Activation, regulation, and effects of the mitochondrial unfolded protein response in endothelial cells
Authors: Al Zaidi M , Eschweiler J , Shakeri F , Sylvester M , Repges E , Nickenig G , Zimmer S , Aksoy A 
Published: October 2024

Abstract

Introduction
Endoplasmic reticulum stress and the resulting unfolded protein response (UPRER) play significant roles in endothelial dysfunction and atherosclerosis. However, the understanding of the mitochondrial unfolded protein response (UPRMito) in endothelial dysfunction is limited. This study aims to characterize the activation, regulation, and effects of the UPRMito to provide a comprehensive understanding of endothelial UPRMito.

Methods and Results
RNA sequencing and quantitative proteomic analysis were employed to investigate the impact of stressors on mitochondrial (MitoBlock-6, CDDO) and endoplasmic (Tunicamycin) protein homeostasis on the transcriptome and proteome of Human Coronary Artery Endothelial cells (HCAEC, Fig.1). The analysis identified 12,745 unique transcripts and 7,891 unique proteins. Mitocarta annotation highlighted the regulatory effect induced by these stressors, particularly on mitochondrial proteins and transcripts (Fig. 2). Geneset enrichment analyses revealed upregulation of pathways regulated by activating transcription factors 4/5 (ATF4/5) and the integrated stress response marker CHOP. Analysis of protein expression changes unveiled that 21 proteins were upregulated by both CDDO and MitoBlock-6, including essential mitochondrial chaperones (HSP10 and HSP60). In contrast, the UPRER stressor Tunicamycin did not induce canonical UPRMito activation. Additionally, 142 proteins were commonly downregulated, affecting crucial biological processes such as mitochondrial translation, mitochondrial gene expression, and ribosome biogenesis. Importantly, NAD+ADP-ribosyltransferase activity was highly downregulated, involving PARP proteins with roles in DNA damage repair.
On a functional level, mitochondrial stressors led to dose-dependent alterations in HCAEC viability, apoptosis, and reactive oxygen species formation (ROS) measured by resazurin-based assays, caspase 3/7 activity, and MitoSox staining, respectively. Low doses and short incubations with mitochondrial stressors promoted endothelial cell health through cytoprotective UPRMito pathways, while prolonged incubation led to unresolvable mitochondrial stress (exemplarily shown in Fig. 3). SiRNA-mediated gene silencing of the UPRMito regulators ATF4 and ATF5 promoted mitochondrial ROS formation in cells treated with mitochondrial stressors. These effects were particularly more pronounced after ATF5-Knockdown, suggesting that ATF5 is the main regulator of the UPRMito in HCAEC (Fig. 4). Moreover, high-dose UPRMito stressors led to an impairment in mitochondrial membrane integrity with cytosolic release of pro-inflammatory mtDNA.

Conclusion
UPRMito and UPRER lead to a different stress response. Depending on the underlying conditions, both stress responses can exert both cytoprotective and cytotoxic effects. Further studies are required to analyze the in vivo relevance and possible therapeutic exploitation of the UPRMito in endothelial dysfunction and atherosclerosis.Figures