Cell-based calcification assays – magnesium overrules calcium phosphate saturation in overall outcome

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

Abstract Soft tissue calcification, a common cause of cardiovascular mortality in conditions like chronic kidney disease (CKD), is influenced by magnesium (Mg). However, the exact mechanism, whether extracellular chemistry-mediated or cell-mediated, remains unclear. Here we focused on the extracellular milieu. Using fluorescence-labelled fetuin-A and a live imaging platform, we found that Mg reduced spontaneous mineral precipitation by stabilizing calcium and phosphate as colloidal protein-mineral particles (CPP) in calcification media containing up to 10 mM calcium and phosphate. Addition of 1.25-5 mM Mg progressively stabilized the protein-mineral particles in the fluid phase, increasing calcification. Dynamic light scattering revealed smaller and more numerous CPP at up to 4 mM Mg. At 10 mM Mg, no calcification occurred even with 10 mM added calcium and phosphate. This suggested a critical Mg concentration range in supersaturated calcification media, where Mg paradoxically enhanced calcification by stabilizing mineral precursors and increasing their availability. These findings demonstrate Mg’s dual role: an inhibitor of protein-mineral complex aggregation at high concentrations and a facilitator of mineral availability from colloidal protein-mineral phases at concentrations up to about 5 mM Mg. This highlights the role of Mg in providing highly dynamic, mineral-rich, stable protein-mineral complexes driving matrix calcification, yet preventing mineral precipitation.

Authors 9

1. Aaron M. Morgan Aachen Biointerface Laboratory

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Biointerface Laboratory, RWTH Aachen University Hospital, Aachen, Germany;

2. Christian Hasberg Aachen Biointerface Laboratory

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Biointerface Laboratory, RWTH Aachen University Hospital, Aachen, Germany;

3. Camilla Franziska Winkler Aachen Biointerface Laboratory

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Biointerface Laboratory, RWTH Aachen University Hospital, Aachen, Germany;

4. Robert Dzhanaev Aachen Biointerface Laboratory

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Biointerface Laboratory, RWTH Aachen University Hospital, Aachen, Germany;

5. Steffen Gräber
6. Luca Anna Reicher Aachen Biointerface Laboratory

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Biointerface Laboratory, RWTH Aachen University Hospital, Aachen, Germany;

7. Andrea Gorgels Aachen Biointerface Laboratory

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Biointerface Laboratory, RWTH Aachen University Hospital, Aachen, GermanyDepartment of Internal Medicine I - Cardiology, Medical Faculty, RWTH Aachen University Hospital, 52074 Aachen, Germany;

8. Claudia Goettsch 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, RWTH Aachen University Hospital, 52074 Aachen, GermanyInstitute of Physiology, Medical Faculty Carl Gustav Carus, Technical University Dresden, 01307 Dresden, Germany

9. Willi Jahnen‐Dechent corresponding Aachen Biointerface Laboratory

   Universitätsklinikum Aachen · RWTH Aachen University

   Affiliation as printed

   Helmholtz-Institute for Biomedical Engineering, Biointerface Laboratory, RWTH Aachen University Hospital, Aachen, Germany;