Elisabeth Ehler

Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is ∼800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3–like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH2-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca2+/calmodulin, as well as G protein–coupled signal transduction in the sarcomere.

Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2

During sarcomere contraction skeletal and cardiac muscle cells consume large amounts of energy. To satisfy this demand, metabolic enzymes are associated with distinct regions of the sarcomeres in the I-band and in the M-band, where they help to maintain high local concentrations of ATP. To date, the mechanism by which metabolic enzymes are coupled to the sarcomere has not been elucidated. Here, we show that the four and a half LIM-only protein DRAL/FHL-2 mediates targeting of the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase by interaction with the elastic filament protein titin in cardiomyocytes. Using yeast two-hybrid assays, colocalisation experiments, co-immunoprecipitation and protein pull-down assays, we show that DRAL/FHL-2 is bound to two distinct sites on titin. One binding site is situated in the N2B region, a cardiac-specific insertion in the I-band part of titin, and the other is located in the is2 region of M-band titin. We also show that DRAL/FHL-2 binds to the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase and might target these enzymes to the N2B and is2 regions in titin. We propose that DRAL/FHL-2 acts as a specific adaptor protein to couple metabolic enzymes to sites of high energy consumption in the cardiac sarcomere.

WNT-3, expressed by motoneurons, regulates terminal arborization of neurotrophin-3-responsive spinal sensory neurons

Sensory axons from dorsal root ganglia neurons are guided to spinal targets by molecules differentially expressed along the dorso-ventral axis of the neural tube. NT-3-responsive muscle afferents project ventrally, cease extending, and branch upon contact with motoneurons (MNs), their synaptic partners. We have identified WNT-3 as a candidate molecule that regulates this process. Wnt-3 is expressed by MNs of the lateral motor column at the time when MNs form synapses with sensory neurons. WNT-3 increases branching and growth cone size while inhibiting axonal extension in NT-3- but not NGF-responsive axons. Ventral spinal cord secretes factors with axonal remodeling activity for NT-3-responsive neurons. This activity is present at limb levels and is blocked by a WNT antagonist. We propose that WNT-3, expressed by MNs, acts as a retrograde signal that controls terminal arborization of muscle afferents.

Dilated cardiomyopathy: a disease of the intercalated disc?

The contractile tissue of the heart is composed of individual cells, making specific cell-cell contacts necessary to ensure mechanical and electrochemical coupling during beating. These contact sites, termed the intercalated discs, have gained increased attention recently due to their potential involvement in cardiac disease. This article discusses how the intercalated discs are assembled during heart development and how they are affected in cardiomyopathy, with particular emphasis on dilated cardiomyopathy. A model is proposed to relate the alterations that are seen at a molecular level with changes in function observed in that kind of cardiac disease.

Design of artificial myocardial microtissues

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1). a linear cell number-microtissue size correlation, (2). intermicrotissue superstructures, (3). retention of key cardiomyocyte-specific cell qualities, (4). a sophisticated extracellular matrix, and (5). a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6). myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.

Estrogen receptor alpha up-regulation and redistribution in human heart failure

Clinical and animal studies suggest that estrogen receptors are involved in the development of myocardial hypertrophy and heart failure. In this study, we investigated whether human myocardial estrogen receptor alpha (ERα) expression, localization, and association with structural proteins was altered in end stage‐failing hearts. We found a 1.8‐fold increase in ERα mRNA and protein in end‐stage human dilated cardiomyopathy (DCM, n=41), as compared with controls (n=25). ERα was visualized by confocal immunofluorescence microscopy and localized to the cytoplasm, sarcolemma, intercalated discs and nuclei of cardiomyocytes. Immunofluorescence studies demonstrated colocalization of ERα with β‐catenin at the intercalated disc in control hearts and immunoprecipitation studies confirmed complex formation of both proteins. Interestingly, the ERα/β‐catenin colocalization was lost at the intercalated disc in DCM hearts. Thus, the ERα/β‐catenin colocalization in the intercalated disc may be of functional relevance and a loss of this association may play a role in the progression of heart failure. The increase of total ERα expression may represent a compensatory process to contribute to the stability of cardiac intercalated discs.—Mahmoodzadeh, S., Eder, S., Nordmeyer, J., Ehler, E., Huber, O., Martus, P., Weiske, J., Pregla, R., Hetzer, R., Regitz‐Zagrosek, V. Estrogen receptor alpha up‐regulation and redistribution in human heart failure. FASEB J. 20, 926–934 (2006)

Sequential myofibrillar breakdown accompanies mitotic division of mammalian cardiomyocytes

The contractile tissue of the heart is composed of individual cardiomyocytes. During mammalian embryonic development, heart growth is achieved by cell division while at the same time the heart is already exerting its essential pumping activity. There is still some debate whether the proliferative activity is carried out by a less differentiated, stem cell-like type of cardiomyocytes or whether embryonic cardiomyocytes are able to perform both of these completely different dynamic tasks, contraction and cell division. Our analysis of triple-stained specimen of cultured embryonic cardiomyocytes and of whole mount preparations of embryonic mouse hearts by confocal microscopy revealed that differentiated cardiomyocytes are indeed able to proliferate. However, to go through cell division, a disassembly of the contractile elements, the myofibrils, has to take place. This disassembly occurs in two steps with Z-disk and thin (actin)-filament-associated proteins getting disassembled before disassembly of the M-bands and the thick (myosin) filaments happens. After cytokinesis reassembly of the myofibrillar proteins to their mature cross-striated pattern can be seen. Another interesting observation was that the cell-cell contacts remain seemingly intact during division, probably reflecting the requirement of intact integration sites of the individual cells in the contractile tissue. Our results suggest that embryonic cardiomyocytes have developed an interesting strategy to deal with their major cytoskeletal elements, the myofibrils, during mitosis. The complex disassembly-reassembly process might also provide a mechanistic explanation, why cardiomyocytes cede to divide postnatally.

Expression ofTiam-1in the Developing Brain Suggests a Role for the Tiam-1–Rac Signaling Pathway in Cell Migration and Neurite Outgrowth

Abstract During development proper neuronal migration and neurite extension are essential for the formation of functional neuronal networks. These processes require the reorganization of the cytoskeleton by modifying the dynamics of actin filaments and microtubules. The Rho subfamily of GTPases regulates actin cytoskeletal changes during development. Tiam-1, a GDP–GTP exchange factor for the small GTPase Rac and implicated in tumor invasion and metastasis, is expressed in the developing CNS. To study the function of Tiam-1 in neuronal migration and neurite extension, we examined the pattern ofTiam-1expression inweavermice, in which cerebellar granule cells fail to migrate to their final position and subsequently die.Tiam-1is expressed in wild-type granule cells as they migrate to the internal granular layer and send axons. In contrast,weaverhomozygous animals do not expressTiam-1in premigratory granule cells. Heterozygous animals, in which granule cells exhibit a slow rate of migration, express low levels ofTiam-1.In the cerebral cortex,Tiam-1is also expressed in migrating neurons. Our findings suggest that Tiam-1 contributes to cytoskeletal reorganization required during cell migration and neurite extension in defined neuronal populations, presumably by activation of Rac.

Thymosin β4 facilitates epicardial neovascularization of the injured adult heart

Ischemic heart disease complicated by coronary artery occlusion causes myocardial infarction (MI), which is the major cause of morbidity and mortality in humans (http://www.who.int/cardiovascular_diseases/resources/atlas/en/index.html). After MI the human heart has an impaired capacity to regenerate and, despite the high prevalence of cardiovascular disease worldwide, there is currently only limited insight into how to stimulate repair of the injured adult heart from its component parts. Efficient cardiac regeneration requires the replacement of lost cardiomyocytes, formation of new coronary blood vessels, and appropriate modulation of inflammation to prevent maladaptive remodeling, fibrosis/scarring, and consequent cardiac dysfunction. Here we show that thymosin β4 (Tβ4) promotes new vasculature in both the intact and injured mammalian heart. We demonstrate that limited EPDC‐derived endothelial‐restricted neovascularization constitutes suboptimal “endogenous repair,” following injury, which is significantly augmented by Tβ4 to increase and stabilize the vascular plexus via collateral vessel growth. As such, we identify Tβ4 as a facilitator of cardiac neovascularization and highlight adult EPDCs as resident progenitors which, when instructed by Tβ4, have the capacity to sustain the myocardium after ischemic damage.

Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a frequent genetic cardiac disease and the most common cause of sudden cardiac death in young individuals. Most of the currently known HCM disease genes encode sarcomeric proteins. Previous studies have shown an association between CSRP3 missense mutations and either dilated cardiomyopathy (DCM) or HCM, but all these studies were unable to provide comprehensive genetic evidence for a causative role of CSRP3 mutations. We used linkage analysis and identified a CSRP3 missense mutation in a large German family affected by HCM. We confirmed CSRP3 as an HCM disease gene. Furthermore, CSRP3 missense mutations segregating with HCM were identified in four other families. We used a newly designed monoclonal antibody to show that muscle LIM protein (MLP), the protein encoded by CSRP3, is mainly a cytosolic component of cardiomyocytes and not tightly anchored to sarcomeric structures. Our functional data from both in vitro and in vivo analyses suggest that at least one of MLPs mutated forms seems to be destabilized in the heart of HCM patients harbouring a CSRP3 missense mutation. We also present evidence for mild skeletal muscle disease in affected persons. Our results support the view that HCM is not exclusively a sarcomeric disease and also suggest that impaired mechano-sensory stress signalling might be involved in the pathogenesis of HCM.