Nyarige, Verah

Introduction: Neonatal pig hearts exhibit remarkable regenerative capacity within two days post-birth. In this study, we propose that a comprehensive multiomics analysis encompassing circRNA, miRNA, and mRNA data from neonatal mouse hearts can unveil intricate circRNA-miRNA-mRNA relationships associated with cardiac regeneration. Our aim is to identify new novel circRNAs and miRNAs implicated in cardiomyocyte proliferation through leveraging multiomics analysis. Methods: Utilizing neonatal mouse hearts, we conducted circRNA, miRNA, and mRNA sequencing across four different postnatal days (P1, P3, P7, and P28; n=6, encompassing both males and females). We employed integrative bioinformatics analyses across distinct experimental designs (P1 vs P3, P1 vs P7, and P1 vs P28). Back splice junctions generated by CIRCexplorer2’s parse command was utilized to annotate sequenced circRNAs as known or novel. Validation of circRNA, miRNA, and mRNA expression levels was achieved through qRT-PCR, while protein expression levels were assessed via western blotting. Fluorescence in situ hybridization (FISH) technology was employed to determine circRNA localization. The functional significance of key genes involved in cardiomyocyte proliferation was assessed via siRNA-mediated knockdown of circRNAs and miRNAs, accompanied by cell cycle assessment using bioluminescence assay and immunostaining. Results: Intersection of differentially expressed target genes from circRNA, miRNA, and mRNA analyses at each experimental design facilitated the construction of circRNA-miRNA-mRNA regulatory networks, aiding in the development of cardiac regeneration-related regulatory networks and inference of novel relationships between circRNA, miRNA, and mRNA. Integrative bioinformatics analysis revealed significant alterations in circRNA-miRNA-mRNA expression profiles during postnatal cardiomyocyte maturation in mice. Pathway enrichment analysis identified several differentially expressed circRNAs enriched in cell division and proliferation-related pathways. qPCR and cell cycle studies identified 17 potential circRNAs associated with cardiomyocyte proliferation in mice, with knockdown of these circRNAs significantly impacting cardiomyocyte cell cycle. Conclusion: Our findings suggest a crucial role for circRNAs in the regulation of neonatal myocardial regeneration.

BACKGROUND: Apelin is an endogenous prepropeptide that regulates cardiac homeostasis and various physiological processes. Intravenous injection has been shown to improve cardiac contractility in patients with heart failure. However, its short half-life prevents studying its impact on left ventricular remodeling in the long term. Here, we aim to study whether microparticle-mediated slow release of apelin improves heart function and left ventricular remodeling in mice with myocardial infarction (MI). METHODS: A cardiac patch was fabricated by embedding apelin-containing microparticles in a fibrin gel scaffold. MI was induced via permanent ligation of the left anterior descending coronary artery in adult C57BL/6J mice followed by epicardial patch placement immediately after (acute MI) or 28 days (chronic MI) post-MI. Four groups were included in this study, namely sham, MI, MI plus empty microparticle-embedded patch treatment, and MI plus apelin-containing microparticle-embedded patch treatment. Cardiac function was assessed by transthoracic echocardiography. Cardiomyocyte morphology, apoptosis, and cardiac fibrosis were evaluated by histology. Cardioprotective pathways were determined by RNA sequencing, quantitative polymerase chain reaction, and Western blot. RESULTS: The level of endogenous apelin was largely reduced in the first 7 days after MI induction and it was normalized by day 28. Apelin-13 encapsulated in poly(lactic-co-glycolic acid) microparticles displayed a sustained release pattern for up to 28 days. Treatment with apelin-containing microparticle-embedded patch inhibited cardiac hypertrophy and reduced scar size in both acute and chronic MI models, which is associated with improved cardiac function. Data from cellular and molecular analyses showed that apelin inhibits the activation and proliferation of cardiac fibroblasts by preventing transforming growth factor-β–mediated activation of Smad2/3 (supporessor of mothers against decapentaplegic 2/3) and downstream profibrotic gene expression. CONCLUSIONS: Poly(lactic-co-glycolic acid) microparticles prolonged the apelin release time in the mouse hearts. Epicardial delivery of the apelin-containing microparticle-embedded patch protects mice from both acute and chronic MI-induced cardiac dysfunction, inhibits cardiac fibrosis, and improves left ventricular remodeling.

Little is known about the expression patterns and functions of circular RNAs (circRNAs) in the heart of large mammals. In this study, we examined the expression profiles of circRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in neonatal pig hearts. Pig heart samples collected on postnatal days 1 (P1), 3 (P3), 7 (P7), and 28 (P28) were sent for total RNA sequencing. Our data revealed a total of 7,000 circRNAs in the 24 pig hearts. Pathway enrichment analysis of hallmark gene sets demonstrated that differentially expressed circRNAs were engaged in different pathways. The most significant difference was observed between P1 and the other 3 groups (P3, P7, and P28) in pathways related to cell cycle and muscle development. Out of the 10 circRNAs that were validated through real-time quantitative PCR to verify their expression, 6 exhibited significant effects on cell cycle activity in human induced pluripotent stem cell–derived cardiomyocytes following small interfering RNA–mediated knockdown. circRNA-miRNA-mRNA networks were constructed to understand the potential mechanisms of circRNAs in the heart. In conclusion, our study provided a data set for exploring the roles of circRNAs in pig hearts. In addition, we identified several circRNAs that regulate cardiomyocyte cell cycle.

Abstract TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a co-inhibitory receptor, highly expressed by Tregs, and contributes to the suppressive function of Tregs by limiting pro-inflammatory Th1 and Th17 cell responses. Herein, we report that subsets of human regulatory B cells (Bregs) express surface TIGIT and can thus play a critical role in immune regulation. Using data from human B cell surface arrays and RNA-seq, we identified two major TIGIT+ human Bregs subsets, exhibiting CD24hiCD27+CD39hiIgD+IgMhiCD1chi marginal zone (MZ)-like and CD24hiCD27+CD39+IgD−IgM+CD1c+ memory Bregs. We further demonstrated that TIGIT+ Bregs were also capable of suppressing immune responses by their ability to express not only IL-10 and PD-L1, but also granzyme B and TGFb1. In addition, TIGIT+ Bregs can also suppress immune responses by altering dendritic cell functions. In line with these observations, liver allograft recipients with donor-specific alloantibody (DSA) had significantly lower numbers of TIGIT+ Bregs than those without DSA. Moreover, the frequency of TIGIT+ Bregs correlated with that of CD4+CD25+CD127lo Tregs, while it inversely correlated with the frequency of follicular helper T cells. We thus concluded that TIGIT+ Bregs play critical roles in immune regulation in liver allograft recipients.