Impact of magnesium:calcium ratio on calcification of the aortic wall

Abstract

Objective: An inverse relationship between serum magnesium concentration and vascular calcification has been reported following observational clinical studies. Moreover, several studies have been suggesting a protective effect of magnesium on the vascular calcification. However, the exact mechanism remains elusive, and investigators have speculated among a myriad of potential actions. The effect of magnesium on calcification of the aortic wall is yet to be investigated. In the present study, the effects of magnesium and calcium on the metabolism of extracellular PPi, the main endogenous inhibitor of vascular calcification, were investigated in the rat aorta.

Approach and results: Calcium and magnesium have antagonist effects on PPi hydrolysis in the aortic wall. Km and Ki values for PPi hydrolysis in rat aortic rings were 1.1 mmol/L magnesium and 32 μmol/L calcium, respectively, but ATP hydrolysis was not affected with calcium. Calcium deposition in the rat aortic wall dramatically increased when the magnesium concentration was increased (ratio of Mg:Ca = 1:1; 1.5 mmol/L calcium and 1.5 mmol/L magnesium) respect to low magnesium concentration (ratio Mg:Ca = 1:3, 1.5 mmol/L calcium and 0.75 mmol/L magnesium).

Conclusion: Data from observational clinical studies showing that the serum magnesium concentration is inversely correlated with vascular calcification could be reinterpreted as a compensatory regulatory mechanism that reduces both PPi hydrolysis and vascular calcification. The impact of magnesium in vascular calcification in humans could be studied in association with calcium levels, for example, as the magnesium:calcium ratio.

Fig 1. Impact of calcium on the hydrolysis of pyrophosphate and ATP.

Rat aortic ring tissue samples were incubated ex vivo in sHBSS (supplemented with 0.9 mmol/L magnesium) with 1 mmol/L calcium (+CaCl₂) or without calcium (-CaCl₂). (A) Representative time course for the hydrolysis of 5 μmol/L PPi (and 10 μCi/mL ³²PPi as radiotracer) measured by Pi release (³²Pi). (B) Representative time course of the hydrolysis of 1 μmol/L ATP (and 10 μCi/mL [γ³²P]ATP as radiotracer) measured by Pi release (γ³²Pi). Results in (C) and (D) are means ± SEM, after 10 min of incuation, of three independent experiments, with a total of 20 rings per condition (10 different rats). Student’s t-test was used for statistical analysis. ***p <0.001.

Fig 2. Kinetic characterisation of calcium and magnesium on pyrophosphate hydrolysis.

Rat aortic rings were incubated ex vivo in HBSS containing the indicated amount of magnesium and calcium. (A) Michaelian saturation curves were plotted to determinate the K_m value of magnesium for PPi hydrolysis. HBSS contained 5 μmol/L PPi and 10 μCi/mL ³²PPi as radiotracer. (B) Competitive inhibition of PPi hydrolysis by calcium; inset, logarithmic transformation of calcium concentration. HBSS contained 1.1 mmol/L magnesium, 5 μmol/L PPi and 10 μCi/mL ³²PPi as radiotracer. PPi hydrolysis was measured by Pi release. Pi was separated from PPi using the molybdate method as explained in the methods section. Results are represented as means ± SEM of three independent experiments with a total of 6, 7 or 13 rings per point (see S2 Table) and 26 rats in total (A) and two independent experiments with a total of 6 rings per point (see S3 Table) and 12 rats in total (B).

Fig 3. Impact of calcium on ATP hydrolysis.

Rat aortic rings were incubated ex vivo in sHBSS (supplemented with 1 mmol/L magnesium) and with 1 mmol/L calcium (A) or without calcium (B). Hydrolysis of 1 μmol/L ATP (and 10 μCi/mL [α³²P]ATP as radiotracer) showed ADP, AMP and α-Pi production in the indicated times, following separation by thin layer chromatography as described in the methods section. Results are means ± SEM of two independent experiments, with a total of 6 rings per condition (6 different rats). The same ring was used to analyze the hydrolysis of ATP in the absence or presence of calcium.

Fig 4. Impact of calcium and magnesium on ATP hydrolysis products.

Rat aortic rings were incubated ex vivo in HBSS supplemented with 1 mmol/L calcium and 1 mmol/L magnesium as indicated. After 30 min of incubation with 1 μmol/L ATP (and 10 μCi/mL [α³²P]ATP as radiotracer), ADP, AMP and Pi products were separated as described in the methods section. (A) Representative thin layer chromatography showing ADP, AMP and Pi products. (B) Quantification of ATP ([α³²P]ATP), ADP ([α³²P]ADP), AMP ([α³²P]AMP) and Pi (α³²Pi) products obtained from ATP hydrolysis. Results are represented as means ± SEM of three independent experiments with a total of 9 rings from 9 different rats (see S4 Table). The same ring was used to analyze the hydrolysis of ATP in the four conditions. Repeated measured ANOVA and Tukey’s multiple comparison tests were used for statistical analysis. Presence of magnesium and calcium was used as reference. **p <0.01; ***p <0.001.

Fig 5. Impact of calcium and magnesium on aortic wall calcification.

Rat aortic rings were incubated ex vivo in DMEM supplemented with the indicated concentration of magnesium and calcium. The medium was replaced every day and contained 45-calcium as a radiotracer. After 7 days of incubation, aortic rings were dried and radioactivity was measured by liquid scintillation counting. Results are represented as means ± SEM of two independent experiments, with a total of 14 rings per condition and 11 rats in total (see S5 Table). One-way ANOVA and Tukey’s multiple comparison test were used for statistical analysis. *p <0.05; ***p <0.001.