Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection has been associated with severe cardiovascular complications. However, the role of epitranscriptional modulation involved in SARS-CoV-2-infected myocarditis is still unclear. Ten-eleven translocation 2 (TET2), a methylcytosine dioxygenase, plays key roles in DNA demethylation during viral infection and host–virus interactions. Using human-induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) as a platform, our data revealed the epitranscriptomic role of TET2 during SARS-CoV-2 infection. First, our RNA sequencing analysis revealed the alterations of the messenger-RNA-expression profiles of epitranscriptomic regulators, including TET2, in hiPSC-CMs during SARS-CoV-2 infection. Second, silencing TET2 markedly reduced both the messenger RNA and protein levels of the viral nucleocapsid (N) protein, leading to attenuated viral replication in infected hiPSC-CMs. Furthermore, RNA dot-blotting analysis revealed that TET2 knockdown suppressed the levels of 5-hydroxymethylcytosine in SARS-CoV-2-infected hiPSC-CMs. To further explore the therapeutic relevance of TET2 inhibition in suppressing SARS-CoV-2 infection, we screened and compared 3 structurally distinct TET2 enzymatic inhibitors: Bobcat339, TETi76, and TFMB-2HG. Among these, Bobcat339 demonstrated the most potent antiviral effect, markedly suppressing SARS-CoV-2 replication and N-protein expression. Molecular docking analysis revealed that Bobcat339 exhibited a high binding affinity for multiple viral targets, including nsp16, RdRp, and N protein, indicating a multitarget mechanism of action. In addition, our data demonstrated that treatment with Bobcat339 can suppress SARS-CoV-2 infectious activity and N-protein expression in infected hiPSC-CMs. Together, our findings highlight the regulatory role of TET2 in SARS-CoV-2 infection and identify Bobcat339 as a promising therapeutic compound. Understanding TET2-driven epitranscriptomics and the functions of TET-targeting inhibitors may provide a novel strategy for mitigating viral infection in SARS-CoV-2-induced cardiomyopathy.

Background: Induced pluripotent stem cell (iPSC) technology has emerged as a powerful tool for disease modeling, providing an innovative platform for investigating disease mechanisms. iPSC-derived organoids, including retinal organoids, offer patient-specific models that closely replicate in vivo cellular environments, making them ideal for studying retinal neurodegenerative diseases where retinal ganglion cells (RGCs) are impacted. N6-methyladenosine (m6A), a prevalent internal modification in eukaryotic mRNAs, plays a critical role in RNA metabolic processes such as splicing, stability, translation, and transport. Given the high energy demands of RGCs, mitochondrial dysfunction, which leads to impaired adenosine triphosphate (ATP) production and increased reactive oxygen species (ROS) levels, is often central to the progression of retinal neurodegenerative disorders. However, the epigenetic mechanisms underlying m6A modification and their contributions to these conditions remain unclear. Methods: Patient-specific iPSCs were generated from individuals with Leber hereditary optic neuropathy (LHON) and differentiated into RGCs within retinal organoids. To analyze m6A methylation, we used quantitative polymerase chain reaction (PCR) and focused on differential expression of key m6A-modifying enzymes. Results: iPSC-derived retinal organoids are adaptable for studying and investigating the epigenetic mechanisms of retinal neurodegenerative diseases. Our data demonstrated the profiling of global m6A-related gene expression levels in LHON patient-derived iPSC-RGCs compared with controls, highlighting specific disruptions in m6A modification pathways. Conclusion: These findings suggest that differential m6A modifications may play pivotal roles in the pathogenesis of retinal neurodegenerative diseases and affect the progression of the disease in affected individuals.

Cystic fibrosis arises from loss-of-function mutations in the CFTR gene, disrupting epithelial ion homeostasis and impairing airway mucus clearance. While missense mutations typically lead to minor conformational alterations that can be rectified with pharmacological interventions, nonsense mutations pose a more significant therapeutic challenge. In this research, we established an in vitro model of cystic fibrosis (CF) employing patient-specific induced pluripotent stem cells (iPSCs) that contain the CFTR-S308X nonsense mutation. We generated CF patient-specific iPSC-derived 3D airway organoids (AOs) that exhibit phenotypic characteristics akin to those of CF, including airway epithelial cells, basal cells, and goblet cells. By employing a single-cell RNA sequencing (scRNA-seq) approach, we analyzed the cellular composition of CFTR-S308X-mutated CF-AOs to explore the potential molecular pathogenesis. Multimodal scRNA-seq analyses were conducted using CellChat to evaluate intercellular communication and RNA velocity to assess transcriptomic dynamics. The CellChat analysis indicated that the TGF-β and EGF signaling pathways may facilitate inflammatory interactions among goblet cells and other cellular subpopulations. The RNA velocity analysis suggested that TNF-α-mediated processes, including autophagy, renin secretion, and relaxin signaling, were upregulated. Deconvolution of bulk RNA sequencing data from patients with CF expressing nonsense mutations corroborated the persistent activation of the TGF-β, TNF-α, and EGF-driven inflammatory pathways in CFTR-S308X-AOs, particularly within the goblet cell subpopulation. Utilizing multimodal single-cell transcriptomics with the ASGARD framework, we found that nintedanib, N-acetylcysteine, and losartan were associated with attenuation of the TNF-α pathway in CF with nonsense mutations. This CF-3D airway organoid model recapitulates aspects of pathogenesis at the single-cell level and serves as a platform for evaluating therapeutics targeting CFTR nonsense mutations.

The growth hormone secretagogue receptor-1a (GHSR1a) is the endogenous receptor for ghrelin. Activation of GHSR1a participates in many physiological processes including energy homeostasis and eating behavior. Due to its transitory half-life, the efficacy of ghrelin treatment in patients is restricted; hence the development of new adjuvant therapy is an urgent need. This study aimed to establish a cell line stably expressing GHSR1a, which could be employed to screen potential ghrelin agonists from natural compounds. First, by means of lentiviral transduction, the genome of a human HEK293T cell was modified, and a cell platform stably overexpressing GHSR1a was successfully established. In this platform, GHSR1a was expressed as a fusion protein tagged with mCherry, which allowed the monitoring of the dynamic cellular distribution of GHSR1a by fluorescent microscopy. Subsequently, the authenticity of the GHSR1a mediated signaling was further characterized by using ghrelin and teaghrelin, two molecules known to stimulate GHSR1a. The results indicated that both ghrelin and teaghrelin readily activated GHSR1a mediated signaling pathways, presumably via increasing phosphorylation levels of ERK. The specific GHSR1a signaling was further validated by using SP-analog, an antagonist of GHSR1a as well as using a cell model with the knockdown expression of GHSR1a. Molecular modeling predicted that crocin might be a potential ghrelin agonist, and this prediction was further confirmed by the established platform.