Introduction & Hypothesis: The molecular mechanisms underlying right ventricle (RV) hypertrophic remodeling (both adaptive and maladaptive) in response to sustained pressure overload, such as that experienced during pulmonary hypertension (PH), are inadequately understood. Our previous work demonstrating a role for the cytosolic organizer protein p47phox in pressure overload-induced RV hypertrophy and our recent publication on a direct interaction between p47phox and ERM-binding cytoskeletal protein Nherf1 lead us to hypothesize that Nherf1 is a regulator of RV cardiomyocyte hypertrophy.
Methods & Results: Cardiomyocyte-derived H9C2 cells and RV rat neonatal cardiomyocytes isolated from 1-day-old pup hearts (RV-RNCM) were subjected to neurohormonal hypertrophic stimulation using angiotensin II (AngII, 1 & 10 μM). RV pressure overload was induced in mice by pulmonary artery banding (PAB; 3wk). AngII treatment resulted in H9C2 and RV-RNCM hypertrophy and induced Nherf1 protein expression. The AngII-induced hypertrophy was attenuated by Nherf1 gene knockdown using siRNA. Moreover, Co-IP studies revealed an Nherf1-p47phox association in H9C2 cells that is enhanced by pro-hypertrophic AngII-treatment. Sequential loss of function strategies educated by novel in silico gene network analyses of PH gene intersected with cardiomyopathy gene networks, combined with a careful consideration of our previous findings and the literature, identified potential involvement of Hippo pathway transcriptional co-activators Yap/Taz, water channel aquaporin1 (Aqp1), and apoptosis signal-regulating kinase 1 (Ask1) in this pathway. In vitro, co-IP studies revealed a Nherf1-Yap association and interruption of Ask1 or Aqp1 reduced AngII-induced cardiomyocyte cellular hypertrophy. In vivo, PAB induced an increase in Nherf1 expression and association with p47phox.
Conclusion: The present study supports a new role for Nherf1 in mediating pro-hypertrophic cardiomyocyte cellular responses and implicates a molecular network previously not connected to either the RV or Nherf1. These data identify potential therapeutic targets for RV dysfunction in PH and shed light on novel molecular mechanisms so far untested in the pressure overloaded right heart.