Matthias Linke

In vitro Modeling of Ryanodine Receptor 2 Dysfunction Using Human Induced Pluripotent Stem Cells

Background/Aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. Methods: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. Results: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca2+ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca2+-induced Ca2+-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. Conclusion: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies.

Sirtuin-6-dependent genetic and epigenetic alterations are associated with poor clinical outcome in hepatocellular carcinoma patients.

Sirtuin 6 (SIRT6) is a member of the sirtuin family of NAD+–dependent deacetylases. Genetic deletion of Sirt6 in mice results in a severe degenerative phenotype with impaired liver function and premature death. The role of SIRT6 in development and progression of hepatocellular carcinoma is currently unknown. We first investigated SIRT6 expression in 153 primary human liver cancers and in normal and cirrhotic livers using microarray analysis. SIRT6 was significantly down‐regulated in both cirrhotic livers and cancer. A Sirt6 knockout (KO) gene expression signature was generated from primary hepatoctyes isolated from 3‐week‐old Sirt6‐deficient animals. Sirt6‐deficient hepatocytes showed up‐regulation of established hepatocellular carcinoma (HCC) biomarkers alpha‐fetoprotein (Afp), insulin‐like growth factor 2 (Igf2), H19, and glypican‐3. Furthermore, decreased SIRT6 expression was observed in hepatoma cell lines that are known to be apoptosis‐insensitive. Re‐expression of SIRT6 in HepG2 cells increased apoptosis sensitivity to CD95‐stimulation or chemotherapy treatment. Loss of Sirt6 was characterized by oncogenic changes, such as global hypomethylation, as well as metabolic changes, such as hypoglycemia and increased fat deposition. The hepatocyte‐specific Sirt6‐KO signature had a prognostic impact and was enriched in patients with poorly differentiated tumors with high AFP levels as well as recurrent disease. Finally, we demonstrated that the Sirt6‐KO signature possessed a predictive value for tumors other than HCC (e.g., breast and lung cancer). Conclusion: Loss of SIRT6 induces epigenetic changes that may be relevant to chronic liver disease and HCC development. Down‐regulation of SIRT6 and genes dysregulated by loss of SIRT6 possess oncogenic effects in hepatocarcinogenesis. Our data demonstrate that deficiency in one epigenetic regulator predisposes a tumorigenic phenotype that ultimately has relevance for outcome of HCC and other cancer patients. (Hepatology 2013;53:1054–1064)

The disease-specific phenotype in cardiomyocytes derived from induced pluripotent stem cells of two long QT syndrome type 3 patients.

Long QT syndromes (LQTS) are heritable diseases characterized by prolongation of the QT interval on an electrocardiogram, which often leads to syncope and sudden cardiac death. Here we report the generation of induced pluripotent stems (iPS) cells from two patients with LQTS type 3 carrying a different point mutation in a sodium channel Nav1.5 (p.V240M and p.R535Q) and functional characterization of cardiomyocytes (CM) derived from them. The iPS cells exhibited all characteristic properties of pluripotent stem cells, maintained the disease-specific mutation and readily differentiated to CM. The duration of action potentials at 50% and 90% repolarization was longer in LQTS-3 CM as compared to control CM but this difference did not reach statistical significance due to high variations among cells. Sodium current recordings demonstrated longer time to peak and longer time to 90% of inactivation of the Na+ channel in the LQTS-3 CM. This hints at a defective Na+ channel caused by deficiency in open-state inactivation of the Na+ channel that is characteristic of LQTS-3. These analyses suggest that the effect of channel mutation in the diseased CM is demonstrated in vitro and that the iPS cell-derived CM can serve as a model system for studying the pathophysiology of LQTS-3, toxicity testing and design of novel therapeutics. However, further improvements in the model are still required to reduce cell-to-cell and cell line-to-cell line variability.

Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes.

To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.

Transcriptome Fingerprint of Bovine 2-Cell Stage Blastomeres Is Directly Correlated with the Individual Developmental Competence of the Corresponding Sister Blastomere

ABSTRACT To date, gene expression profiles of bovine preimplantation embryos have only been indirectly related to developmental potential due to the invasive nature of such procedures. This study sought to find a direct correlation between transcriptome fingerprint of blastomeres of bovine 2-cell stage embryos with developmental competence of the corresponding sister blastomeres. Isolated blastomeres were classified according to the sister blastomeres development into three groups: two groups displayed developmental incompetency, including those blastomeres whose corresponding sister blastomeres either stopped cleaving after separation (2CB) or were blocked after two additional cleavages before embryonic genome activation (8CB). In the third group were competent blastomeres, which were defined as those whose sister blastomeres developed to the blastocyst stage (BL). As a result, developmental capacity of corresponding sister blastomeres was highly similar. Microarray analysis revealed 77 genes to be commonly differentially regulated among competent and incompetent blastomeres as well as blocked blastomeres. Clustering of differentially expressed genes according to molecular functions and pathways revealed antioxidant activity, NRF2-mediated oxidative stress response, and oxidative phosphorylation to be the main ontologies affected. Expression levels of selected candidate genes were further characterized in an independent model for developmental competence based on the time of first cleavage postfertilization. Moreover, overall results of this study were confirmed by higher developmental rates and more beneficial expression of CAT and PRDX1 when cultured in an antioxidative environment. These results will help us to understand molecular mechanisms defining developmental destination of individual bovine preimplantation embryos.

Stochastic Loss of Silencing of the Imprinted Ndn/NDN Allele, in a Mouse Model and Humans with Prader-Willi Syndrome, Has Functional Consequences

Genomic imprinting is a process that causes genes to be expressed from one allele only according to parental origin, the other allele being silent. Diseases can arise when the normally active alleles are not expressed. In this context, low level of expression of the normally silent alleles has been considered as genetic noise although such expression has never been further studied. Prader-Willi Syndrome (PWS) is a neurodevelopmental disease involving imprinted genes, including NDN, which are only expressed from the paternally inherited allele, with the maternally inherited allele silent. We present the first in-depth study of the low expression of a normally silent imprinted allele, in pathological context. Using a variety of qualitative and quantitative approaches and comparing wild-type, heterozygous and homozygous mice deleted for Ndn, we show that, in absence of the paternal Ndn allele, the maternal Ndn allele is expressed at an extremely low level with a high degree of non-genetic heterogeneity. The level of this expression is sex-dependent and shows transgenerational epigenetic inheritance. In about 50% of mutant mice, this expression reduces birth lethality and severity of the breathing deficiency, correlated with a reduction in the loss of serotonergic neurons. In wild-type brains, the maternal Ndn allele is never expressed. However, using several mouse models, we reveal a competition between non-imprinted Ndn promoters which results in monoallelic (paternal or maternal) Ndn expression, suggesting that Ndn allelic exclusion occurs in the absence of imprinting regulation. Importantly, specific expression of the maternal NDN allele is also detected in post-mortem brain samples of PWS individuals. Our data reveal an unexpected epigenetic flexibility of PWS imprinted genes that could be exploited to reactivate the functional but dormant maternal alleles in PWS. Overall our results reveal high non-genetic heterogeneity between genetically identical individuals that might underlie the variability of the phenotype.

The impact of ovarian stimulation on the expression of candidate reprogramming genes in mouse preimplantation embryos.

Ovarian stimulation with gonadotrophins is an integral part of assisted reproductive technologies in human subfertility/infertility treatment. Recent findings have associated ovarian stimulation with the increased incidence of imprinting disorders in humans as well as defects in genome-wide methylation reprogramming and, in particular, imprinting in mice. Here, we present the first study that determined the impact of ovarian stimulation on the expression of developmentally important reprogramming genes (Apex1, Lig1, Lig3, Mbd2, Mbd3, Mbd4, and Polb) in single early mouse morula embryos (16-cell stage). Using absolute quantification of mRNA by quantitative real-time PCR, we observed an association of ovarian stimulation with a downregulation of mRNAs encoding the base excision repair proteins APEX1 and POLB as well as the 5-methyl-CpG-binding domain protein MBD3 in individual morula embryos. Whole mount immunofluorescence staining of early and late morula embryos with an antibody against APEX1 revealed individual embryos with lower protein expression levels after ovarian stimulation and a correlation of mRNA expression with protein abundance. Our data argue for a negative impact of ovarian stimulation during female gametogenesis and/or early embryo development affecting the expression of candidate reprogramming factors.

Early-life adversity selectively impairs α2-GABAA receptor expression in the mouse nucleus accumbens and influences the behavioral effects of cocaine

ABSTRACT Haplotypes of the Gabra2 gene encoding the &agr;2‐subunit of the GABAA receptor (GABAAR) are associated with drug abuse, suggesting that &agr;2‐GABAARs may play an important role in the circuitry underlying drug misuse. The genetic association of Gabra2 haplotypes with cocaine addiction appears to be evident primarily in individuals who had experienced childhood trauma. Given this association of childhood trauma, cocaine abuse and the Gabra2 haplotypes, we have explored in a mouse model of early life adversity (ELA) whether such events influence the behavioral effects of cocaine and if, as suggested by the human studies, &agr;2‐GABAARs in the nucleus accumbens (NAc) are involved in these perturbed behaviors. In adult mice prior ELA caused a selective decrease of accumbal &agr;2‐subunit mRNA, resulting in a selective decrease in the number and size of the &agr;2‐subunit (but not the &agr;1‐subunit) immunoreactive clusters in NAc core medium spiny neurons (MSNs). Functionally, in adult MSNs ELA decreased the amplitude and frequency of GABAAR‐mediated miniature inhibitory postsynaptic currents (mIPSCs), a profile similar to that of &agr;2 “knock‐out” (&agr;2−/−) mice. Behaviourally, adult male ELA and &agr;2−/− mice exhibited an enhanced locomotor response to acute cocaine and blunted sensitisation upon repeated cocaine administration, when compared to their appropriate controls. Collectively, these findings reveal a neurobiological mechanism which may relate to the clinical observation that early trauma increases the risk for substance abuse disorder (SAD) in individuals harbouring haplotypic variations in the Gabra2 gene. HighlightsEarly‐life adversity (ELA) is a risk factor for adult psychopathology and drug abuse.Gabra2 gene variations coupled with childhood trauma associate with cocaine abuse.ELA selectively impaired accumbal &agr;2‐GABAAR expression and function in adult mice.ELA and &agr;2−/− mice displayed similar abnormal behavioral responses to cocaine.The findings complement clinical associations of ELA, Gabra2 gene and drug abuse.