Glutaminolysis and α-ketoglutarate-stimulated K3.1 expression contribute to β-adrenoceptor activation-induced myocardial fibrosis in mice.

Heart failure is associated with myocardial fibrosis, a pivotal histopathological feature arising from β-adrenergic receptor (β-AR) stimulation through sympathetic nervous system activation. Augmented glutaminolysis with increased bioavailability of α-ketoglutarate (α-KG) is suggested to contribute to fibrogenesis and changes in cellular gene expression. K3.1 is a calcium-activated potassium channel expressed in fibroblasts and has been implicated in mediating fibrosis, yet the putative interactions between glutaminolysis and K3.1 in β-AR-mediated cardiac fibrosis remain poorly understood. Here, we performed a series of in vitro and in vivo experiments to investigate how α-KG might influence the expression of K3.1 in the context of experimental myocardial fibrosis driven by β-AR activation. In cultured adult mouse cardiac fibroblasts, α-KG exposure resulted in the upregulation of K3.1 mRNA and protein levels that were commensurate with the dose and duration of exposure, and also led to increased K3.1 channel currents. Exposure to α-KG led to a significant decrease in levels of histone methylation (H3K27me3) within the K3.1 promoter, a decrease in the association of the transcription repressor REST from this site, as well as an enrichment of transcription activator AP-1 binding. The exacerbated fibrotic signaling induced by α-KG in cultured fibroblasts was suppressed by functional inhibition of K3.1 or by genetic knockdown of Kcnn4. Moreover, β-AR activation by isoproterenol significantly augmented glutaminolysis mediated by glutaminase 1 (GLS1) and significantly increased α-KG levels detected in the supernatant of cultured fibroblasts and cardiomyocytes. In addition, isoproterenol-induced K3.1 expression in fibroblasts was curtailed by treatment with the GLS1 inhibitor CB-839, or by GLS1 gene knockdown, or by treatment with the selective β-AR antagonist, ICI118551. In mouse models of established cardiac fibrosis evoked by isoproterenol-stimulation or β-AR overexpression, treatment with CB-839 for 4 weeks suppressed the phenotypic features of fibrosis, and this was associated with a decline in α-KG tissue content, a lack of histone demethylation at the K3.1 promoter, as well as suppression of K3.1 expression. Taken together, our study demonstrates for the first time that glutaminolysis contributes to β-AR activation-induced myocardial fibrosis via α-KG-stimulated K3.1 expression. We anticipate that treatments which target the β-AR/GLS1/α-KG/K3.1 signaling pathway might be effective for cardiac fibrosis.