Dietary magnesium supplementation improves lifespan in a mouse model of progeria

Abstract Aging is associated with redox imbalance according to the redox theory of aging. Consistently, a mouse model of premature aging (Lmna G609G/+) showed an increased level of mitochondrial reactive oxygen species (ROS) and a reduced basal antioxidant capacity, including loss of the NADPH‐coupled glutathione redox system. Lmna G609G/+ mice also exhibited reduced mitochondrial ATP synthesis secondary to ROS‐induced mitochondrial dysfunction. Treatment of Lmna G609G/+ vascular smooth muscle cells with magnesium‐enriched medium improved the intracellular ATP level, enhanced the antioxidant capacity, and thereby reduced mitochondrial ROS production. Moreover, treatment of Lmna G609G/+ mice with dietary magnesium improved the proton pumps (complexes I, III, and IV), stimulated extramitochondrial NADH oxidation and enhanced the coupled mitochondrial membrane potential, and thereby increased H+‐coupled mitochondrial NADPH and ATP synthesis, which is necessary for cellular energy supply and survival. Consistently, magnesium treatment reduced calcification of vascular smooth muscle cells in vitro and in vivo, and improved the longevity of mice. This antioxidant property of magnesium may be beneficial in children with HGPS.


Introduction
Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare, sporadic genetic disorder that is characterized by premature aging and accelerated cardiovascular disease progression, including that of vascular calcification (Nair et al, 2004;Salamat et al, 2010;Hanumanthappa et al, 2011). Most HGPS patients carry a de novo noninherited autosomal dominant heterozygous mutation of the LMNA gene (p.G608G in humans; p.G609G in mice) (De Sandre-Giovannoli et al, 2003;Eriksson et al, 2003). This mutation activates a cryptic splice donor site, which causes synthesis of a lamin A mutant that disrupts nuclear membrane architecture and induces multiple cellular defects, including abnormal gene transcription, signal transduction, and DNA damage. HGPS patients die at a mean age of 13-14 years (a mean of~38 weeks old in Lmna G609G/+ mice), typically because of a cardiovascular event (Merideth et al, 2008).
Experimental and observational studies have shown that high magnesium intake has beneficial effects on cardiovascular risk factors, mediated by improvements in insulin-glucose metabolism, endothelium-dependent vasodilation, and the lipid profile, a reduction in vascular calcification, and the induction of anti-hypertensive and anti-inflammatory effects (DiNicolantonio et al, 2018;Rosique-Esteban et al, 2018). For example, vascular calcification in uremic rats is prevented by magnesium supplementation (Diaz-Tocados et al, 2017). However, magnesium also plays diverse roles in the pathogenesis of cardiovascular diseases at the biochemical and cellular levels (DiNicolantonio et al, 2018;Rosique-Esteban et al, 2018).
Magnesium is an essential mineral that serves as a cofactor in more than 300 enzymatic reactions, including those involved in energy metabolism and protein/nucleic acid synthesis. Magnesium is essential for mitochondrial function and particularly for ATP production, and magnesium deficiency is found in cardiovascular disease, type 2 diabetes mellitus, hypertension, heart failure, and ventricular arrhythmia patients (DiNicolantonio et al, 2018;Rosique-Esteban et al, 2018). In addition, magnesium supplementation improves mitochondrial and cardiac diastolic function in diabetic patients (Liu et al, 2019). Vascular calcification has been identified in a mouse model of Hutchinson-Gilford progeria syndrome . The excessive accumulation of calcium in the vessels of HGPS mice (Osorio et al, 2011) is associated with defective extracellular pyrophosphate metabolism, due to a reduction in ATP synthesis secondary to mitochondrial dysfunction .
In the present study, we aimed to determine whether magnesium supplementation ameliorates vascular calcification and improves longevity in Lmna G609G/+ mice.

Magnesium improves Lmna G609G/+ vascular smooth muscle cell (VSMC) viability
Several studies have shown that the accumulation of DNA damage in cells activates DNA damage and replication checkpoints, which attenuate cell-cycle progression and arrest replication (Liu et al, 2005(Liu et al, , 2006Varela et al, 2005;Richards et al, 2011;Sieprath et al, 2015). We first performed a comparative analysis of the proliferative ability of primary vascular smooth muscle cells from Lmna G609G/+ mice and their wild-type littermates. Notably, microscopy images showed an apparent similar cellular morphology in both genotypes during its growth ( Fig EV1A). However, Lmna G609G/+ VSMCs exhibited much lower proliferation than control cells (Fig EV1B). The rate of division per day was significantly lower (by 36%) than that of wild-type control cells (0.36 AE 0.07 versus 0.23 AE 0.06 divisions per day; Fig EV1C; Appendix Table S1).
To determine the status of DNA replication, the replicative incorporation of 5-bromodeoxyuridine (BrdU) was assessed (Fig EV1D; Appendix Table S1). DNA synthesis in Lmna G609G/+ VSMCs occurred at a 44% slower rate than in wild-type cells. Notably, Lmna G609G/+ VSMCs incubated in medium containing a high magnesium concentration showed a significantly higher replication rate, both with respect to the number of divisions per day (0.30 AE 0.05), and the replicative incorporation of BrdU (75% of wild type).
Magnesium ameliorates mitochondrial oxidative stress in Lmna G609G/+ VSMCs Mitochondrial reactive oxygen species (ROS)-mediated cell damage has been implicated in progeria (Richards et al, 2011;Sieprath et al, 2015;Kadoguchi et al, 2020). To evaluate the antioxidant properties of magnesium, ROS concentration was measured using the cell permeant reagent 2 0 ,7 0 -dichlorofluorescin diacetate (DCFDA), a fluorogenic dye that can be used to quantify hydroxyl, peroxyl, and other ROS activities within the cell. Lmna G609G/+ VSMCs showed significantly higher (3-fold) ROS content than wild-type cells (Fig EV2A; Appendix Table S3). In addition, the concentrations of two specific ROSs were also assessed. Mitochondrial superoxide (O 2 À ) and hydrogen peroxide (H 2 O 2 ) were present in significantly higher (1.6-fold and 2.3-fold, respectively) concentrations in Lmna G609G/+ VSMCs than in wild-type cells (Fig EV2B;  Appendix Table S3). Notably, this overproduction of ROS was significantly reduced (by 69% for ROS, by 43% for H 2 O 2 , and by 29% for O 2 À ) in Lmna G609G/+ VSMCs incubated in magnesiumenriched medium. The rate of ROS generation and the cellular defenses against ROS toxicity (which include enzymes, small molecules, and proteins) contribute to the overall level of oxidative stress. The total antioxidant capacity (TAC) can be considered a cumulative index of antioxidant status. To evaluate the overall cellular capacity to counteract ROS, TAC was assessed using a Cu 2+ reduction assay. Lmna G609G/+ VSMCs showed significantly lower TAC (38%) than wild-type VSMCs (Fig EV2C; Appendix Table S3). This reduction was significantly ameliorated (by 27%) in Lmna G609G/+ VSMCs incubated in magnesium-enriched medium.
Reduced glutathione (GSH) is the major detoxifying redox buffer in cells and participates in the defense against ROS and the repair of mitochondrial oxidative damage, by being both a potent antioxidant itself and a substrate for antioxidant enzymes, including the glutathione reductase redox systems. Notably, total glutathione, A C B Figure 1. Magnesium improves ATP synthesis in Lmna G609G/+ VSMCs.
A-C (A) Oxygen consumption ratio, (B) mitochondrial ATP synthesis, and (C) mitochondrial membrane potential (MP), in the indicated VSMC types. Results are presented as the mean AE SD of three independent experiments (four wells per experiment). One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001.
Source data are available online for this figure.
2 of 13 EMBO Molecular Medicine 12: e12423 | 2020 ª 2020 The Author which includes GSH and oxidized glutathione (GSSG), and glutathione reductase (GR) activity were significant lower in Lmna G609G/+ VSMCs than in wild-type cells (Fig EV2D and E; Appendix Table S3). In addition, the ratio of reduced glutathione to oxidized glutathione (GSH:GSSG) was measured to assess the oxidative profile of the cells. Lmna G609G/+ VSMCs showed a significantly lower (51%) GSH:GSSG ratio than wild-type VSMCs ( Fig EV2D).
Notably, this reduction was significantly ameliorated (by 51%) in treated Lmna G609G/+ VSMCs, although the GR activity and total glutathione concentration were similar in treated and untreated Lmna G609G/+ VSMCs. GR uses reduced nicotinamide adenine dinucleotide phosphate (NADPH) to maintain the GSH redox state. Notably, although both types of VSMCs contained similar amounts of total nicotinamide adenine dinucleotide phosphate (NADPH and its oxidized form, NADP + ), Lmna G609G/+ VSMCs had a significantly lower (48%) NADPH:NADP + ratio than wild-type cells (Fig EV2F and G; Appendix Table S3). However, the NADPH:NADP + ratio was significantly improved (by 45%) by magnesium treatment of Lmna G609G/+ VSMCs.
Intracellular acidification can lead to cytosolic and mitochondrial calcium overload, which depolarizes DΨ m to limit ATP production and stimulates mitochondrial ROS generation and permeability transition (Brookes et al, 2004;Görlach et al, 2015;Santulli et al, 2015). Lmna G609G/+ VSMCs incubated with 45 Ca 2+ as a radiotracer showed significantly higher (55%) mitochondrial calcium than wild-type cells, which was significantly reduced (by 21%) in treated Lmna G609G/+ VSMCs ( Fig 2D). In addition, Lmna G609G/+ VSMCs showed significantly lower (35%) mitochondrial magnesium than wild-type cells, which was significantly increased (by 37%) in treated Lmna G609G/+ VSMCs (Fig 2E and F).  Table S5). Untreated Lmna G609G/+ VSMCs showed 11-fold higher (in live cells) and 17fold higher (in fixed cells) calcium deposition after 7 days of incubation in phosphate-calcifying medium. However, treated living Lmna G609G/+ VSMCs showed significantly lower calcium accumulation (7.3-fold), although the calcium content in fixed cells was similar in treated and untreated Lmna G609G/+ VSMCs (17-fold). The addition of pyrophosphate or phosphonoformic acid (two known inhibitors of calcium phosphate crystal deposition) (Villa-Bellosta & Sorribas, 2009) to the phosphate-calcifying medium completely prevented calcium accumulation in both fixed/living and treated/untreated Lmna G609G/+ VSMCs. Notably, magnesium supplementation of the phosphate-calcifying medium significantly reduced (by 38% in untreated and 51% in treated cells) calcium deposition in living cells. By contrast, magnesium supplementation did not reduce calcium deposition in either treated or untreated fixed VSMCs. Taken together, these results suggest that magnesium prevents calcium phosphate deposition by a cellular activitydependent mechanism, and not by direct binding to calcium phosphate crystals, preventing their formation and growth. Finally, the capacity to inhibit calcification (DCa 2+ ) was calculated as the difference in calcium deposition in living versus fixed cells (Ca 2+ in fixed cells À Ca 2+ in living cells). The DCa 2+ in treated cells was significantly higher than that in untreated cells ( Fig 3G). Importantly, magnesium supplementation of the phosphate-calcifying medium caused significant increases in DCa 2+ in both treated and untreated cells.

Magnesium prevents vascular calcification in HGPS mice
Clinically, plasma magnesium is usually measured despite the fact that less than 1% of magnesium exists extracellularly. Hence, plasma magnesium levels do not always accurately reflect total body magnesium stores. In fact, plasma magnesium levels can be normal despite depletion of the total body magnesium content. Notably, plasma magnesium levels were in the normal range in both wild-type and Lmna G609G/+ mice, although they were significantly lower in 21-and 34-week-old Lmna G609G/+ mice than in wild-type littermates (Table EV1).
To assess the effect of supplemental magnesium on Lmna G609G/+ mice, their drinking water was supplemented with MgCl 2 . Thereafter, the consumption of food and water was measured in the mice between 8 and 34 weeks of age. The median food and water consumption of untreated and treated Lmna G609G/+ mice was similar (3.46 AE 0.77 versus 3.53 AE 0.72 g/day/mouse and 3.96 AE 0.62 versus 4.01 AE 0.73 ml/day/mouse, respectively). Therefore, the total magnesium intake by treated Lmna G609G/+ mice was significantly higher (4.6-fold) than that by untreated Lmna  Table S6). Finally, the total calcium content of aortas obtained from treated Lmna G609G/+ mice was significantly lower than that of aortas obtained from untreated Lmna G609G/+ mice (401.5 AE 77.7 versus 741.9 AE 101.6 lg/g aorta; Fig 4C; Appendix Table S6).

Magnesium improves ATP synthesis in HGPS mice
Liver homogenates from untreated Lmna G609G/+ mice showed significantly lower (55%) intracellular ATP, which was 65% higher in treated mice (Fig 5A; Appendix Table S8). Moreover, isolated mitochondria showed 89% higher calcium content in untreated Lmna G609G/+ mice relative to wild-type mice, but this was 34% lower in treated mice ( Fig 5B; Appendix Table S8). In contrast, isolated mitochondria showed 33% lower magnesium content in untreated Lmna G609G/+ mice relative to wild-type mice, but this was 35% higher in treated mice (Fig 5C; Appendix Table S8). Moreover, the activities of complexes I, III, IV, and V were significantly lower in untreated Lmna G609G/+ than wild-type mice, but these defects were significantly ameliorated in treated Lmna G609G/+ mice (Fig 5D and E; Appendix Table S8), Notably, the subunits of these mitochondrial complexes are encoded by mitochondrial DNA.
Magnesium improves mitochondrial ATP synthesis ATP synthesis in isolated mitochondria was significantly lower in untreated Lmna G609G/+ mice than in wild-type mice, in media containing either 0.1 mM magnesium (109.9 AE 29.6 versus 244.8 AE 78.8 nmol/min/mg protein, respectively) or 1 mM magnesium (226 AE 75.1 versus 503.7 AE 104.4 nmol/min/mg protein, The boxed scheme describes the mitochondrial calcium overload hypothesis. Lactic acidosis forces the Na + /H + exchanger (NHX) to import Na + , resulting in cytosolic Na + overload. Subsequently, the Na + /Ca 2+ exchanger (NCX) is forced into reverse mode to dispose of excess Na + , resulting in cytosolic calcium overload. This Ca 2+ is then taken up by mitochondria, resulting in mitochondrial calcium overload. 2-DG (2-deoxyglucose; 50 mM) blocks glycolysis through competitive hexokinase inhibition, whereas oligomycin (10 lM) inhibits mitochondrial ATP synthase. G-6-P: glucose-6-phosphate.
Data information: Results are presented as the mean AE SD of three independent experiments (four wells per experiment). One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure.
4 of 13 EMBO Molecular Medicine 12: e12423 | 2020 ª 2020 The Author respectively; Fig EV4A; Appendix Table S9). In both media, ATP synthesis was significantly higher (57% and 54%, respectively) for treated Lmna G609G/+ mice than for untreated Lmna G609G/+ mice. Notably, ATP synthesis in all the experimental groups was 2-fold higher when mitochondria were assessed in incubation media containing 1 mM magnesium compared to incubation media containing 0.1 mM magnesium, which implies that ATP synthase is simulated by magnesium independently of the effect of treatment.

Magnesium improves extramitochondrial NADH oxidation
Oxidation of exogenous NADH by mitochondria in the presence of added extramitochondrial cytochrome c has been described previously (Bodrova et al, 1998;Lemeshko, 2000). Oxidation was shown to be insensitive to rotenone, antimycin A, and was suppressed by cyanide (Bodrova et al, 1998). The external NADHcytochrome c reductase electron transport system of the outer membrane of mitochondria is known to have a very high activity (Lemeshko, 2000). Notably, rotenone-insensitive oxidation of external NADH in isolated mitochondria was significantly higher in both treated (4.9-fold) and untreated (4.4-fold) Lmna G609G/+ and wild-type (4.2-fold) mice in media containing magnesium compared to media in the absence of magnesium (Fig EV4B; Appendix Table S9). This stimulation of NADH oxidation by Mg 2+ ions was enhancer by addition of exogenous cytochrome c and suppressed by cyanide (Fig EV4B). The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells (DCa 2+ ).
Data information: Results are presented as the mean AE SD of three independent experiments (four wells per condition). One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. **P < 0.01; ***P < 0.001. Source data are available online for this figure.
While the exact mechanisms whereby magnesium prevents calcification remain to be determined, our data seem to exclude a physicochemical role of magnesium in altering calcium phosphate crystal growth, as evidenced by our finding showing similar calcium deposition in fixed Lmna G609G/+ VSMCs incubated with high magnesium to those incubated in medium containing a standard concentration of magnesium (Fig 3). Thus, the beneficial role of magnesium in attenuating vascular calcification is likely to be linked to an active cellular role.
Consistent with this, a synergistic effect has been demonstrated when magnesium and ATP are used together in solution to delay the conversion of a slurry of amorphous calcium phosphate to crystalline hydroxyapatite (Blumenthal et al, 1977). Moreover, ATP has also been found to prevent vascular calcification by directly Data information: Results are presented as the mean AE SD. Statistical analyses were performed using Student's t-test (A, B, D), log-rank test (E), or one-way ANOVA and Tukey's multiple comparison post hoc test (C). **P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure.
6 of 13  Neill, 2018). We have shown increases in ATP availability, in both treated Lmna G609G/+ VSMCs and mice, which could be explained by increases in the synthesis of ATP in mitochondria and glycolysis. Mg 2+ is an important divalent cation in cells that stabilizes nucleic acid and protein structure, and mediates magnesium-dependent enzymatic reactions as a cofactor, including enzymatic reactions involving ATP (Pilchova et al, 2017). The mammalian mitochondrial ATP synthase (complex V) catalyzes ATP synthesis from ADP, phosphate, and magnesium using energy generated by an electrochemical gradient of protons produced by the electron transport chain (Chen et al, 2006). The mitochondria also generate ROS as a consequence of inefficiencies in the electron transport chain (Murphy, 2009), which cause oxidative stress, DNA damage, and cellular senescence, molecular defects that are found in the premature aging syndrome HGPS (Viteri et al, 2010;Gordon et al, 2014).
Notably, HGPS fibroblasts generate higher concentrations of ROS than normal fibroblasts (Richards et al, 2011). Furthermore, the basal expression of antioxidant enzymes, which defend cells against ROS-induced damage, is also lower in HGPS fibroblasts (Yan et al, 1999). Moreover, in HGPS fibroblasts, a marked downregulation of mitochondrial oxidative phosphorylation proteins, accompanied by severe mitochondrial dysfunction, has been observed, along with a marked reduction in COX activity (cytochrome c oxidase; mitochondrial complex IV) and a significant increase in glycolytic dependency Aliper et al, 2015). Therefore, the higher oxidative stress in HGPS cells could be as result of greater ROS formation (see Fig 6), due to defective mitochondrial oxidative phosphorylation, as well as lower ROS-counteracting antioxidative capacity (Kubben et al, 2016). Furthermore, emerging evidence suggests that COX dysfunction is invariably associated with greater mitochondrial ROS generation (Srinivasan & Avadhani, 2012;Kadoguchi et al, 2020). Moreover, Ca 2+ accumulation can impair mitochondrial function, leading to lower ATP production and greater release of ROS . E The boxed scheme shows the five mitochondrial complexes involved in the electron transport chain and their known inhibitors. Data information: Results are presented as mean AE SD (n = 16). One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001.
Source data are available online for this figure.
ª 2020 The Author EMBO Molecular Medicine 12: e12423 | 2020 addition, Mg 2+ deficiency is associated with greater production of ROS and the induction of immune and inflammatory reactions (Bussière et al, 2002). Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary factor responsible for the protection of cells from oxidative stress, which it does by regulating cytoprotective gene expression, including that of the antioxidant glutathione pathway (Harvey et al, 2009). Interestingly, repression of the antioxidant NRF2 pathway has been found in HGPS (Kubben et al, 2016). Consistent with this, we have shown significantly lower total glutathione synthesis and GR activity, which were not improved in the presence of a high magnesium concentration. By contrast, both the GSH:GSSG and NADPH:NAD + ratios, which are indicative of antioxidant status, were improved by magnesium treatment.
According to the redox theory of aging, aging is associated with redox imbalance (Sohal & Orr, 2012;Go & Jones, 2017). Thus, both increases in mitochondrial ROS and a deterioration in antioxidant status stimulate aging (see Fig 6). The reductions in cytoplasmic and mitochondrial NADPH:NADP + ratio with aging are associated with reductions in the activities of cytoplasmic and mitochondrial GRs (and thioredoxin reductases), which lead to greater oxidization of the glutathione redox couple (GSH:GSSG), resulting in lower activities of glutathione peroxidases (and thioredoxin peroxidases) (Bradshaw, 2019). This oxidation of the NADPH-linked redox systems with aging also causes the oxidation of ascorbate (vitamin C) and tocopherols (vitamin E) (Ren et al, 2017). In the present study, we found a significant reduction in the total antioxidant capacity in both Lmna G609G/+ cells and mice, which was ameliorated by magnesium treatment.
The uptake of Ca 2+ ions by mitochondria should depolarize mitochondrial membranes (DΨ m ) (Chalmers & McCarron, 2008), which is required for both H + -coupled mitochondrial ATP synthase to generate ATP (Saraste, 1999) and NADPH transhydrogenase to generate NADPH (Rydström, 2006). Furthermore, magnesium increases the activities of mitochondrial dehydrogenases (Fig EV1E), including pyruvate dehydrogenase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase (Pilchova et al, 2017). Therefore, mitochondrial NADP + -dependent isocitrate dehydrogenase could also contribute to the increase in NADPH availability. Moreover, magnesium can reverse the effects of calcium-induced DΨ m depolarization (Racay, 2008) and inhibit mitochondrial ROS generation (Kowaltowski et al, 1998). Furthermore, several studies (including the present study) have shown that magnesium has a stimulatory effect on the NADH-cytochrome c reductase systems located in the outer mitochondrial membrane (Bodrova et al, 1998;Lemeshko, 2000). Therefore, magnesium treatment can also improve exogenous NADH oxidation and the coupled DΨ m . Consistent with this, the present study has shown improvements in mitochondrial membrane potential and NADPH-coupled glutathione redox system following magnesium treatment. In addition, we have shown that Lmna G609G/+ VSMCs and mice treated with high magnesium concentrations have lower ROS concentrations, improvements in both mitochondrial function and mitochondrial ATP synthesis, and thus greater ATP availability, which is necessary for cellular energy supply and survival.
Finally, several studies have shown the beneficial effect of dietary magnesium supplementation in several diseases, including atherosclerosis, diabetes, and heart failure. These studies support the effect of magnesium at the molecular level independently of progerin production and its interactions with nuclear membrane proteins. However, magnesium could interact with nuclear proteins, including telomerase and lamins A, B, and C, which may improve or attenuate its reported beneficial effects. These molecular mechanisms, including other metabolic pathways, signaling pathways, and enzyme activities, will be evaluated in future studies.

Conclusion
Plasma magnesium levels do not always accurately reflect total body magnesium stores. In fact, plasma magnesium levels can be normal despite depletion of the total body magnesium content. However, several studies have shown a connection between magnesium deficiency and aging. In addition, a statistically significant inverse correlation between the level of magnesium in drinking water and cardiovascular mortality has been reported in observational epidemiological studies (Rosique-Esteban et al, 2018). Therefore, a lack of magnesium in drinking water and food may underlie the aging-associated progressive deterioration of physiological functions, including the redox balance, senescence, and vascular calcification, while high magnesium intake may delay aging. Consistently, the current study demonstrated that addition of magnesium to drinking water significantly extended longevity in progeroid mice. Therefore, dietary magnesium supplementation may be beneficial in children with HGPS, even those who appear to be normomagnesemic (Merideth et al, 2008). Further experiments are needed to test the effect of magnesium supplement in human HGPS context and validate the results obtained in mouse HGPS model.

Animals
Male Lmna G609G/+ and wild-type (C57/BL6) littermates were used at the indicated age. Lmna G609G/+ was designed by Carlos López-Otín research group (Oviedo University, Spain) in close collaboration with two French teams (one lead by Nicolas Lévy and the other by Bernard Malissen). The protocol was approved by ethics committees both the FIIS-FJD (Fundación Instituto de Investigación Sanitaria, Fundación Jiménez Díaz) and Madrid Community (PROEX177/15); and conformed to directive 2010/63EU and recommendation 2007/526/EC regarding the protection of animals used for experimental and other scientific purposes, enforced in Spanish law under RD1201/2005. Sample size for animal studies was estimated based on our previous experience and mouse availability. Animals were grouped by genotype. No blinding was performed.

Aorta isolation
Lmna G609G/+ mice were euthanized via carbon dioxide inhalation and thoracic aorta tissue was perfused with saline and removed according to previously published protocol (Villa-Bellosta & Hamczyk, 2015).

Quantification of aortic calcification
To quantify the calcium content, mice aortas were dried, weighed, and treated with 0.6 M HCl 24 h. Then, calcium was quantified using a colorimetric QuantiChrom Calcium Assay Kit (BioAssay Systems, Hayward, CA).

Plasma magnesium levels
Blood was collected in heparin-containing tubes and separated into plasma by centrifugation at 5,000 g for 10 min at 4°C. Mg was determined with the QuantiChrom Magnesium Assay Kit (BioAssay System) by the manufacturer's instructions.

Cell proliferation
The replicative incorporation of 5-bromodeoxyuridine (BrdU) was measured using BrdU Cell Proliferation ELISA Kit (Abcam, ab126556) by the manufacturer's instructions.

Cell viability
The cell viability was assessed measuring the mitochondrial activity by using tetrazolium salt which in cleavage to formazan by cellular mitochondria dehydrogenase, (WST-1 Assay Kit, ab65475, Abcam). Water-soluble WST-1 was added to each well, and the absorbance was measured using a scanning multiwell microplate according to the manufacturer's protocol.

b-Gal activity
Senescence-associated b-galactosidase activity was measured in cell lysates by plate reader using a fluorescent probe (b-gal Activity Assay Kit, BioVision), according to the manufacturer's protocols. Beta-galactosidase hydrolyzes a non-fluorescent substrate to generate a strong fluorescent product, which was measured (Ex/ Em = 480/520) in two time points (0 and 60 min).

ATP quantification
ATP was measured by a coupled luciferin/luciferase reaction with an ATP Determination Kit (Invitrogen). Cells were treated with lysis buffer (50 Tris-HCl mM, 150 NaCl mM, 1% Triton X-100 containing inhibitor cocktail, pH 7.4). VSMCs or liver lysates (intracellular ATP) and ATP standards were measured, according to the manufacturer's instructions (Villa-Bellosta, 2019). For mitochondrial ATP measurement, VSMCs were previously incubated with or without oligomycin (10 lM) for 15 min. Mitochondrial ATP was calculated by the subtraction of intracellular ATP levels (with oligomycin) from total ATP (without oligomycin).

Mitochondrial membrane potential (DΨ m ) measurement
The DΨ m was assessed by plate reader using a fluorescent probe (JC-10; ab112134, Abcam) by following the manufacturer's instruction. When mitochondria are polarized electrically, JC-10 forms Jaggregates that emit orange-red fluorescence. J-monomers, indicating depolarized mitochondria, emit green fluorescence. The DΨ m was calculated by a ratio of red/green fluorescence, indicating mitochondria depolarization with smaller ratio.

ROS detection
Reactive oxygen species (ROS) were measured using the cell permeant reagent 2 0 ,7 0 -dichlorofluorescin diacetate (DCFDA) according to the manufacturer's protocol (Abcam, ab113851). DCFDA is deacetylated by cellular esterases and oxidized by ROS into a highly fluorescent compound which was measured using a fluorescence microplate reader (excitation/emission wavelength of 488/535 nm). Amplex â Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) was used to detect hydrogen peroxide (H 2 O 2 ). In the presence of peroxidase, the Amplex â Red reagent reacts with H 2 O 2 to produce the red-fluorescent oxidation product, resorufin, which was measured using fluorescence microplate reader (excitation/ emission wavelength of 430/590 nm), according to the manufacturer's protocol (A22188, Invitrogen). To detect production of mitochondrial superoxide radical in live cells, the Mitochondrial Superoxide Detection Kit (ab219943, Abcam) was used, according to the manufacturer's protocols. Superoxide was measured using fluorescence microplate reader (excitation/emission wavelength of 540/590 nm).

Total antioxidant capacity
Total antioxidant capacity (TAC) was determined using a commercially available assay kit (Abcam, ab65329) which utilizes the conversion of Cu 2+ ions to Cu + through endogenous protein and small molecule antioxidants, standardized to Trolox equivalents. VSMCs and liver were used for analysis of TAC according to the manufacturer's protocols. Colorimetric activity was measured by optimal density at 570 nm.

NADPH-coupled glutathione redox system assay
Reduced/oxidized glutathione (GSH/GSSG) ratio, NADPH/NADP ratio, and glutathione reductase (GR) activity in both VSMCs and liver lysates were measured with commercials kit (Abcam, ab138881, ab176724, and ab83461, respectively), using a 96-well plate reader, according to the manufacturer's protocols. Liver samples were measured in triplicate on the same plate, and fluorescence/colorimetric values were normalized to micrograms of protein loaded in the assay per sample. Protein was measured with the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, USA), according to the manufacturer's protocols. GSH and total glutathione were determined by changes in fluorescence intensity (excitation/emission wavelength of 490/520 nm). 20 mg of liver tissue or VSMCs (5 × 10 6 ) were homogenized/lysed in 400/100 ll cold lysis buffer, respectively. Homogenates/lysates were centrifuged at top speed for 15 min at 4°C. Supernatants were deproteinized using Deproteinizing Sample Preparation Kit-TCA (Abcam, ab204708) according to the manufacturer's protocol.
NADPH and total nicotinamide adenine dinucleotide phosphate (NADPH + NADP + ) were determined by changes in fluorescence intensity (excitation/emission wavelength of 540/590 nm). 20 mg of liver tissue or VSMCs (5 × 10 6 ) were homogenized/lysed in 400/ 100 ll lysis buffer, respectively. Homogenates/lysates were centrifuged at 2,500/1,500 rpm, respectively, for 5 min at RT. GR activity was measured by optimal density at 405 nm. 20 mg of liver tissue or 1 × 10 6 VSMCs were homogenized/lysed in 200 ll cold assay buffer and centrifuged at 10,000 × g for 15 min at 4°C. Supernatant was pre-treated to destroy GSH before the assay, according to the manufacturer's protocol.

Mitochondrial and cytosolic ATP synthesis
Liver mitochondria were isolated by the standard procedure of differential centrifugation using the isolation medium composed of 250 mM sucrose, 2 mM EGTA, and 5 mM MOPS-KOH (pH 7.4). Mitochondria were washed and finely suspended in the medium composed of 120 mM KCl, 20 mM MOPS, and 0.5 mM EGTA (KME medium). Mitochondria protein was measured with the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, USA), according to the manufacturer's protocols. Mitochondria (1 mg of protein/ml) were incubated in KME medium containing 5 mM succinate, 2 lM rotenone, 10 mM NaCl, 800 pmol of A23187/mg of protein, and 5 mM phosphate (10 lCi/ml of 32 Pi as radiotracer) at 37°C. After 5 min, 1.2 mM ADP was added, and the reaction was stopped 30 s later by addition of 200 ll of 30% (w/v) cold trichloroacetic acid. Orthophosphate ( 32 Pi) was separated from ATP ([c 32 P]ATP) by molybdate method, as previously described (Villa-Bellosta, 2018. Briefly, 20 ll of sample was mixed with 400 lL of ammonium molybdate (to bind the orthophosphate) and 0.75 M sulfuric acid. Samples were then extracted with 800 ll of isobutanol/petroleum ether (4:1) to separate the phosphomolybdate from pyrophosphate and ATP. Next, 400 ll of the aqueous phase containing ATP was removed and subjected to radioactivity counting (Tri-Carb 2810TR, PerkinElmer).

Glycolysis assay
Extracellular acidification and lactate were measured with fluorometric kits (Abcam, ab197244 and ab169557, respectively), using a 96-well plate reader, according to the manufacturer's protocols. Extracellular acidification was determined in VSMCs (5 × 10 5 cells/ well) by changes in fluorescence intensity (excitation/emission wavelength of 380/615 nm), using a water-soluble and cellimpermeable pH-sensitive reagent. L-lactate was determined by changes in fluorescence intensity (excitation/emission wavelength of 535/587 nm). VSMCs (1 × 10 6 ) were homogenized with 110 ll cold lactate assay buffer on ice and centrifuged at 14,000 g for 5 min. Supernatant was measured in duplicate on the same plate.

Mitochondrial calcium and magnesium
For calcium accumulation by mitochondria, VSMCs were incubated in MEM containing 10 lCi/ml calcium-45 ( 45 Ca 2+ ) as a radiotracer. After 24 h, VSMCs were washed five times in MEM. Mitochondria were isolated from VSMCs by method of homogenization followed by low-and high-speed centrifugation at 4°C. The homogenate was centrifuged at 1,000 × g for 10 min. Mitochondria were sedimented at 3,500 × g for 15 min. The pellet contains the isolated mitochondria was washed and centrifuged at 12,000 × g for 15 min. The isolation medium contained 250 mM sucrose, 0.5 mM EGTA, and 5 mM MOPS-KOH (pH 7.4). EGTA was excluded from washing medium. Mitochondria pellets was resuspended in liquid scintillation counting (UltraGold, 6013329, PerkinElmer) and subjected to radioactivity counting (Tri-Carb 2810TR, PerkinElmer).
Liver mitochondria were isolated by the method of homogenization followed by low (1,000 × g, 10 min)-and high (3,500 × g, 15 min)-speed centrifugation at 4°C. Mitochondria were washed and finely suspended in dH 2 O. Mitochondria protein was measured with the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, USA), according to the manufacturer's protocols. Calcium was measured using a colorimetric QuantiChrom Calcium Assay Kit (BioAssay System, Hayward, CA) according to the manufacturer's protocols.
Mitochondrial magnesium concentration was measured in isolated mitochondria using mag-fura 2-AM (Thermo Fisher) as described previously (Kolisek et al, 2003). Briefly, mitochondria were loaded with 5 lM mag-fura 2-AM for 40 min at 25°C and washed twice to remove excess dye. Magnesium concentration was determined by measuring the fluorescence of the probe-loaded mitochondria with excitation at 340 and 380 nm, and emission at 510 nm. Mitochondrial magnesium was calculated from the 340/ 380 nm ratio according to the formula of Grynkiewicz et al (Grynkiewicz et al, 1985). The minimum (R min ) and maximum (R max ) ratios were obtained at the end of each experiment. R max was obtained by the addition of SDS (10% w/v) and MgCl 2 (25 mM). R min was detected by addition of EDTA (50 mM, pH 8).

Magnesium treatment
To assess the effect of supplemental magnesium on Lmna G609G/+ mice, their drinking water (containing 39 mg/l magnesium) was supplemented with 15 g/l MgCl 2 . Thereafter, the consumption of food (containing 0.17% magnesium) and that of both untreated and treated water was measured in the mice between 8 and 34 weeks of age. Magnesium intake was measured twice a week. Average daily food and water intake was calculated per day and by weight of the mouse in each cage.

Statistical analyses
Results are presented as means AE SD. The Kolmogorov-Smirnov test was used to assess the normality of the data. Student's t-test or one-way ANOVA and Tukey's multiple comparison posttest were used for statistical analysis. Asterisks near the top of the columns compare untreated or treated cells/mice with respect to control (wild type). Longevity was assessed by the Kaplan-Meier methods. All statistical analyses were performed using GraphPad Prism 5 software. Differences were considered significant at P < 0.05. Randomization or blinding was not applicable in this study.

Data availability
All source data of this study are available in the supplementary material of the article. Other data that support the findings of The paper explained Problem Loss of antioxidant capacity, excessive generation of reactive oxygen species (ROS) and mitochondrial dysfunction contribute to the main symptoms observed in premature aging associated to Hutchinson-Gilford progeria syndrome (HGPS).

Results
Here, we show that treatment with exogenous magnesium improved the mitochondrial function and reduced oxidative stress both in HGPS mice and vascular smooth muscle cells. Magnesium treatment improved mitochondrial ATP synthesis, and thus greater ATP availability, which is necessary for cellular energy supply and survival. Consistently, magnesium treatment improved mice longevity and reduced vascular calcification.

Impact
This study shows antioxidant properties of magnesium and its capacity to increase the ATP viability in a mouse model of HGPS, which in turn suggest novel possibilities for treating children with HGPS.
this study are available from the corresponding authors upon request.
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