Cardiovascular Protective Effects of AT2R Activation by Peptide Drug NP‐6A4

Abstract

Introduction and ObjectiveLiterature shows that elevated Angiotensin II Type 2 receptor (AT2R) expression is cardiovascular reparative and mediates protection from hypertension, as well as vascular and renal injury. AT2R is a member of the anti‐inflammatory branch of the renin‐angiotensin system (RAS). Clinically, loss of AT2R expression in men due to the intronic G1675A or A1818T polymorphism is associated with impaired kidney function, pulse pressure and increased arterial stiffness. Given the pathological consequences of the loss of AT2R expression there is need for a drug that can increase AT2R gene (Agtr2) expression to mitigate cardiovascular damage. Currently there are no FDA‐approved drugs that meet these parameters. NP‐6A4 is a peptide agonist for AT2R, recently developed by Novopyxis Inc. We have shown that NP‐6A4 increases the viability of nutrient‐stressed mouse and human cardiovascular cells. Chemotherapeutic agents such as anthracyclins are inducers of cardiotoxicity. Therefore we investigated whether NP‐6A4 can protect cardiac cells from Doxorubicin (Dox; an anthracyclin) induced toxicity. To test the translational potential of a treatment paradigm that utilizes NP‐6A4 to improve cardiac functions and mitigate cardiac structural damage, we investigated the effects of NP‐6A4 treatment in Male Zucker Obese (ZO‐M) rats, a model for heart failure with preserved ejection fraction and cardiomyopathy.MethodsMTS assay (BioVision) was used to determine the effect of Dox treatment (0.5–2 μM) with and without co‐treatment with NP‐6A4 (1μM) on the survival of H9c2 cardiomyoblasts. To evaluate the effect of NP‐6A4 on cardiac structure and function, we treated 11‐week‐old ZO‐M rats that had fully developed cardiovascular disease with NP‐6A4 (1.8mg/kg/day by subcutaneous delivery). Echocardiography was used to assess cardiac function parameters. Cardiac architecture, hypertrophy, capillary density and fibrosis were assessed by staining using hematoxylin and eosin, Helix pomatia agglutinin (HPA), isolectin B4 (IB4) and Picrosirius Red (PSR) respectively. Expression levels of AT2R in the heart were assessed by quantitative RT‐PCR.Results24‐hour Dox treatment of H9c2 cardiomyoblasts suppressed cell viability (78% for 2μM vs untreated) and co‐treatment with NP‐6A4 restored cell survival. NP‐6A4 improved several cardiac parameters including endocardial circumferential strain (p≤0.05), myocardial performance index (MPI) (p≤0.005), and E/E' ratio (p≤0.002). NP‐6A4 reduced cardiomyocyte hypertrophy and fibrosis, and increased capillary density (p≤0.05). Importantly, cardiac Agtr2 (AT2R) mRNA levels were increased by about 9‐fold in NP‐6A4 treated rats vs. saline treated rats. Histopathology staining revealed regions of micro‐infarcts present in the saline group which were largely absent in the NP‐6A4 treated animals.ConclusionNP‐6A4 could mitigate Dox‐induced cardiac cell toxicity in vitro. NP‐6A4 also improved cardiac functions in a murine model of heart failure with preserved ejection fraction and cardiomyopathy, decreased risk of cardiac event (as measured by E/E'), mitigated myocardial structural damage, and improved cardiac vascularization. To our knowledge, NP‐6A4 is the first AT2R agonist that can increase cardiac AT2R expression. Based on these observations, we propose that this therapeutic candidate has the potential to treat chemotherapy‐induced cardiotoxicity as well as heart failure.Support or Funding InformationThis work was supported by NIH grant 1R01HL118376‐01 (LP), a small grant from Novopyxis Inc (LP), and Research Services facilities of HSTMVH.