Asxl1 clonal hematopoiesis exacerbates heart failure via the NEAT1-inflammasome axis

Background Clonal hematopoiesis of indeterminate potential (CHIP) promotes inflammation and exacerbates various cardiovascular diseases, including non-ischemic heart failure. Although CHIP is recognized as a contributor to adverse cardiovascular outcomes, its molecular pathophysiology remains incompletely understood, limiting the development of effective therapeutic strategies to mitigate its pathogenic effects. Purpose The purpose of this study is to elucidate the mechanism by which CHIP exacerbates heart failure using single-nucleus RNA sequencing (snRNA-seq) of human heart tissue and a mouse model of DCM with or without CHIP. Methods We conducted snRNA-seq of human heart tissue samples including both CHIP positive and negative patients with DCM. The subset of myeloid cell populations was extracted and clustered based on gene expression. We generated a DCM mouse model by introducing a heterozygous Ttn F28051Ifs*15 mutation (Ttn-MT). Eight- to 10-week-old Ttn-MT mice were lethally irradiated (total dose: 9Gy), followed by bone marrow transplantation (BMT) from mice with a heterozygous Asxl1 G643Wfs*12 mutation (Asxl1-MT) or wild-type mice, to establish DCM with Asxl1-CHIP model. Ttn-MT mice with BMT from Asxl1-MT; Neat1 knockout mice were used to examine the role of Neat1 in Asxl1-CHIP-mediated exacerbation of heart failure. Eight weeks after BMT, transthoracic echocardiography was performed, and the mice were euthanized to obtain cardiac tissue for histopathological, mRNA, protein, and flow cytometric analyses. Bone marrow-derived macrophages (BMDMs) were used to validate findings from the mouse model. Results snRNA-seq of human DCM heart tissue revealed that various inflammatory signaling pathways were upregulated in cardiac myeloid cells of CHIP-positive DCM patients. We identified NEAT1, a long non-coding RNA, as a functional marker gene that defines the characteristics of myeloid cell populations from CHIP-positive DCM patients (Figure A). Consistent with the data from human studies, DCM with Asxl1-CHIP mice exhibited significant cardiac dysfunction and increased left ventricular fibrosis compared with controls (Figure B). Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of sorted cardiac macrophages revealed a significant increase in Il1b mRNA expression. RNA in situ hybridization and RT-qPCR showed increased expression of Neat1 in the DCM with Asxl1-CHIP model (Figure B). In vitro assays using BMDMs demonstrated that Asxl1 mutation induced up-regulation and cytoplasmic translocation of Neat1, and activated the NLRP3 inflammasome. Furthermore, BMT from Asxl1-MT; Neat1 knock-out mice rescued the cardiomyopathic phenotype in DCM with Asxl1-CHIP mice. (Figure C). Conclusion CHIP exacerbates heart failure via the Neat1-Inflammasome axis. Targeting Neat1 may emerge as a novel therapeutic strategy to mitigate CHIP-associated worsening of heart failure.