Sarcoplasmic reticulum Ca2+ uptake and leak properties, and SERCA isoform expression, in type I and type II fibres of human skeletal muscle

Abstract

The Ca(2+) uptake properties of the sarcoplasmic reticulum (SR) were compared between type I and type II fibres of vastus lateralis muscle of young healthy adults. Individual mechanically skinned muscle fibres were exposed to solutions with the free [Ca(2+)] heavily buffered in the pCa range (-log10[Ca(2+)]) 7.3-6.0 for set times and the amount of net SR Ca(2+) accumulation determined from the force response elicited upon emptying the SR of all Ca(2+). Western blotting was used to determine fibre type and the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoform present in every fibre examined. Type I fibres contained only SERCA2 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.8, whereas type II fibres contained only SERCA1 and displayed half-maximal Ca(2+) uptake rate at ∼pCa 6.6. Maximal Ca(2+) uptake rate was ∼0.18 and ∼0.21 mmol Ca(2+) (l fibre)(-1) s(-1) in type I and type II fibres, respectively, in good accord with previously measured SR ATPase activity. Increasing free [Mg(2+)] from 1 to 3 mM had no significant effect on the net Ca(2+) uptake rate at pCa 6.0, indicating that there was little or no calcium-induced calcium release occurring through the Ca(2+) release channels during uptake in either fibre type. Ca(2+) leakage from the SR at pCa 8.5, which is thought to occur at least in part through the SERCA, was ∼2-fold lower in type II fibres than in type I fibres, and was little affected by the presence of ADP, in marked contrast to the larger SR Ca(2+) leak observed in rat muscle fibres under the same conditions. The higher affinity of Ca(2+) uptake in the type I human fibres can account for the higher relative level of SR Ca(2+) loading observed in type I compared to type II fibres, and the SR Ca(2+) leakage characteristics of the human fibres suggest that the SERCAs are regulated differently from those in rat and contribute comparatively less to resting metabolic rate.

Figure 1

Force responses elicited in type I fibre (A) and type II fibre (B) upon emptying the SR of all releasable Ca²⁺ after loading under indicated conditions. First response (‘Endo’) produced when releasing the endogenous SR Ca²⁺. Subsequent responses elicited after loading SR (for 5–20 s) in solutions with free [Ca²⁺] heavily buffered at indicated pCa and with amount of CaEGTA set at 5 mm to ensure a large constant rate of Ca²⁺ diffusion into the skinned fibre (see Methods and text). Last full release response elicited after loading for 180 s in solution at pCa 6.7 with moderate level of buffering (0.5 mm CaEGTA–0.5 mm EGTA) (labelled with *); time integral of response indicative of maximal SR Ca²⁺ capacity. Last two responses in each panel are the maximum Ca²⁺-activated force elicited by directly activating the contractile apparatus (with solution at pCa 4.7 with 50 mm CaEGTA) and the force response to a solution with pSr 5.2. Time scale in B: 5 s during SR Ca²⁺ release responses and 10 s during maximum activation and pSr 5.2 exposure. Inset in A: western blots for MHC and SERCA isoforms in same two fibres.

Figure 2

Analysis of data for two fibres shown in Fig. 1. Left-hand panels plot the relative amount of Ca²⁺ found in SR after loading for the specified time in a solution with 5 mm CaEGTA at indicated pCa (see Methods). Values represent the time integral of the force responses expressed relative to that obtained after maximal loading of the SR (at pCa 6.7 for 180 s, see Fig. 1). Back-extrapolation of the line of best fit for the data sets obtained when loading at pCa 7.0, 6.7 and 6.4 all gave a similar intercept value on the ordinate axis (mean ± SEM: −2.9 ± 1.8% and −19.5 ± 0.4% in A and B, respectively); all data then refitted with lines constrained to this mean intercept value. This negative intercept in effect indicates the true origin and hence how much Ca²⁺ has to be loaded into the SR and released in order to elicit any measurable force (see text). Right-hand panels plot the relative slope of the SR Ca²⁺ content versus load time fits shown in left-hand panels, with values expressed relative to that found at pCa 6.0. Best-fit Hill curves to these data gave the pCa for half-maximal uptake rate and Hill coefficient (h) as 6.86 and 2.1 for the type I fibre (A), and 6.59 and 1.4 for the type II fibre (B).

Figure 3

Superposition of average (continuous lines) and individual single fibre cases (dashed lines) of Hill curve fits to SR Ca²⁺ uptake data for all type I (red) and type II (blue) muscle fibres examined, determined as in Fig. 2. The mean values for the fits to the individual fibre data gave pCa_50(SR), the pCa for half-maximal SR Ca²⁺ uptake rate, as 6.79 ± 0.02 and 6.60 ± 0.02 in type I and type II fibres, respectively, with similar coefficients in the two cases (1.73 ± 0.09 and 1.68 ± 0.08, respectively). n denotes number of fibres and N the number of subjects.

Figure 4

Time required in load solution at pCa 7.0 to reload SR from empty back to original endogenous level in human type I fibres (•) and in type II fibres (▪). Heavily Ca²⁺-buffered load solution with 5 mm CaEGTA. Amount of endogenous Ca²⁺ initially present in SR ascertained in each fibre as in Fig. 1. n denotes number of fibres and N the number of subjects. Same fibres as in Fig. 3. Horizontal bars indicate mean.

Figure 5

Mean (+SEM) of relative amount of SR Ca²⁺ uptake in human type I and type II muscle fibres when loading at pCa 6.0 in presence of 1 mm (control), 3 mm or 10 mm free Mg²⁺. Loading examined at each [Mg²⁺] in each of four type I and four type II fibres (N = 2 subjects in both cases). Experiments performed similarly to that shown in Fig. 1 using 5 s loading in pCa 6.0 solution with 5 mm CaEGTA present. Amount of Ca²⁺ uptake normalized to that found with 1 mm Mg²⁺ (i.e. standard load conditions) in same fibre. No significant difference between Ca²⁺ uptake with 1 and 3 Mg²⁺.

Figure 6

Representative force responses in a type I (A) and in a type II (B) human muscle fibre when emptying the SR of all releasable Ca²⁺ with the full release solution, with or without a preceding 1 min leakage period in leak solution at pCa 8.5 and various [ADP] (see Methods). Before each leak–release cycle the SR was loaded for 1 min in solution at pCa 6.7 (0.5 mm CaEGTA). Maximum Ca²⁺-activated force (at pCa 4.7) and response of contractile apparatus to Sr²⁺ (at pSr 5.2) determined at end of experiment, as in Fig. 1. Time scale: 5 or 10 s during SR Ca²⁺ release, and 30 s for maximum and Sr²⁺-activated force.

Figure 7

Mean (±SEM) of relative amount of Ca²⁺ remaining in SR following a 1 min leak period at pCa 8.5 in presence of indicated [ADP] (∼0.1 μm, 31 μm or 1 mm) in type I and type II fibres from human vastus lateralis muscle and from rat soleus and EDL muscles. Experiments carried out as in Fig. 6. Content values expressed relative to that found with no leakage period, determined from bracketing responses in each fibre. n denotes number of fibres and N the number of human subjects or rats. *Significantly different from data for 0.1 μm ADP. #Significantly different from data for 31 μm ADP.