GRP75 inhibition attenuates arterial calcification

Atherosclerosis, vol. 409, pp. 119243

Abstract

BACKGROUND AND AIMS: Arterial calcification is a risk factor for cardiovascular mortality. The calcification process is driven by the osteogenic transition of vascular smooth muscle cells (SMCs), which release extracellular vesicles (EVs) that act as mineralization nucleation sites. While mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in arterial calcification, the role of their contact sites remains unknown. Mitochondria-associated membranes (MAMs) are inter-organelle contacts connecting the outer mitochondrial membrane to the ER membrane through protein-protein interactions. This study investigated the role of Glucose-regulated protein 75 (GRP75), a MAM linker protein, in SMC calcification and EV cargo. METHODS: Human coronary artery SMCs were cultured in osteogenic medium to induce calcification. MAMs were isolated from SMCs and human carotid artery by subcellular fractionation and visualized using transmission electron microscopy. SMC-derived EVs were isolated from the conditioned culture medium by ultracentrifugation. GRP75 inhibition was achieved using silencing RNA or the inhibitor MKT-077. Mitochondrial respiration and ER stress were analyzed using Seahorse analysis and Western blotting. RESULTS: Calcifying SMCs expressed higher GRP75 mRNA (2.2-fold ± 0.7, p = 0.043) and protein (1.3-fold ± 0.2, p = 0.008) levels compared to control SMCs. GRP75 was enriched at MAMs, and electron microscopy imaging demonstrated closer mitochondria-ER contacts in both calcifying SMCs in vitro and human calcified carotid artery specimens. GRP75 inhibition by silencing RNA (-35 % ± 13 %, p < 0.001) or MKT-077 (-57 % ± 3 %, p < 0.001) attenuated matrix mineralization and reduced close mitochondria-ER contacts along with attenuating mitochondrial respiration capacity. Additionally, GRP75 was enriched in EVs released by calcifying SMCs (1.3-fold ± 0.1, p = 0.040). CONCLUSIONS: Our findings demonstrate that MAMs are altered in calcifying SMCs. GRP75 inhibition disrupted close mitochondria-ER contact formation, decreased mitochondrial respiration, modulated the osteogenic transition of SMCs, and reduced vascular calcification. Therefore, GRP75 could serve as a potential target for preventing arterial calcification.

Authors 8

1. Jonas Heyn Aachen Department of Internal Medicine I-Cardiology Medical Faculty

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Department of Internal Medicine I - Cardiology, Medical Faculty, Uniklinik RWTH Aachen, RWTH Aachen University, 52074, Aachen, Germany

2. Nicolas Hense Aachen Department of Internal Medicine I-Cardiology Medical Faculty

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Department of Internal Medicine I - Cardiology, Medical Faculty, Uniklinik RWTH Aachen, RWTH Aachen University, 52074, Aachen, Germany

3. Alexander Gombert Aachen Department of Vascular Surgery Medical Faculty

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Department of Vascular Surgery, Medical Faculty, Uniklinik RWTH Aachen, RWTH Aachen University, 52074, Aachen, Germany

4. Eva Miriam Buhl Aachen Electron Microscopy Facility Institute of Pathology, Medical Faculty

   RWTH Aachen University

   Affiliation as printed

   Electron Microscopy Facility, Institute of Pathology, Medical Faculty, RWTH Aachen University, 52074, Aachen, Germany

5. Sonja Vondenhoff Aachen Institute for Molecular Cardiovascular Research Medical Faculty

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Institute for Molecular Cardiovascular Research (IMCAR), Medical Faculty, Uniklinik RWTH Aachen, RWTH Aachen University, 52074, Aachen, Germany

6. Heidi Noels Aachen Institute for Molecular Cardiovascular Research Medical Faculty

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Institute for Molecular Cardiovascular Research (IMCAR), Medical Faculty, Uniklinik RWTH Aachen, RWTH Aachen University, 52074, Aachen, Germany

7. Nikolaus Marx Aachen Department of Internal Medicine I-Cardiology Medical Faculty

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Department of Internal Medicine I - Cardiology, Medical Faculty, Uniklinik RWTH Aachen, RWTH Aachen University, 52074, Aachen, Germany

8. Claudia Goettsch corresponding Aachen Department of Internal Medicine I-Cardiology Medical Faculty

   Universitätsklinikum Aachen · Technische Universität Dresden · RWTH Aachen University

   Affiliation as printed

   Department of Internal Medicine I - Cardiology, Medical Faculty, Uniklinik RWTH Aachen, RWTH Aachen University, 52074, Aachen, Germany; Institute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden, 01307, Dresden, Germany. Electronic address: cgoettsch@ukaachen.de