Single nucleus transcriptomics supports a role for CCNA2-induced human adult cardiomyocyte cytokinesis.

Cyclin A2 (CCNA2) is a master regulatory gene of the cell cycle that is normally silenced in postnatal mammalian cardiomyocytes. We have previously demonstrated that it can induce significant cardiac repair in both small and large animals when delivered to the heart via a viral vector. To date, whether CCNA2 gene delivery can induce cytokinesis in isolated cardiomyocytes from adult human hearts has not been investigated. Therefore, we designed a human gene therapy vector featuring a replication-deficient, E1/E3-deleted human adenovirus five encoding human CCNA2 driven by the cardiac Troponin T promoter to enable the expression of CCNA2 in freshly isolated human cardiomyocytes. Utilizing time-lapse microscopy live imaging of cultured adult human cardiomyocytes isolated from a 21-year-old male, 41-year-old female, and 55-year-old male, we now report that human adult cardiomyocytes can be induced to undergo complete cytokinesis in response to CCNA2 gene delivery with preservation of sarcomere integrity in the resulting daughter cells. To elucidate the mechanistic underpinnings of CCNA2-dependent gene regulation in governing cardiomyocyte cytokinesis, we conducted single nucleus transcriptomics (snRNA-seq, 10X Genomics) analysis in hearts isolated from adult transgenic mice that constitutively express CCNA2 in cardiomyocytes (CCNA2-Tg) and non-transgenic mice (nTg). Remarkably, we identified a subpopulation of cardiomyocytes enriched with cytokinesis, proliferative, and reprogramming genes in hearts obtained from CCNA2-Tg mice as compared to hearts obtained from nTg mice. We also performed bulk RNA sequencing of human adult and fetal hearts, and we identified key reprogramming genes that are involved in CCNA2-induced cytokinesis. These results provide a compelling path forward for the clinical development of cardiac regenerative therapy based on strategic manipulation of the cardiomyocyte cell cycle.

EV Cargo Sorting in Therapeutic Development for Cardiovascular Disease.

Cardiovascular disease remains the leading cause of morbidity and mortality in the world. Thus, therapeutic interventions to circumvent this growing burden are of utmost importance. Extracellular vesicles (EVs) actively secreted by most living cells, play a key role in paracrine and endocrine intercellular communication via exchange of biological molecules. As the content of secreted EVs reflect the physiology and pathology of the cell of their origin, EVs play a significant role in cellular homeostasis, disease pathogenesis and diagnostics. Moreover, EVs are gaining popularity in clinics as therapeutic and drug delivery vehicles, transferring bioactive molecules such as proteins, genes, miRNAs and other therapeutic agents to target cells to treat diseases and deter disease progression. Despite our limited but growing knowledge of EV biology, it is imperative to understand the complex mechanisms of EV cargo sorting in pursuit of designing next generation EV-based therapeutic delivery systems. In this review, we highlight the mechanisms of EV cargo sorting and methods of EV bioengineering and discuss engineered EVs as a potential therapeutic delivery system to treat cardiovascular disease.

Intracellular IRF5 Dimerization Assay.

The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5 dimers, THP-1 cells are incubated in the presence or absence of the IRF5-targeted CPP (IRF5-CPP) inhibitor for 30 min then the cells are stimulated with R848 for 1 h. Cell lysates are separated by native-polyacrylamide gel electrophoresis (PAGE) and IRF5 dimers are detected by immunoblotting with IRF5 antibodies. To detect endogenous interactions between IRF5 and FITC-labeled IRF5-CPP, an in-cell fluorescence resonance energy transfer (FRET) assay is used. In this assay, THP-1 cells are left untreated or treated with FITC-IRF5-CPP conjugated inhibitors for 1 h. Next, cells are fixed, permeabilized, and stained with anti-IRF5 and TRITC-conjugated secondary antibodies. Transfer of fluorescence can be measured and calculated as FRET units. These methods provide rapid and accurate assays to detect IRF5 molecular interactions.