Title: Dietary magnesium supplementation improves lifespan in a mouse model of progeria
Abstract: Article16 August 2020Open Access Source DataTransparent process Dietary magnesium supplementation improves lifespan in a mouse model of progeria Ricardo Villa-Bellosta Corresponding Author Ricardo Villa-Bellosta [email protected] orcid.org/0000-0002-1680-552X Fundación Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Ricardo Villa-Bellosta Corresponding Author Ricardo Villa-Bellosta [email protected] orcid.org/0000-0002-1680-552X Fundación Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Author Information Ricardo Villa-Bellosta *,1 1Fundación Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Madrid, Spain *Corresponding author. Tel: +34 91 550 48 97; E-mail: [email protected] EMBO Mol Med (2020)12:e12423https://doi.org/10.15252/emmm.202012423 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Aging is associated with redox imbalance according to the redox theory of aging. Consistently, a mouse model of premature aging (LmnaG609G/+) 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. LmnaG609G/+ mice also exhibited reduced mitochondrial ATP synthesis secondary to ROS-induced mitochondrial dysfunction. Treatment of LmnaG609G/+ 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 LmnaG609G/+ 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. Synopsis A mouse model of Hutchinson-Gilford progeria syndrome (HGPS) exhibited reduced ATP availability and elevated oxidative stress, two hallmarks of aging. Treatment with dietary magnesium improved the longevity of HGPS mice. The mitochondrial membrane potential is essential for H+ -coupled ATP and NADPH synthesis, which is necessary for survival. Dietary magnesium treatment improved mitochondrial ATP synthesis and the NADPH-dependent glutathione redox system in HGPS mice. Magnesium acted as an antioxidant in mitochondria, thereby reducing the synthesis of reactive oxygen species and increasing the antioxidant capacity, which in turn improved mitochondrial dysfunction. Magnesium treatment reduced vascular calcification, a hallmark of HGPS, in HGPS mice. 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. 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 non-inherited 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 LmnaG609G/+ 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 (Villa-Bellosta et al, 2013). 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 (Villa-Bellosta et al, 2013). In the present study, we aimed to determine whether magnesium supplementation ameliorates vascular calcification and improves longevity in LmnaG609G/+ mice. Results Magnesium improves LmnaG609G/+ 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, 2006; Varela 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 LmnaG609G/+ mice and their wild-type littermates. Notably, microscopy images showed an apparent similar cellular morphology in both genotypes during its growth (Fig EV1A). However, LmnaG609G/+ 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 ± 0.07 versus 0.23 ± 0.06 divisions per day; Fig EV1C; Appendix Table S1). Click here to expand this figure. Figure EV1. Magnesium improves LmnaG609G/+ VSMC viabilityVSMCs were incubated in MEM containing 10% FBS and 0.8 mM magnesium (wild-type and untreated LmnaG609G/+ VSMCs) or 1.8 mM magnesium (treated LmnaG609G/+ VSMCs) from passage 1 to passage 8 (P8). A. Representative microscopy images (10x; scale bar: 100 μm) of wild-type and LmnaG609G/+ VSMCs at passage 10. B. Number of replicative cells at the indicated times. Cell count begins at passage 8 and ends after 60 days. C. Mean number of divisions per day over the first 30 days. D. Replicative incorporation of 5-bromodeoxyuridine (BrdU) into DNA. E. Cell viability measured using the cleavage of tetrazolium salt by cellular mitochondria dehydrogenases. F. Intracellular ATP content. G. β-galactosidase (β-Gal) activity. Data information: Results are presented as the mean ± 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. Download figure Download PowerPoint To determine the status of DNA replication, the replicative incorporation of 5-bromodeoxyuridine (BrdU) was assessed (Fig EV1D; Appendix Table S1). DNA synthesis in LmnaG609G/+ VSMCs occurred at a 44% slower rate than in wild-type cells. Notably, LmnaG609G/+ 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 ± 0.05), and the replicative incorporation of BrdU (75% of wild type). Cellular activity, measured as cellular mitochondrial dehydrogenase activity, was significantly lower (by 35%) in LmnaG609G/+ VSMCs than in control cells (Fig EV1E; Appendix Table S1). Moreover, LmnaG609G/+ VSMCs had significantly lower (40%) intracellular ATP concentrations versus control cells (Fig EV1F; Appendix Table S1). In addition, senescence-associated β-galactosidase (β-gal) activity was significantly higher (3-fold) in LmnaG609G/+ VSMCs than in wild-type cells (Fig EV1G; Appendix Table S1). Notably, LmnaG609G/+ VSMCs treated with magnesium-enriched medium showed significantly higher intracellular ATP (24%) and cellular activity (21%) than untreated LmnaG609G/+ VSMCs. In contrast, LmnaG609G/+ VSMCs treated with magnesium-enriched medium showed significantly lower β-gal activity (33%) than untreated LmnaG609G/+ VSMCs. Magnesium improves mitochondrial ATP synthesis in LmnaG609G/+ VSMCs Previous studies have demonstrated mitochondrial dysfunction in progeria (Rivera-Torres et al, 2013; Villa-Bellosta et al, 2013; Aliper et al, 2015). Both oxygen consumption ratio (OCR) and ATP synthesis were significantly lower (by 41% and 39%, respectively) in LmnaG609G/+ VSMCs than in wild-type cells (Fig 1A and B; Appendix Table S2). Moreover, LmnaG609G/+ VSMCs had significantly lower mitochondrial membrane potential (ΔΨm; 37%), assessed using the red-to-green ratio of JC-10 fluorescence, than wild-type cells (Fig 1C; Appendix Table S2). Notably, LmnaG609G/+ VSMCs showed significant higher ΔΨm (32%), OCR (37%), and mitochondrial ATP synthesis (31%) when incubated in a magnesium-enriched medium. Figure 1. Magnesium improves ATP synthesis in LmnaG609G/+ 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 ± 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. Source Data for Figure 1 [emmm202012423-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Magnesium ameliorates mitochondrial oxidative stress in LmnaG609G/+ 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′,7′-dichlorofluorescin diacetate (DCFDA), a fluorogenic dye that can be used to quantify hydroxyl, peroxyl, and other ROS activities within the cell. LmnaG609G/+ 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 (O2−) and hydrogen peroxide (H2O2) were present in significantly higher (1.6-fold and 2.3-fold, respectively) concentrations in LmnaG609G/+ 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 H2O2, and by 29% for O2−) in LmnaG609G/+ VSMCs incubated in magnesium-enriched medium. Click here to expand this figure. Figure EV2. Magnesium ameliorates oxidative stress in LmnaG609G/+ VSMCs A, B. (A) Reactive oxygen species, and (B) superoxide and hydrogen peroxide radicals generated by the indicated VSMC types. C, D. (C) Total antioxidant capacity and (D) total glutathione (which includes reduced -GSH- and oxidized -GSSG- glutathione), reduced glutathione (GHS), and the ratio of reduced and oxidized glutathione (GSSG) in the indicated cell types. E, F. (E) Glutathione reductase (GR) activity, and (F) NADPH:NADP+ ratio and total NADPH (NADPH + NADP+) in the indicated VSMC types. G. The boxed scheme shows the NADPH-coupled glutathione redox systems, H+-coupled ATP synthesis by mitochondrial ATP synthase, and H+-coupled synthesis of NADPH by mitochondrial NADPH transhydrogenase (NNT). ΔΨm: mitochondrial membrane potential. Data information: Results are presented as the mean ± 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. Download figure Download PowerPoint 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 Cu2+ reduction assay. LmnaG609G/+ VSMCs showed significantly lower TAC (38%) than wild-type VSMCs (Fig EV2C; Appendix Table S3). This reduction was significantly ameliorated (by 27%) in LmnaG609G/+ 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, which includes GSH and oxidized glutathione (GSSG), and glutathione reductase (GR) activity were significant lower in LmnaG609G/+ 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. LmnaG609G/+ 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 LmnaG609G/+ VSMCs, although the GR activity and total glutathione concentration were similar in treated and untreated LmnaG609G/+ 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+), LmnaG609G/+ 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 LmnaG609G/+ VSMCs. Magnesium ameliorates acidification-induced mitochondrial calcium overload An increase in glycolysis that compensates for the loss of mitochondrial ATP synthesis has previously been shown in patient cells (Rivera-Torres et al, 2013). LmnaG609G/+ VSMCs showed higher cytosolic ATP synthesis (1.8-fold, Fig 2A; Appendix Table S4), lactate production (1.9-fold, Fig 2B; Appendix Table S4), and extracellular acidification (2.1-fold, Fig 2C; Appendix Table S4) than wild-type cells. However, LmnaG609G/+ VSMCs incubated in a magnesium-enriched medium showed significantly lower intracellular lactate concentration (19%) and extracellular acidification (14%). In contrast, cytosolic ATP synthesis was 21% higher in treated LmnaG609G/+ VSMCs than in untreated cells. This result is consistent with the notion that magnesium increases the activities of the ATP-coupled glycolytic enzymes hexokinase, phosphofructokinase, phosphoglycerate kinase, and pyruvate kinase (Pilchova et al, 2017). Figure 2. Magnesium reduces acidification-induced mitochondrial calcium overload A. Cytosolic ATP synthesis in the indicated VSMC types, measured by incorporation of phosphate-32 (32Pi) into ADP. [32Pi]-ATP was separated from 32Pi using the molybdate method, as explained in the Materials and Methods section. B, C. (B) Intracellular lactate concentration and (C) external acidification in the indicated VSMC types. D. Calcium accumulation in mitochondria after 24 h of incubation in MEM containing 10 μCi/ml calcium-45 (45Ca2+) as a radiotracer. E. Magnesium concentration in isolated mitochondria. F. 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+/Ca2+ exchanger (NCX) is forced into reverse mode to dispose of excess Na+, resulting in cytosolic calcium overload. This Ca2+ 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 μM) inhibits mitochondrial ATP synthase. G-6-P: glucose-6-phosphate. Data information: Results are presented as the mean ± 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. Source Data for Figure 2 [emmm202012423-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Intracellular acidification can lead to cytosolic and mitochondrial calcium overload, which depolarizes ΔΨm to limit ATP production and stimulates mitochondrial ROS generation and permeability transition (Brookes et al, 2004; Görlach et al, 2015; Santulli et al, 2015). LmnaG609G/+ VSMCs incubated with 45Ca2+ as a radiotracer showed significantly higher (55%) mitochondrial calcium than wild-type cells, which was significantly reduced (by 21%) in treated LmnaG609G/+ VSMCs (Fig 2D). In addition, LmnaG609G/+ VSMCs showed significantly lower (35%) mitochondrial magnesium than wild-type cells, which was significantly increased (by 37%) in treated LmnaG609G/+ VSMCs (Fig 2E and F). Magnesium prevents phosphate-induced LmnaG609G/+ VSMC calcification Previous studies show that calcification can occur without cellular activity, both in cultured devitalized aortas (Villa-Bellosta, 2018) and in fixed smooth muscle cells (Villa-Bellosta & Sorribas, 2009; Villa-Bellosta et al, 2011). To determine the effect of magnesium on vascular calcification, treated and untreated LmnaG609G/+ VSMCs were incubated in 2 mM phosphate-calcifying medium. Phosphate-induced calcification was then assessed in both living and fixed cells (Fig 3A–G; Appendix Table S5). Untreated LmnaG609G/+ VSMCs showed 11-fold higher (in live cells) and 17-fold higher (in fixed cells) calcium deposition after 7 days of incubation in phosphate-calcifying medium. However, treated living LmnaG609G/+ VSMCs showed significantly lower calcium accumulation (7.3-fold), although the calcium content in fixed cells was similar in treated and untreated LmnaG609G/+ 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 LmnaG609G/+ 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 activity-dependent mechanism, and not by direct binding to calcium phosphate crystals, preventing their formation and growth. Finally, the capacity to inhibit calcification (ΔCa2+) was calculated as the difference in calcium deposition in living versus fixed cells (Ca2+ in fixed cells − Ca2+ in living cells). The ΔCa2+ 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 ΔCa2+ in both treated and untreated cells. Figure 3. Magnesium improves LmnaG609G/+ vascular smooth muscle cell calcification A. Scheme showing the principle of the measurement. LmnaG609G/+ VSMCs were incubated in MEM (containing 0.8 mM magnesium; untreated) or in magnesium-enriched MEM (containing 1.8 mM magnesium; treated) from passage 1 to passage 8. Then, cells were incubated overnight in MEM containing 0.1% FBS and some cells were fixed, as described in the Materials and Methods section. Then, cells were incubated in MEM (containing 0.1% FBS) with 1 or 2 mM phosphate, 1.8 mM magnesium (+Mg), 100 μM pyrophosphate (+PPi), or 500 μM phosphonoformic acid (+PFA). After 7 days of incubation, during which the media were replaced daily, the calcium content was measured as described in the Materials and Methods section. B. Representative time-course of 2 mM phosphate on calcification of LmnaG609G/+ VSMCs (up). Calcification was visualized with Alizarin red. Representative microscopic images (10x; scale bar: 100 μm) showing calcification of treated and untreated LmnaG609G/+ VSMCs (down). C–F. Measures of calcium in treated living LmnaG609G/+ VSMCs (C), treated fixed LmnaG609G/+ VSMCs (D), untreated living LmnaG609G/+ VSMCs (E), and untreated fixed LmnaG609G/+ VSMCs (F). G. The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells (ΔCa2+). Data information: Results are presented as the mean ± 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. Source Data for Figure 3 [emmm202012423-sup-0006-SDataFig3.pdf] Download figure Download PowerPoint 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 LmnaG609G/+ mice, although they were significantly lower in 21- and 34-week-old LmnaG609G/+ mice than in wild-type littermates (Table EV1). To assess the effect of supplemental magnesium on LmnaG609G/+ mice, their drinking water was supplemented with MgCl2. 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 LmnaG609G/+ mice was similar (3.46 ± 0.77 versus 3.53 ± 0.72 g/day/mouse and 3.96 ± 0.62 versus 4.01 ± 0.73 ml/day/mouse, respectively). Therefore, the total magnesium intake by treated LmnaG609G/+ mice was significantly higher (4.6-fold) than that by untreated LmnaG609G/+ mice (976.2 ± 261.7 versus 213.9 ± 45.0 mg/day/kg; Fig 4A, see Materials and Methods section). Notably, the plasma magnesium concentration was significantly higher in treated LmnaG609G/+ mice than in untreated LmnaG609G/+ mice (1.02 ± 0.06 versus 0.96 ± 0.05 mM; Fig 4B; Appendix Table S6). Finally, the total calcium content of aortas obtained from treated LmnaG609G/+ mice was significantly lower than that of aortas obtained from untreated LmnaG609G/+ mice (401.5 ± 77.7 versus 741.9 ± 101.6 μg/g aorta; Fig 4C; Appendix Table S6). Figure 4. Oral magnesium treatment improves the longevity of LmnaG609G/+ mice A, B. (A) Magnesium intake and (B) plasma magnesium concentration in 34-week-old untreated and treated LmnaG609G/+ mice (n = 16). C. Calcium content of aortas obtained from 34-week-old wild-type mice and untreated and treated LmnaG609G/+ mice (n = 20). D. Body masses of 34-week-old untreated and treated LmnaG609G/+ mice. E. Kaplan–Meier graph for untreated and treated LmnaG609G/+ mice (n = 16). F. Representative photographs of 40-wk-old wild-type, untreated, and treated LmnaG609G/+ mice. Data information: Results are presented as the mean ± 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. Source Data for Figure 4 [emmm202012423-sup-0007-SDataFig4.pdf] Download figure Download PowerPoint Magnesium improves the longevity of HGPS mice The body mass of 34-wk-old treated LmnaG609G/+ mice was significantly higher (10%) than that of untreated LmnaG609G/+ mice (26.5 ± 1.1 versus 24.0 ± 2.4 g; Fig 4D). Moreover, the median survival time of treated LmnaG609G/+ mice was extended from 38.2 weeks to 42.9 weeks (Fig 4E and F). Magnesium improves the antioxidant status of HGPS mice Liver homogenates from LmnaG609G/+ mice had 42% lower TAC (Fig EV3A; Appendix Table S7), 38% lower total glutathione (Fig EV3B; Appendix Table S7), 48% lower GSH:GSSG ratio (Fig EV3C; Appendix Table S7), 55% lower NADPH:NAD+ ratio (Fig EV3D; Appendix Table S7), and 43% lower GR activity (Fig EV3E; Appendix Table S7) than wild-type mice, implying the presence of an impairment in the NADPH-coupled GR redox system. Notably, treated LmnaG609G/+ mice showed significant improvements in TAC (26%), GSH:GSSG ratio (52%), and NADPH:NAD ratio (45%) compared with untreated LmnaG609G/+ mice. However, total glutathione and GR activity were not significantly better in treated LmnaG609G/+ mice versus untreated LmnaG609G/+ mice. Click here to expand this figure. Figure EV3. Magnesium improves the NADPH-coupled glutathione redox status in LmnaG609G/+ miceLiver homogenates were obtained from 34-week-old wild-type, untreated, or treated LmnaG609G/+ mice. A–C. (A) Total antioxidant capacity, (B) total glutathione (which includes reduced and oxidized glutathione), and (C) the ratio of reduced (GSH) and oxidized (GSSG) in the indicated experimental mouse groups. D, E. (D) The NADPH:NADP+ ratio and (E) glutathione reductase (GR) activity. Data information: Results are presented as mean ± SD (n = 16). One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05; ***P < 0.001. Source data are available online for this figure. Download figure Download PowerPoint Magnesium improves ATP synthesis in HGPS mice Liver homogenates from untreated LmnaG609G/+ 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 LmnaG609G/+ 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 LmnaG609G/+ 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 LmnaG609G/+ than wild-type mice, but these defects were significantly ameliorated in treated LmnaG609G/+ mice (Fig 5D and E; Appendix Table S8), Notably, the subunits of these mitochondrial complexes are encoded by mitochondrial DNA. Figure 5. Magnesium improves mitochondrial ATP synthesis in LmnaG609G/+ mice A. ATP concentration in liver homogenates obtained from 34-week-old wild-type, untreated, or treated LmnaG609G/+ mice. B. Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups. C. Magnesium concentration in isolated mitochondria. D. Activities of the indicated mitochondrial complexes (I, II, III, IV, and V) in the absence or presence of rotenone (2 μM), 2-theno