Norbert Huebner

The complete mouse nebulin gene sequence and the identification of cardiac nebulin

Nebulin is a giant (M(r) 750-850kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin (thin) filaments in vertebrate skeletal muscles. Nebulins potential role as a molecular template is based on its structural and biochemical properties. Its central approximately 700kDa portion associates with actin along the entire length of the thin filament, its N-terminal region extends to thin filament pointed ends, and approximately 80kDa of its C-terminal region integrates within the Z-line lattice. Here, we determined the exon/intron organization of the entire mouse nebulin gene, which contains 165 exons in a 202kb segment. We identified 16 novel exons, 15 of which encode nebulin-repeat motifs (12 from its central region and 3 from its Z-line region). One novel exon shares high sequence homology to the 20 residue repeats of the tight-junction protein, ZO-1. RT-PCR analyses revealed that all 16 novel exons are expressed in mouse skeletal muscle. Surprisingly, we also amplified mRNA transcripts from mouse and human heart cDNA using primers designed along the entire length of nebulin. The expression of cardiac-specific nebulin transcripts was confirmed by in situ hybridization in fetal rat cardiomyocytes and in embryonic Xenopus laevis (frog) heart. On the protein level, antibodies specific for skeletal muscle nebulins N and C-terminal regions stained isolated rat cardiac myofibrils at the pointed and barbed ends of thin filaments, respectively. These data indicate a conserved molecular layout of the nebulin filament systems in both cardiac and skeletal myofibrils. We propose that thin filament length regulation in cardiac and skeletal muscles may share conserved nebulin-based mechanisms, and that nebulin isoform diversity may contribute to thin filament length differences in cardiac and skeletal muscle.

Human Engineered Heart Tissue: Analysis of Contractile Force

Summary Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered heart tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human heart tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling.

Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives

BackgroundMouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency.ResultsBy gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed.ConclusionWe have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.

Characterization of mouse embryonic stem cell differentiation into the pancreatic lineage in vitro by transcriptional profiling, quantitative RT-PCR and immunocytochemistry

We have previously shown that mouse embryonic stem (ES) cells differentiate into insulin-positive cells via multi-lineage progenitors. Here, we used Affymetrix chips and quantitative RT-PCR analysis to determine transcriptional profiles of undifferentiated wildtype (wt) and Pax4 expressing (Pax4+) ES cells and differentiated cells of committed progenitor and advanced stages. From undifferentiated to the committed stage, 237 (wt) and 263 (Pax4+) transcripts were 5- or more-fold up-regulated, whereas from the committed to the advanced stage, 28 (wt) and 5 (Pax4+) transcripts, respectively, were two- or more-fold up-regulated. Transcripts were classified into main subclasses including transcriptional regulation, signalling/growth factors, adhesion/extracellular matrix, membrane/transport, metabolism and organogenesis. Remarkably, endoderm-specific Sox17 and early pancreas-specific Isl1 transcripts were up-regulated at an earlier stage of multi-lineage progenitors, whereas highly up-regulated probe sets and transcripts of genes involved in endoderm, pancreatic, hepatic, angiogenic and neural differentiation were detected at the committed progenitor stage. Pax4+ cells showed specific differences in transcript up-regulation and a lower amount of up-regulated neural-specific transcripts in comparison to wt cells, but no enhanced gene expression complexity. Immunocytochemical analysis of selected proteins involved in endoderm and pancreatic differentiation, such as chromogranin B, transthyretin, Foxa1 and neuronatin revealed co-expression with insulin- or C-peptide-positive cells. The comparison of transcript profiles of ES cells differentiating in vitro with those of the embryonic and adult pancreas in vivo suggested that in vitro differentiated cells resemble an embryonal stage of development, supporting the view that ES-derived pancreatic cells are unable to complete pancreatic differentiation in vitro.

Characterization of the genomic structure and function of regions influencing renin and angiogenesis in the SS rat

Impaired regulation of renin in Dahl salt-sensitive rats (SS/JRHsdMcwi, SS) contributes to attenuated angiogenesis in this strain. This study examined angiogenic function and genomic structure of regions surrounding the renin gene using subcongenic strains of the SS and BN/NHsdMcwi (BN) rat to identify important genomic variations between SS and BN involved in angiogenesis. Three candidate regions on Chr 13 were studied: two congenic strains containing 0.89 and 2.62 Mb portions of BN Chr 13 that excluded the BN renin allele and a third strain that contained a 2.02 Mb overlapping region that included the BN renin allele. Angiogenesis induced by electrical stimulation of the tibialis anterior muscle was attenuated in the SS compared with the BN. Congenics carrying the SS renin allele had impaired angiogenesis, while strains carrying the BN renin allele had angiogenesis restored. The exception was a congenic including a region of BN genome 0.4 Mb distal to renin that restored both renin regulation and angiogenesis. This suggests that there is a distant regulatory element in the BN capable of restoring normal regulation of the SS renin allele. The importance of ANG II in the restored angiogenic response was demonstrated by blocking with losartan. Sequencing of the 4.05 Mb candidate region in SS and BN revealed a total of 8,850 SNPs and other sequence variants. An analysis of the genes and their variants in the region suggested a number of pathways that may explain the impaired regulation of renin and angiogenesis in the SS rat.

Increased Prevalence of Microangiopathic Brain Lesions among Siblings of Patients with Lacunar Stroke

Background: Family and twin studies suggest predisposing genetic factors in stroke. Lacunar infarcts represent a homogeneous phenotype, which is a prerequisite for genetic analyses. Applying an affected sib -pair analysis, we prospectively assessed the prevalence of microangiopathic brain lesions (MBL) and associated risk factors among siblings of patients with lacunar stroke. Methods: Index patients fulfilled clinical criteria of a lacunar stroke in combination with a corresponding MBL on CT or MRI. Siblings were characterized as affected if MBL demonstrated on MRI. The prevalence of MBL was compared with population prevalence data extracted from other studies. Results: From 784 patients screened, 81 index patients with lacunar stroke and 97 siblings were recruited, of which 42% were identified as affected. Compared with data from unselected historical controls and stratified by age groups, prevalence was between 2 and 5 times higher. Conclusions: Our results indicate that genetic stroke studies are feasible even in subtypes of ischemic stroke. The high prevalence of MBL among siblings of patients with lacunar infarct might suggest a familial aggregation. However, due to the small sample size these results need to be interpreted with caution and require confirmation by planned genetic analyses.

The role of NFκB in spheroid formation of human breast cancer cells cultured on the Random Positioning Machine

Human MCF-7 breast cancer cells were exposed to a Random Positioning Machine (RPM). After 24 hours (h) the cells grew either adherently within a monolayer (AD) or within multicellular spheroids (MCS). AD and MCS populations were separately harvested, their cellular differences were determined performing qPCR on genes, which were differently expressed in AD and MCS cells. Gene array technology was applied to detect RPM-sensitive genes in MCF-7 cells after 24 h. Furthermore, the capability to form multicellular spheroids in vitro was compared with the intracellular distribution of NF-kappaB (NFκB) p65. NFκB was equally distributed in static control cells, but predominantly localized in the cytoplasm in AD cells and nucleus in MCS cells exposed to the RPM. Gene array analyses revealed a more than 2-fold change of only 23 genes including some whose products are affected by oxygen levels or regulate glycolysis. Significant upregulations of the mRNAs of enzymes degrading heme, of ANXA1, ANXA2, CTGF, CAV2 and ICAM1, as well as of FAS, Casp8, BAX, p53, CYC1 and PARP1 were observed in MCS cells as compared with 1g-control and AD cells. An interaction analysis of 47 investigated genes suggested that HMOX-1 and NFκB variants are activated, when multicellular spheroids are formed.

Thyroid cancer cells in space during the TEXUS-53 sounding rocket mission – The THYROID Project

Human follicular thyroid cancer cells (FTC-133) were sent to space via a sounding rocket during the TEXUS-53 mission to determine the impact of short-term microgravity on these cells. To enable cell culture and fixation in real microgravity, an automated experiment container (EC) was constructed. In order to ensure safe cell culture, cell-chambers consisting of polycarbonate (PC) material were used. They were highly biocompatible as proved by measuring cell survival using Annexin V flow cytometry. In the follow-up experiment, FTC-133 cells were sent to space via a sounding rocket and were fixed before and after the microgravity (µg) phase with RNAlater. In addition, cells were tested for reactions on hypergravity (hyper-g) as much as 18 g to determine whether worst case acceleration during launch can have an influence on the cells. We investigated genes belonging to biological processes such as cytoskeleton, cell adhesion, tumor growth, angiogenesis and apoptosis. Pathway analyses revealed central functions of VEGFA and EGF. EGF upregulates aspartate beta-hydroxylase (ASPH) which is influencing CASP3. Hyper-g induced a significant up-regulation of TUBB1, VIM, RDX, CAV1, VEGFA and BCL2. FTC-133 cells grown in an automated EC exposed to µg revealed moderate gene expression changes indicating their survival in orbit.

Pathway Analysis Hints Towards Beneficial Effects of Long-Term Vibration on Human Chondrocytes

Background/Aims: Spaceflight negatively influences the function of cartilage tissue in vivo. In vitro human chondrocytes exhibit an altered gene expression of inflammation markers after a two-hour exposure to vibration. Little is known about the impact of long-term vibration on chondrocytes. Methods: Human cartilage cells were exposed for up to 24 h (VIB) on a specialised vibration platform (Vibraplex) simulating the vibration profile which occurs during parabolic flights and compared to static control conditions (CON). Afterwards, they were investigated by phase-contrast microscopy, rhodamine phalloidin staining, microarray analysis, qPCR and western blot analysis. Results: Morphological investigations revealed no changes between CON and VIB chondrocytes. F-Actin staining showed no alterations of the cytoskeleton in VIB compared with CON cells. DAPI and TUNEL staining did not identify apoptotic cells. ICAM-1 was elevated and vimentin, beta-tubulin and osteopontin proteins were significantly reduced in VIB compared to CON cells. qPCR of cytoskeletal genes, ITGB1, SOX3, SOX5, SOX9 did not reveal differential regulations. Microarray analysis detected 13 differentially expressed genes, mostly indicating unspecific stimulations. Pathway analyses demonstrated interactions of PSMD4 and CNOT7 with ICAM. Conclusions: Long-term vibration did not damage human chondrocytes in vitro. The reduction of osteopontin protein and the down-regulation of PSMD4 and TBX15 gene expression suggest that in vitro long-term vibration might even positively influence cultured chondrocytes.