High intracellular [Ca²⁺] alters sarcoplasmic reticulum function in skinned skeletal muscle fibres of the rat

Abstract

1. The effect on sarcoplasmic reticulum (SR) function of exposure to high intracellular [Ca¥] was studied in mechanically skinned fibres from the extensor digitorum longus muscle of the rat, using caffeine to assay the SR Ca¥ content. 2. A 15 s exposure to 50 ìÒ Ca¥ irreversibly reduced the ability of the SR to loadÏretain Ca¥ and completely abolished depolarization-induced Ca¥ release, whereas a 90 s exposure to 10 ìÒ Ca¥ had no detectable effect on either function. The reduction in net SR Ca¥ uptake: (a) was near-maximal with treatment at 50 ìÒ Ca¥, (b) was unrelated to voltage-sensor function, and (c) persisted unchanged for > 20 min. The reduction was primarily due to a threefold increase in leakage of Ca¥ out of the SR. This increased leakage was not substantially blocked by the presence of 10 mÒ Mg¥ or 2 ìÒ Ruthenium Red. 3. The adverse effect on SR function of exposure to high [Ca¥] could also be observed by the reduction in the ability of the SR to maintain a low [Ca¥] within the skinned fibre in the face of elevated [Ca¥] METHODS Isolation of skinned fibresLong-Evans hooded rats (Rattus norvegicus) aged 14 to 20 weeks were anaesthetized by halothane (2 % vÏv) and killed by asphyxiation in accordance with guidelines of the La Trobe University Animal Ethics Committee. As described previously (Lamb & Stephenson, 1994), both extensor digitorum longus muscles were then removed and placed in paraffin oil and kept cool on ice. Single muscle fibres were dissected free at one end from the muscle and mechanically skinned. The skinned fibre segment was mounted onto a force transducer (AME875, SensoNor, Horten, Norway), and stretched to 120% of its resting length. The fibre was then placed into a 2 ml Perspex bath containing a potassium or sodium 1,6-diaminohexane-N,N,N' ,N'-tetraacetic acid (HDTA) solution to equilibrate before being stimulated by rapid substitution of an appropriate solution. All experiments were performed at room temperature (23 ± 2°C). SolutionsAll chemicals were obtained from Sigma unless stated otherwise. The make-up of the solutions used in the different parts of the experiments is set out below. All solutions had a pH of 7·10 ± 0·01 and an osmolality of 295 ± 5 mosmol kg¢, and unless stated otherwise had 1 mÒ free Mg¥ and 8 mÒ total ATP. Free [Ca¥] at ü 0·1 ìÒ was verified with a Ca¥-sensitive electrode (Orion, Cambridge, MA, USA) . Leak solution. K-HDTA solution with 2 mÒ total EGTA (pCa > 8), but otherwise similar to pre-equilibration solution. Used to allow Ca¥ leakage from the SR in the absence of Ca¥ uptake. Additional matching solutions were also made with 10 mÒ free Mg¥ (22·7 mÒ total magnesium) or with all K¤ replaced with Na¤ (for chronic T-system depolarization).Contractile apparatus experiments Relaxing solution. Potassium solution with 50 mÒ EGTA (pCa > 9), which is similar to the polarizing solution with all HDTA replaced with EGTA (see Stephenson & Williams, 1981).Maximum-activation solution. Same as Relaxing solution but with 50 mÒ CaEGTA (pCa 4·5). Referred to as 'M...

ATP consumption by sarcoplasmic reticulum Ca2+ pumps accounts for 50% of resting metabolic rate in mouse fast and slow twitch skeletal muscle

“…The amount of slippage/leakage of SERCA pumps increases with increasing temperature (16) and ADP concentration (38). Compared with the skinned fiber study by Lamb and Cellini (35), both the experimental temperature (23 vs. 30°C) and the ADP concentration in the fibers (ϳ0.1 vs. ϳ10 M) were higher in the present study. Higher coupling ratios have been reported for rabbit skeletal muscle preparations, and, in contrast to our calculations, the Ca 2ϩ -to-ATP ratio was higher in slow-twitch muscle SR vesicles (i.e., 0.98 Ca 2ϩ /1 ATP) than in fast-twitch skeletal muscle SR vesicles (i.e., 0.37 Ca 2ϩ /1 ATP) (44).…”

“…However, our values for ATP turnover by SERCAs strongly suggest that the Ca 2ϩ -to-ATP coupling ratio is actually much lower than 2:1 under physiological conditions. If the coupling ratio was 2 Ca 2ϩ to 1 ATP, the rate of Ca 2ϩ uptake (which is equal to the rate of SR Ca 2ϩ leakage in resting muscle) would be ϳ318 nmol·g Ϫ1 ·s Ϫ1 in EDL and ϳ418 nmol·g Ϫ1 ·s Ϫ1 in soleus, which is ϳ22-to 26-fold higher than what has been measured in mechanically skinned EDL and soleus fibers from the rat (35,38,39). Taking into account the ATP turnover rates that we measured in this study and the SR Ca 2ϩ leak rate reported by Lamb and Cellini (35) for rat EDL (which would be equal in soleus), we can calculate coupling ratios of ϳ0.09 Ca 2ϩ /1 ATP in EDL and ϳ0.07 Ca 2ϩ /1 ATP in soleus.…”

Sarcoplasmic reticulum function in slow- and fast-twitch skeletal muscles from mdx mice

“…Furthermore, the higher activity of Ca 2+ -activated potassium channels in mdx than control mice is clear functional evidence in favour of a basal subsarcolemmal increase of Ca 2+ [31]. Some authors have suggested that exposure of fibres to high [Ca 2+ ] reduces the ability of the sarcoplasmic reticulum to load and retain Ca 2+ [26]. This sarcoplasmic reticulum Ca 2+ efflux mechanism is not well understood and could occur by reversal of the Ca 2+ -ATPase, via a distinct channel or through the ryanodine receptor/Ca 2+ -release channels [15,41].…”

Effect of endurance training and acute exercise on sarcoplasmic reticulum function in rat fast- and slow-twitch skeletal muscles

“…This decline in function, referred to as fatigue, has a complex etiology which can involve various factors, e.g., reduced availability of energy, build-up of metabolites, and dysfunction of excitation-contraction coupling (for review see Fitts 1994). Although suggested already in 1976 by Eberstein and Sandow, alterations in the function of the sarcoplasmic reticulum (SR), which regulates the intracellular Ca 2+ concentration, have only recently been considered, as a major contributor to fatigue (Lamb and Cellini 1999).…”