Unlocking Signaling Mechanisms That Underlie Persistent Anti‐Fibrotic Effects of Transient ACE Inhibition

Abstract

Fibrosis plays a major role in the progression of heart failure. Angiotensin II (Ang II) is a potent inducer of cardiac remodeling involving hypertrophy and fibrosis, which may be mediated in part through increases in reactive oxygen species (ROS). There is currently no drug treatment that can reverse fibrosis, however angiotensin converting enzyme inhibitors (ACEI) are able to slow the progression of fibrosis. Moreover, we have shown that protection against fibrotic remodeling persists even after ACEI treatment has been stopped. The mechanism underlying this long‐term protection has not yet been elucidated. In this study, we aimed to determine whether the beneficial effects of prior ACEI treatment are related to a more favorable oxidative stress profile in response to Ang II treatment. We hypothesize that hearts from hypertensive rats previously treated with ACEI will show decreased pro‐oxidant and increased antioxidant enzymes in response to Ang II. Adult (11‐week‐old) male and female spontaneously hypertensive rats were treated with vehicle or ACEI (enalapril, 30mg/kg/day) for two weeks, followed by a two‐week washout period. A subset of vehicle treated rats and all of the ACEI treated rats were then administered Ang II (200ng/kg/min) for 2 or 4 weeks (n=3/group per sex). Protein levels of pro‐oxidant (NOX2 and NOX4) and antioxidant (SOD1, SOD2, catalase) enzymes in the left ventricle (LV) were measured by western blotting. Ang II treatment caused a significant increase in LV hypertrophy after 4 weeks of Ang II. Overall, pro‐oxidant NOX4 levels were higher, while NOX2 levels were lower, in females compared to males regardless of treatment (p<0.05). This suggests alternative sources of ROS are implicated in the male vs. female hypertensive hearts. Additionally, Ang II treatment significantly increased NOX4 expression in males but not females (p<0.05) highlighting a sex‐specific responsiveness to Ang II. Antioxidant protein catalase expression is lower in females compared to males (p<0.05), while both sexes exhibited significant increases in response to Ang II (p<0.05). Ang II significantly decreased SOD1 expression in males with or without ACEI (p<0.05), while SOD2 tended to increase in females. ACEI treatment in males prevented Ang II‐induced effects in catalase and NOX2, but not other proteins, suggesting ACEI‐sensitive pathways. In summary, our findings reveal sex differences in both overall levels of NOX2, NOX4, and catalase, as well as responsiveness to Ang II and ACEI. Altered modulation of antioxidants may contribute to or compensate for sex‐ and Ang II‐dependent inflammation with hypertension. Determining whether oxidative stress is altered by prior ACEI treatment will allow for a better understanding of the mechanisms, and therefore therapeutic targets for slowing or preventing cardiac remodeling and heart failure. The unexpected finding of sex differences in oxidative protein profiles builds on a growing body of evidence that sex‐specific targeted therapies may be necessary for cardiovascular disease.Support or Funding InformationAmerican Physiological Society UGSRF program; NHLBI R56HL141165 (TMH); AHA 19AIREA34460000 (TMH); AHA 19POST34410055 (AMG)