Magnesium deficiency accelerates cellular senescence in cultured human fibroblasts

Abstract

Magnesium inadequacy affects more than half of the U.S. population and is associated with increased risk for many age-related diseases, yet the underlying mechanisms are unknown. Altered cellular physiology has been demonstrated after acute exposure to severe magnesium deficiency, but few reports have addressed the consequences of long-term exposure to moderate magnesium deficiency in human cells. Therefore, IMR-90 human fibroblasts were continuously cultured in magnesium-deficient conditions to determine the long-term effects on the cells. These fibroblasts did not demonstrate differences in cellular viability or plating efficiency but did exhibit a decreased replicative lifespan in populations cultured in magnesium-deficient compared with standard media conditions, both at ambient (20% O(2)) and physiological (5% O(2)) oxygen tension. The growth rates for immortalized IMR-90 fibroblasts were not affected under the same conditions. IMR-90 fibroblast populations cultured in magnesium-deficient conditions had increased senescence-associated beta-galactosidase activity and increased p16(INK4a) and p21(WAF1) protein expression compared with cultures from standard media conditions. Telomere attrition was also accelerated in cell populations from magnesium-deficient cultures. Thus, the long-term consequence of inadequate magnesium availability in human fibroblast cultures was accelerated cellular senescence, which may be a mechanism through which chronic magnesium inadequacy could promote or exacerbate age-related disease.

Fig. 1.

Long-term exposure to magnesium deficiency causes accelerated cellular senescence in primary but not immortalized IMR-90 cells. Cells were cultured in full medium (black circles) or magnesium-deficient media repleted to 13% (red circles), 50% (yellow circles), or 100% (green circles) of normal magnesium content. (A) Representative lifespan curves show change in PD over time cultured at ambient (20%) oxygen levels with data fit by point-to-point function. (B) Representative lifespan curves show change in PD over time cultured at physiologic (5%) oxygen levels with data fit by point-to-point function. (C) Average loss of PD (mean ± SD) for five independent lifespan curves from cells cultured at ambient (20%) oxygen levels is shown. The 99% confidence intervals were −1.46–5.54 for 100% Mg, 1.53–6.55 for 50% Mg, and 3.13–8.17 for 13% Mg conditions; asterisks indicate loss of PD values outside the 99% confidence interval of full medium condition. (D) Average loss of PD (mean ± SD) for four independent lifespan curves from cells cultured at physiologic (5%) oxygen levels is shown. The 99% confidence intervals were −0.43–3.61 for 100% Mg, −4.55–9.45 for 50% Mg, and 2.44–6.76 for 13% Mg conditions; asterisks indicate loss of PD values outside the 99% confidence interval of full medium condition. (E) Representative (n = 4) growth curves of SV-IMR-90 cells for comparable time of IMR-90 cells show change in PD over time cultured at ambient (20%) oxygen levels. Data for all conditions were fit to a linear regression function that converged with r² > 0.98 and indicated that slopes for each condition were not statistically different. (F) Representative (n = 2) growth curves of SV-IMR-90 cells for comparable time of IMR-90 cells show change in PD over time cultured at physiologic (5%) oxygen levels. Data for all conditions were fit to a linear regression function that converged with r² > 0.98 and indicated that slopes for each condition were not statistically different.

Fig. 2.

Long-term exposure to magnesium deficiency causes greater expression of senescence-associated β-galactosidase activity in IMR-90 cells. Cells were cultured in full medium or magnesium-deficient media repleted to 13% (13% Mg), 50% (50% Mg), or 100% (100% Mg) of normal magnesium content at ambient oxygen levels. (A) Representative micrographs show senescence-associated β-galactosidase activity as a function of media condition in late PD IMR-90 cells; four replicates at pH 6 (senescence-associated β-galactosidase activity) and one control at pH 7 (negative control) are shown for each media condition. (B) Percentage of cells that are positive senescence-associated β-galactosidase activity in three independent IMR-90 populations from early, middle, and late PD are shown. Asterisks indicate significant elevation over full medium control (P < 0.05, one-way ANOVA, Dunnett's multiple comparison post hoc test).

Fig. 3.

Long-term exposure to magnesium deficiency causes greater expression of senescence-associated protein expression in IMR-90 cells. Cells were cultured in different media conditions at ambient oxygen levels. (A) Autoradiographs show hybridization of p16^INK4a, p21^WAF1, or actin as a function of PD indicated across the top. (B) Expression levels of p16^INK4a and p21^WAF1 in relative densitometric units (RDU) were normalized to actin levels as a loading control, illustrating increased expression as a function of PD. (C) Representative autoradiographs show hybridization of p16^INK4a, p21^WAF1, or actin as a function of media condition in a population set with PD ranging from 40 to 45. Cells were cultured in full medium (F) or magnesium-deficient media repleted to 13% (13), 50% (50), or 100% (100) of normal magnesium content across the top; positive control peptide for antibody indicated by “+.” (D) Relative expression levels of p16^INK4a and p21^WAF1 expression were normalized to actin levels as a loading control and expressed as a percentage (mean ± SD, n = 2) of youngest PD (25), illustrating increased expression with decreasing magnesium condition.

Fig. 4.

Long-term exposure to magnesium deficiency causes accelerated telomere attrition in IMR-90 cells. Cells were cultured in different media conditions at ambient oxygen levels. (A) Representative autoradiograph shows mean TRF as a function of media condition (lanes 2–5) and PD (lanes 6–7) in IMR-90 cells. Cells were cultured in full medium (FULL) or magnesium-deficient media repleted to 13% (13% Mg), 50% (50% Mg), or 100% (100% Mg) of normal magnesium content (lanes 2–5) with PD ranging from 35 to 38. High (10.2 kbp) and low (3.9 kbp) TRF DNA standard was used as positive control for TRF quantitation. (B) The average telomere attrition rate derived from seven independent experiments with a wide range of PD (black circles) is shown; the solid line indicates fit to a linear regression function (r² = 0.72) and the dotted line corresponds to a 90% prediction band. The attrition rate was determined at 105 ± 17 bp lost per population doubling. Four independent experiments using populations from magnesium-deficient media repleted to 13% (red stars), 50% (yellow stars), or 100% (green stars) at early, middle, and late PD are overlaid. Mean TRF from middle and late PD populations cultured in 10% Mg medium conditions were outside the 90% prediction band (dotted line).