Deficiency of pituitary adenylate cyclase-activating peptide and its receptor PAC1 confers interstitial cardiac fibrosis in a murine model of metabolic stress
Authors: Schubart J M , Schaefer M K H , Bonaterra G A , Mey L , Luesebrink U , Schieffer B , Eiden L E , Kinscherf R , Weihe E , Waechter C 
Published: October 2022

Abstract

Background
Fibrosis is a hallmark of cardiac remodeling and is present in a variety of cardiac diseases. In particular, in the highly prevalent heterogeneous group of cardiac dysfunctions with preserved left ventricular ejection fraction (LVEF), interstitial fibrosis has been identified as a pivotal pathophysiological factor. Current evidence suggests that comorbidities, mainly from the metabolic spectrum, promote chronic systemic low-grade inflammation with secondary affection of the heart, leading to fibrosis and thus impairing cardiac structure and function. In contrast to cardiac dysfunction with reduced LVEF, which is predominantly of ischemic origin, effective antifibrotic treatment options are very limited emphasizing the urgent need for new therapeutic targets. In this context, the recent report of an antifibrotic effect of the pleiotropic neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) in irradiation-induced myocardial damage [1] renders it an intriguing new candidate.

Purpose
The aim of this study is to elucidate the role of PACAP and of PAC1 receptor in cardiac fibrosis in a murine model of metabolic stress.

Methods
PACAP−/− and PAC1−/− C57/Bl6 J mice were crossbred with an ApoE−/− strain and metabolic stress was induced by feeding a cholesterol-enriched diet (CED) for 10 weeks. Standard chow (SD) served as control diet. Tissue samples of the ventricles were processed for FFPE histological analysis with hematoxylin/eosin, Giemsa and picrosirius red stains and subjected to morphometric quantification of cardiomyocyte cross diameters and fibrosis using a software-based image analysis approach.

Results
After 10 weeks of CED, a statistically significant higher extent of myocardial fibrosis was detected in PACAP−/−/ApoE−/− and PAC1−/−/ApoE−/− compared to ApoE−/− mice (Fig. 1). The pattern of fibrous deposition was consistent with that of reactive interstitial fibrosis and affected both ventricles uniformly. Morphometric analysis revealed no macroscopic or cellular hypertrophy in the PACAP−/−/ApoE−/− and PAC1−/−/ApoE−/− mice compared with ApoE−/− mice after CED. All morphological changes were exclusively present after feeding CED and were not detectable with SD feeding.

Conclusions
This study provides novel evidence for a significant involvement of PACAP and its receptor PAC1 in the development of metabolically induced cardiac fibrosis. The antifibrotic effect of PACAP, which can be inferred in this context, is mediated to a significant extent via the PAC1 receptor, and only becomes apparent under metabolic stress conditions. The absence of macroscopic and cellular cardiac hypertrophy in PACAP deficient or PAC1 deficient mice as demonstrated here precludes relevant hemodynamic effects of PACAP and PAC1 receptor, respectively. According to the results of our pilot study, PACAP analogues or PAC1 receptor agonists may offer a therapeutic potential to attenuate metabolically triggered cardiac fibrosis.

Funding Acknowledgement
Type of funding sources: Foundation. Main funding source(s): This research was partly supported by the Von Behring-Röntgen-Stiftung Schloss 1 D-35037 Marburg, Germany.