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.

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.

A selectable all-in-one CRISPR prime editing piggyBac transposon allows for highly efficient gene editing in human cell lines.

CRISPR prime-editors are emergent tools for genome editing and offer a versatile alternative approach to HDR-based genome engineering or DNA base-editors. However, sufficient prime-editor expression levels and availability of optimized transfection protocols may affect editing efficiencies, especially in hard-to-transfect cells like hiPSC. Here, we show that piggyBac prime-editing (PB-PE) allows for sustained expression of prime-editors. We demonstrate proof-of-concept for PB-PE in a newly designed lentiviral traffic light reporter, which allows for estimation of gene correction and defective editing resulting in indels, based on expression of two different fluorophores. PB-PE can prime-edit more than 50% of hiPSC cells after antibiotic selection. We also show that improper design of pegRNA cannot simply be overcome by extended expression, but PB-PE allows for estimation of effectiveness of selected pegRNAs after few days of cultivation time. Finally, we implemented PB-PE for efficient editing of an amyotrophic lateral sclerosis-associated mutation in the SOD1-gene of patient-derived hiPSC. Progress of genome editing can be monitored by Sanger-sequencing, whereas PB-PE vectors can be removed after editing and excised cells can be enriched by fialuridine selection. Together, we present an efficient prime-editing toolbox, which can be robustly used in a variety of cell lines even when non-optimized transfection-protocols are applied.

SARS-CoV-2 receptor ACE2-dependent implications on the cardiovascular system: From basic science to clinical implications.

The current COVID-19 pandemic started several months ago and is still exponentially growing in most parts of the world - this is the most recent and alarming update. COVID-19 requires the collaboration of nearly 200 countries to curb the spread of SARS-CoV-2 while gaining time to explore and improve treatment options especially for cardiovascular disease (CVD) and immunocompromised patients, who appear to be at high-risk to die from cardiopulmonary failure. Currently unanswered questions are why elderly people, particularly those with pre-existing comorbidities seem to exhibit higher mortality rates after SARS-CoV-2 infection and whether intensive care becomes indispensable for these patients to prevent multi-organ failure and sudden death. To face these challenges, we here summarize the molecular insights into viral infection mechanisms and implications for cardiovascular disease. Since the infection starts in the upper respiratory system, first flu-like symptoms develop that spread throughout the body. The wide range of affected organs is presumably based on the common expression of the major SARS-CoV-2 entry-receptor angiotensin-converting enzyme 2 (ACE2). Physiologically, ACE2 degrades angiotensin II, the master regulator of the renin-angiotensin-aldosterone system (RAAS), thereby converting it into vasodilatory molecules, which have well-documented cardio-protective effects. Thus, RAAS inhibitors, which may increase the expression levels of ACE2, are commonly used for the treatment of hypertension and CVD. This, and the fact that SARS-CoV-2 hijacks ACE2 for cell-entry, have spurred controversial discussions on the role of ACE2 in COVID-19 patients. In this review, we highlight the state-of-the-art knowledge on SARS-CoV-2-dependent mechanisms and the potential interaction with ACE2 expression and cell surface localization. We aim to provide a list of potential treatment options and a better understanding of why CVD is a high risk factor for COVID-19 susceptibility and further discuss the acute as well as long-term cardiac consequences.

Single-molecule imaging correlates decreasing nuclear volume with increasing TF-chromatin associations during zebrafish development.

Zygotic genome activation (ZGA), the onset of transcription after initial quiescence, is a major developmental step in many species, which occurs after ten cell divisions in zebrafish embryos. How transcription factor (TF)-chromatin interactions evolve during early development to support ZGA is largely unknown. We establish single molecule tracking in live developing zebrafish embryos using reflected light-sheet microscopy to visualize two fluorescently labeled TF species, mEos2-TBP and mEos2-Sox19b. We further develop a data acquisition and analysis scheme to extract quantitative information on binding kinetics and bound fractions during fast cell cycles. The chromatin-bound fraction of both TFs increases during early development, as expected from a physical model of TF-chromatin interactions including a decreasing nuclear volume and increasing DNA accessibility. For Sox19b, data suggests the increase is mainly due to the shrinking nucleus. Our single molecule approach provides quantitative insight into changes of TF-chromatin associations during the developmental period embracing ZGA.

Regulatory T Cells Know What Is Needed to Regenerate.

Adaptive immunity has been suggested to limit regeneration in mammals. However, in this issue of Developmental Cell, Hui et al. (2017) report that regulatory T cells are required for regeneration of heart, spinal cord, and retina in the zebrafish. Intriguingly, in each organ system, Treg cells secrete organ-specific regeneration factors.

Zebrafish fin and heart: what's special about regeneration?

Many organs regenerate well in adult zebrafish, but most research has been directed toward fin and heart regeneration. Cells have been found to remain generally lineage-restricted during regeneration, and proliferative regenerative progenitors can be formed by dedifferentiation from differentiated cells. Recent studies begin to shed light on the molecular underpinnings of differences between development and regeneration. Retinoic acid, BMP and NF-κB signaling are emerging as regulators of cellular dedifferentiation. Reactive oxygen species promote regeneration, and the dynamics of ROS signaling might help explain differences between wound healing and regeneration. Finally, the heart has been added to those organs that require a nerve supply to regenerate, and a trade-off between regeneration and tumor suppression has been proposed to help explain why mammals regenerate poorly.

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration.

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta-catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

FOXP1 potentiates Wnt/ß-catenin signaling in diffuse large B cell lymphoma.

The transcription factor FOXP1 (forkhead box protein P1) is a master regulator of stem and progenitor cell biology. In diffuse large B cell lymphoma (DLBCL), copy number amplifications and chromosomal translocations result in overexpression of FOXP1. Increased abundance of FOXP1 in DLBCL is a predictor of poor prognosis and resistance to therapy. We developed a genome-wide, mass spectrometry-coupled, gain-of-function genetic screen, which revealed that FOXP1 potentiates ß-catenin-dependent, Wnt-dependent gene expression. Gain- and loss-of-function studies in cell models and zebrafish confirmed that FOXP1 was a general and conserved enhancer of Wnt signaling. In a Wnt-dependent fashion, FOXP1 formed a complex with ß-catenin, TCF7L2 (transcription factor 7-like 2), and the acetyltransferase CBP [CREB (adenosine 3',5'-monophosphate response element-binding protein)-binding protein], and this complex bound the promoters of Wnt target genes. FOXP1 promoted the acetylation of ß-catenin by CBP, and acetylation was required for FOXP1-mediated potentiation of ß-catenin-dependent transcription. In DLBCL, we found that FOXP1 promoted sensitivity to Wnt pathway inhibitors, and knockdown of FOXP1 or blocking ß-catenin transcriptional activity slowed xenograft tumor growth. These data connect excessive FOXP1 with ß-catenin-dependent signal transduction and provide a molecular rationale for Wnt-directed therapy in DLBCL.