Sarcoplasmic reticulum Ca2+ release declines in muscle fibers from aging mice

This study hypothesized that decline in sarcoplasmic reticulum (SR) Ca²⁺ release and maximal SR-releasable Ca²⁺ contributes to decreased specific force with aging. To test it, we recorded electrically evoked maximal isometric specific force followed by 4-chloro-m-cresol (4-CmC)-evoked maximal contracture force in single intact fibers from the mouse flexor digitorum brevis muscle. Significant differences in tetanic, but not in 4-CmC-evoked, contracture forces were recorded in fibers from aging mice as compared to younger mice. Peak intracellular Ca²⁺ in response to 4-CmC did not differ significantly. SR Ca²⁺ release was recorded in whole-cell patch-clamped fibers in the linescan mode of confocal microscopy using a low-affinity Ca²⁺ indicator (Oregon green bapta-5N) with high-intracellular ethylene glycol-bis(α-aminoethyl ether)-N,N,N′N′-tetraacetic acid (20 mM). Maximal SR Ca²⁺ release, but not voltage dependence, was significantly changed in fibers from old compared to young mice. Increasing the duration of fiber depolarization did not increase the maximal rate of SR Ca²⁺ release in fibers from old compared to young mice. Voltage-dependent inactivation of SR Ca²⁺ release did not differ significantly between fibers from young and old mice. These findings indicate that alterations in excitation-contraction coupling, but not in maximal SR-releasable Ca²⁺, account for the age-dependent decline in intracellular Ca²⁺ mobilization and specific force.     

o-Phthalaldehyde activates the Ca(2+) release mechanism from skeletal muscle sarcoplasmic reticulum.

o-Phthalaldehyde (OPA) is a bifunctional reagent that forms an isoindole derivative by reacting with cysteine and lysine residues separated by approximately 0.3 nm. OPA inhibits sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity at low micromolar concentrations and induces Ca(2+) release from actively loaded SR vesicles by activating the ryanodine receptor from fast twitch skeletal muscle. Both ryanodine binding and single-channel activity show a biphasic concentration dependence. At low OPA concentrations (<100 microM), ryanodine binding and single channel activity are stimulated, while at higher concentrations, a time-dependent sequential activation and inhibition of receptor binding is observed. Activation is characterized by a Ca(2+)-independent increase in maximal receptor occupancy. Data are presented to support a model in which Ca(2+) channel and ryanodine binding activity are enhanced due to an intramolecular cross-linking of nearby lysine and nonhyperreactive cysteine residues. OPA complexation with endogenous lysine residue(s) is critical for receptor activation.     

Luminal Ca(2+) and the activity of sarcoplasmic reticulum Ca(2+) pumps modulate histamine-induced all-or-none Ca(2+) release in smooth muscle cells

We have studied histamine (HA)-evoked intracellular Ca(2+) release in single, freshly isolated myocytes from the guinea pig urinary bladder. Short applications of histamine (5 s) produced a thapsigargin (TG)-sensitive transient increase in intracellular calcium concentration ([Ca(2+)](i)). It was established that histamine and caffeine (Caff) released Ca(2+) from the same intracellular stores in these cells. Reducing the Ca(2+) content of internal stores by incubating cells with U-73343 or cyclopiazonic acid (CPA) inhibited the histamine-evoked Ca(2+) release in 69% and 60% of cells, respectively. Under these conditions, all cells released Ca(2+) in response to either caffeine or acetylcholine (ACh). However, decreasing internal Ca(2+) stores by removing external Ca(2+) inhibited histamine-induced Ca(2+) mobilization in only 22% of cells. A similar small fraction of cells was inhibited when sarcoplasmic reticulum (SR) Ca(2+) pumps were quickly blocked to avoid a significant reduction of luminal Ca(2+). In conclusion, lowering the luminal Ca(2+) content in combination with an impairment of the SR Ca(2+) pump activity significantly diminishes the ability of histamine to evoke an all-or-none intracellular Ca(2+) release.     

Reduced Ca(2+)-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum at low pH.

The purpose of this investigation was to determine the effects of reduced pH on Ca(2+)-induced Ca2+ release (CICR) from skeletal muscle sarcoplasmic reticulum (SR). Frog semitendinosus fiber bundles (1-3/bundle) were chemically skinned via saponin treatment (50 micrograms/mL, 20 min), which removes the sarcolemma and leaves the SR functional. The SR was first depleted of Ca2+ then loaded for 2 min at pCa (log free Ca2+ concentration) 6.6. CICR was then evoked by exposing the fibers to pCa 5-7 for 5-60 s. CICR was evoked both in the absence of ATP and Mg2+ and in the presence of beta, gamma-methyleneadenosine-5'-triphosphate (AMPPCP, a nonhydrolyzable form of ATP) and Mg2+. Ca2+ remaining in the SR was then assayed via caffeine (25 mM) contracture. In all cases, CICR evoked at pH 6.5 resulted in larger caffeine contractures than that evoked at 7.0, suggesting that more Ca2+ was released during CICR at the higher pH. Accordingly, rate constants for CICR were significantly greater at pH 7.0 than at pH 6.5. These results indicate that reduced pH depresses CICR from skeletal muscle SR.     

Sarcoplasmic reticulum interacts with the Ca(2+) indicator precursor fura-2-am

Fura-2-am, the pentaester precursor of the fluorescent Ca(2+) indicator fura-2, is modified when it is exposed to isolated skeletal muscle sarcoplasmic reticulum vesicles. The modified fura-2-am has enhanced fluorescence, is not sensitive to Ca(2+), and is partially bound to the SR membrane. The isolated product is further converted into fura-2 by esterase. It is suggested that the SR-induced modification is a selective enzymatic hydrolysis of only some of the five ester moieties on fura-2-am. A structure is proposed to account for the results. The potential for this effect of SR on fura-2-am to cause complications in measurements of in vivo intracellular free [Ca(2+)] is noted.     

Sarcoplasmic reticulum Ca(2+)-ATPase of sea cucumber smooth muscle: regulation by K(+) and ATP

Although several Ca(2+)-ATPase isoforms have been described in vertebrates, little is known about Ca(2+)-transport in the muscle of invertebrates. In the microsomal fraction obtained from the sea cucumber (Ludwigothurea grisea) longitudinal body wall smooth muscle, we identified a Ca(2+)-transport ATPase that is able to transport Ca(2+) at the expense of ATP hydrolysis. This enzyme has a high affinity for both Ca(2+) and ATP, an optimum pH around 7.0, and - different from the vertebrate sarcoplasmic reticulum Ca(2+)-ATPases isoforms so far described - is activated 3- to 5-fold by K(+) but not by Li(+), at all temperatures, Ca(2+) and ATP concentrations tested. Calcium accumulation by the sea cucumber microsomes is inhibited by Mg/ATP concentrations >1 mM and the accumulated Ca(2+) is released to the medium when the ATP concentration is raised from 0.1 to 4.0 mM.     

Effects of fatigue and training on sarcoplasmic reticulum Ca(2+) regulation in human skeletal muscle.

Little is known about fatigue and training effects on sarcoplasmic reticulum (SR) function in human muscle, and we therefore investigated this in eight untrained controls (UT), eight endurance-trained (ET), and eight resistance-trained athletes (RT). Muscle biopsies (vastus lateralis) taken at rest and after 50 maximal quadriceps contractions (180 degrees/s, 0.5 Hz) were analyzed for fiber composition, metabolites and maximal SR Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase activity. Fatigue reduced (P < 0.05) Ca(2+) release (42.1 +/- 3.8%, 43.4 +/- 3.9%, 31.3 +/- 6.1%), Ca(2+) uptake (43.0 +/- 5.2%, 34.1 +/- 4.6%, 28.4 +/- 2.8%), and Ca(2+)-ATPase activity (38.6 +/- 4.2%, 48.5 +/- 5.7%, 29.6 +/- 5.0%), in UT, RT, and ET, respectively. These decreases were correlated with fatigability and with type II fiber proportion (P < 0.05). Resting SR measures were correlated with type II proportion (r > or = 0.51, P < 0.05). ET had lower resting Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase (P < 0.05) than UT and RT (P < 0.05), probably because of their lower type II proportion; only minor effects were found in RT. Thus SR function is markedly depressed with fatigue in controls and in athletes, is dependent on fiber type, and appears to be minimally affected by chronic training status.     

Impaired sarcoplasmic reticulum Ca(2+) release rate after fatiguing stimulation in rat skeletal muscle.

The purpose of the study was to characterize the sarcoplasmic reticulum (SR) function and contractile properties before and during recovery from fatigue in the rat extensor digitorum longus muscle. Fatiguing contractions (60 Hz, 150 ms/s for 4 min) induced a reduction of the SR Ca(2+) release rate to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/dt) and relaxation (-dF/dt) were depressed by approximately 80% after stimulation. Recovery occurred in two phases: an initial phase, in which during the first 0.5-1 h the metabolic state recovered to resting levels, and a slow phase from 1-3 h characterized by a rather slow recovery of the mechanical properties. The recovery of SR Ca(2+) release rate was closely correlated to +dF/dt during the slow phase of recovery (r(2) = 0.51; P < 0.05). Despite a slowing of the relaxation rate, we did not find any significant alterations in the SR Ca(2+) uptake function. These data demonstrate that the Ca(2+) release mechanism of SR is sensitive to repetitive in vitro muscle contraction. Moreover, the results indicate that +dF/dt to some extent depends on the rate of Ca(2+) release during the slow phase of recovery.     

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel.

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method. Single channels were recorded in symmetric 0.25 M KCl media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from < 0.1 microM to approximately 250 microM, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 microM lumenal Ca2+ was also observed when the channel was minimally activated by < 1 microM cytosolic Ca2+ in the absence of ATP. With microM cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2 or MgCl2 to the lumenal side, which contained 50 microM Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the "fast" Ca2+ buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cls chamber increased cytosolic ATP-, lumenal Ca(2+)-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in "BAPTA-inaccessible" and "BAPTA-accessible" spaces, respectively.     

A mechanism for both capacitative Ca(2+) entry and excitation-contraction coupled Ca(2+) release by the sarcoplasmic reticulum of skeletal muscle cells

We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca(2+) uptake from outside cells increases when the endoplasmic reticulum (sarcoplasmic reticulum in muscle, or SR) store is decreased. Evidence for CCE rested on the use of thapsigargin (Tg), an inhibitor of the SR CaATPase and consequently transport of Ca(2+) from cytosol to SR, and measurements of cytosolic Ca(2+). When Ca(2+) is added to Ca(2+)-free cells in the presence of Tg, the measured cytosolic Ca(2+) rises. This has been universally interpreted to mean that as SR Ca(2+) is depleted, exogenous Ca(2+) crosses the plasma membrane, but accumulates in the cytosol due to CaATPase inhibition. Our goal in the present study was to examine CCE in more detail by measuring Ca(2+) in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca(2+) in the presence of Tg showed an increase in luminal Ca(2+) concentration in response to added exogenous Ca(2+). While we were able to reproduce the conventional demonstration of CCE-an increase of Ca(2+) in the cytosol in the presence of thapsigargin-we found that this process was inhibited by the prior addition of ryanodine (Ry), which inhibits the SR Ca(2+) release channel, the ryanodine receptor (RyR). This was also unexpected if Ca(2+) enters the cytosol first. When Ca(2+) was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca(2+) and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca(2+) transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca(2+) inhibited its own uptake, a known feature of the RyR. We confirmed that Ca(2+) enters the cells through the dihydropyridine receptor (DHPR, also known as the L-channel) by demonstrating inhibition by diltiazem. A previous suggestion to the contrary had used Mn(2+) in place of direct Ca(2+) measurements; we showed that Mn(2+) was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca(2+) flow in muscle cells. Our findings are entirely explained by a new model whereby Ca(2+) enters the SR from the extracellular space directly through a combined channel formed from the DHPR and the RyR. These are known to be in close proximity in skeletal muscle. Ca(2+) subsequently appears in the cytosol by egress through a separate, unoccupied RyR, explaining Ry inhibition. We suggest that upon excitation, the DHPR, in response to the electrical field of the plasma membrane, shifts to an erstwhile-unoccupied receptor, and Ca(2+) is released from the now open RyR to trigger contraction. We discuss how this model also resolves existing paradoxes in the literature, and its implications for other cell types.     

Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training

To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% (P < 0.01) after training. The 5-wk sprint training induced a significantly higher (P < 0.05) peak rate of AgNO(3)-stimulated Ca(2+) release from 709 (range 560-877; before) to 774 (596-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P < 0.05) increase in total number of RyR. No significant differences in Ca(2+) uptake rate and Ca(2+)-ATPase capacity were observed following the training, despite that the relative density of Ca(2+)-ATPase isoforms SERCA1 and SERCA2 had increased 41% and 55%, respectively (P < 0.05). These data suggest that high-intensity training induces an enhanced peak SR Ca(2+) release, due to an enhanced total volume of SR, whereas SR Ca(2+) sequestration function is not altered.     

Effects of imperatoxin A on local sarcoplasmic reticulum Ca(2+) release in frog skeletal muscle

We have investigated the effects of imperatoxin A (IpTx(a)) on local calcium release events in permeabilized frog skeletal muscle fibers, using laser scanning confocal microscopy in linescan mode. IpTx(a) induced the appearance of Ca(2+) release events from the sarcoplasmic reticulum that are approximately 2 s and have a smaller amplitude (31 +/- 2%) than the "Ca(2+) sparks" normally seen in the absence of toxin. The frequency of occurrence of long-duration imperatoxin-induced Ca(2+) release events increased in proportion to IpTx(a) concentrations ranging from 10 nM to 50 nM. The mean duration of imperatoxin-induced events in muscle fibers was independent of toxin concentration and agreed closely with the channel open time in experiments on isolated frog ryanodine receptors (RyRs) reconstituted in planar lipid bilayer, where IpTx(a) induced opening of single Ca(2+) release channels to prolonged subconductance states. These results suggest involvement of a single molecule of IpTx(a) in the activation of a single Ca(2+) release channel to produce a long-duration event. Assuming the ratio of full conductance to subconductance to be the same in the fibers as in bilayer, the amplitude of a spark relative to the long event indicates involvement of at most four RyR Ca(2+) release channels in the production of short-duration Ca(2+) sparks.     

Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.     

Dynamic regulation of sarcoplasmic reticulum Ca(2+) content and release by luminal Ca(2+)-sensitive leak in rat ventricular myocytes.

In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.