Alexander Heinick

Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO-1)

To elucidate the function of Omega class glutathione transferases (GSTs) (EC 2.5.1.18) in multicellular organisms, the GSTO‐1 from Caenorhabditis elegans (GSTO‐1; C29E4.7) was investigated. Disc diffusion assays using Escherichia coli overexpressing GSTO‐1 provided a test of resistance to long‐term exposure under oxidative stress. After affinity purification, the recombinant GSTO‐1 had minimal catalytic activity toward classic GST substrates but displayed significant thiol oxidoreductase and dehydroascorbate reductase activity. Microinjection of the GSTO‐1‐promoter green fluorescent protein construct and immunolocalization by electron microscopy localized the protein exclusively in the intestine of all postembryonic stages of C. elegans. Deletion analysis identified an ~300‐nucleotide sequence upstream of the ATG start site necessary for GSTO‐1 expression. Site‐specific mutagenesis of a GATA transcription factor binding motif in the minimal promoter led to the loss of reporter expression. Similarly, RNA interference (RNAi) of Elt‐2 indicated the involvement of this gut‐specific transcription factor in GSTO‐1 expression. Transcriptional up‐regulation under stress conditions of GSTO‐1 was confirmed by analyzing promoter‐reporter constructs in transgenic C. elegans strains. To investigate the function of GSTO‐1 in vivo, transgenic animals overexpressing GSTO‐1 were generated exhibiting an increased resistance to juglone‐, paraquat‐, and cumene hydroperoxide‐induced oxidative stress. Specific silencing of the GSTO‐1 by RNAi created worms with an increased sensitivity to several prooxidants, arsenite, and heat shock. We conclude that the stress‐responsive GSTO‐1 plays a key role in counteracting environmental stress.—Burmeister, C., Lüersen, K., Heinick, A., Hussein, A., Domagalski, M., Walter, R. D., Liebau, E. Oxidative stress in Caenorhabditis elegans: protective effects of the Omega class glutathione transferase (GSTO‐1). FASEB J. 22, 343–354 (2008)

The MAP kinase JNK‐1 of Caenorhabditis elegans: Location, activation, and influences over temperature‐dependent insulin‐like signaling, stress responses, and fitness

The mitogen‐activated protein kinase (MAPK) pathways and insulin‐like signaling play pivotal roles in cellular stress response. Using an anti‐phospho‐SAPK/JNK antibody and a daf‐16::GFP‐based reporter assay, the present study shows in Caenorhabditis elegans that ambient temperature (1–37°C) specifically influences the activation (phosphorylation) of the MAP kinase JNK‐1 as well as the nuclear translocation of DAF‐16, the main downstream target of insulin‐like signaling. Activated JNK‐1 was detected only in neuronal cells, and JNK‐1 was found to be controlled by the MAPK JKK‐1 under heat stress. Comparative analyses on the wildtype and a jnk‐1 deletion mutant revealed a promoting influence of JNK‐1 on both nuclear DAF‐16 translocations and DAF‐16 target gene (superoxide dismutase 3, sod‐3) expressions within peripheral, non‐neuronal tissue. Consequently, the mutant exhibited a reduced thermal tolerance and reproductive fitness at higher temperatures. These results provide evidence of indirect interactions between neuronal MAPK and peripheral insulin‐like signaling in response to environmental stimuli (temperature, H2O2). J. Cell. Physiol. 214: 721–729, 2008.

Universal cardiac induction of human pluripotent stem cells in two and three-dimensional formats: implications for in vitro maturation.

Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large‐scale production of cardiomyocytes (CMs) for translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high‐efficiency procedure for generating CMs both in two‐dimensional (2D) and three‐dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost‐efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin‐free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time‐course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture‐induced maturation of the resulting CMs. This suggested that hPSC‐CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC‐CMs to functional readouts and thus define the cornerstones of culture‐induced maturation. Stem Cells 2015;33:1456–1469

Caenorhabditis elegans P5B-type ATPase CATP-5 operates in polyamine transport and is crucial for norspermidine-mediated suppression of RNA interference

Physiological polyamines are required in various biological processes. In the current study, we used norspermidine, a structural analog of the natural polyamine spermidine, to investigate polyamine uptake in the model organism Caenorhabditis elegans. Norsper‐midine was found to have two remarkable effects: it is toxic for the nematode, without affecting its food, Escherichia coli; and it hampers RNA interference. By characterizing a norspermidine‐resistant C. elegans mutant strain that has been isolated in a genetic screen, we demonstrate that both effects, as well as the uptake of a fluorescent polyamine‐conjugate, depend on the transporter protein CATP‐5, a novel P5B‐type ATPase. To our knowledge, CATP‐5 represents the first P5‐type ATPase that is associated with the plasma membrane, being expressed in the apical membrane of intestinal cells and the excretory cell. Moreover, genetic interaction studies using C. elegans polyamine synthesis mutants indicate that CATP‐5 has a function redundant to polyamine synthesis and link reduced polyamine levels to retarded postembryonic development, reduced brood size, shortened life span, and small body size. We suggest that CATP‐5 represents a crucial component of the pharmacologically important polyamine transport system, the molecular nature of which has not been identified so far in metazoa.—Heinick, A., Urban, K., Roth, S., Spies, D., Nunes, F., Phanstiel IV, O., Liebau, E., Lüersen, K. Caenorhabditis elegans P5B‐type ATPase CATP‐5 operates in polyamine transport and is crucial for norspermidine‐mediated suppression of RNA interference. FASEB J. 24, 206–217 (2010). www.fasebj.org

Tropisetron suppresses collagen synthesis in skin fibroblasts via α7 nicotinic acetylcholine receptor and attenuates fibrosis in a scleroderma mouse model

OBJECTIVE There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT(3/4) receptor-modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo. METHODS Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca(2+) measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunohistochemical analysis. RESULTS Tropisetron suppressed collagen synthesis induced by transforming growth factor β1 (TGFβ1). This effect was independent of 5-HT(3/4) receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFβ1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin. CONCLUSION Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma.

Protein phosphatase 2A is regulated by protein kinase Cα (PKCα)-dependent phosphorylation of its targeting subunit B56α at Ser41.

Background: PP2A activity and intracellular targeting are regulated by post-translational modifications of B56 phosphoprotein subunits. Results: PP2A is inhibited by a PKCα-dependent phosphorylation of B56α at Ser41 leading to downstream functional effects. Conclusion: This inhibition may represent an important signaling pathway regulated by stimuli that activate PKCα. Significance: Our data focus B56α on a dynamic role in the interplay between protein kinases and PP2A. Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser41 of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca2+ release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser41. This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca2+ homeostasis.

Reproductive fitness and dietary choice behavior of the genetic model organism Caenorhabditis elegans under semi-natural conditions

Laboratory breeding conditions of the model organism C. elegans do not correspond with the conditions in its natural soil habitat. To assess the consequences of the differences in environmental conditions, the effects of air composition, medium and bacterial food on reproductive fitness and/or dietary-choice behavior of C. elegans were investigated. The reproductive fitness of C. elegans was maximal under oxygen deficiency and not influenced by a high fractional share of carbon dioxide. In media approximating natural soil structure, reproductive fitness was much lower than in s tandard laboratory media. I n seminatural media, the reproductive fitness of C. elegans was low with the standard laboratory food bacterium E. coli (γ-Proteobacteria), but significantly higher with C. arvensicola (Bacteroidetes) and B. tropica (β-Proteobacteria) as food. Dietary-choice experiments in semi-natural media revealed a low preference of C. elegans for E. coli but significantly higher preferences for C. arvensicola and B. tropica (among other bacteria). Dietary-choice experiments under quasi-natural conditions, which were feasible by fluorescence in situ hybridization (FISH) of bacteria, showed a high preference of C. elegans for Cytophaga-Flexibacter-Bacteroides, Firmicutes, and β-Proteobacteria, but a low preference for γ-Proteobacteria. The results show that data on C. elegans under standard laboratory conditions have to be carefully interpreted with respect to their biological significance.

Universal Cardiac Induction of Human Pluripotent Stem Cells in 2D and 3D formats - Implications for In-Vitro Maturation

Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large‐scale production of cardiomyocytes (CMs) for translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high‐efficiency procedure for generating CMs both in two‐dimensional (2D) and three‐dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost‐efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin‐free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time‐course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture‐induced maturation of the resulting CMs. This suggested that hPSC‐CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC‐CMs to functional readouts and thus define the cornerstones of culture‐induced maturation. Stem Cells 2015;33:1456–1469

Cardiac expression of the CREM repressor isoform CREM-IbΔC-X in mice leads to arrhythmogenic alterations in ventricular cardiomyocytes

Chronic β-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to β-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the β-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca2+ releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca2+ waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca2+ transport and NCX1 protein level in TG. An increase in INCX and decrease in Ito and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.

Modulation of SR Ca2+ release by the triadin-to-calsequestrin ratio in ventricular myocytes

Calsequestrin (CSQ) is a Ca(2+) storage protein that interacts with triadin (TRN), the ryanodine receptor (RyR), and junctin (JUN) to form a macromolecular tetrameric Ca(2+) signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). Heart-specific overexpression of CSQ in transgenic mice (TG(CSQ)) was associated with heart failure, attenuation of SR Ca(2+) release, and downregulation of associated junctional SR proteins, e.g., TRN. Hence, we tested whether co-overexpression of CSQ and TRN in mouse hearts (TG(CxT)) could be beneficial for impaired intracellular Ca(2+) signaling and contractile function. Indeed, the depressed intracellular Ca(2+) concentration ([Ca](i)) peak amplitude in TG(CSQ) was normalized by co-overexpression in TG(CxT) myocytes. This effect was associated with changes in the expression of cardiac Ca(2+) regulatory proteins. For example, the protein level of the L-type Ca(2+) channel Ca(v)1.2 was higher in TG(CxT) compared with TG(CSQ). Sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) expression was reduced in TG(CxT) compared with TG(CSQ), whereas JUN expression and [(3)H]ryanodine binding were lower in both TG(CxT) and TG(CSQ) compared with wild-type hearts. As a result of these expressional changes, the SR Ca(2+) load was higher in both TG(CxT) and TG(CSQ) myocytes. In contrast to the improved cellular Ca(2+), transient co-overexpression of CSQ and TRN resulted in a reduced survival rate, an increased cardiac fibrosis, and a decreased basal contractility in catheterized mice, working heart preparations, and isolated myocytes. Echocardiographic and hemodynamic measurements revealed a depressed cardiac performance after isoproterenol application in TG(CxT) compared with TG(CSQ). Our results suggest that co-overexpression of CSQ and TRN led to a normalization of the SR Ca(2+) release compared with TG(CSQ) mice but a depressed contractile function and survival rate probably due to cardiac fibrosis, a lower SERCA2a expression, and a blunted response to β-adrenergic stimulation. Thus the TRN-to-CSQ ratio is a critical modulator of the SR Ca(2+) signaling.