魚崎 英毅

CRISPR/Cas9-mediated genome editing has expanded the possibilities for precise gene modifications; however, the efficiency of targeted insertion remains suboptimal. In this study, we describe a triple-reporter system in mouse embryonic stem cells that simultaneously tracks double-strand break (DSB) induction, homology-directed repair (knock-in), and end-joining-mediated targeted insertion (EJ-TI). Using both plasmid and adeno-associated virus (AAV) donor vectors, our results demonstrate that ataxia telangiectasia and Rad3-related kinase (ATR) activity is essential for knock-in regardless of the donor type, whereas ataxia telangiectasia mutated (ATM) inhibition exhibits a donor-dependent role. In cells receiving circular plasmid donors, ATM inhibition with AZD1390 markedly reduced the knock-in and EJ-TI efficiencies, consistent with its canonical role in DSB repair. In contrast, with linear AAV donors, ATM inhibition enhanced the knock-in efficiency by suppressing the overactivation of the ATM-p53-caspase 3 apoptotic pathway and partially suppressing classical non-homologous end-joining. These findings highlight the critical influence of donor DNA configuration on DNA damage response signaling and provide a strategy for optimizing genome editing efficiency by selectively modulating the ATM pathways, an approach that may have significant implications for gene therapy, cell engineering, and other applications.

UNLABELLED: Krüppel-like factor 5 (KLF5) is an intrinsically disordered transcription factor involved in cardiac remodeling, cancer, and metabolic diseases. Targeting KLF5 has been a persistent challenge in drug development due to its structural inaccessibility. We investigated cardioprotective effects of NC114, a rationally designed small molecule that mimics a short, hydrophobic α-helical motif in KLF5, thereby disrupting its protein–protein interactions. Adult C57BL/6J male mice underwent transverse aortic constriction (TAC) or sham surgery, followed by administration of NC114 or vehicle. NC114-treated TAC mice exhibited preserved cardiac function, reduced heart weight-to-body weight ratio, and markedly attenuated interstitial fibrosis. Gene expression analysis demonstrated decreased cardiac expression of Klf5, Nppb, Tgfb1, PAI-1, Col1a1, and Fn1. NC114 also suppressed oxidative stress and reduced phosphorylation of PKCδ and expression of HIF-1α during the early phase post-TAC. Metabolomic profiling revealed that NC114 treatment reversed TAC-induced accumulation of organic and amino acids. NC114, a novel peptidomimetic molecule, targets the undruggable transcription factor KLF5 to attenuate cardiac hypertrophy, fibrosis, and metabolic dysregulation in pressure overload-induced heart failure. This study highlights the potential of KLF5 inhibition as a therapeutic strategy in cardiovascular disease. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-32155-y.

Induced pluripotent stem cell-derived cardiomyocytes have shown promise to be an essential tool for studying genetic cardiac diseases. However, their limited maturity remains a barrier to reaching their full potential. Many have challenged this problem; however, it is difficult to compare the results because the parameters for cardiomyocyte maturation are diverse, mostly relying on physiological experiments that display significant lab-to-lab variations and are labor-intensive, and are not comparable to maturing cardiomyocytes in vivo. Here, we propose a transcriptome-based scoring method for cardiomyocyte maturation. We first established the maturation score based on transcriptome of mouse ventricles from embryonic (day 11) to adult (10-month-old) ventricles. We then demonstrated that known maturation conditions increased the maturation scores of mouse embryonic stem cell-derived cardiomyocytes. We finally performed expression screening of 92 candidate transcriptional factors (TFs) and identified pro-maturation TFs, including peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), PGC1β, and estrogen-related receptor alpha (ERRα). These results support that the transcriptome-based maturation score is a quantitative and reliable approach for identifying pro-maturation factors for cardiomyocytes.

BACKGROUND: Cardiomyocyte loss occurs in acute and chronic cardiac injury, including cardiotoxicity due to chemotherapeutics like doxorubicin, and contributes to heart failure development. There is a pressing need for cardiac-specific therapeutics that target cardiomyocyte loss, preventing chemotherapy complications without compromising chemotherapeutic efficacy. OBJECTIVES: The authors employed massively parallel combinatorial genetic screening to find microRNA (miRNA) combinations that promote cardiomyocyte survival. METHODS: CombiGEM (combinatorial genetics en masse) screening in a cardiomyocyte cell line was followed by validation in the original cell type and screening in primary cardiomyocytes. The top combination was tested in mouse and developing zebrafish models of doxorubicin cardiotoxicity. RNA sequencing provided insight into possible mechanisms. RESULTS: Multiple miRNA combinations protected cardiomyocytes from doxorubicin in vitro. The most effective (miR-222+miR-455) appeared to act synergistically, and mitigated doxorubicin cardiotoxicity phenotypes in murine and zebrafish in vivo models. RNA sequencing revealed overlapping and synergistic regulation of relevant genes and biological processes in cardiomyocytes, including mitochondrial homeostasis, oxidative stress, muscle contraction, and others. CONCLUSIONS: We identified miR-222 and miR-455 as a combination with potential therapeutic applications for cardioprotection. This study furthers our knowledge of the cardiac effects of miRNAs and their combinations and demonstrates the potential of CombiGEM for cardioprotective combinatorial therapeutic discovery.