Abstract Body (Do not enter title and authors here): Background: Myofibroblasts mediate tissue fibrosis and are a driver of disease progression in heart failure (HF). Reactive oxygen species (ROS) generated by NADPH oxidase-4 (NOX4) are required for transforming growth factor-beta (TGFb)-induced myofibroblast activation. Therapeutic targeting of NOX4 inhibited fibrosis development and reversed existing fibrosis in an aging model of lung fibrosis. However, the cell-specific roles of NOX4 in the heart have not been fully explored.
Objective: We sought to define the cardiac cell-type specific expression and role of NOX4 activation in the progression to HF.
Methods: Mice carrying a mutation in the phospholamban gene (PLNR9C) develop progressive dilated cardiomyopathy (DCM), fibrosis and HF. Cardiac cells were isolated using the Langendorff hanging heart method followed by flow cytometry. Single nucleus RNA-sequencing (snRNA-seq) was performed to define cell-specific gene expression. Immortalized mouse cardiac fibroblasts were treated with TGFb to establish longitudinal gene expression patterns with differentiation to myofibroblasts.
Results: Nox4 was upregulated in hearts (mRNA 8.5-fold; protein 3.1-fold; n=4, p<0.01) and isolated cardiac fibroblasts (mRNA 6.1-fold; protein 2.5-fold; n=3, p<0.01) from PLNR9C mice with HF but not age-matched wild type (WT) mice or young PLNR9C mice prior to the onset of DCM. Using snRNA-seq, we identified fibroblasts as the primary cell type expressing Nox4 in the heart in WT and PLNR9C mice; notably there was almost no expression of Nox4 in cardiomyocytes. This was consistent with published data in mouse heart analyzed from the NCBI GEO database. Fibroblasts from PLNR9C hearts with DCM had elevated ROS levels (Amplex Red assay 2.5-fold, p<0.01, n=3) and displayed a senescent phenotype, with elevated levels of the senescence markers p16 (2.1-fold, p<0.01, n=3) and b-galactosidase (1.7-fold, p<0.01, n=3), compared to fibroblasts from age-matched WT mice. In immortalized cardiac fibroblasts activated with TGFb, Nox4 increased 12 hours prior to expression of myofibroblast-specific genes (Postn, Col3a1). Co-treatment with siRNA targeting SMAD3 or the bromodomain and extraterminal (BET) inhibitor JQ1 blocked TGFb-induced Nox4 expression and fibroblast to myofibroblast differentiation.
Conclusions: These data identify NOX4 as a putative proximal mediator of cardiac fibroblast activation in mice with genetic DCM and suggest that cardiac fibroblasts develop a premature aging phenotype in HF.