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.

Encapsulating In Vitro Transcribed circRNA into Lipid Nanoparticles Via Microfluidic Mixing.

This chapter serves as a guide for researchers embarking on circular RNA-based translational studies. It provides a foundation for the successful encapsulation of circular RNA into lipid nanoparticles (LNPs) and facilitates progress in this emerging field. Crucial scientific methods and techniques involved in the formulation process, particle characterization, and downstream processing of circ-LNPs are covered. The production of in vitro transcribed circular RNA-containing LNPs based on a commercially available lipid mix is provided, in addition to the fundamentals for successful encapsulation based on lipid mixes composed of single components. Furthermore, the transfection and validation protocols for the identification of a functional and potentially therapeutic circRNA candidate for initial in vitro verification, before subsequent LNP studies, are explained.

A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity.

AIMS: Cardiotoxicity leading to heart failure (HF) is a growing problem in many cancer survivors. As specific treatment strategies are not available, RNA discovery pipelines were employed and a new and powerful circular RNA (circRNA)-based therapy was developed for the treatment of doxorubicin-induced HF. METHODS AND RESULTS: The circRNA sequencing was applied and the highly species-conserved circRNA insulin receptor (Circ-INSR) was identified, which participates in HF processes, including those provoked by cardiotoxic anti-cancer treatments. Chemotherapy-provoked cardiotoxicity leads to the down-regulation of Circ-INSR in rodents and patients, which mechanistically contributes to cardiomyocyte cell death, cardiac dysfunction, and mitochondrial damage. In contrast, Circ-INSR overexpression prevented doxorubicin-mediated cardiotoxicity in both rodent and human cardiomyocytes in vitro and in a mouse model of chronic doxorubicin cardiotoxicity. Breast cancer type 1 susceptibility protein (Brca1) was identified as a regulator of Circ-INSR expression. Detailed transcriptomic and proteomic analyses revealed that Circ-INSR regulates apoptotic and metabolic pathways in cardiomyocytes. Circ-INSR physically interacts with the single-stranded DNA-binding protein (SSBP1) mediating its cardioprotective effects under doxorubicin stress. Importantly, in vitro transcribed and circularized Circ-INSR mimics also protected against doxorubicin-induced cardiotoxicity. CONCLUSION: Circ-INSR is a highly conserved non-coding RNA which is down-regulated during cardiotoxicity and cardiac remodelling. Adeno-associated virus and circRNA mimics-based Circ-INSR overexpression prevent and reverse doxorubicin-mediated cardiomyocyte death and improve cardiac function. The results of this study highlight a novel and translationally important Circ-INSR-based therapeutic approach for doxorubicin-induced cardiac dysfunction.

Mouse Colonic Epithelial Cells Functionally Express the Histamine H4 Receptor.

We hypothesized that, in mice, histamine via the histamine receptor subtype 4 (H4R) on colon epithelial cells affects epithelial barrier integrity, perturbing physiologic function of the colonic mucosa and thus aggravating the severity of colitis. To test this hypothesis, bone marrow-chimeric mice were generated from H4R knockout (H4R-/-) and wild-type (WT) BALB/cJ mice and subjected to the dextrane sodium sulfate (DSS)-induced acute colitis model. Clinical symptoms and pathohistological derangements were scored. Additionally, total RNA was extracted from either mouse whole-colon homogenates or primary cell preparations enriched for epithelial cells, and gene expression was analyzed by real-time quantitative polymerase chain reaction. The impact of the H4R on epithelial barrier function was assessed by measurement of transepithelial electrical resistence of organoid-derived two-dimensional monolayers from H4R-/- and WT mice using chopstick electrodes. Bone marrow-chimeric mice with genetic depletion of the H4R in nonhematopoietic cells exhibited less severe DSS-induced acute colitis symptoms compared with WT mice, indicating a functional proinflammatory expression of H4R in nonimmune cells of the colon. Analysis of H4R expression revealed the presence of H4R mRNA in colon epithelial cells. This expression could be confirmed and complemented by functional analyses in organoid-derived epithelial cell monolayers. Thus, we conclude that the H4R is functionally expressed in mouse colon epithelial cells, potentially modulating mucosal barrier integrity and intestinal inflammatory reactions, as was demonstrated in the DSS-induced colitis model, in which presence of the H4R on nonhematopoietic cells aggravated the inflammatory phenotype. SIGNIFICANCE STATEMENT: The histamine H4 receptor (H4R) is functionally expressed on mouse colon epithelial cells, thereby aggravating dextrane sodium sulfate-induced colitis in BALB/cJ mice. Histamine via the H4R reduces transepithelial electrical resistance of colon epithelial monolayers, indicating a function of H4R in regulation of epithelial barrier integrity.