Pulmonary hypertension (PH) is a serious complication of left heart disease (LHD), characterized by increased pulmonary vascular pressures and resistance and extensive vascular remodeling. Recently, we demonstrated that upregulation of Runt-related transcription factor 2 (RUNX2) in pulmonary artery smooth muscle cells (PASMC) contributes to disease progression by promoting osteogenic transformation, calcification, and vascular stiffening. Accordingly, pharmacological inhibition of RUNX2 effectively reversed PH in preclinical models (Liu et al., 2024). Yet, the mechanisms driving increased RUNX2 expression in PH-LHD are poorly understood.
Here, we investigated potential regulatory mechanisms of RUNX2 at the transcriptional, post-transcriptional, and post-translational levels. Using bulk RNA sequencing of pulmonary artery (PA) samples from PH-LHD patients, we identified bone morphogenetic protein 2 (BMP2) as transcriptional regulator that was upregulated in PH-LHD. Treatment of PASMC with BMP2 increased RUNX2 levels as detected by Western blotting. By small RNA sequencing we identified microRNA-320 (miR-320) as post-transcriptional regulator of RUNX2 that targets 3' UTR at four predicted binding sites. miR-320 was downregulated in PAs of PH-LHD patients compared to LHD without PH. Inhibition of miR-320 increased RUNX2 expression in healthy PASMC, while miR-320 mimic decreased RUNX2 protein levels.
Finally, we explored post-translational modifications of RUNX2, focusing on acetylation and lactylation. Acetylation of nuclear RUNX2 was significantly increased in PH-LHD PAs and correlated with elevated pulmonary artery systolic pressure and transpulmonary pressure gradient. Although lactylation levels were not significantly elevated in PH-LHD, they correlated positively with acetylation, indicating potential synergistic effects on RUNX2 activity.
These findings uncover novel transcriptional, post-transcriptional, and post-translational regulators of RUNX2 in PH-LHD, thus providing new mechanistic insights and identifying therapeutic targets for further exploration.
This research was supported by the German Center for Cardiovascular Research (DZHK), the Federal Ministry of Education and Research (BMBF), the German Research Foundation (DFG; Project ID: 549161294), and the Collaborative Research Center 1470 (Project ID: 437531118, Sub-project A04).
