of inflammatory prominin-1 expressing progenitors from pro-fibrotic into the F4/80 expressing macrophage phenotype protects from myocarditis progression, cardiac fibrosis, and heart failure. These findings offer a modern therapeutic model and challenge former concepts, which attributed macrophages a detrimental role in inflammatory cardiomyopathy progression.

Protein-binding site at the immunoglobulin mu membrane polyadenylylation signal: possible role in transcription termination.

mRNAs specifying immunoglobulin mu and delta heavy chains are encoded by a single large, complex transcription unit (mu + delta gene). The transcriptional activity of delta gene segments in terminally differentiated, IgM-secreting B lymphocytes is 10-20 times lower than in earlier B-lineage cells expressing delta mRNA. We find that transcription of the mu + delta gene in IgM-secreting murine myeloma cells terminates within a region of 500-1000 nucleotides immediately following the mu membrane (mu m) polyadenylylation site. Transcription decreases only minimally through this region in murine cell lines representative of earlier stages in B-cell development. A DNA fragment containing the mu m polyadenylylation signal gives protein-DNA complexes with different mobilities in gel retardation assays with nuclear extracts from myeloma cells than with nuclear extracts from earlier B-lineage cells. However, using a recently developed "footprinting" procedure in which protein-DNA complexes resolved in gel retardation assays are subjected to nucleolytic cleavage while still in the polyacrylamide gel, we find that the DNA sequences protected by factors from the two cell types are indistinguishable. The factor-binding site on the DNA is located 5' of the mu m polyadenylylation signal AATAAA and includes the 15-nucleotide-long A + T-rich palindrome CTGTAAACAAATGTC. This type of palindromic binding site exhibits orientation-dependent activity consistent with the reported properties of polymerase II termination signals. This binding site is followed by two sets of directly repeated DNA sequences with different helical conformation as revealed by their reactivity with the chemical nuclease 1,10-phenanthroline-copper. The close proximity of these features to the signals for mu m mRNA processing may reflect a linkage of the processes of developmentally regulated mu m polyadenylylation and transcription termination.

Cardiac Lineage Protein-1 (CLP-1) Regulates Cardiac Remodeling via Transcriptional Modulation of Diverse Hypertrophic and Fibrotic Responses and Angiotensin II-transforming Growth Factor β (TGF-β1) Signaling Axis.

It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-β1 and renin-angiotensin system signaling alters the fibroblast phenotype by promoting its differentiation into morphologically distinct pathological myofibroblasts, which potentiates collagen synthesis and fibrosis and causes enhanced extracellular matrix deposition. However, the atrial natriuretic peptide, which is induced during left ventricular hypertrophy, plays an anti-fibrogenic and anti-hypertrophic role by blocking, among others, the TGF-β-induced nuclear localization of Smads. It is not clear how the hypertrophic and fibrotic responses are transcriptionally regulated. CLP-1, the mouse homolog of human hexamethylene bis-acetamide inducible-1 (HEXIM-1), regulates the pTEFb activity via direct association with pTEFb causing inhibition of the Cdk9-mediated serine 2 phosphorylation in the carboxyl-terminal domain of RNA polymerase II. It was recently reported that the serine kinase activity of Cdk9 not only targets RNA polymerase II but also the conserved serine residues of the polylinker region in Smad3, suggesting that CLP-1-mediated changes in pTEFb activity may trigger Cdk9-dependent Smad3 signaling that can modulate collagen expression and fibrosis. In this study, we evaluated the role of CLP-1 in vivo in induction of left ventricular hypertrophy in angiotensinogen-overexpressing transgenic mice harboring CLP-1 heterozygosity. We observed that introduction of CLP-1 haplodeficiency in the transgenic α-myosin heavy chain-angiotensinogen mice causes prominent changes in hypertrophic and fibrotic responses accompanied by augmentation of Smad3/Stat3 signaling. Together, our findings underscore the critical role of CLP-1 in remodeling of the genetic response during hypertrophy and fibrosis.

A phenotypical study of vascular smooth muscle cells in human arterial and venous stenosis

Abstract Introduction The primary function of the contractile vascular smooth muscle cells (cVSMCs) is the regulation of the vascular contractility which means the adaptation of the vascular tonus in response to the modulation of the blood pressure and blood flow. The cVSMCs are essentially quiescent, and therefore their synthesis rate is very limited. They are characterized by the expression of contractile proteins specific to the muscular tissue including myosin, h-­‐caldesmon and <-­‐smooth muscle actin (〈-­‐SMA). These contractile cells are strongly represented in the media layer of the arterial wall and, in a smaller proportion, of the vein wall. Their typical stretched-­‐out morphology allows recognizing them by a histological analysis. They do not produce any extracellular matrix (ECM), and do not migrate through the different layers of the vessel wall, and are not directly involved in the development of intimal hyperplasia (IH). Neointimal formation occurs after endothelial disruption leading to complex molecular and biological mechanisms. The de-­‐differentiation of cVSMCs into synthetic VSMCs (sVSMCs) is mentioned as a key element. These non mature cells are able to proliferate and produce ECM. The characterization of the vascular smooth muscle cells (VSMCs) from healthy and stenosed vascular tissues will contribue to the understanding of the different biological processes leading to IH and will be useful for the development of new therapies to interfere with the cVSMCs growth and migration. The aim of our research was to quantify the proportion of cVSMCs and sVSMCs into the healthy and pathologic human blood vessel wall and to characterize their phenotype. Methods We selected 23 specimens of arterial and venous segments from 18 patients. All these specimens were stored in the biobank from the thoracic and vascular surgery departement. 4 groups were designed (group 1 :arteries without lesions (n=3) ;group 2 : veins without lesions (n=1); group 3: arteries with stenosis (n=9); group 4: veins with stenosis (n=10)). Histology: 5µm-­‐sections were made from each sample embedded in paraffin wax and further stained with hematoxylin & eosin (HE), Van Gieson's stain (VGEL) and Masson's Trichrome (TMB). Pathologic tissues were defined using the label that was given to the macroscopic samples by the surgeon and also, based on the histological analysis with HE and VGEL evaluating the presence of a thickened intima. The same was done to the control samples evaluating the absence of thickening. Immunohistochemistry : The primary antibodies were used :〈-­‐SMA, vimentin, h-­‐ caldesmon, calponin, smooth muscle-myosin heavy chain (SM-­‐MHC), tropomyosin-­‐4, retinol binding protein-­‐1 (RBP-­‐1), nonmuscle-­‐myosin heavy chain-­‐B (NM-­‐MHC-­‐B), Von Willebrand factor (VWF). A semi-­‐quantitative assessment of the intensity of each sample stained was performed. Western Blot : Segments of arteries and veins were analyzed using the following primary antibodies :〈-­‐SMA, Calponin, SM-­‐MHC, NM-­‐MHC-­‐B. The given results were then normalized with tubulin. Results Our data showed that, when using immunohistochemistry analysis we found that〈-­‐SMA was mostly expressed in control arteries, whereas NM-­‐MHC-­‐B in the pathologic ones. Using SM-­‐MHC, calponin, vimentin and caldesmon we found no significative differences in the expression of these proteins in the control and in the pathologic samples. Western Blot analysis showed an inverse correlation between healthy and pathological samples as <-­‐ SMA was more expressed in the pathological samples, while NM-­‐MHC-­‐B in the control group; SM-­‐MHC and calponin were mostly expressed in the pathologic samples. Conclusion Our study showed no clear differences between stenotic and control arterial and venous segments using semi-­‐quantitative assessement by immunohistochemistry. Western Blot showed a significant increased expression of 〈-­‐SMA, calponin and SM-­‐MHC in the arteries with stenosis, while NM-­‐MHC-­‐B was mostly expressed in the arteries without lesions. Further studies are needed to track the lineage of VSMCs to understand the mechanisms leading toIH.

Innate signaling promotes formation of regulatory nitric oxide-producing dendritic cells limiting T-cell expansion in experimental autoimmune myocarditis.

BACKGROUND: Activation of innate pattern-recognition receptors promotes CD4+ T-cell-mediated autoimmune myocarditis and subsequent inflammatory cardiomyopathy. Mechanisms that counterregulate exaggerated heart-specific autoimmunity are poorly understood. METHODS AND RESULTS: Experimental autoimmune myocarditis was induced in BALB/c mice by immunization with α-myosin heavy chain peptide and complete Freund's adjuvant. Together with interferon-γ, heat-killed Mycobacterium tuberculosis, an essential component of complete Freund's adjuvant, converted CD11b(hi)CD11c(-) monocytes into tumor necrosis factor-α- and nitric oxide synthase 2-producing dendritic cells (TipDCs). Heat-killed M. tuberculosis stimulated production of nitric oxide synthase 2 via Toll-like receptor 2-mediated nuclear factor-κB activation. TipDCs limited antigen-specific T-cell expansion through nitric oxide synthase 2-dependent nitric oxide production. Moreover, they promoted nitric oxide synthase 2 production in hematopoietic and stromal cells in a paracrine manner. Consequently, nitric oxide synthase 2 production by both radiosensitive hematopoietic and radioresistant stromal cells prevented exacerbation of autoimmune myocarditis in vivo. CONCLUSIONS: Innate Toll-like receptor 2 stimulation promotes formation of regulatory TipDCs, which confine autoreactive T-cell responses in experimental autoimmune myocarditis via nitric oxide. Therefore, activation of innate pattern-recognition receptors is critical not only for disease induction but also for counterregulatory mechanisms, protecting the heart from exaggerated autoimmunity.

RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBFB-MYH11 transcripts.

As acute nonlymphocytic leukemia (ANLL) with inv(16) (p13q22) or t(16;16)(p13;q22) has been shown to result from the fusion of transcription factor subunit core binding factor (CBFB) to a myosin heavy chain (MYH11), we sought to design methods to detect this rearrangement using reverse transcriptase-polymerase chain reaction (RT-PCR). In all of 27 inv(16)(p13q22) and four t(16;16)(p13;q22) cases tested, a chimeric CBFB-MYH11 transcript coding for an in-frame fusion protein was detected. In a more extensive RT-PCR analysis with different primer pairs, we detected a second new chimeric CBFB-MYH11 transcript in 10 of 11 patients tested. The CBFB-MYH11 reading frame of the second transcript was maintained in one patient but not in the others. We show that the different CBFB-MYH11 transcripts in one patient arise from alternative splicing. Translation of the transcript in which the CBFB-MYH11 reading frame is not maintained leads to a slightly truncated CBFB protein.

Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells.

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.

Induction of the alternative NF-κB pathway by lymphotoxin αβ (LTαβ) relies on internalization of LTβ receptor.

Several tumor necrosis factor receptor (TNFR) family members activate both the classical and the alternative NF-κB pathways. However, how a single receptor engages these two distinct pathways is still poorly understood. Using lymphotoxin β receptor (LTβR) as a prototype, we showed that activation of the alternative, but not the classical, NF-κB pathway relied on internalization of the receptor. Further molecular analyses revealed a specific cytosolic region of LTβR essential for its internalization, TRAF3 recruitment, and p100 processing. Interestingly, we found that dynamin-dependent, but clathrin-independent, internalization of LTβR appeared to be required for the activation of the alternative, but not the classical, NF-κB pathway. In vivo, ligand-induced internalization of LTβR in mesenteric lymph node stromal cells correlated with induction of alternative NF-κB target genes. Thus, our data shed light on LTβR cellular trafficking as a process required for specific biological functions of NF-κB.

Cardiac raptor ablation impairs adaptive hypertrophy, alters metabolic gene expression, and causes heart failure in mice.

Background- Cardiac hypertrophy involves growth responses to a variety of stimuli triggered by increased workload. It is an independent risk factor for heart failure and sudden death. Mammalian target of rapamycin (mTOR) plays a key role in cellular growth responses by integrating growth factor and energy status signals. It is found in 2 structurally and functionally distinct multiprotein complexes called mTOR complex (mTORC) 1 and mTORC2. The role of each of these branches of mTOR signaling in the adult heart is currently unknown. Methods and Results- We generated mice with deficient myocardial mTORC1 activity by targeted ablation of raptor, which encodes an essential component of mTORC1, during adulthood. At 3 weeks after the deletion, atrial and brain natriuretic peptides and β-myosin heavy chain were strongly induced, multiple genes involved in the regulation of energy metabolism were altered, but cardiac function was normal. Function deteriorated rapidly afterward, resulting in dilated cardiomyopathy and high mortality within 6 weeks. Aortic banding-induced pathological overload resulted in severe dilated cardiomyopathy already at 1 week without a prior phase of adaptive hypertrophy. The mechanism involved a lack of adaptive cardiomyocyte growth via blunted protein synthesis capacity, as supported by reduced phosphorylation of ribosomal S6 kinase 1 and 4E-binding protein 1. In addition, reduced mitochondrial content, a shift in metabolic substrate use, and increased apoptosis and autophagy were observed. Conclusions- Our results demonstrate an essential function for mTORC1 in the heart under physiological and pathological conditions and are relevant for the understanding of disease states in which the insulin/insulin-like growth factor signaling axis is affected such as diabetes mellitus and heart failure or after cancer therapy.

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Cardiac raptor deletion impairs adaptive hypertrophy, alters metabolic gene expression and causes heart failure in mice

Background: Mammalian target of rapamycin (mTOR), a central regulator of cell growth, is found in two structurally and functionally distinct multiprotein complexes called mTOR complex (mTORC)1 and mTORC2. The specific roles of each of these branches of mTOR signaling have not been dissected in the adult heart. In the present study, we aimed to bring new insights into the function of cardiac mTORC1-mediated signaling in physiological as well as pathological situations.Methods: We generated mice homozygous for loxP-flanked raptor and positive for the tamoxifen-inducible Cre recombinase (MerCreMer) under control of the α- myosin heavy chain promoter. The raptor gene encodes an essential component of mTORC1. Gene ablation was induced at the age of 10-12 weeks, and two weeks later the raptor cardiac-knockout (raptor-cKO) mice started voluntary cagewheel exercise or were subjected to transverse aortic constriction (TAC) to induce pressure overload.Results: In sedentary raptor-cKO mice, ejection fractions gradually decreased, resulting in significantly reduced values at 38 days (P < 0.001). Raptor-cKO mice started to die during the fifth week after the last tamoxifen injection. At that time, the mortality rate was 36% in sedentary (n = 11) and 64% in exercising (n = 14) mice. TAC-induced pressure overload resulted in severe cardiac dysfunction already at earlier timepoints. Thus, at 7-9 days after surgery, ejection fraction and fractional shortening values were 22.3% vs 43.5% and 10.2% vs 21.5% in raptor-cKO vs wild-type mice, respectively. This was accompanied by significant reductions of ventricular wall and septal thickness as well as an increase in left ventricular internal diameter. Moreover, ventricular weight to tibial length ratios were increased in wild-type, but not in the raptor-cKO TAC mice. Together, this shows that raptor-cKO mice rapidly developed dilated cardiomyopathy without going through a phase of adaptive hypertrophy. Expression of ANP and β-MHC was induced in all raptor-cKO mice irrespective of the cardiac load conditions. Consistent with reduced mTORC1 activity, phosphorylation of ribosomal S6 kinase and 4E-BP1 was blunted, indicating reduced protein synthesis. Moreover, expression of multiple genes involved in the regulation of energy metabolism was altered, and followed by a shift from fatty acid to glucose oxidation.Conclusion: Our study suggests that mTORC1 coordinates protein and energy metabolic pathways in the heart. Moreover, we demonstrate that raptor is essential for the cardiac adaptation to increased workload and importantly, also for normal physiological cardiac function.

Absence of nonhematopoietic MHC class II expression protects mice from experimental autoimmune myocarditis.

Experimental autoimmune myocarditis (EAM) is a CD4(+) T-cell-mediated model of human inflammatory dilated cardiomyopathies. Heart-specific CD4(+) T-cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation-induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV-/- K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α-myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV-/- K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4(+) T cells and in expression of IFN-γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN-γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN-γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis.