CRISPR-mediated Bmpr2 point mutation exacerbates late pulmonary vasculopathy and reduces survival in rats with experimental pulmonary hypertension

<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Patients with pulmonary arterial hypertension (PAH) carrying bone morphogenetic protein receptor type 2 (<jats:italic>Bmpr2</jats:italic>) mutations present earlier with severe hemodynamic compromise and have poorer survival outcomes than those without mutation. The mechanism underlying the worsening clinical phenotype of PAH with <jats:italic>Bmpr2</jats:italic> mutations has been largely unaddressed in rat models of pulmonary hypertension (PH) because of the difficulty in reproducing progressive PH in mice and genetic modification in rats. We tested whether a clinically-relevant <jats:italic>Bmpr2</jats:italic> mutation affects the progressive features of monocrotaline (MCT) induced-PH in rats.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>A monoallelic single nucleotide insertion in exon 1 of <jats:italic>Bmpr2</jats:italic> (+/44insG) was generated in rats using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9, then PH, pulmonary vascular disease (PVD) and survival after MCT injection with or without a phosphodiesterase type 5 inhibitor, tadalafil, administration were assessed.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>The +/44insG rats had reduced BMPR2 signalling in the lungs compared with wild-type. PH and PVD assessed at 3-weeks after MCT injection were similar in wild-type and +/44insG rats. However, survival at 4-weeks after MCT injection was significantly reduced in +/44insG rats. Among the rats surviving at 4-weeks after MCT administration, +/44insG rats had increased weight ratio of right ventricle to left ventricle plus septum (RV/[LV + S]) and % medial wall thickness (MWT) in pulmonary arteries (PAs). Immunohistochemical analysis showed increased vessels with Ki67-positive cells in the lungs, decreased mature and increased immature smooth muscle cell phenotype markers in the PAs in +/44insG rats compared with wild-type at 3-weeks after MCT injection. Contraction of PA in response to prostaglandin-F2α and endothelin-1 were significantly reduced in the +/44insG rats. The +/44insG rats that had received tadalafil had a worse survival with a significant increase in RV/(LV + S), %MWT in distal PAs and RV myocardial fibrosis compared with wild-type.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>The present study demonstrates that the <jats:italic>Bmpr2</jats:italic> mutation promotes dedifferentiation of PA smooth muscle cells, late PVD and RV myocardial fibrosis and adversely impacts both the natural and post-treatment courses of MCT-PH in rats with significant effects only in the late stages and warrants preclinical studies using this new genetic model to optimize treatment outcomes of heritable PAH.</jats:p> </jats:sec>