Cardiac aging is regulated by autophagic disorder mediated by chronic inflammation via the Rubicon/NOX4 pathway
Authors: Kamihara T , Bando Y.K , Murohara T 
Published: November 2020

Abstract

Background and introduction
Aging is known to one of the primary causes of heart failure, in particular in the case of heart failure with preserved left-ventricular ejection fraction (HFpEF). Interestingly, recent evidences demonstrated that DNA damage occurred in aging causes autophagic disorder that contributes to aging phenotype via Rubicon (Run domain Beclin-1 interacting and cysteine-rich containing protein) in non-cardiac tissues.

Purpose
To elucidate whether aging-related autophagic disorder may lead to myocardial remodeling via chronic inflammation by Rubicon activation.

Methods
As an aging model with DNA injury, we employed mice model of progeria (mouse model of Werner syndrome that was generated by amino acid mutation exhibiting the functional deletion of DNA helixase; WRN-K577M).

Results
Cardiac aging markers (PARP-1, p53 and γ H2AX), and apoptosis (TUNEL) were augmented in WRN-K577M (male 18 week-old) as compared to the wild type counterpart. Consistently, cardiac oxidative stress that was measured by DHE was elevated in WRN-K577M with significant increase in oxidative stress enhancer NOX4. WRN-K577M exhibited cardiomegaly and diastolic left-ventricular (LV) dysfunction with preserved systolic LV function. Histological analysis revealed that WRN-K577M exhibited enhanced cardiac fibrosis and cardiomyocyte hypertrophy. Consistently, DNA microarray revealed significant upregulation of sixteen genes, of which ontology was hypertrophy, fibrosis, inflammation. Changes in autophagic activity was assessed by use of LC3 turnover assay and autophagic flux was evaluated by application of pharmacological inhibitor of autolysosome fusion (chloroquine) and fluorescence indicators for monitoring turnover of autophagosome (DAP green) and autolysosome (DAL green) to specify the essential step(s) of the aging-induced changes in the autophagic flux of heart. The LC3 turnover assay revealed that autophagic turnover was pathologically increased in myocardium of WRN-K577M at baseline and chloroquine (50 microg/g body weight) had no effect in WRN-K577M. Furthermore, DAL-positive spots were decreased in cardiomyocytes of WRN-K577M at rest, indicating that the impairment of autophagic flux particularly via impaired lysosome fusion may be responsible for the augmented autophagic turnover in WRN-K577M. Furthermore, we tested the impact of Rubicon. Rubicon was upregulated in heart of WRN-K577M and, more interestingly, Rubicon was found to be co-localized specifically with NOX4 in heart. Using CRISPER-CAS9 system, we generated Rubicon-KD H9C2 and Rubicon-activated HEK293. Upregulation of Rubicon revealed augmented LC3II, p62 and Beclin1 similar to WRN-K577M and augmented oxidative stress and, in contrast, downregulation of Rubicon had no effect on autophagy markers.

Conclusion(s)
Rubicon, the dual regulator of autophagy and inflammation is essential for autophagic disorder occurred in cardiac aging and the related oxidative stress via NOX4.

Funding Acknowledgement
Type of funding source: None