Rosenthal GroupPublications

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Comments on Point:Counterpoint: IGF is/is not the major physiological regulator of muscle mass. IGF-1 is a major regulator of muscle mass during growth but not for adult myofiber hypertrophy.
Vinciguerra, M., Hede, M. & Rosenthal, N.
J Appl Physiol. 2010 Jun;108(6):1829-30.

Class 2 IGF-1 isoforms are dispensable for viability, growth and maintenance of IGF-1 serum levels.
Temmerman, L., Slonimsky, E. & Rosenthal, N.
Growth Horm IGF Res. 2010 Jun;20(3):255-63. Epub 2010 Apr 9.

Insulin-like growth factor 1 (IGF-1) is a pleiotropic factor involved in growth, cell survival and cellular differentiation. It exerts its functions through endocrine, paracrine or autocrine mechanisms. Circulating IGF-1 is essential for normal fetal and postnatal growth, although the published phenotypes of IGF-1 null animals have been only partially penetrant, presumably due to mixed genetic backgrounds. Molecular dissection of IGF-1 action is complicated by the existence of at least nine different IGF-1 isoforms, generated in both humans and rodents by usage of alternate promoters, differential splicing and different post-translational modifications. Several lines of evidence suggest that the Class 2 IGF-1 isoform is specifically destined for circulation, supporting an endocrine role of IGF-1 in normal growth processes. Using Cre/LoxP conditional gene targeting of exon 2 of the IGF-1 gene, we have generated a Class 2 IGF-1 knockout mouse line in a pure C57/Bl6 genetic background, where the specific removal of exon 2 ablated Class 2 IGF-1 isoform. Class 2 IGF-1 knockout mice exhibited normal development and postnatal growth patterns and had normal IGF-1 circulating levels, due to compensatory upregulation of Class 1 transcripts. In contrast, progeny of a total IGF-1 knockout line lacking exon 3 in the same genetic background were predictably smaller, displayed dramatically reduced IGF-1 receptor phosphorylation and all died perinatally, apparently due to respiratory failure. These results confirm that Class 2 signal peptide is not necessary for systemic circulation of IGF-1, revealing an internal compensation system for maintaining IGF-1 serum concentrations. We also uncover a vital requirement of IGF-1 for perinatal viability, previously obscured by modifiers in heterogeneous genetic backgrounds.

Comments on Point:Counterpoint: IGF is/is not the major physiological regulator of muscle mass.
Spangenburg, E.E., Phillips, S.M., Yang, S.Y., Musaro, A., Shenkman, B.S., Kachaeva, E., Turtikova, O., Leinsoo, T., Lysenko, E., Baar, K., Hamilton, D.L., Philp, A., Barton, E.R., Svensson, J., Loughna, P.T., Harridge, S.D., Shavlakadze, T., Grounds, M.D., Vinciguerra, M., Hede, M., Rosenthal, N., Esser, K.A., Song, Y.H. & Ameredes, B.T.
J Appl Physiol. 2010 Jun;108(6):1825-31.

The consequence of natural selection on genetic variation in the mouse.
Reuveni, E., Birney, E. & Gross, C.T.
Genomics. 2010 Apr;95(4):196-202. Epub 2010 Feb 18.

Laboratory mouse strains are known to have emerged from recent interbreeding between individuals of Mus musculus isolated populations. As a result of this breeding history, the collection of polymorphisms observed between laboratory mouse strains is likely to harbor the effects of natural selection between reproductively isolated populations. Until now no study has systematically investigated the consequences of this breeding history on gene evolution. Here we have used a novel, unbiased evolutionary approach to predict the founder origin of laboratory mouse strains and to assess the balance between ancient and newly emerged mutations in the founder subspecies. Our results confirm a contribution from at least four distinct subspecies. Additionally, our method allowed us to identify regions of relaxed selective constraint among laboratory mouse strains. This unique structure of variation is likely to have significant consequences on the use of mouse to find genes underlying phenotypic variation.

A growth stimulus is needed for IGF-1 to induce skeletal muscle hypertrophy in vivo.
Shavlakadze, T., Chai, J., Maley, K., Cozens, G., Grounds, G., Winn, N., Rosenthal, N. & Grounds, M.D.
J Cell Sci. 2010 Mar 15;123(Pt 6):960-71. Epub 2010 Feb 23.

Here, we characterise new strains of normal and dystrophic (mdx) mice that overexpress Class 2 IGF-1 Ea in skeletal myofibres. We show that transgenic mice have increased muscle levels of IGF-1 (approximately 13-26 fold) and show striking muscle hypertrophy (approximately 24-56% increase in mass). Adult normal muscles were resistant to elevated IGF-1; they reached adult steady state and maintained the same mass from 3 to 12 months. By contrast, dystrophic muscles from mdx/IGF-1(C2:Ea) mice continued to increase in mass during adulthood. IGF-1 signalling was evident only in muscles that were growing as a result of normal postnatal development (23-day-old mice) or regenerating in response to endogenous necrosis (adult mdx mice). Increased phosphorylation of Akt at Ser473 was not evident in fasted normal adult transgenic muscles, but was 1.9-fold higher in fasted normal young transgenic muscles compared with age-matched wild-type controls and fourfold higher in fasted adult mdx/IGF-1(C2:Ea) compared with mdx muscles. Muscles of adult mdx/IGF-1(C2:Ea) mice showed higher p70(S6K)(Thr421/Ser424) phosphorylation and both young transgenic and adult mdx/IGF-1(C2:Ea) mice had higher phosphorylation of rpS6(Ser235/236). The level of mRNA encoding myogenin was increased in normal young (but not adult) transgenic muscles, indicating enhanced myogenic differentiation. These data demonstrate that elevated IGF-1 has a hypertrophic effect on skeletal muscle only in growth situations.

Insights into the organization of dorsal spinal cord pathways from an evolutionarily conserved raldh2 intronic enhancer.
Castillo, H.A., Cravo, R.M., Azambuja, A.P., Simoes-Costa, M.S., Sura-Trueba, S., Gonzalez, J., Slonimsky, E., Almeida, K., Abreu, J.G., de Almeida, M.A., Sobreira, T.P., de Oliveira, S.H., de Oliveira, P.S., Signore, I.A., Colombo, A., Concha, M.L., Spengler, T.S., Bronner-Fraser, M., Nobrega, M., Rosenthal, N. & Xavier-Neto, J.
Development. 2010 Feb;137(3):507-18.

Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.

Antioxidant amelioration of dilated cardiomyopathy caused by conditional deletion of NEMO/IKKgamma in cardiomyocytes.
Kratsios, P., Huth, M., Temmerman, L., Salimova, E., Al Banchaabouchi, M., Sgoifo, A., Manghi, M., Suzuki, K., Rosenthal, N. & Mourkioti, F.
Circ Res. 2010 Jan 8;106(1):133-44. Epub 2009 Oct 22.

RATIONALE: Insight into the function of nuclear factor (NF)-kappaB in the adult heart has been hampered by the embryonic lethality of constitutive NF-kappaB inactivation. OBJECTIVE: The goal of the present study was therefore to gain insights into the role of NF-kappaB pathway specifically in mouse cardiomyocytes by conditional deletion of the NF-kappaB essential modulator (NEMO). METHODS AND RESULTS: Using a Cre/loxP system, we disrupted the Nemo gene in a cardiomyocyte-specific manner in the heart, which simulated gene expression changes underlying human heart failure and caused adult-onset dilated cardiomyopathy accompanied by inflammation and apoptosis. Pressure overload challenges of NEMO-deficient young hearts precociously induced the functional decrements that develop spontaneously in older knockout animals. Moreover, oxidative stress in NEMO-deficient cardiomyocytes is a critical pathological component that can be attenuated with antioxidant diet in vivo. CONCLUSIONS: These results reveal an essential physiological role for NEMO-mediated signaling in the adult heart to maintain cardiac function in response to age-related or mechanical challenges, in part through modulation of oxidative stress.

Redeployment of Notch1 embryonic signaling mediates cardiac morphogenesis and promotes myocardial repair in the adult.
Kratsios, P., Catela, C., Salimova, E., Huth, M., Berno, V., Rosenthal, N. Mourkioti, F.
Circ. Research, in press

Distinct Roles for Cell-Autonomous Notch Signaling in Cardiomyocytes of the Embryonic and Adult Heart.
Kratsios, P., Catela, C., Salimova, E., Huth, M., Berno, V., Rosenthal, N. & Mourkioti, F.
Circ Res. 2009 Dec 10.

Rationale: The Notch signaling pathway is important for cell-cell communication that controls tissue formation and homeostasis during embryonic and adult life, but the precise cell targets of Notch signaling in the mammalian heart remain poorly defined. Objective: To investigate the functional role of Notch signaling in the cardiomyocyte compartment of the embryonic and adult heart. Methods and Results: Here, we report that either conditional overexpression of Notch1 intracellular domain (NICD1) or selective silencing of Notch signaling in the embryonic cardiomyocyte compartment results in developmental defects and perinatal lethality. In contrast, augmentation of endogenous Notch reactivation after myocardial infarction in the adult, either by inducing cardiomyocyte-specific Notch1 transgene expression or by intramyocardial delivery of a Notch1 pseudoligand, increases survival rate, improves cardiac functional performance, and minimizes fibrosis, promoting antiapoptotic and angiogenic mechanisms. Conclusions: These results reveal a strict requirement for cell-autonomous modulation of Notch signaling during heart morphogenesis, and illustrate how the same signaling pathway that promotes congenital heart defects when perturbed in the embryo can be therapeutically redeployed for the treatment of adult myocardial damage.

A CREB-C/EBPbeta cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair.
Ruffell, D., Mourkioti, F., Gambardella, A., Kirstetter, P., Lopez, R.G., Rosenthal, N. & Nerlov, C.
Proc Natl Acad Sci U S A. 2009 Oct 13;106(41):17475-80. Epub 2009 Sep 24.

Macrophages play an essential role in the resolution of tissue damage through removal of necrotic cells, thus paving the way for tissue regeneration. Macrophages also directly support the formation of new tissue to replace the injury, through their acquisition of an anti-inflammatory, or M2, phenotype, characterized by a gene expression program that includes IL-10, the IL-13 receptor, and arginase 1. We report that deletion of two CREB-binding sites from the Cebpb promoter abrogates Cebpb induction upon macrophage activation. This blocks the downstream induction of M2-specific Msr1, Il10, II13ra, and Arg-1 genes, whereas the inflammatory (M1) genes Il1, Il6, Tnfa, and Il12 are not affected. Mice carrying the mutated Cebpb promoter (betaDeltaCre) remove necrotic tissue from injured muscle, but exhibit severe defects in muscle fiber regeneration. Conditional deletion of the Cebpb gene in muscle cells does not affect regeneration, showing that the C/EBPbeta cascade leading to muscle repair is muscle-extrinsic. While betaDeltaCre macrophages efficiently infiltrate injured muscle they fail to upregulate Cebpb, leading to decreased Arg-1 expression. CREB-mediated induction of Cebpb expression is therefore required in infiltrating macrophages for upregulation of M2-specific genes and muscle regeneration, providing a direct genetic link between these two processes.

Post-publication sharing of data and tools.
Schofield, P.N., Bubela, T., Weaver, T., Portilla, L., Brown, S.D., Hancock, J.M., Einhorn, D., Tocchini-Valentini, G., Hrabe de Angelis, M. & Rosenthal, N.
Nature. 2009 Sep 10;461(7261):171-3.

Despite existing guidelines on access to data and bioresources, good practice is not widespread. A meeting of mouse researchers in Rome proposes ways to promote a culture of sharing.

Multiple congenital malformations of Wolf-Hirschhorn syndrome are recapitulated in Fgfrl1 null mice.
Catela, C., Bilbao-Cortes, D., Slonimsky, E., Kratsios, P., Rosenthal, N. & te Welscher, P.
Dis Model Mech. 2009 May-Jun;2(5-6):283-94. Epub 2009 Apr 21.

Wolf-Hirschhorn syndrome (WHS) is caused by deletions in the short arm of chromosome 4 (4p) and occurs in about one per 20,000 births. Patients with WHS display a set of highly variable characteristics including craniofacial dysgenesis, mental retardation, speech problems, congenital heart defects, short stature and a variety of skeletal anomalies. Analysis of patients with 4p deletions has identified two WHS critical regions (WHSCRs); however, deletions targeting mouse WHSCRs do not recapitulate the classical WHS defects, and the genes contributing to WHS have not been conclusively established. Recently, the human FGFRL1 gene, encoding a putative fibroblast growth factor (FGF) decoy receptor, has been implicated in the craniofacial phenotype of a WHS patient. Here, we report that targeted deletion of the mouse Fgfrl1 gene recapitulates a broad array of WHS phenotypes, including abnormal craniofacial development, axial and appendicular skeletal anomalies, and congenital heart defects. Fgfrl1 null mutants also display a transient foetal anaemia and a fully penetrant diaphragm defect, causing prenatal and perinatal lethality. Together, these data support a wider role for Fgfrl1 in development, implicate FGFRL1 insufficiency in WHS, and provide a novel animal model to dissect the complex aetiology of this human disease.

Models for financial sustainability of biological databases and resources.
Chandras, C., Weaver, T., Zouberakis, M., Smedley, D., Schughart, K., Rosenthal, N., Hancock, J.M., Kollias, G., Schofield, P.N. & Aidinis, V.
Database (Oxford). 2009;2009:bap017. Epub 2009 Oct 23.

Following the technological advances that have enabled genome-wide analysis in most model organisms over the last decade, there has been unprecedented growth in genomic and post-genomic science with concomitant generation of an exponentially increasing volume of data and material resources. As a result, numerous repositories have been created to store and archive data, organisms and material, which are of substantial value to the whole community. Sustained access, facilitating re-use of these resources, is essential, not only for validation, but for re-analysis, testing of new hypotheses and developing new technologies/platforms. A common challenge for most data resources and biological repositories today is finding financial support for maintenance and development to best serve the scientific community. In this study we examine the problems that currently confront the data and resource infrastructure underlying the biomedical sciences. We discuss the financial sustainability issues and potential business models that could be adopted by biological resources and consider long term preservation issues within the context of mouse functional genomics efforts in Europe.

Overexpression of mIGF-1 in keratinocytes improves wound healing and accelerates hair follicle formation and cycling in mice.
Semenova, E., Koegel, H., Hasse, S., Klatte, J.E., Slonimsky, E., Bilbao, D., Paus, R., Werner, S. & Rosenthal, N.
Am J Pathol. 2008 Nov;173(5):1295-310. Epub 2008 Oct 2.

Insulin-like growth factor 1 (IGF-1) is an important regulator of growth, survival, and differentiation in many tissues. It is produced in several isoforms that differ in their N-terminal signal peptide and C-terminal extension peptide. The locally acting isoform of IGF-1 (mIGF-1) was previously shown to enhance the regeneration of both muscle and heart. In this study, we tested the therapeutic potential of mIGF-1 in the skin by generating a transgenic mouse model in which mIGF-1 expression is driven by the keratin 14 promoter. IGF-1 levels were unchanged in the sera of hemizygous K14/mIGF-1 transgenic animals whose growth was unaffected. A skin analysis of young animals revealed normal architecture and thickness as well as proper expression of differentiation and proliferation markers. No malignant tumors were formed. Normal homeostasis of the putative stem cell compartment was also maintained. Healing of full-thickness excisional wounds was accelerated because of increased proliferation and migration of keratinocytes, whereas inflammation, granulation tissue formation, and scarring were not obviously affected. In addition, mIGF-1 promoted late hair follicle morphogenesis and cycling. To our knowledge, this is the first work to characterize the simultaneous, stimulatory effect of IGF-1 delivery to keratinocytes on two types of regeneration processes within a single mouse model. Our analysis supports the use of mIGF-1 for skin and hair regeneration and describes a potential cell type-restricted action.

Expression of follistatin-related genes is altered in heart failure.
Lara-Pezzi, E., Felkin, L.E., Birks, E.J., Sarathchandra, P., Panse, K.D., George, R., Hall, J.L., Yacoub, M.H., Rosenthal, N. & Barton, P.J.
Endocrinology. 2008 Nov;149(11):5822-7. Epub 2008 Jul 10.

Follistatins play roles in diverse biological processes including cell proliferation, wound healing, inflammation, and skeletal muscle growth, yet their role in the heart is currently unknown. We have investigated the myocardial expression profile and cellular distribution of follistatin (FST) and the FST-like genes FSTL1 and FSTL3 in the normal and failing heart. Expression was further analyzed in the novel setting of recovery from heart failure in myocardium obtained from patients who received combined mechanical (left ventricular assist device) and pharmacological therapy. Real-time PCR revealed that FSTL1 and FSTL3 expression was elevated in heart failure but returned to normal after recovery. FSTL3 expression levels correlated with molecular markers of disease severity and FSTL1 with the endothelial cell marker CD31, suggesting a potential link with vascularization. FSTL1 levels before treatment correlated with cardiac function after recovery, suggesting initial levels may influence long-term outcome. Immunohistochemistry revealed that FST was primarily localized to fibroblasts and vascular endothelium within the heart, whereas FSTL1 was localized to myocytes, endothelium, and smooth muscle cells and FSLT3 to myocytes and endothelium. Microarray analysis revealed that FST and FSTL1 were associated with extracellular matrix-related and calcium-binding proteins, whereas FSTL3 was associated mainly with cell signaling and transcription. These data show for the first time that elevated myocardial expression of FST-like genes is a feature of heart failure and may be linked to both disease severity and mechanisms underlying recovery, revealing new insight into the pathogenesis of heart failure and offering novel therapeutic targets.

Skeletal muscle is a primary target of SOD1G93A-mediated toxicity.
Dobrowolny, G., Aucello, M., Rizzuto, E., Beccafico, S., Mammucari, C., Boncompagni, S., Belia, S., Wannenes, F., Nicoletti, C., Del Prete, Z., Rosenthal, N., Molinaro, M., Protasi, F., Fano, G., Sandri, M. & Musaro, A.
Cell Metab. 2008 Nov;8(5):425-36.

The antioxidant enzyme superoxide dismutase 1 (SOD1) is a critical player of the antioxidative defense whose activity is altered in several chronic diseases, including amyotrophic lateral sclerosis. However, how oxidative insult affects muscle homeostasis remains unclear. This study addresses the role of oxidative stress on muscle homeostasis and function by the generation of a transgenic mouse model expressing a mutant SOD1 gene (SOD1(G93A)) selectively in skeletal muscle. Transgenic mice developed progressive muscle atrophy, associated with a significant reduction in muscle strength, alterations in the contractile apparatus, and mitochondrial dysfunction. The analysis of molecular pathways associated with muscle atrophy revealed that accumulation of oxidative stress served as signaling molecules to initiate autophagy, one of the major intracellular degradation mechanisms. These data demonstrate that skeletal muscle is a primary target of SOD1(G93A) -mediated toxicity and disclose the molecular mechanism whereby oxidative stress triggers muscle atrophy.

Analysis of CRE-mediated recombination driven by myosin light chain 1/3 regulatory elements in embryonic and adult skeletal muscle: a tool to study fiber specification.
Mourkioti, F., Slonimsky, E., Huth, M., Berno, V. & Rosenthal, N.
Genesis. 2008 Aug;46(8):424-30.

An increasing number of genes have been implicated in skeletal muscle fiber diversity. To study the contribution of diverse genetic elements to the regulation of fiber-type composition, we generated a transgenic mouse in which CRE recombinase expression is driven by muscle-specific regulatory sequences of the myosin light chain 1/3 locus (MLC). Using ROSA26 conditional reporter mice, we detected expression of the MLC-Cre transgene starting from embryonic day 12.5 (E12.5). By E15, recombination was detected in all muscle-derived structures. Immunohistochemical analysis revealed CRE activity was restricted to fast-twitch (type II) and excluded from slow-twitch (type I) fibers of skeletal muscle. The MLC-Cre transgenic mouse can be used in conjunction with conditional alleles to study both developmental patterning and maintenance of fast fiber-type phenotypes.

Interleukin-10 from transplanted bone marrow mononuclear cells contributes to cardiac protection after myocardial infarction.
Burchfield, J.S., Iwasaki, M., Koyanagi, M., Urbich, C., Rosenthal, N., Zeiher, A.M. & Dimmeler, S.
Circ Res. 2008 Jul 18;103(2):203-11. Epub 2008 Jun 19.

Bone marrow mononuclear cells (BM-MNCs) have successfully been used as a therapy for the improvement of left ventricular (LV) function after myocardial infarction (MI). It has been suggested that paracrine factors from BM-MNCs may be a key mechanism mediating cardiac protection. We previously performed microarray analysis and found that the pleiotropic cytokine interleukin (IL)-10 was highly upregulated in human progenitor cells in comparison with adult endothelial cells and CD14+ cells. Moreover, BM-MNCs secrete significant amounts of IL-10, and IL-10 could be detected from progenitor cells transplanted in infarcted mouse hearts. Specifically, intramyocardial injection of wild-type BM-MNCs led to a significant decrease in LV end-diastolic pressure (LVEDP) and LV end-systolic volume (LVESV) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. Furthermore, intramyocardial injection of wild-type BM-MNCs led to a significant increase in stroke volume (SV) and rate of the development of pressure over time (+dP/dt) compared to hearts injected with either diluent or IL-10 knock-out BM-MNCs. The IL-10-dependent improvement provided by transplanted cells was not caused by reduced infarct size, neutrophil infiltration, or capillary density, but rather was associated with decreased T lymphocyte accumulation, reactive hypertrophy, and myocardial collagen deposition. These results suggest that BM-MNCs mediate cardiac protection after myocardial infarction and this is, at least in part, dependent on IL-10.

NF-kappaB signaling in skeletal muscle: prospects for intervention in muscle diseases.
Mourkioti, F. & Rosenthal, N.
J Mol Med. 2008 Jul;86(7):747-59. Epub 2008 Feb 2.

Muscle remodeling is an important physiological process that promotes adaptive changes in cytoarchitecture and protein composition after exercise, aging, or disease conditions. Numerous transcription factors have been reported to regulate skeletal muscle homeostasis. NF-kappaB is a major pleiotropic transcription factor modulating immune, inflammatory, cell survival, and proliferating responses; however, its role in muscle development, physiology, and disease has just started to be elucidated. The current review article aims to summarize the literature on the role of NF-kappaB signaling in skeletal muscle pathophysiology, investigated over the last years using in vitro and more recently in vivo systems. Understanding the exact role of NF-kappaB in muscle cells will allow better therapeutic manipulations in the setting of human muscle diseases.

Reliability, Robustness and Reproducibility in mouse behavioral phenotyping: a cross-laboratory study.
Mandillo, S., Tucci, V., Holter, S.M., Meziane, H., Al Banchaabouchi, M., Kallnik, M., Lad, H.V., Nolan, P.M., Ouagazzal, A.M., Coghill, E.L., Gale, K., Golini, E., Jacquot, S., Krezel, W., Parker, A., Riet, F., Schneider, I., Marazziti, D., Auwerx, J.H., Brown, S.D., Chambon, P., Rosenthal, N., Tocchini-Valentini, G. & Wurst, W.
Physiol Genomics. 2008 May 27;.

Establishing standard operating procedures (SOPs) as tools for the analysis of behavioural phenotypes is fundamental to mouse functional genomics. It is essential that the tests designed provide reliable measures of the process under investigation but most importantly that these are reproducible across both time and laboratories. For this reason, we devised and tested a set of SOPs to investigate mouse behaviour. Five research centres were involved across France, Germany, Italy and the UK in this study, as part of the EUMORPHIA program. All the procedures underwent a cross-validation experimental study to investigate the robustness of the designed protocols. Four inbred reference strains (C57BL/6J, C3HeB/FeJ, BALB/cByJ, 129S2/SvPas), reflecting their use as common background strains in mutagenesis programmes, were analysed to validate these tests. We demonstrate that the operating procedures employed, which includes open field, SHIRPA, grip-strength, rotarod, Y-maze, pre-pulse inhibition and tail flick tests, generated reproducible results between laboratories for a number of the test output parameters. However, we also identified several uncontrolled variables that constitute confounding factors in behavioral phenotyping. The EUMORPHIA SOPs described here are an important start-point for the ongoing development of increasingly robust phenotyping platforms and their application in large-scale, multi-centre mouse phenotyping programmes. Key words: inbred mouse strains, behavioral phenotyping, test battery.

Derive and conquer: sourcing and differentiating stem cells for therapeutic applications.
Klimanskaya, I., Rosenthal, N. & Lanza, R.
Nat Rev Drug Discov. 2008 Feb;7(2):131-42.

Although great progress has been made in the isolation and culture of stem cells, the future of stem-cell-based therapies and their productive use in drug discovery and regenerative medicine depends on two key factors: finding reliable sources of multipotent and pluripotent cells and the ability to control their differentiation to generate desired derivatives. It is essential for clinical applications to establish reliable sources of pathogen-free human embryonic stem cells (ESCs) and develop suitable differentiation techniques. Here, we address some of the problems associated with the sourcing of human ESCs and discuss the current status of stem-cell differentiation technology.

Follistatin gene expression in heart failure and myocardial recovery following LVAD combination therapy.
Lara-Pezzi, E; Felkin, LE; Sarathchandra, P; George, R; Hall, JL; Yacoub, MH; Rosenthal, N.; Birks, EJ; Barton, PJ
Journal of Heart and Lung Transplantation 2008 27(2) S220

A naturally occurring calcineurin variant inhibits FoxO activity and enhances skeletal muscle regeneration.
Lara-Pezzi, E., Winn, N., Paul, A., McCullagh, K., Slominsky, E., Santini, M.P., Mourkioti, F., Sarathchandra, P., Fukushima, S., Suzuki, K. & Rosenthal, N.
J Cell Biol. 2007 Dec 17;179(6):1205-18.

The calcium-activated phosphatase calcineurin (Cn) transduces physiological signals through intracellular pathways to influence the expression of specific genes. Here, we characterize a naturally occurring splicing variant of the CnAbeta catalytic subunit (CnAbeta1) in which the autoinhibitory domain that controls enzyme activation is replaced with a unique C-terminal region. The CnAbeta1 enzyme is constitutively active and dephosphorylates its NFAT target in a cyclosporine-resistant manner. CnAbeta1 is highly expressed in proliferating myoblasts and regenerating skeletal muscle fibers. In myoblasts, CnAbeta1 knockdown activates FoxO-regulated genes, reduces proliferation, and induces myoblast differentiation. Conversely, CnAbeta1 overexpression inhibits FoxO and prevents myotube atrophy. Supplemental CnAbeta1 transgene expression in skeletal muscle leads to enhanced regeneration, reduced scar formation, and accelerated resolution of inflammation. This unique mode of action distinguishes the CnAbeta1 isoform as a candidate for interventional strategies in muscle wasting treatment.

The mouse ascending: perspectives for human-disease models.
Rosenthal, N. & Brown, S.
Nat Cell Biol. 2007 Sep;9(9):993-9.

The laboratory mouse is widely considered the model organism of choice for studying the diseases of humans, with whom they share 99% of their genes. A distinguished history of mouse genetic experimentation has been further advanced by the development of powerful new tools to manipulate the mouse genome. The recent launch of several international initiatives to analyse the function of all mouse genes through mutagenesis, molecular analysis and phenotyping underscores the utility of the mouse for translating the information stored in the human genome into increasingly accurate models of human disease.

ROCK2 and its alternatively spliced isoform ROCK2m positively control the maturation of the myogenic program.
Pelosi, M., Marampon, F., Zani, B.M., Prudente, S., Perlas, E., Caputo, V., Cianetti, L., Berno, V., Narumiya, S., Kang, S.W., Musaro, A. & Rosenthal, N.
Mol Cell Biol. 2007 Sep;27(17):6163-76. Epub 2007 Jul 2.

Signal transduction cascades involving Rho-associated kinases (ROCK), the serine/threonine kinases downstream effectors of Rho, have been implicated in the regulation of diverse cellular functions including cytoskeletal organization, cell size control, modulation of gene expression, differentiation, and transformation. Here we show that ROCK2, the predominant ROCK isoform in skeletal muscle, is progressively up-regulated during mouse myoblast differentiation and is highly expressed in the dermomyotome and muscle precursor cells of mouse embryos. We identify a novel and evolutionarily conserved ROCK2 splicing variant, ROCK2m, that is preferentially expressed in skeletal muscle and strongly up-regulated during in vivo and in vitro differentiation processes. The specific knockdown of ROCK2 or ROCK2m expression in C2C12 myogenic cells caused a significant and selective impairment of the expression of desmin and of the myogenic regulatory factors Mrf4 and MyoD. We demonstrate that in myogenic cells, ROCK2 and ROCK2m are positive regulators of the p42 and p44 mitogen-activated protein kinase-p90 ribosomal S6 kinase-eucaryotic elongation factor 2 intracellular signaling pathways and, thereby, positively regulate the hypertrophic effect elicited by insulin-like growth factor 1 and insulin, linking the multifactorial functions of ROCK to an important control of the myogenic maturation.

Phenostat: visualization and statistical tool for analysis of phenotyping data.
Reuveni, E., Carola, V., Banchaabouchi, M.A., Rosenthal, N., Hancock, J.M. & Gross, C.
Mamm Genome. 2007 Aug 3;.

The effective extraction of information from multidimensional data sets derived from phenotyping experiments is a growing challenge in biology. Data visualization tools are important resources that can aid in exploratory data analysis of complex data sets. Phenotyping experiments of model organisms produce data sets in which a large number of phenotypic measures are collected for each individual in a group. A critical initial step in the analysis of such multidimensional data sets is the exploratory analysis of data distribution and correlation. To facilitate the rapid visualization and exploratory analysis of multidimensional complex trait data, we have developed a user-friendly, web-based software tool called Phenostat. Phenostat is composed of a dynamic graphical environment that allows the user to inspect the distribution of multiple variables in a data set simultaneously. Individuals can be selected by directly clicking on the graphs and thus displaying their identity, highlighting corresponding values in all graphs, allowing their inclusion or exclusion from the analysis. Statistical analysis is provided by R package functions. Phenostat is particularly suited for rapid distribution and correlation analysis of subsets of data. An analysis of behavioral and physiologic data stemming from a large mouse phenotyping experiment using Phenostat reveals previously unsuspected correlations. Phenostat is freely available to academic institutions and nonprofit organizations and can be used from our website at http://www.bioinfo.embl.it/phenostat/ .

Immune response to stem cells and strategies to induce tolerance.
Batten, P., Rosenthal, N.A. & Yacoub, M.H.
Philos Trans R Soc Lond B Biol Sci. 2007 Aug 29;362(1484):1343-56.

Although recent progress in cardiovascular tissue engineering has generated great expectations for the exploitation of stem cells to restore cardiac form and function, the prospects of a common mass-produced cell resource for clinically viable engineered tissues and organs remain problematic. The refinement of stem cell culture protocols to increase induction of the cardiomyocyte phenotype and the assembly of transplantable vascularized tissue are areas of intense current research, but the problem of immune rejection of heterologous cell type poses perhaps the most significant hurdle to overcome. This article focuses on the potential advantages and problems encountered with various stem cell sources for reconstruction of the damaged or failing myocardium or heart valves and also discusses the need for integrating advances in developmental and stem cell biology, immunology and tissue engineering to achieve the full potential of cardiac tissue engineering. The ultimate goal is to produce 'off-the-shelf' cells and tissues capable of inducing specific immune tolerance.

Enhancing repair of the mammalian heart.
Santini, M.P., Tsao, L., Monassier, L., Theodoropoulos, C., Carter, J., Lara-Pezzi, E., Slonimsky, E., Salimova, E., Delafontaine, P., Song, Y.H., Bergmann, M., Freund, C., Suzuki, K. & Rosenthal, N.
Circ Res. 2007 Jun 22;100(12):1732-40. Epub 2007 May 24.

The injured mammalian heart is particularly susceptible to tissue deterioration, scarring, and loss of contractile function in response to trauma or sustained disease. We tested the ability of a locally acting insulin-like growth factor-1 isoform (mIGF-1) to recover heart functionality, expressing the transgene in the mouse myocardium to exclude endocrine effects on other tissues. supplemental mIGF-1 expression did not perturb normal cardiac growth and physiology. Restoration of cardiac function in post-infarct mIGF-1 transgenic mice was facilitated by modulation of the inflammatory response and increased antiapoptotic signaling. mIGF-1 ventricular tissue exhibited increased proliferative activity several weeks after injury. The canonical signaling pathway involving Akt, mTOR, and p70S6 kinase was not induced in mIGF-1 hearts, which instead activated alternate PDK1 and SGK1 signaling intermediates. The robust response achieved with the mIGF-1 isoform provides a mechanistic basis for clinically feasible therapeutic strategies for improving the outcome of heart disease.

Identification of novel peptide hormones in the human proteome by hidden Markov model screening.
Mirabeau, O., Perlas, E., Severini, C., Audero, E., Gascuel, O., Possenti, R., Birney, E., Rosenthal, N. & Gross, C.
Genome Res. 2007 Mar;17(3):320-7. Epub 2007 Feb 6.

Peptide hormones are small, processed, and secreted peptides that signal via membrane receptors and play critical roles in normal and pathological physiology. The search for novel peptide hormones has been hampered by their small size, low or restricted expression, and lack of sequence similarity. To overcome these difficulties, we developed a bioinformatics search tool based on the hidden Markov model formalism that uses several peptide hormone sequence features to estimate the likelihood that a protein contains a processed and secreted peptide of this class. Application of this tool to an alignment of mammalian proteomes ranked 90% of known peptide hormones among the top 300 proteins. An analysis of the top scoring hypothetical and poorly annotated human proteins identified two novel candidate peptide hormones. Biochemical analysis of the two candidates, which we called spexin and augurin, showed that both were localized to secretory granules in a transfected pancreatic cell line and were recovered from the cell supernatant. Spexin was expressed in the submucosal layer of the mouse esophagus and stomach, and a predicted peptide from the spexin precursor induced muscle contraction in a rat stomach explant assay. Augurin was specifically expressed in mouse endocrine tissues, including pituitary and adrenal gland, choroid plexus, and the atrio-ventricular node of the heart. Our findings demonstrate the utility of a bioinformatics approach to identify novel biologically active peptides. Peptide hormones and their receptors are important diagnostic and therapeutic targets, and our results suggest that spexin and augurin are novel peptide hormones likely to be involved in physiological homeostasis.

Local expression of IGF-1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines.
Pelosi, L., Giacinti, C., Nardis, C., Borsellino, G., Rizzuto, E., Nicoletti, C., Wannenes, F., Battistini, L., Rosenthal, N., Molinaro, M. & Musaro, A.
FASEB J. 2007 Jan 30;.

Muscle regeneration following injury is characterized by myonecrosis accompanied by local inflammation, activation of satellite cells, and repair of injured fibers. The resolution of the inflammatory response is necessary to proceed toward muscle repair, since persistence of inflammation often renders the damaged muscle incapable of sustaining efficient muscle regeneration. Here, we show that local expression of a muscle-restricted insulin-like growth factor (IGF)-1 (mIGF-1) transgene accelerates the regenerative process of injured skeletal muscle, modulating the inflammatory response, and limiting fibrosis. At the molecular level, mIGF-1 expression significantly down-regulated proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, and modulated the expression of CC chemokines involved in the recruitment of monocytes/macrophages. Analysis of the underlying molecular mechanisms revealed that mIGF-1 expression modulated key players of inflammatory response, such as macrophage migration inhibitory factor (MIF), high mobility group protein-1 (HMGB1), and transcription NF-kappaB. The rapid restoration of injured mIGF-1 transgenic muscle was also associated with connective tissue remodeling and a rapid recovery of functional properties. By modulating the inflammatory response and reducing fibrosis, supplemental mIGF-1 creates a qualitatively different environment for sustaining more efficient muscle regeneration and repair.--Pelosi, L., Giacinti, C., Nardis, C., Borsellino, G., Rizzuto, E., Nicoletti, C., Wannenes, F., Battistini, L., Rosenthal, N., Molinaro, M., Musaro, A. Local expression of IGF-1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines.

The neuroprotective effects of a locally acting IGF-1 isoform.
Musaro, A., Dobrowolny, G. & Rosenthal, N.
Exp Gerontol. 2007 Jan-Feb;42(1-2):76-80. Epub 2006 Jun 19.

In the last decade, dramatic progress has been made in elucidating the molecular defects underlying a number of neuromuscular diseases. With the characterization of mutations responsible for muscle and nerve dysfunction in several inherited pathologies, and the identification of novel signaling pathways, in which subtle alterations can lead to significant defects in tissue metabolism, the field is poised to devise successful strategies for treatment of this debilitating and often fatal group of human ailments. Yet progress in therapeutic application has been slow despite our newly gained knowledge of basic biology. Hence, where direct therapeutic approaches to address the primary diseases are still sub-optimal, it may be more effective to focus on strategies for improving neuromuscular function. Among potential candidates, insulin-like growth factor (IGF-1) has been involved in several anabolic pathways in both skeletal muscle and the nervous system and it is a promising candidate to attenuate neuromuscular diseases. In this review, we will discuss the role of IGF-1 isoforms in neuromuscular diseases and the contribution of muscle-produced IGF-1 (mIGF-1) to motor neuron survival and activity.

Regenerative medicine in cardiovascular research: of molecules, cells and scaffolds
Santini, MP, Lara-Pezzi, E., Rosenthal, N.
Brit. Soc. Cardiovasc. Res. 2007 20:4-12

Targeted ablation of IKK2 improves skeletal muscle strength, maintains mass, and promotes regeneration.
Mourkioti, F., Kratsios, P., Luedde, T., Song, Y.H., Delafontaine, P., Adami, R., Parente, V., Bottinelli, R., Pasparakis, M. & Rosenthal, N.
J Clin Invest. 2006 Nov 1;116(11):2945-2954.

NF-kappaB is a major pleiotropic transcription factor modulating immune, inflammatory, cell survival, and proliferative responses, yet the relevance of NF-kappaB signaling in muscle physiology and disease is less well documented. Here we show that muscle-restricted NF-kappaB inhibition in mice, through targeted deletion of the activating kinase inhibitor of NF-kappaB kinase 2 (IKK2), shifted muscle fiber distribution and improved muscle force. In response to denervation, IKK2 depletion protected against atrophy, maintaining fiber type, size, and strength, increasing protein synthesis, and decreasing protein degradation. IKK2-depleted mice with a muscle-specific transgene expressing a local Igf-1 isoform (mIgf-1) showed enhanced protection against muscle atrophy. In response to muscle damage, IKK2 depletion facilitated skeletal muscle regeneration through enhanced satellite cell activation and reduced fibrosis. Our results establish IKK2/NF-kappaB signaling as an important modulator of muscle homeostasis and suggest a combined role for IKK inhibitors and growth factors in the therapy of muscle diseases.

The future of regenerative therapy in patients with chronic heart failure.
Yacoub, M., Suzuki, K. & Rosenthal, N.
Nat Clin Pract Cardiovasc Med. 2006 Mar;3 Suppl 1:S133-5.

Regenerative therapy is a rapidly growing branch of science and medicine, which could have an important impact on the treatment of heart failure, a major cause of disability and death. Regeneration of the damaged myocardium in heart failure can be achieved through different strategies aimed at 'reviving' existing malfunctioning cells, repopulating the myocardium by new cells from exogenous or endogenous sources, altering the extracellular matrix, or increasing blood supply by enhancing vasculogenesis. To date, the clinical application of some of these strategies has had minimal or no impact on the global epidemic of chronic heart failure. However, several small clinical trials have reported varying degrees of functional improvement which could be considerable in some cases. We here review recent progress in the field, suggest an integrated approach, and outline the many gaps in our knowledge which need to be resolved by intensive laboratory research if regenerative therapy for chronic heart failure is to achieve its future potential.

Growth factor enhancement of cardiac regeneration.
Rosenthal, N., Santini, M.P. & Musaro, A.
Cell Transplant. 2006;15 Suppl 1:S41-5.

The potential for endogenous or supplementary stem cells to restore the form and function of damaged tissues is particularly promising for overcoming the restricted regenerative capacity of the mammalian heart. To maintain blood circulation, this essential organ needs to launch a rapid response to repair damage of the muscle wall and to prevent muscle loss. The capacity of growth factors to supplement the repair process has been successfully applied to restore the integrity of damaged skeletal muscle, reducing the fibrotic response to injury, and recruiting local populations of self-renewing precursor cells and circulating stem cells. We review the recent evidence that extension of growth factor supplementation to the heart may overcome its inherent regenerative impediments through improvement of the local tissue environment and stimulation of cell replacement, and we speculate on future research directions for treatment of myocardial damage.

Growth factor enhancement of mammalian regeneration.
Rosenthal, N.
Kidney Int 2005 Nov;68(5):1965-6.

The remarkable potential of lower vertebrates to rebuild heart and skeletal muscle contrasts with relatively poor regenerative capacity in mammals, which may not retain a sufficiently robust progenitor cell population into adulthood. Regeneration of the mammalian myocardium is perhaps the most recalcitrant, having apparently lost the ability to activate localized dedifferentiation of postmitotic cells that accompanies heart regeneration in lower vertebrates. Myogenic progenitors were enhanced in regenerating transgenic mouse muscle expressing a local insulin-like growth factor-1 (mIGF-1) isoform, which maintains tissue integrity during exercise and aging, counters muscle decline in degenerative disease and cachexia, and enhances healing following injury. After cardiotoxin-induced damage, regenerating mIGF-1 transgenic muscles rapidly down-regulated markers of inflammation, suggesting that resolution of the inflammatory response is an important component of efficient regeneration. In hearts expressing the same mIGF-1 transgene under the control of a cardiac-specific promoter, heightened expression of physiologic but not pathologic markers of cardiac growth and hypertrophy in postnatal mIGF-1 hearts was accompanied by sustained increases in protein translational components and decreases in specific markers of inflammation. The regenerative capacity of mIGF-1 transgene expression was directly compared in heart and skeletal muscle by myocardial infarction of wild-type and transgenic mice, producing localized damage, cell death, and massive inflammation in both genotypes. In contrast to wild-type controls, transgenic mIGF-1 expression resulted in enhanced repair of the injured heart with reduced scar formation and displayed increased proliferation in the area of damage after 1 month. Down-regulation of specific inflammatory cytokines suggests that IGF-1 improves cardiac regeneration in part by modulation of the inflammatory response. Since supplementary expression of mIGF-1 does not alter normal heart development or long-term postnatal cardiac growth and function, the enhancement of cardiac regeneration by localized expression of this growth factor suggests novel and clinically feasible therapeutic strategies to decrease inflammation and increase cell proliferation.

IGF-1, inflammation and stem cells: interactions during muscle regeneration.
Mourkioti, F. & Rosenthal, N.
Trends Immunol 2005 Oct;26(10):535-542.

Insulin-like growth factor-I (IGF-1) is an important mediator in numerous developmental processes, such as proliferation, differentiation, survival, growth, apoptosis and regeneration. Mouse genetics have provided important insights into the signalling mechanisms that are necessary for the coordination of muscle repair. Recent studies on the role of IGF-1 in the promotion of cell recruitment to the injured muscle and the subsequent resolution of the inflammatory response have unveiled new perspectives into local repair mechanisms.

Transgenic overexpression of locally acting insulin-like growth factor-1 inhibits ubiquitin-mediated muscle atrophy in chronic left-ventricular dysfunction.
Schulze, P.C., Fang, J., Kassik, K.A., Gannon, J., Cupesi, M., MacGillivray, C., Lee, R.T. & Rosenthal, N.
Circ Res 2005 Sep 2;97(5):418-26. Epub 2005 Jul 28.

Metabolic abnormalities develop in various chronic diseases and lead to progressive catabolism with decrements in the skeletal musculature that result in muscle atrophy. We investigated pathways of skeletal muscle proteolysis using an experimental model of chronic left-ventricular dysfunction. Skeletal muscle atrophy developed in wild-type mice 12 weeks following myocardial infarction accompanied by an increase in total protein ubiquitination and enhanced proteasome activity, activation of Foxo transcription factors, and robust induction of the ubiquitin-protein ligase atrogin-1/MAFbx. Further studies identified skeletal muscle myosin as a specific target of ubiquitin-mediated degradation in muscle atrophy. In contrast, transgenic overexpression of a local isoform of insulin-like growth factor-1 prevented muscle atrophy and increased proteasome activity, inhibited skeletal muscle activation primarily of Foxo4, and blocked the expression of atrogin-1/MAFbx. These results suggest that skeletal muscle atrophy occurs through increased activity of the ubiquitin-proteasome pathway. The inhibition of muscle atrophy by local insulin-like growth factor-1 provides a promising therapeutic avenue for the prevention of skeletal muscle wasting in chronic heart failure and potentially other chronic diseases associated with skeletal muscle atrophy.

Vasopressin-dependent myogenic cell differentiation is mediated by both Ca2+/calmodulin-dependent kinase and calcineurin pathways.
Scicchitano, B.M., Spath, L., Musaro, A., Molinaro, M., Rosenthal, N., Nervi, C. & Adamo, S.
Mol Biol Cell 2005 Aug;16(8):3632-41. Epub 2005 Jun 1.

Arg8-vasopressin (AVP) promotes the differentiation of myogenic cell lines and mouse primary satellite cells by mechanisms involving the transcriptional activation of myogenic bHLH regulatory factors and myocyte enhancer factor 2 (MEF2). We here report that AVP treatment of L6 cells results in the activation of calcineurin-dependent differentiation, increased expression of MEF2 and GATA2, and nuclear translocation of the calcineurin target NFATc1. Interaction of these three factors occurs at MEF2 sites of muscle specific genes. The different kinetics of AVP-dependent expression of early (myogenin) and late (MCK) muscle-specific genes correlate with different acetylation levels of histones at their MEF2 sites. The cooperative role of calcineurin and Ca2+/calmodulin-dependent kinase (CaMK) in AVP-dependent differentiation is demonstrated by the effect of inhibitors of the two pathways. We show here, for the first time, that AVP, a "novel" myogenesis promoting factor, activates both the calcineurin and the CaMK pathways, whose combined activation leads to the formation of multifactor complexes and is required for the full expression of the differentiated phenotype. Although MEF2-NFATc1 complexes appear to regulate the expression of an early muscle-specific gene product (myogenin), the activation of late muscle-specific gene expression (MCK) involves the formation of complexes including GATA2.

Youthful prospects for human stem-cell therapy. In another few decades, revised attitudes toward stem cells could lead to disease prevention and life extension.
Rosenthal, N.
EMBO Rep. 2005 Jul;6 Spec No:S30-4.

Insulin-like growth factor I-mediated skeletal muscle hypertrophy is characterized by increased mTOR-p70S6K signaling without increased Akt phosphorylation.
Song, Y.H., Godard, M., Li, Y., Richmond, S.R., Rosenthal, N. & Delafontaine, P.
J Investig Med 2005 Apr;53(3):135-42.

BACKGROUND: Insulin-like growth factor I (IGF-I) is an anabolic hormone that is known to induce skeletal muscle hypertrophy. However, the signaling pathways mediating IGF-I's hypertrophic effect in vivo are unknown. METHOD: The phosphorylation of 46 proteins was investigated by Kinetworks proteomic analysis in the gastrocnemius muscle of transgenic mice overexpressing IGF-I myosin light chain/muscle specific IGF-I (MLC/mlgf-I) and wild-type littermates. RESULTS: In the hypertrophic muscle of MLC/mlgf-I mice, we observed increased phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1; 53% increase), the mammalian target of rapamycin (mTOR; 112% increase), and p70 S6 kinase (p70S6K) (254% increase) but no significant change in Akt phosphorylation (4% decrease). Furthermore, we found reduced phosphorylation of MAP kinase kinase 1 and 2 (MEK1/2) (60% decrease) and of mitogen-activated protein kinase kinases 3 and 6 (MKK3/6) (50% decrease) in muscle from transgenic mice, suggesting that the hypertrophic and mitogenic effects of IGF-I are mediated via distinct signaling pathways in skeletal muscle and that inhibition of the mitogen-activated protein (MAP) kinase pathway may be required for the IGF-I-induced hypertrophic effect. Single-fiber analysis revealed a trend toward a higher percentage of the fast twitch fibers (IIb and IIx) in the transgenic mice. CONCLUSION: Persistent overexpression of IGF-I in mice skeletal muscle results in hypertrophy, which is likely mediated via the mTOR/p70S6K pathway, potentially via an Akt-independent signaling pathway.

Muscle-specific expression of IGF-1 blocks angiotensin II-induced skeletal muscle wasting.
Song, Y.H., Li, Y., Du, J., Mitch, W.E., Rosenthal, N. & Delafontaine, P.
J Clin Invest 2005 Feb;115(2):451-8.

Advanced congestive heart failure is associated with activation of the renin-angiotensin system and skeletal muscle wasting. We previously showed that angiotensin II infusion in rats produces cachexia secondarily to increased muscle proteolysis and also decreases levels of circulating and skeletal muscle IGF-1. Here we show that angiotensin II markedly downregulates phospho-Akt and activates caspase-3 in skeletal muscle, leading to actin cleavage, an important component of muscle proteolysis, and to increased apoptosis. These changes are blocked by muscle-specific expression of IGF-1, likely via the Akt/mTOR/p70S6K signaling pathway. We also demonstrate that mRNA levels of the ubiquitin ligases atrogin-1 and muscle ring finger-1 are upregulated in angiotensin II-infused WT, but not in IGF-1-transgenic, mice. These findings strongly suggest that angiotensin II downregulation of IGF-1 in skeletal muscle is causally related to angiotensin II-induced wasting. Because the renin-angiotensin system is activated in many catabolic conditions, our findings have broad implications for understanding mechanisms of skeletal muscle wasting and provide a rationale for new therapeutic approaches.

Reconciling data from transgenic mice that overexpress IGF-I specifically in skeletal muscle.
Shavlakadze, T., Winn, N., Rosenthal, N. & Grounds, M.D.
Growth Horm IGF Res 2005 Feb;15(1):4-18. Epub 2005 Jan 21.

Transgenic mice that overexpress insulin-like growth factor-1 (IGF-I) specifically in skeletal muscle have generated much information about the role of this factor for muscle growth and remodelling and provide insight for therapeutic applications of IGF-I for different pathological states and ageing. However, difficulties arise when attempting to critically compare the significance of data obtained in vivo by using different genetically engineered mouse lines and various experimental models. Complications arise due to complexity of the IGF-I system, since multiple transcripts of the IGF-I gene encode different isoforms generated by alternate promoter usage, differential splicing and post-translational modification, and how IGF-I gene expression relates to its diverse autocrine, paracrine and endocrine modes of action in vivo has still to be elucidated. In addition, there are problems related to specification of the exact IGF-I isoform used, expression patterns of the promoters, and availability of the transgene product under different experimental conditions. This review discusses the factors that must be considered when reconciling data from cumulative studies on IGF-I in striated muscle growth and differentiation using genetically modified mice. Critical evaluation of the literature focuses specifically on: (1) the importance of detailed information about the IGF-I isoforms and their mode of action (local, systemic or both); (2) expression pattern and strength of the promoters used to drive transgenic IGF-I in skeletal muscle cells (mono and multi-nucleated); (3) local compared with systemic action of the transgene product and possible indirect effects of transgenic IGF-I due to upregulation of other genes within skeletal muscle; (4) re-interpretation of these results in light of the most recent approaches to the dissection of IGF-I function. Full understanding of these complex in vivo issues is essential, not only for skeletal muscle but for many other tissues, in order to effectively extend observations derived from transgenic studies into potential clinical situations.

Human hepatocytes in mice receiving pre-immune injection with human cord blood cells.
Turrini, P., Monego, G., Gonzalez, J., Cicuzza, S., Bonanno, G., Zelano, G., Rosenthal, N., Paonessa, G., Laufer, R. & Padron, J.
Biochem Biophys Res Commun 2005 Jan 7;326(1):66-73.

It is well established that certain subpopulations of human adult stem cells can generate hepatocyte-like cells when transplanted into adult immunosuppressed mice. In the present study, we wanted to explore whether xeno-transplantation of human cord blood CD34(+) (hCBCD34(+)) cells during pre-immune stages of development in immunocompetent mice might also lead to human-mouse liver chimerism. Freshly isolated hCBCD34(+) cells were xeno-transplanted into non-immunosuppressed mice by both intra-blastocyst and intra-fetal injections. One and four weeks after birth, immunostaining for different human-specific hepatocyte markers: human hepatocyte-specific antigen, human serum albumin, and human alpha-1-antitrypsin indicated the presence of human hepatocyte-like cells in the livers of transplanted animals. Detection of human albumin mRNA further corroborated the development of pre-immune human-mouse chimeras. The current report, besides providing new evidence of the potential of hCBCD34(+) cells to generate human hepatocyte-like cells, suggests novel strategies for generating immunocompetent mice harboring humanized liver.

The Eumorphia consortium EMPReSS: standardised phenotype screens for functional annotation of the mouse genome.
Rosenthal. N.
Nature Genetics 2005 37:1155

Enhanced muscle hypertrophy protects motor neurons in an ALS mouse model
Dobrowolny, G., Giancinti, C., Pelosi, L., Nicoletti, C., Berberi, L, Molinaro, M., Rosenthal, N., a Musarò, A.
J. Cell. Biol. 2005, 168:193-199

The European dimension for the mouse genome mutagenesis program.
Auwerx, J., Avner, P., Baldock, R., Ballabio, A., Balling, R., Barbacid, M., Berns, A., Bradley, A., Brown, S., Carmeliet, P., Chambon, P., Cox, R., Davidson, D., Davies, K., Duboule, D., Forejt, J., Granucci, F., Hastie, N., de Angelis, M.H., Jackson, I., Kioussis, D., Kollias, G., Lathrop, M., Lendahl, U., Malumbres, M., von Melchner, H., Muller, W., Partanen, J., Ricciardi-Castagnoli, P., Rigby, P., Rosen, B., Rosenthal, N., Skarnes, B., Stewart, A.F., Thornton, J., Tocchini-Valentini, G., Wagner, E., Wahli, W. & Wurst, W.
Nat Genet 2004 Sep;36(9):925-7.

The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.

The stem cell challenge.
Lanza, R. & Rosenthal, N.
Sci Am 2004 Jun;290(6):92-9.

Stem cell-mediated muscle regeneration is enhanced by local isoform of insulin-like growth factor 1.
Musaro, A., Giacinti, C., Borsellino, G., Dobrowolny, G., Pelosi, L., Cairns, L., Ottolenghi, S., Cossu, G., Bernardi, G., Battistini, L., Molinaro, M. & Rosenthal, N.
Proc Natl Acad Sci U S A 2004 Feb 3;101(5):1206-10. Epub 2004 Jan 26.

We investigated the mechanism whereby expression of a transgene encoding a locally acting isoform of insulin-like growth factor 1 (mIGF-1) enhances repair of skeletal muscle damage. Increased recruitment of proliferating bone marrow cells to injured MLC/mIgf-1 transgenic muscles was accompanied by elevated bone marrow stem cell production in response to distal trauma. Regenerating MLC/mIgf-1 transgenic muscles contained increased cell populations expressing stem cell markers, exhibited accelerated myogenic differentiation, expressed markers of regeneration and readily converted cocultured bone marrow to muscle. These data implicate mIGF-1 as a powerful enhancer of the regeneration response, mediating the recruitment of bone marrow cells to sites of tissue damage and augmenting local repair mechanisms.

Targeted expression of insulin-like growth factor-1 reduces early myofiber necrosis in dystrophic mdx mice.
Shavlakadze, T., White, J., Hoh, J.F., Rosenthal, N. & Grounds, M.D.
Mol. Ther. 2004 10 829-843

Stem cells and the regenerating heart.
Rosenthal, N. & Santini, M.P.
In "Stem Cell Handbook" Elsevier, 2004

A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field.
Hochgreb, T., Linhares, V.L., Menezes, D.C., Sampaio, A.C., Yan, C.Y., Cardoso, W.V., Rosenthal, N. & Xavier-Neto, J.
Development. 2003 Nov;130(22):5363-74. Epub 2003 Sep 16.

Establishment of anteroposterior (AP) polarity is one of the earliest decisions in cardiogenesis and plays an important role in the coupling between heart and blood vessels. Recent research implicated retinoic acid (RA) in the communication of AP polarity to the heart. We utilized embryo culture, in situ hybridization, morphometry, fate mapping and treatment with the RA pan-antagonist BMS493 to investigate the relationship between cardiac precursors and RA signalling. We describe two phases of AP signalling by RA, reflected in RALDH2 expression. The first phase (HH4-7) is characterized by increasing proximity between sino-atrial precursors and the lateral mesoderm expressing RALDH2. In this phase, RA signalling is consistent with diffusion of the morphogen from a large field rather than a single hot spot. The second phase (HH7-8) is characterized by progressive encircling of cardiac precursors by a field of RALDH2 originating from a dynamic and evolutionary-conserved caudorostral wave pattern in the lateral mesoderm. At this phase, cardiac AP patterning by RA is consistent with localized action of RA by regulated activation of the Raldh2 gene within an embryonic domain. Systemic treatment with BMS493 altered the cardiac fate map such that ventricular precursors were found in areas normally devoid of them. Topical application of BMS493 inhibited atrial differentiation in left anterior lateral mesoderm. Identification of the caudorostral wave of RALDH2 as the endogenous source of RA establishing cardiac AP fates provides a useful model to approach the mechanisms whereby the vertebrate embryo confers axial information on its organs.

Prometheus's vulture and the stem-cell promise.
Rosenthal, N.
N Engl J Med 2003 Jul 17;349(3):267-74.

Developmental basis of evolutionary digit loss in the Australian lizard Hemiergis.
Shapiro, M.D., Hanken, J. & Rosenthal, N.
J Exp Zoolog Part B Mol Dev Evol 2003 Jun 15;297(1):48-56.

Loss of limb skeletal elements is a recurring theme in tetrapod evolution, but the developmental mechanisms underlying this phenomenon remain largely unknown. The Australian lizard genus Hemiergis offers an excellent model system to study limb reduction among closely related, naturally occurring populations with different numbers of digits. Evolutionary digit loss in Hemiergis does not result from simple truncation of a pentadactyl skeletal developmental program. Rather, the duration of embryonic expression of the patterning molecule Sonic hedgehog (SHH) is shortened in limbs with reduced numbers of digits, and is correlated with decreased cell proliferation in the posterior aspect of the limb. Moreover, this comparative analysis suggests an early role for SHH in specification of digit identity and later importance in maintaining cell proliferation and survival. Subtle changes in spatial or temporal regulation of SHH may alter proliferation and patterning of the developing limb, thereby effecting divergence in adult limb morphology among closely related species. In contrast, expression of MSX and Distal-less proteins were similar among embryos from different populations.

Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes.
Prevot, V., Rio, C., Cho, G.J., Lomniczi, A., Heger, S., Neville, C.M., Rosenthal, N.A., Ojeda, S.R. & Corfas, G.
J Neurosci 2003 Jan 1;23(1):230-9.

The initiation of mammalian puberty requires the activation of hypothalamic neurons secreting the neuropeptide luteinizing hormone-releasing hormone (LHRH). It is thought that this activation is caused by changes in trans-synaptic input to LHRH neurons. More recently, it has been postulated that the pubertal increase in LHRH secretion in female animals also requires neuron-glia signaling mediated by growth factors of the epidermal growth factor (EGF) family and their astrocytic erbB receptors. Although it appears clear that functional astrocytic erbB1 receptors are necessary for the timely advent of puberty, the physiological contribution that erbB4 receptors may make to this process has not been established. To address this issue, we generated transgenic mice expressing a dominant-negative erbB4 receptor (DN-erbB4) under the control of the GFAP promoter, which targets transgene expression to astrocytes. DN-erbB4 expression is most abundant in hypothalamic astrocytes, where it blocks the ligand-dependent activation of glial erbB4 and erbB2 receptors, without affecting erbB1 (EGF) receptor signaling. Mice carrying the transgene exhibit delayed sexual maturation and a diminished reproductive capacity in early adulthood. These abnormalities are related to a deficiency in pituitary gonadotropin hormone secretion, caused by impaired release of LHRH, the hypothalamic neuropeptide that controls sexual development. In turn, the reduction in LHRH release is caused by the inability of hypothalamic astrocytes to respond to neuregulin (NRG) with production of prostaglandin E(2), which in wild-type animals mediates the stimulatory effect of astroglial erbB receptor activation on neuronal LHRH release. Thus, neuron-astroglia communication via NRG-erbB4/2 receptor signaling appears to be essential for the timely unfolding of the developmental program by which the brain controls mammalian sexual maturation.

Machinations of the marrow.
Rosenthal, N.
J Clin Invest 2003 Jan;111(1):29-30.

All at the tip of a needle.
Rosenthal, N.
Development. 2003, 130:5566-7

Gene therapy for cardiac cachexia?
Rosenthal, N. & Musaro, A.
Int J Cardiol 2002 Sep;85(1):185-91.

The prevention or attenuation of disease-related skeletal muscle degeneration has been a common goal in the treatment of cardiac cachexia. Cell-based therapies are complicated by insufficient numbers of autologous myoblasts and by ineffective incorporation into host muscle. Pharmacological administration of growth hormone in a variety of clinical conditions characterized by an increase in catabolic rate have been associated with increases in mortality and morbidity, resulting in a decrease in the clinical use of growth hormone and its downstream effector, insulin-like growth factor-1 and a decline in general research into anabolic treatment strategies. In mouse models, however, the selective expression of a muscle-specific transgene encoding a locally acting IGF-1 isoform induces muscle hypertrophy, prevents age- or disease-related atrophy, by increasing stem cell recruitment to injured or degenerating tissue. This gene-based approach avoids hypertrophic effects on distal organs such as the heart, and eliminates risk of possible neoplasms induced by inappropriate high expression levels of circulating IGF-1. The potential therapeutic role of locally expressed IGF-1 is discussed in the context of current strategies for the attenuation of cardiac cachexia.

Taking stock of our models: the function and future of stock centres.
Rosenthal, N. & Ashburner, M.
Nat Rev Genet 2002 Sep;3(9):711-7.

Stock centres for our animal models are as important as other scientific resources, such as the primary literature or genome databases. But they need forward planning, international cooperation and secure funding to keep pace with the explosion in functional genomics that relies so heavily on them.

The role of stem cells in skeletal and cardiac muscle repair.
Grounds, M.D., White, J.D., Rosenthal, N. & Bogoyevitch, M.A.
J Histochem Cytochem 2002 May;50(5):589-610.

In postnatal muscle, skeletal muscle precursors (myoblasts) can be derived from satellite cells (reserve cells located on the surface of mature myofibers) or from cells lying beyond the myofiber, e.g., interstitial connective tissue or bone marrow. Both of these classes of cells may have stem cell properties. In addition, the heretical idea that post-mitotic myonuclei lying within mature myofibers might be able to re-form myoblasts or stem cells is examined and related to recent observations for similar post-mitotic cardiomyocytes. In adult hearts (which previously were not considered capable of repair), the role of replicating endogenous cardiomyocytes and the recruitment of other (stem) cells into cardiomyocytes for new cardiac muscle formation has recently attracted much attention. The relative contribution of these various sources of precursor cells in postnatal muscles and the factors that may enhance stem cell participation in the formation of new skeletal and cardiac muscle in vivo are the focus of this review. We concluded that, although many endogenous cell types can be converted to skeletal muscle, the contribution of non-myogenic cells to the formation of new postnatal skeletal muscle in vivo appears to be negligible. Whether the recruitment of such cells to the myogenic lineage can be significantly enhanced by specific inducers and the appropriate microenvironment is a current topic of intense interest. However, dermal fibroblasts appear promising as a realistic alternative source of exogenous myoblasts for transplantation purposes. For heart muscle, experiments showing the participation of bone marrow-derived stem cells and endothelial cells in the repair of damaged cardiac muscle are encouraging.

Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice.
Barton, E.R., Morris, L., Musaro, A., Rosenthal, N. & Sweeney, H.L.
J Cell Biol 2002 Apr 1;157(1):137-48.

Duchenne muscular dystrophy is an X-linked degenerative disorder of muscle caused by the absence of the protein dystrophin. A major consequence of muscular dystrophy is that the normal regenerative capacity of skeletal muscle cannot compensate for increased susceptibility to damage, leading to repetitive cycles of degeneration-regeneration and ultimately resulting in the replacement of muscle fibers with fibrotic tissue. Because insulin-like growth factor I (IGF-I) has been shown to enhance muscle regeneration and protein synthetic pathways, we asked whether high levels of muscle-specific expression of IGF-I in mdx muscle could preserve muscle function in the diseased state. In transgenic mdx mice expressing mIgf-I (mdx:mIgf+/+), we showed that muscle mass increased by at least 40% leading to similar increases in force generation in extensor digitorum longus muscles compared with those from mdx mice. Diaphragms of transgenic mdx:mIgf+/+ exhibited significant hypertrophy and hyperplasia at all ages observed. Furthermore, the IGF-I expression significantly reduced the amount of fibrosis normally observed in diaphragms from aged mdx mice. Decreased myonecrosis was also observed in diaphragms and quadriceps from mdx:mIgf+/+ mice when compared with age-matched mdx animals. Finally, signaling pathways associated with muscle regeneration and protection against apoptosis were significantly elevated. These results suggest that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin.

Targeted deletion of the MLC1f/3f downstream enhancer results in precocious MLC expression and mesoderm ablation.
Jiang, P., Song, J., Gu, G., Slonimsky, E., Li, E. & Rosenthal, N.
Dev Biol 2002 Mar 15;243(2):281-93.

The expression of skeletal muscle contractile proteins is tightly regulated during embryonic development. In the mouse, the myosin light chain (MLC) 1f/3f gene locus is not activated until E9.5, exclusively in skeletal muscle precursor cells. A potent enhancer downstream of the MLC1f/3f locus confers correct temporal and spatial activation of linked reporter gene in transgenic mouse embryos. To examine roles of the MLC downstream enhancer (MLCE) in its native context of the MLC1f/3f gene locus, we eliminated a 1.5-kb DNA segment containing the enhancer from the mouse genome by targeted deletion, leaving no exogenous sequences at the deletion site. Mouse embryos homozygous for the MLCE deletion were smaller and developmentally delayed, formed no mesoderm by E7.5, and were resorbed almost completely at E8.5. In situ hybridization and RT-PCR analyses of affected mutant embryos at E7.5 revealed ectopic MLC transcripts, whose products would be predicted to interfere with a variety of nonmuscle cell functions determining differentiation of mesoderm. These results suggest that the MLC downstream enhancer and its flanking sequences include negative regulatory elements which block precocious activation of MLC expression in mesodermal precursors during a critical window of development, as well as positive elements which subsequently permit tissue-restricted MLC transcription in differentiating skeletal muscles. (C)2002 Elsevier Science (USA).

Different modes of hypertrophy in skeletal muscle fibers.
Paul, A.C. & Rosenthal, N.
J Cell Biol 2002 Feb 18;156(4):751-60.

Skeletal muscles display a remarkable diversity in their arrangement of fibers into fascicles and in their patterns of innervation, depending on functional requirements and species differences. Most human muscle fascicles, despite their great length, consist of fibers that extend continuously from one tendon to the other with a single nerve endplate band. Other mammalian muscles have multiple endplate bands and fibers that do not insert into both tendons but terminate intrafascicularly. We investigated whether these alternate structural features may dictate different modes of cell hypertrophy in two mouse gracilis muscles, in response to expression of a muscle-specific insulin-like growth factor (IGF)-1 transgene (mIGF-1) or to chronic exercise. Both hypertrophic stimuli independently activated GATA-2 expression and increased muscle cross-sectional area in both muscle types, with additive effects in exercising myosin light chain/mIGF transgenic mice, but without increasing fiber number. In singly innervated gracilis posterior muscle, hypertrophy was characterized by a greater average diameter of individual fibers, and centralized nuclei. In contrast, hypertrophic gracilis anterior muscle, which is multiply innervated, contained longer muscle fibers, with no increase in average diameter, or in centralized nuclei. Different modes of muscle hypertrophy in domestic and laboratory animals have important implications for building appropriate models of human neuromuscular disease.

Insulin-like growth factor isoforms in skeletal muscle aging, regeneration, and disease.
Winn, N., Paul, A., Musaro, A. & Rosenthal, N.
Cold Spring Harb Symp Quant Biol. 2002;67:507-18.

The role of Insulin-like Growth Factor-1 isoforms in the physiopathology of skeletal muscle.
Musaro, A. & Rosenthal, N.
Current Genomics 2002 3(3) 149-162

Retinoid signaling and cardiac anteroposterior segmentation.
Xavier-Neto, J., Rosenthal, N., Silva, F.A., Matos, T.G., Hochgreb, T. & Linhares, V.L.
Genesis 2001 Nov;31(3):97-104.

Establishment of anterior-posterior polarity is one of the earliest decisions in cardiogenesis. Specification of anterior (outflow) and posterior (inflow) structures ensures proper connections between venous system and inflow tract and between arterial tree and outflow tract. The last few years have witnessed remarkable progress in our understanding of cardiac anteroposterior patterning. Molecular cloning and subsequent studies on RALDH2, the key embryonic retinaldehyde dehydrogenase in retinoic acid (RA) synthesis, provided the missing link between teratogenic studies on RA deficiency and excess and normal chamber morphogenesis. We discuss work establishing the foundations of our current understanding of the mechanisms of cardiac anteroposterior segmentation, the reasons why early evidence pointing to the role of RA in anteroposterior segmentation was overlooked, and the key experiments unraveling the role of RA in cardiac anteroposterior segmentation. We have also integrated recent experiments in a model of cardiac anteroposterior patterning in which RALDH2 expression determines anteroposterior boundaries in the heart field.

High hopes for the heart.
Rosenthal, N.
N Engl J Med 2001 Jun 7;344(23):1785-7.

Helping the heart to heal with stem cells.
Rosenthal, N. & Tsao, L.
Nat Med 2001 Apr;7(4):412-3.

Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression.
Bruneau, B.G., Bao, Z.Z., Fatkin, D., Xavier-Neto, J., Georgakopoulos, D., Maguire, C.T., Berul, C.I., Kass, D.A., Kuroski-de Bold, M.L., de Bold, A.J., Conner, D.A., Rosenthal, N., Cepko, C.L., Seidman, C.E. & Seidman, J.G.
Mol Cell Biol 2001 Mar;21(5):1730-6.

To define the role of Irx4, a member of the Iroquois family of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designated Irx4(Delta ex2/Delta ex2)) was normal during embryogenesis and in early postnatal life. Adult Irx4(Delta ex2/Delta ex2) mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy, Irx4(Delta ex2/Delta ex2) hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increased Irx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and alpha-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic markers BNP and beta-myosin heavy chain accompanied adult-onset cardiac hypertrophy. Cardiac expression of Irx1, Irx2, and Irx5 may partially compensate for loss of Irx4 function. We conclude that Irx4 is not sufficient for ventricular chamber formation but is required for the establishment of some components of a ventricle-specific gene expression program. In the absence of genes under the control of Irx4, ventricular function deteriorates and cardiomyopathy ensues.

Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle.
Musaro, A., McCullagh, K., Paul, A., Houghton, L., Dobrowolny, G., Molinaro, M., Barton, E.R., Sweeney, H.L. & Rosenthal, N.
Nat Genet 2001 Feb;27(2):195-200.

Aging skeletal muscles suffer a steady decline in mass and functional performance, and compromised muscle integrity as fibrotic invasions replace contractile tissue, accompanied by a characteristic loss in the fastest, most powerful muscle fibers. The same programmed deficits in muscle structure and function are found in numerous neurodegenerative syndromes and disease-related cachexia. We have generated a model of persistent, functional myocyte hypertrophy using a tissue-restricted transgene encoding a locally acting isoform of insulin-like growth factor-1 that is expressed in skeletal muscle (mIgf-1). Transgenic embryos developed normally, and postnatal increases in muscle mass and strength were not accompanied by the additional pathological changes seen in other Igf-1 transgenic models. Expression of GATA-2, a transcription factor normally undetected in skeletal muscle, marked hypertrophic myocytes that escaped age-related muscle atrophy and retained the proliferative response to muscle injury characteristic of younger animals. The preservation of muscle architecture and age-independent regenerative capacity through localized mIgf-1 transgene expression suggests clinical strategies for the treatment of age or disease-related muscle frailty.

From the bottom of the heart: anteroposterior decisions in cardiac muscle differentiation.
Rosenthal, N. & Xavier-Neto, J.
Curr Opin Cell Biol 2000 Dec;12(6):742-6.

Recently, studies on specification of axes in the developing embryo have focused on the heart, which is the first functional organ to form and probably responds to common cues controlling positional information in surrounding tissues. The early differentiation of heart cells affords an opportunity to link the acquisition of regional identity with the signals underlying terminal differentiation. In the past year, a wealth of information on these signals has emerged, elucidating the general pathways controlling body axes in the context of the developing heart.

Sequential programs of retinoic acid synthesis in the myocardial and epicardial layers of the developing avian heart.
Xavier-Neto, J., Shapiro, M.D., Houghton, L. & Rosenthal, N.
Dev Biol 2000 Mar 1;219(1):129-41.

Endogenous patterns of retinoic acid (RA) signaling in avian cardiac morphogenesis were characterized by localized expression of a key RA-synthetic enzyme, RALDH2, which displayed a biphasic pattern during heart development. RALDH2 immunoreactivity was initially apparent posterior to Hensen's node of stage 5-6 embryos and subsequently in somites and unsegmented paraxial and lateral plate mesoderm overlapping atrial precursors in the cardiogenic plate of stage 9- embryos. Initial RALDH2 synthesis in the posterior myocardium coincided with activation of the AMHC1 gene, a RA-responsive marker of inflow heart segments. A wave of RALDH2 synthesis then swept the myocardium in a posterior-to-anterior direction, reaching the outflow tract by stage 13, then fading from the myocardial layer. The second phase of RALDH2 expression, initiated at stage 18 in the proepicardial organ, persisted in migratory epicardial cells that completely enveloped the heart by stage 24. Early restriction of RALDH2 expression to the posterior cardiogenic plate, overlapping RA-inducible gene activation, provides evidence for commitment of posterior avian heart segments by localized production of RA, whereas subsequent RALDH2 expression exclusively in the migratory epicardium suggests a role for the morphogen in ventricular expansion and morphogenesis of underlying myocardial tissues.

Revisiting calcineurin and human heart failure.
Tsao, L., Neville, C., Musaro, A., McCullagh, K.J. & Rosenthal, N.
Nat Med 2000 Jan;6(1):2-3.

Single allele mutations at the heart of congenital disease.
Rosenthal, N. & Harvey, R.P.
J Clin Invest. 1999 Dec;104(11):1483-4.

Regulation of a muscle-specific transgene by persistent expression of Hox genes in postnatal murine limb muscle.
Houghton, L. & Rosenthal, N.
Dev Dyn. 1999 Dec;216(4-5):385-97.

Homeobox genes are necessary for the generation of the embryonic body plan in both invertebrate and vertebrate organisms. To investigate the potential function of homeodomain proteins in normal and regenerating skeletal muscle, we analyzed patterns of clustered homeobox gene expression in neonatal and adult muscle tissue. Transcripts encoding 5' genes in the HoxA cluster were detected in muscles from both the fore- and hindlimbs of neonatal and adult mice, whereas expression of HoxC gene transcripts was generally restricted to the muscles of the hindlimb. In contrast, transcripts encoding genes of the HoxB or HoxD clusters were not detected in muscles from either fore- or hindlimbs. Although ectopic expression of select HOX proteins in muscle cell cultures had modest effects upon the activity of a co-transfected myosin light chain (MLC) enhancer, mutation of a Hox binding site in this enhancer elicited increased linked reporter gene expression. Induction of muscle damage and regeneration was accompanied by the down-regulation of at least one Hox gene, concurrent with the activation of the regenerative program. Moreover, targeted ablation of the Hoxc-8 gene, normally expressed in mature fore- and hindlimb muscles, resulted in reduced expression of an MLC enhancer-driven transgene only in specific leg muscles. These results indicate that members of the HoxA and C clusters may, in combination, mediate various aspects of differentiation and patterning in adult musculature.

An E box comprises a positional sensor for regional differences in skeletal muscle gene expression and methylation.
Ceccarelli, E., McGrew, M.J., Nguyen, T., Grieshammer, U., Horgan, D., Hughes, S.H. & Rosenthal, N.
Dev Biol 1999 Sep 1;213(1):217-29.

To dissect the molecular mechanisms conferring positional information in skeletal muscles, we characterized the control elements responsible for the positionally restricted expression patterns of a muscle-specific transgene reporter, driven by regulatory sequences from the MLC1/3 locus. These sequences have previously been shown to generate graded transgene expression in the segmented axial muscles and their myotomal precursors, fortuitously marking their positional address. An evolutionarily conserved E box in the MLC enhancer core, not recognized by MyoD, is a target for a nuclear protein complex, present in a variety of tissues, which includes Hox proteins and Zbu1, a DNA-binding member of the SW12/SNF2 gene family. Mutation of this E box in the MLC enhancer has only a modest positive effect on linked CAT gene expression in transfected muscle cells, but when introduced into transgenic mice the same mutation elevates CAT transgene expression in skeletal muscles, specifically releasing the rostral restriction on MLC-CAT transgene expression in the segmented axial musculature. Increased transgene activity resulting from the E box mutation in the MLC enhancer correlates with reduced DNA methylation of the distal transgenic MLC1 promoter as well as in the enhancer itself. These results identify an E box and the proteins that bind to it as a positional sensor responsible for regional differences in axial skeletal muscle gene expression and accessibility.

IGF-1 induces skeletal myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1.
Musaro, A., McCullagh, K.J., Naya, F.J., Olson, E.N. & Rosenthal, N.
Nature 1999 Aug 5;400(6744):581-5.

Localized synthesis of insulin-like growth factors (IGFs) has been broadly implicated in skeletal muscle growth, hypertrophy and regeneration. Virally delivered IGF-1 genes induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy, restoring and improving muscle mass and strength in mice. Here we show that the molecular pathways underlying the hypertrophic action of IGF-1 in skeletal muscle are similar to those responsible for cardiac hypertrophy. Transfected IGF-1 gene expression in postmitotic skeletal myocytes activates calcineurin-mediated calcium signalling by inducing calcineurin transcripts and nuclear localization of calcineurin protein. Expression of activated calcineurin mimics the effects of IGF-1, whereas expression of a dominant-negative calcineurin mutant or addition of cyclosporin, a calcineurin inhibitor, represses myocyte differentiation and hypertrophy. Either IGF-1 or activated calcineurin induces expression of the transcription factor GATA-2, which accumulates in a subset of myocyte nuclei, where it associates with calcineurin and a specific dephosphorylated isoform of the transcription factor NF-ATc1. Thus, IGF-1 induces calcineurin-mediated signalling and activation of GATA-2, a marker of skeletal muscle hypertrophy, which cooperates with selected NF-ATc isoforms to activate gene expression programs.

A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart.
Xavier-Neto, J., Neville, C.M., Shapiro, M.D., Houghton, L., Wang, G.F., Nikovits W, J.r, Stockdale, F.E. & Rosenthal, N.
Development 1999 Jun;126(12):2677-87.

To study the specification of inflow structures in the heart we generated transgenic animals harboring the human alkaline phosphatase (HAP) gene driven by the proximal 840 bp of a quail SMyHC3 promoter. In transgenic mice, the SMyHC3-HAP reporter was expressed in posterior heart precursors at 8.25 dpc, in sinus venosa and in the atrium at 8.5 and 9.0 dpc, and in the atria from 10.5 dpc onwards. SMyHC3-HAP transgene expression overlapped synthesis and endogenous response to retinoic acid (RA) in the heart, as determined by antibodies directed against a key RA synthetic enzyme and by staining of RAREhsplacZ transgenic animals. A single pulse of all-trans RA administered to pregnant mice at 7.5, but not after 8.5, dpc induced cardiac dismorphology, ranging from complete absence of outflow tract and ventricles to hearts with reduced ventricles expressing both SMyHC3-HAP and ventricular markers. Blockade of RA synthesis with disulfiram inhibited RA-induced transcription and produced hearts lacking the atrial chamber. This study defines a novel marker for atrial-restricted transcription in the developing mouse heart. It also suggests that atrial-specific gene expression is controlled by localized synthesis of RA, and that exclusion of RA from ventricular precursors is essential for correct specification of the ventricles.

Transgenic mouse models of muscle aging.
Musaro, A. & Rosenthal, N.
Exp Gerontol. 1999 Apr;34(2):147-56.

In the last decade transgenic animals have been become a powerful and exciting research model to study the molecular mechanisms underlying the cellular and physiological processes such as cell growth, differentiation, apoptosis, and the regulation of specific gene expression. In the context of skeletal muscle development, transgenic mice and gene-targeting approaches have led to the definition of specific roles for Muscle Regulatory Factors (MRFs) during embryogenesis, although less is known about the molecular mechanism underlying skeletal muscle aging. Recent studies using specific models of transgenic mice have added new insights into the muscle aging process, providing a baseline for designing appropriate strategies to attenuate or to reverse the cumulative effects of aging. In this review we discuss some of the transgenic models currently available to address the molecular mechanisms of skeletal muscle senescence. Given the complexity of the aging process, this review should be regarded as a presentation of works in progress rather than a comprehensive description of muscle aging.

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.
Musaro, A. & Rosenthal, N.
Mol Cell Biol 1999 Apr;19(4):3115-24.

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.

Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function.
Barton-Davis, E.R., Shoturma, D.I., Musaro, A., Rosenthal, N. & Sweeney, H.L.
Proc Natl Acad Sci U S A 1998 Dec 22;95(26):15603-7.

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart.
Moss, J.B., Xavier-Neto, J., Shapiro, M.D., Nayeem, S.M., McCaffery, P., Drager, U.C. & Rosenthal, N.
Dev Biol 1998 Jul 1;199(1):55-71.

Retinoic acid (RA) has been implicated in cardiac morphogenesis by its teratogenic effects on the heart, although its role in normal cardiogenesis remains unknown. To define the parameters of RA action in cardiac morphogenesis, we analyzed the patterns of ligand synthesis, response, and inactivation in the developing mouse heart. Activation of a lacZ transgene controlled by an RA response element (RARE) was compared to the localization of the retinaldehyde-oxidizing dehydrogenase RALDH2, the earliest RA synthetic enzyme in the mouse embryo, and to the expression of a gene encoding an RA-degrading enzyme (P450RA). We observed that RALDH2 localization and RA response were virtually superimposable throughout heart development. Initially, both RALDH2 and RARE-LacZ activity were restricted to the sinus venosa in unlooped hearts, but were high in the dorsal mesocardium, while P450RA expression was restricted to the endocardium. Later stages were characterized by a sequential, noncontiguous progression of RALDH2 accumulation and RA response, from the sinus venosa to atria, dorsal-medial conotruncus, aortic arches, and the epicardium. This dynamic pattern of RA response was a direct result of localized RALDH2, since hearts of cultured embryos were uniformly competent to respond to an exogenous RA challenge. These observations support a model in which the influence of endogenous RA on heart development depends upon localized presentation of the ligand, with only limited diffusion from the source of its synthesis.

In search of perverse polymorphisms.
Rosenthal, N. & Schwartz, R.S.
N Engl J Med. 1998 Jan 8;338(2):122-4.

Ectopic expression of phospholamban in fast-twitch skeletal muscle alters sarcoplasmic reticulum Ca2+ transport and muscle relaxation.
Slack, J.P., Grupp, I.L., Ferguson, D.G., Rosenthal, N. & Kranias, E.G.
J Biol Chem. 1997 Jul 25;272(30):18862-8.

There are three isoforms of the sarcoplasmic reticulum Ca2+-ATPase; they are known as SERCA1, SERCA2, and SERCA3. Phospholamban is present in tissues that express the SERCA2 isoform and is an inhibitor of the affinity of SERCA2 for calcium. In vitro reconstitution and cell culture expression studies have shown that phospholamban can also regulate SERCA1, the fast-twitch skeletal muscle isoform. To determine whether regulation of SERCA1 by phospholamban can be of physiological relevance, we generated transgenic mice that ectopically express phospholamban in fast-twitch skeletal muscle, a tissue normally devoid of phospholamban. Ectopic expression of phospholamban was associated with a decrease in the affinity of SERCA1 for calcium. Assessment of isometric twitch contractions of intact fast-twitch skeletal muscles revealed depressed rates of relaxation in transgenic mice compared with wild-type cohorts. Furthermore, the prolongation of muscle relaxation appeared to correlate with the levels of phospholamban expressed in two transgenic mouse lines. These findings indicate that ectopic expression of phospholamban in fast-twitch skeletal muscle is associated with inhibition of SERCA1 activity and decreased relaxation rates of this muscle.

Developmental regulation of Zbu1, a DNA-binding member of the SWI2/SNF2 family.
Gong, X., Kaushal, S., Ceccarelli, E., Bogdanova, N., Neville, C., Nguyen, T., Clark, H., Khatib, Z.A., Valentine, M., Look, A.T. & Rosenthal, N.
Dev Biol. 1997 Mar 15;183(2):166-82.

The SWI2/SNF2 gene family has been implicated in a wide variety of processes, involving regulation of DNA structure and chromatin configuration, mitotic chromosome segregation, and DNA repair. Here we report the characterization of the Zbu1 gene, also known as HIP116, located on human chromosome band 3q25, which encodes a DNA-binding member of this superfamily. Zbu1 was isolated in this study by its affinity for a site in the myosin light chain 1/3 enhancer. The protein has single-stranded DNA-dependent ATPase activity, includes seven helicase motifs, and a RING finger motif that is shared exclusively by the RAD5, spRAD8, and RAD16 family members. During mouse embryogenesis, Zbu1 transcripts are detected relatively late in fetal development and increase in neonatal stages, whereas the protein accumulates asynchronously in heart, skeletal muscle, and brain. In adult human tissues, alternatively spliced Zbu1 transcripts are ubiquitous with highest expression in these tissues. Gene expression is also dramatically induced in human tumor lines and in Li-Fraumeni fibroblast cultures, suggesting that it is aberrantly regulated in malignant cells. The developmental profile of Zbu1 gene expression and the association of the protein with a tissue-specific transcriptional regulatory element distinguish it from other members of the SWI2/SNF2 family and suggest novel roles for the Zbu1 gene product.

Skeletal muscle cultures.
Neville, C., Rosenthal, N., McGrew, M., Bogdanova, N. & Hauschka, S.
Methods Cell Biol. 1997;52:85-116.

DNA transfection of cultured muscle cells.
Neville, C., Rosenthal, N. & Hauschka, S.
Methods Cell Biol. 1997;52:405-22.

Proliferation precedes differentiation in IGF-I-stimulated myogenesis.
Engert, J.C., Berglund, E.B. & Rosenthal, N.
J Cell Biol. 1996 Oct;135(2):431-40.

The insulin-like growth factors (IGFs) have dramatic and complex effects on the growth of many tissues and have been implicated in both the proliferation and differentiation of skeletal muscle cells. A detailed analysis of gene expression was performed in L6E9 myoblast cultures treated with IGF-I to dissect the early events leading to the stimulation of myogenic differentiation by this growth factor. A time course of transcript accumulation in confluent L6E9 myoblasts treated with defined media containing IGF-I revealed an initial transient decrease in myogenic factors, accompanied by an increase in cell cycle markers and cell proliferation. This pattern was reversed at later time points, when the subsequent activation of myogenic factors resulted in a net increase in structural gene expression and larger myotubes. The data presented here support the hypothesis that IGF-I activates proliferation first, and subsequently stimulates events leading to the expression of muscle-specific genes in myogenic cell cultures.

Distinct gene expression patterns in skeletal and cardiac muscle are dependent on common regulatory sequences in the MLC1/3 locus.
McGrew, M.J., Bogdanova, N., Hasegawa, K., Hughes, S.H., Kitsis, R.N. & Rosenthal, N.
Mol Cell Biol. 1996 Aug;16(8):4524-34.

The myosin light-chain 1/3 locus (MLC1/3) is regulated by two promoters and a downstream enhancer element which produce two protein isoforms in fast skeletal muscle at distinct stages of mouse embryogenesis. We have analyzed the expression of transcripts from the internal MLC3 promoter and determined that it is also expressed in the atria of the heart. Expression from the MLC3 promoter in these striated muscle lineages is differentially regulated during development. In transgenic mice, the MLC3 promoter is responsible for cardiac-specific reporter gene expression while the downstream enhancer augments expression in skeletal muscle. Examination of the methylation status of endogenous and transgenic promoter and enhancer elements indicates that the internal promoter is not regulated in a manner similar to that of the MLC1 promoter or the downstream enhancer. A GATA protein consensus sequence in the proximal MLC3 promoter but not the MLC1 promoter binds with high affinity to GATA-4, a cardiac muscle- and gut-specific transcription factor. Mutation of either the MEF2 or GATA motifs in the MLC3 promoter attenuates its activity in both heart and skeletal muscles, demonstrating that MLC3 expression in these two diverse muscle types is dependent on common regulatory elements.

Modular elements of the MLC 1f/3f locus confer fiber-specific transcription regulation in transgenic mice.
Neville, C., Gonzales, D., Houghton, L., McGrew, M.J. & Rosenthal, N.
Dev Genet. 1996;19(2):157-62.

The two proteins encoded by the fast alkali myosin light chain (MLC) 1f/3f locus are developmentally regulated, muscle specific, and expressed exclusively in fast-twitch fibers. Their expression is independently regulated by two separate promoters and a downstream enhancer. Previous studies showed a reporter gene directed by the rat MLC If promoter and MLC enhancer to exhibit correct skeletal muscle-specific expression in transgenic mice during development and to be preferentially expressed in fast-twitch Type IIB fibers [Donoghue et al., (1991) J. Cell B.ol. 115:423-434]. The MLC 3f promoter also directed muscle-specific expression of a CAT reporter gene in adult transgenic mice and showed little dependence upon the enhancer. Here, we show that the MLC 3f promoter also directs transgene expression in the fast-twitch fibers of adult skeletal muscle, but almost exclusively to fiber Types IIA and IIX. MLC 3f transgene expression occurs in only a subset of the fiber types that express the endogenous locus, indicating modular elements included in the transgene confer fiber-specific transcription regulation. MyoD protein was also found to be restricted to fiber Types IIA and IIX, providing evidence for its possible role in mediating fiber-specific gene expression.

Molecular control of muscle diversity and plasticity.
Buonanno, A. & Rosenthal, N.
Dev Genet. 1996;19(2):95-107.