Circular RNA circZFPM2 regulates cardiomyocyte hypertrophy and survival.

Hypertrophic cardiomyopathy (HCM) constitutes the most common genetic cardiac disorder. However, current pharmacotherapeutics are mainly symptomatic and only partially address underlying molecular mechanisms. Circular RNAs (circRNAs) are a recently discovered class of non-coding RNAs and emerged as specific and powerful regulators of cellular functions. By performing global circRNA-specific next generation sequencing in cardiac tissue of patients with hypertrophic cardiomyopathy compared to healthy donors, we identified circZFPM2 (hsa_circ_0003380). CircZFPM2, which derives from the ZFPM2 gene locus, is a highly conserved regulatory circRNA that is strongly induced in HCM tissue. In vitro loss-of-function experiments were performed in neonatal rat cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and HCM-patient-derived hiPSC-CMs. A knockdown of circZFPM2 was found to induce cardiomyocyte hypertrophy and compromise mitochondrial respiration, leading to an increased production of reactive oxygen species and apoptosis. In contrast, delivery of recombinant circZFPM2, packaged in lipid-nanoparticles or using AAV-based overexpression, rescued cardiomyocyte hypertrophic gene expression and promoted cell survival. Additionally, HCM-derived cardiac organoids exhibited improved contractility upon CM-specific overexpression of circZFPM2. Multi-Omics analysis further promoted our hypothesis, showing beneficial effects of circZFPM2 on cardiac contractility and mitochondrial function. Collectively, our data highlight that circZFPM2 serves as a promising target for the treatment of cardiac hypertrophy including HCM.

The long non-coding RNA NRON promotes the development of cardiac hypertrophy in the murine heart.

Physiological and pathological cardiovascular processes are tightly regulated by several cellular mechanisms. Non-coding RNAs, including long non-coding RNAs (lncRNAs), represent one important class of molecules involved in regulatory processes within the cell. The lncRNA non-coding repressor of NFAT (NRON) was described as a repressor of the nuclear factor of activated T cells (NFAT) in different in vitro studies. Although the calcineurin/NFAT-signaling pathway is one of the most important pathways in pathological cardiac hypertrophy, a potential regulation of hypertrophy by NRON in vivo has remained unclear. Applying subcellular fractionation and RNA fluorescence in situ hybridization (RNA-FISH), we found that, unlike what is known from T cells, in cardiomyocytes, NRON predominantly localizes to the nucleus. Hypertrophic stimulation in neonatal mouse cardiomyocytes led to a downregulation of NRON, while NRON overexpression led to an increase in expression of hypertrophic markers. To functionally investigate NRON in vivo, we used a mouse model of transverse aortic constriction (TAC)-induced hypertrophy and performed NRON gain- and loss-of-function experiments. Cardiomyocyte-specific NRON overexpression in vivo exacerbated TAC-induced hypertrophy, whereas cardiomyocyte-specific NRON deletion attenuated cardiac hypertrophy in mice. Heart weight, cardiomyocyte cell size, hypertrophic marker gene expression, and left ventricular mass showed a NRON-dependent regulation upon TAC-induced hypertrophy. In line with this, transcriptome profiling revealed an enrichment of anti-hypertrophic signaling pathways upon NRON-knockout during TAC-induced hypertrophy. This set of data refutes the hypothesized anti-hypertrophic role of NRON derived from in vitro studies in non-cardiac cells and suggests a novel regulatory function of NRON in the heart in vivo.

In vitro modelling of familial amyloidotic polyneuropathy allows quantitative detection of transthyretin amyloid fibril-like structures in hepatic derivatives of patient-specific induced pluripotent stem cells.

The transthyretin protein is thermodynamically destabilised by mutations in the transthyretin gene, promoting the formation of amyloid fibrils in various tissues. Consequently, impaired autonomic organ function is observed in patients suffering from transthyretin-related familial amyloidotic polyneuropathy (FAP). The influence of individual genetic backgrounds on fibril formation as a potential cause of genotype-phenotype variations needs to be investigated in order to ensure efficient patient-specific therapies. We reprogrammed FAP patient fibroblasts to induced pluripotent stem (iPS) cells and differentiated these cells into transthyretin-expressing hepatocyte-like cells (HLCs). HLCs differentiated from FAP iPS cells and healthy control iPS cells secreted the transthyretin protein in similar concentrations. Mass spectrometry revealed the presence of mutant transthyretin protein in FAP HLC supernatants. In comparison to healthy control iPS cells, we demonstrated the formation of transthyretin amyloid fibril-like structures in FAP HLC supernatants using the amyloid-specific dyes Congo red and thioflavin T. These dyes were also applicable for the quantitative determination of in vitro formed transthyretin fibril-like structures. Moreover, we confirmed the inhibition of fibril formation by the TTR kinetic stabiliser diclofenac. Thioflavin T fluorescence intensity measurements even allowed the quantification of amyloid fibril-like structures in 96-well plate formats as a prerequisite for patient-specific drug screening approaches.

Biphasic modulation of Wnt signaling supports efficient foregut endoderm formation from human pluripotent stem cells.

Pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are of great promise in regenerative medicine, including molecular studies of disease mechanisms, if the affected cell type can be authentically generated during in vitro differentiation. Most existing protocols aim to mimic embryonic development steps by the supplementation of specific cytokines and small molecules, but the involved signaling pathways need further exploration. In this study, we investigated enhanced initial activation of Wnt signaling for definitive endoderm formation and subsequent rapid shutdown of Wnt signaling for proper foregut endoderm specification using 3 µM CHIR99021 and 0.5 µg/mL of secreted frizzled-related protein 5 (sFRP-5) for biphasic modulation of the Wnt pathway. The definitive endoderm and foregut endoderm differentiation capabilities of Wnt pathway-modulated cells were determined based on the expression levels of the endodermal transcription factors SOX17 and FOXA2 and those of the transcription activator GATA4 and the α-fetoprotein (AFP) gene, respectively. Furthermore, the resulting biphasic Wnt pathway modulation was investigated at the protein level by analyzing phosphorylation of glycogen synthase kinase 3 beta (GSK3ß) and ß-catenin. Finally, Wnt target gene expression was determined using an improved lentiviral reporter construct that enabled robust T-cell transcription factor 4 (TCF4)/lymphoid enhancer-binding factor 1 (LEF1)-mediated luciferase expression in differentiating pluripotent stem cells. In conclusion, we demonstrated robust, homogeneous, and efficient derivation of foregut endodermal cells by inducing a biphasic modulation of the Wnt signaling pathway.

CRISPR/Cas9-based GLA knockout to generate the female Fabry disease human induced pluripotent stem cell line MHHi001-A-15.

The X-linked lysosomal storage disorder Fabry disease originates from GLA gene mutations causing α-galactosidase A enzyme deficiency. Here we generated the GLA knockout hiPSC line MHHi001-A-15 (GLA-KOhiPSC) as an in vitro Fabry disease model by targeting exon 2 of the GLA gene by CRISPR/Cas9 in the established control hiPSC line MHHi001-A. GLA-KOhiPSCs retained the expression of pluripotency markers, trilineage differentiation potential, as well as normal karyotype and stem cell morphology but lacked α-galactosidase A enzyme activity. The GLA-KOhiPSCs represent a potent resource to not only study the Fabry disease manifestation but also screen for novel treatment options.

Generation of human induced pluripotent stem cell line MHHi029-A from a male Fabry disease patient carrying c.959A > T mutation.

Fabry disease (FD) is a rare and inherited monogenetic disease caused by mutations in the X-chromosomal alpha-galactosidase A gene GLA concomitant with accumulation of its substrate globotriaosylceramide (Gb3) and multi-organ symptoms. We derived an induced pluripotent stem cell line, MHHi029-A, from a male FD patient carrying a c.959A > T missense mutation in the GLA gene. The hiPSCs show a normal karyotype, expression of pluripotency markers and trilineage differentiation capacity. Importantly, they present the patient-specific mutation in the GLA gene and are therefore a valuable resource for investigating the FD mechanism and identifying novel therapies.

The Long Non-coding RNA Cyrano Is Dispensable for Pluripotency of Murine and Human Pluripotent Stem Cells.

Pluripotency is tightly regulated and is crucial for stem cells and their implementation for regenerative medicine. Non-coding RNAs, especially long non-coding RNAs (lncRNAs) emerged as orchestrators of versatile (patho)-physiological processes on the transcriptional and post-transcriptional level. Cyrano, a well-conserved lncRNA, is highly expressed in stem cells suggesting an important role in pluripotency, which we aimed to investigate in loss-off-function (LOF) experiments. Cyrano was described previously to be essential for the maintenance of mouse embryonic stem cell (ESC) pluripotency. In contrast, using different genetic models, we here found Cyrano to be dispensable in murine and human iPSCs and in human ESCs. RNA sequencing revealed only a moderate influence of Cyrano on the global transcriptome. In line, Cyrano-depleted iPSCs retained the potential to differentiate into the three germ layers. In conclusion, different methods were applied for LOF studies to rule out potential off-target effects. These approaches revealed that Cyrano does not impact pluripotency.

Circulating microRNAs in Fabry Disease.

Fabry disease is an X-linked deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-Gal). This results in an accumulation of globotriaosylceramide (GL-3/Gb3) in a variety of cells with subsequent functional impairment. The continuous progress of FD often leads to decreased quality of life and premature death caused by multi-organic complications. The overall aim of our study was to determine the amount of circulating miRNAs in Fabry patients and to test whether ERT would alter the level of individual circulating miRNAs. We used miRNA sequencing by the HTG EdgeSeq System to identify the circulating miRNA pool from Fabry patients with and without enzyme replacement therapy (n = 6). In total, 296 miRNAs in serum of patients were identified. Among them 9 miRNAs were further evaluated in extra serum samples (n = 31) using real-time qPCR and 6 of them showed significant differential expression. The resulting miRNA pattern may help to better understand mechanisms involved in the beneficial effects of ERT and these new miRNA markers could help to estimate the efficacy of ERT or to identify Fabry patients with specific need for ERT.

MicroRNA-29 impairs the early phase of reprogramming process by targeting active DNA demethylation enzymes and Wnt signaling.

Somatic cell reprogramming by transcription factors and other modifiers such as microRNAs has opened broad avenues for the study of developmental processes, cell fate determination, and interplay of molecular mechanisms in signaling pathways. However, many of the mechanisms that drive nuclear reprogramming itself remain yet to be elucidated. Here, we analyzed the role of miR-29 during reprogramming in more detail. Therefore, we evaluated miR-29 expression during reprogramming of fibroblasts transduced with lentiviral OKS and OKSM vectors and we show that addition of c-MYC to the reprogramming factor cocktail decreases miR-29 expression levels. Moreover, we found that transfection of pre-miR-29a strongly decreased OKS-induced formation of GFP+-colonies in MEF-cells from Oct4-eGFP reporter mouse, whereas anti-miR-29a showed the opposite effect. Furthermore, we studied components of two pathways which are important for reprogramming and which involve miR-29 targets: active DNA-demethylation and Wnt-signaling. We show that inhibition of Tet1, Tet2 and Tet3 as well as activation of Wnt-signaling leads to decreased reprogramming efficiency. Moreover, transfection of pre-miR-29 resulted in elevated expression of ß-Catenin transcriptional target sFRP2 and increased TCF/LEF-promoter activity. Finally, we report that Gsk3-ß is a direct target of miR-29 in MEF-cells. Together, our findings contribute to the understanding of the molecular mechanisms by which miR-29 influences reprogramming.