Ca(2+) leakage out of the sarcoplasmic reticulum is increased in type I skeletal muscle fibres in aged humans

Abstract

Key points: The amount of Ca(2+) stored in the sarcoplasmic reticulum (SR) of muscle fibres is decreased in aged individuals, and an important question is whether this results from increased Ca(2+) leakage out through the Ca(2+) release channels (ryanodine receptors; RyRs). The present study examined the effects of blocking the RyRs with Mg(2+), or applying a strong reducing treatment, on net Ca(2+) accumulation by the SR in skinned muscle fibres from Old (∼70 years) and Young (∼24 years) adults. Raising cytoplasmic [Mg(2+)] and reducing treatment increased net SR Ca(2+) accumulation in type I fibres of Old subjects relative to that in Young. The densities of RyRs and dihydropyridine receptors were not significantly changed in the muscle of Old subjects. These findings indicate that oxidative modification of the RyRs causes increased Ca(2+) leakage from the SR in muscle fibres in Old subjects, which probably deleteriously affects normal muscle function both directly and indirectly.

Abstract: The present study examined whether the lower Ca(2+) storage levels in the sarcoplasmic reticulum (SR) in vastus lateralis muscle fibres in Old (70 ± 4 years) relative to Young (24 ± 4 years) human subjects is the result of increased leakage of Ca(2+) out of the SR through the Ca(2+) release channels/ryanodine receptors (RyRs) and due to oxidative modification of the RyRs. SR Ca(2+) accumulation in mechanically skinned muscle fibres was examined in the presence of 1, 3 or 10 mm cytoplasmic Mg(2+) because raising [Mg(2+)] strongly inhibits Ca(2+) efflux through the RyRs. In type I fibres of Old subjects, SR Ca(2+) accumulation in the presence of 1 mm Mg(2+) approached saturation at shorter loading times than in Young subjects, consistent with Ca(2+) leakage limiting net uptake, and raising [Mg(2+)] to 10 mm in such fibres increased maximal SR Ca(2+) accumulation. No significant differences were seen in type II fibres. Treatment with dithiothreitol (10 mm for 5 min), a strong reducing agent, also increased maximal SR Ca(2+) accumulation at 1 mm Mg(2+) in type I fibres of Old subjects but not in other fibres. The densities of dihydropyridine receptors and RyRs were not significantly different in muscles of Old relative to Young subjects. These findings indicate that Ca(2+) leakage from the SR is increased in type I fibres in Old subjects by reversible oxidative modification of the RyRs; this increased SR Ca(2+) leak is expected to have both direct and indirect deleterious effects on Ca(2+) movements and muscle function.

Figure 1. Effect of cytoplasmic [Mg²⁺] on SR Ca²⁺ accumulation in a type I muscle fibre from an Old and a Young subject

Force responses in type I fibres from Old (A) and Young (B) subjects. In each case, the skinned fibre was subjected to repeated load–release cycles in which the SR was loaded with Ca²⁺ for the indicated time in a load solution at pCa 6.7 with 1, 3 or 10 mm cytoplasmic free Mg²⁺ and then the SR Ca²⁺ released by exposing the fibre to ‘full release solution’ (see Methods). The first response in each sequence was that elicited upon releasing the endogenous SR Ca²⁺ (‘Endo’). The final response in each sequence shows the maximum force elicited by directly activating the contractile apparatus in a heavily Ca²⁺‐buffered solution at pCa 4.7 (see Methods). Time scale: 10 s during SR Ca²⁺ release; 30 s for maximum Ca²⁺‐activated force.

Figure 2. SR Ca²⁺ loading characteristics in type I fibre from an Old and a Young subject

Plots of relative SR Ca²⁺ content versus loading time when Ca²⁺ loading a type I fibre from an Old (A) or Young (B) subjects in the presence of 1, 3 or 10 mm Mg²⁺, derived from records in Figs 1 A and 1 B, respectively. Each value is expressed as a percentage of maximal SR Ca²⁺ capacity found with 180 s of loading in 1 mm Mg²⁺. Data sets fitted with best‐fit single exponential function. Data have been adjusted for the negative ordinate intercept resulting from releasing SR Ca²⁺ in the presence of 0.5 mm EGTA (see Methods). Half‐time for Ca²⁺ loading with 1 mm Mg²⁺ present was ∼16 s and ∼23 s in (A) and (B), respectively. Open circle symbols on fitted functions for 1 mm Mg²⁺ indicate the relative force–time integral found upon releasing the endogenous Ca²⁺ content, and correspond to ∼49% and 51% of the maximum content in fibres in (A) and (B), respectively.

Figure 3. Measure of maximum SR Ca²⁺ loading in 1 mm Mg²⁺ in fibres from Young (Y) and Old (O) subjects

Mean ± SD of the time integral of the force response upon full release of SR Ca²⁺ after loading fibres for 180 s in the presence of 1 mm Mg²⁺ (Fig. 1); values normalized to maximum Ca²⁺‐activated force measured in same fibre. n, number of fibres; N, number of subjects. ^#Value in the Old group significantly different from the matching value in the Young group (Student's paired two‐tailed t test).

Figure 4. Effect of raised [Mg²⁺] on maximal SR Ca²⁺ accumulation in individual fibres

Values denote SR Ca²⁺ content accumulated after 180 s of loading in the presence of 10 mm cytoplasmic Mg²⁺, expressed as a percentage of that accumulated in the same fibre in 1 mm Mg²⁺, measured as in Figs 1 and 2. Each particular symbol labels fibres obtained from a given subject: nine Old (O) and nine Young (Y) subjects. Horizontal lines indicate median value in each case. The presence of 10 mm Mg²⁺ resulted in greater SR Ca²⁺ accumulation in type I fibres of Old subjects compared to that in type I fibres of Young subjects.

Figure 5. Inverse relationship between SR Ca²⁺ leak measure and half‐time for SR Ca²⁺ accumulation in type I fibres of Old subjects

Relative SR Ca²⁺ accumulation in presence of 10 mm cytoplasmic Mg²⁺ (expressed as a percentage of that accumulated in 1 mm Mg²⁺) in individual type I fibres from Young and Old subjects versus half‐time of SR Ca²⁺ loading in presence of 1 mm Mg²⁺ (measured as in Fig 2). Pearson's correlation coefficient (r) was –0.56 for the fibres from the Old subjects (P < 0.05) indicating a negative correlation, and −0.35 for the Young type I fibres (P > 0.05, no significant correlation). The line of best fit is shown for each case.

Figure 6. Effect of reducing treatment (10 mm DTT) on SR Ca²⁺ accumulation

Mean ± SD of maximum SR Ca²⁺ accumulation (180 s of loading time) in 1 mm Mg²⁺ in type I and type II fibres from Young (Y) and Old (O) subjects following DTT treatment (10 mm for 5 min), expressed as a percentage of that accumulated in the same fibre before DTT treatment. n, number of fibres; N, number of subjects. #Value significantly greater than 100% (P < 0.025, Student's one‐tailed t test). Note that a small decrease in the amount of Ca²⁺ accumulation is observed upon successive measurements even without DTT treatment.

Figure 7. DHPR and RyR density in muscle homogenates from Old and Young subjects

Upper panels: western blots for DHPR α1 subunit and RyR1 in muscle homogenates from seven Old (O) and five Young (Y) subjects. Right‐hand lanes contained 1–8 μl of a mixed homogenate of muscle from all subjects, used for signal calibration. Bottom panel: myosin band on Coomassie‐stained post‐transfer gel. 4–12% BIS/TRIS Criterion precast gel.

Figure 8. DHPR and RyR density in single muscle fibres from Old and Young subjects

Western blots for DHPRα1, RyR1, SERCA2a, actin, MHCI and MHCIIa in individual fibres from Young (Y) and Old (O) subjects (single skinned fibre segment in each lane), with four right‐hand lanes containing 1–10 μl of a mixed homogenate of muscle from all subjects, used for signal calibration. Labels I and II denotes fibres containing predominantly MHCI and MHCIIa respectively; label II/I denotes a mixed fibre containing substantial amounts of both MHCIIa and MHCI. 4–12% Bis/Tris Criterion precast gel. Top panel: myosin band on Coomassie‐stained post‐transfer gel.