Activation of Ca(2+)-dependent protein kinase II during repeated contractions in single muscle fibres from mouse is dependent on the frequency of sarcoplasmic reticulum Ca(2+) release.

AIM: To investigate the importance and contribution of calmodulin-dependent protein kinase II (CaMKII) activity on sarcoplasmic reticulum (SR) Ca(2+)-release in response to different work intensities in single, intact muscle fibres. METHODS: CaMKII activity was blocked in single muscle fibres using either the inhibitory peptide AC3-I or the pharmacological inhibitor KN-93. The effect on tetanic force production and [Ca(2+)](i) was determined during work of different intensities. The activity of CaMKII was assessed by mathematical modelling. RESULTS: Using a standard protocol to induce fatigue (50x 70 Hz, 350 ms duration, every 2 s) the number of stimuli needed to induce fatigue was decreased from 47 +/- 3 contractions in control to 33 +/- 3 with AC3-I. KN-93 was a more potent inhibitor, decreasing the number of contractions needed to induce fatigue to 15 +/- 3. Tetanic [Ca(2+)](i) was 100 +/- 11%, 97 +/- 11% and 67 +/- 11% at the end of stimulation in control, AC3-I and KN-93 respectively. A similar inhibition was obtained using a high intensity protocol (20x 70 Hz, 200 ms duration, every 300 ms). However, using a long interval protocol (25x 70 Hz, 350 ms duration, every 5 s) no change was observed in either tetanic [Ca(2+)](i) or force when inhibiting CaMKII. A mathematical model used to investigate the activation pattern of CaMKII suggests that there is a threshold of active CaMKII that has to be surpassed in order for CaMKII to affect SR Ca(2+) release. CONCLUSION: Our results show that CaMKII is crucial for maintaining proper SR Ca(2+) release and that this is regulated in a work intensity manner.

Effects of a myosin-II inhibitor (N-benzyl-p-toluene sulphonamide, BTS) on contractile characteristics of intact fast-twitch mammalian muscle fibres.

We have examined the effects of N-benzyl-p-toluene sulphonamide (BTS), a potent and specific inhibitor of fast muscle myosin-II, using small bundles of intact fibres or single fibres from rat foot muscle. BTS decreased tetanic tension reversibly in a concentration-dependent manner with half-maximal inhibition at approximately approximately 2 microM at 20 degrees C. The inhibition of tension with 10 microM BTS was marked at the three temperatures examined (10, 20 and 30 degrees C), but greatest at 10 degrees C. BTS decreased active muscle stiffness to a lesser extent than tetanic tension indicating that not all of the tension inhibition was due to a reduced number of attached cross-bridges. BTS-induced inhibition of active tension was not accompanied by any change in the free myoplasmic Ca2+ transients. The potency and specificity of BTS make it a very suitable myosin inhibitor for intact mammalian fast muscle and should be a useful tool for the examination of outstanding questions in muscle contraction.

Prolonged force increase following a high-frequency burst is not due to a sustained elevation of [Ca2+]i.

A brief high-frequency burst of action potentials results in a sustained force increase in skeletal muscle. The present study investigates whether this force potentiation is the result of a sustained increase of the free myoplasmic [Ca2+] ([Ca2+]i). Single fibers from mouse flexor brevis muscles were stimulated with three impulses at 150 Hz (triplet) at the start of a 350-ms tetanus or in the middle of a 700-ms tetanus; the stimulation frequency of the rest of the tetanus ranged from 20 to 60 Hz. After the triplet, force was significantly (P < 0.05) increased between 17 and 20% when the triplet was given at the start of the tetanus and between 5 and 18% when the triplet was given in the middle (n = 7). However, during this potentiation, [Ca2+]i was not consistently increased. Hence, the increased force following a high-frequency burst is likely due to changes in the myofibrillar properties.

The role of Ca2+ and calmodulin in insulin signalling in mammalian skeletal muscle.

The role of Ca2+ in mediating effects of insulin on skeletal muscle has been widely debated. It is believed that in skeletal muscle Ca2+ has a permissive role, necessary but not of prime importance in mediating the stimulatory actions of insulin. In this review, we present evidence that insulin causes a localized increase in the concentration of Ca2+. Specifically, insulin induces a rise in near-membrane Ca2+ but not the bulk Ca2+ in the myoplasm. The rise in near-membrane Ca2+ is because of an influx through channels that can be blocked by L-type Ca2+ channel inhibitors. Calcium appears to exert some of its subsequent effects via calmodulin-dependent processes as calmodulin inhibitors block the translocation of glucose transporters and other enzymes as well as the insulin-stimulated increase in glucose transport.

Contraction and intracellular Ca(2+) handling in isolated skeletal muscle of rats with congestive heart failure.

A decreased exercise tolerance is a common symptom in patients with congestive heart failure (CHF). This decrease has been suggested to be partly due to altered skeletal muscle function. Therefore, we have studied contractile function and cytoplasmic free Ca(2+) concentration ([Ca(2+)](i), measured with the fluorescent dye indo 1) in isolated muscles from rats in which CHF was induced by ligation of the left coronary artery. The results show no major changes of the contractile function and [Ca(2+)](i) handling in unfatigued intact fast-twitch fibers isolated from flexor digitorum brevis muscles of CHF rats, but these fibers were markedly more susceptible to damage during microdissection. Furthermore, CHF fibers displayed a marked increase of baseline [Ca(2+)](i) during fatigue. Isolated slow-twitch soleus muscles of CHF rats displayed slower twitch contraction and tetanic relaxation than did muscles from sham-operated rats; the slowing of relaxation became more pronounced during fatigue in CHF muscles. Immunoblot analyses of sarcoplasmic reticulum proteins and sarcolemma Na(+),K(+)-ATPase showed no difference in flexor digitorum brevis muscles of sham-operated versus CHF rats. In conclusion, functional impairments can be observed in limb muscle isolated from rats with CHF. These impairments seem to mainly involve structures surrounding the muscle cells and sarcoplasmic reticulum Ca(2+) pumps, the dysfunction of which becomes obvious during fatigue.

Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions.

The present study investigated mitochondrial Ca2+ uptake and release in intact living skeletal muscle fibres subjected to bouts of repetitive activity. Confocal microscopy was used in conjunction with the Ca2+-sensitive dye Rhod-2 to monitor changes in mitochondrial Ca2+ in single Xenopus or mouse muscle fibres. A marked increase in the mitochondrial Ca2+ occurred in Xenopus fibres after 10 tetani applied at 4 s intervals. The mitochondrial Ca2+ continued to increase with increasing number of tetani. After the end of tetanic stimulation, mitochondrial Ca2+ declined to 50% of the maximal increase within 10 min and thereafter took up to 60 min to return to its original value. Depolarization of the mitochondria with FCCP greatly attenuated the rise in the mitochondrial Ca2+ evoked by repetitive tetanic stimulation. In addition, FCCP slowed the rate of decay of the tetanic Ca2+ transient which in turn led to an elevation of resting cytosolic Ca2+. Accumulation of Ca2+ in the mitochondria was accompanied by a modest mitochondrial depolarization. In contrast to the situation in Xenopus fibres, mitochondria in mouse toe muscle fibres did not show any change in the mitochondrial Ca2+ during repetitive stimulation and FCCP had no effect on the rate of decay of the tetanic Ca2+ transient. It is concluded that in Xenopus fibres, mitochondria play a role in the regulation of cytosolic Ca2+ and contribute to the relaxation of tetanic Ca2+ transients. In contrast to their important role in Xenopus fibres, mitochondria in mouse fast-twitch skeletal fibres play little role in Ca2+ homeostasis.

Functional significance of Ca2+ in long-lasting fatigue of skeletal muscle.

Repeated activation of skeletal muscle causes fatigue, which involves a reduced ability to produce force and slowed contraction regarding both the speed of shortening and relaxation. One important component in skeletal muscle fatigue is a reduced sarcoplasmic reticulum (SR) Ca2+ release. In the present review we will describe different types of fatigue-induced inhibition of SR Ca2+ release. We will focus on a type of long-lasting failure of SR Ca2+ release which is called low-frequency fatigue, because this type of fatigue may be involved in the muscle dysfunction and chronic pain experienced by computer workers. Paradoxically it appears that the Ca2+ released from the SR, which is required for contraction, may actually be responsible for the failure of SR Ca2+ release during low-frequency fatigue. We will also discuss the relationship between gross morphological changes in muscle fibres and long-lasting failure of SR Ca2+ release. Finally, a model linking muscle cell dysfunction and muscle pain is proposed.

Vacuole formation in fatigued skeletal muscle fibres from frog and mouse: effects of extracellular lactate.

Isolated, living muscle fibres from either Xenopus or mouse were observed in a confocal microscope and t-tubules were visualized with sulforhodamine B. Observations were made before and after fatiguing stimulation. In addition, experiments were performed on fibres observed in an ordinary light microscope with dark-field illumination. In Xenopus fibres, recovering after fatigue, t-tubules started to show dilatations 2-5 min post-fatigue. These swellings increased in size over the next 10-20 min to form vacuoles. After 2-3 h of recovery the appearance of the fibres was again normal and force production, which had been markedly depressed 10-40 min post-fatigue, was close to control. Vacuoles were not observed in mouse fibres, fatigued with the same protocol and allowed to recover. In Xenopus fibres, fatigued in normal Ringer solution and allowed to recover in Ringer solution with 30-50 mM L-lactate substituting for chloride (lactate-Ringer), the number and size of vacuoles were markedly reduced. Also, force recovery was significantly faster. Replacement of chloride by methyl sulphate or glucuronate had no effect on vacuolation. Resting Xenopus fibres exposed to 50 mM lactate-Ringer and transferred to normal Ringer solution displayed vacuoles within 5-10 min, but to a smaller extent than after fatigue. Vacuolation was not associated with marked force reduction. Mouse fibres, fatigued in 50 mM lactate-Tyrode (L-lactate substituting for chloride in Tyrode solution) and recovering in normal Tyrode solution, displayed vacuoles for a limited period post-fatigue. Vacuolation had no effect on force production. The results are consistent with the view that lactate, formed during fatigue, is transported into the t-tubules where it attracts water and causes t-tubule swelling and vacuolation. This vacuolation may be counteracted in vivo due to a gradual extracellular accumulation of lactate during fatigue.

Frog skeletal muscle fibers recovering from fatigue have reduced charge movement.

Following prolonged exercise, muscle force production is often impaired. One possible cause of this force deficit is impaired intracellular activation. We have used single skeletal muscle fibers from the lumbrical muscle of Xenopus laevis to study the effects of fatigue on excitation-contraction coupling. Fatigue was induced in 13 intact fibers. Five fibers recovered in normal Ringer only (fatigued-only fibers). The remaining eight fibers were subjected to a brief hypotonic treatment (F-H fibers) that is known to prolong the effects of fatigue. Intramembrane charge movement, changes in intracellular calcium concentration ([Ca2+]i) and force transients were measured in a single Vaseline gap chamber under voltage clamp. In F-H fibers, membrane capacitance was reduced. Confocal microscopy showed that this was not due to closure of the transverse tubules. The amount of normalized intramembrane charge was reduced from 21.0 +/- 2.8 nC/microF (n = 10) in rested fibers to 12.2 +/- 1.1 nC/microF in F-H fibers. However, the voltage dependence of intramembrane charge movement was unchanged. In F-H fibers, force production was virtually abolished. This was the consequence of the greatly reduced [Ca2+]i accompanying a depolarizing pulse. In recovering fatigued-only fibers, while the maximal available charge was not significantly smaller (18.3 +/- 1.1 nC/ microF), both calcium and force were reduced, albeit to a lesser extent than in F-H fibers. The data are consistent with a model where fatigue reduces the number of voltage sensors in the t-tubules and, in addition, alters the coupling between the remaining functional voltage sensors and the calcium channels of the sarcoplasmic reticulum.

Vacuole formation in fatigued single muscle fibres from frog and mouse.

Force recovery from fatigue in skeletal muscle may be very slow. Gross morphological changes with vacuole formation in muscle cells during the recovery period have been reported and it has been suggested that this is the cause of the delayed force recovery. To study this we have used confocal microscopy of isolated, living muscle fibres from Xenopus and mouse to visualise transverse tubules (t-tubules) and mitochondria and to relate possible fatigue-induced morphological changes in these to force depression. T-tubules were stained with either RH414 or sulforhodamine B and mitochondrial staining was with either rhodamine 123 or DiOC6(3). Fatigue was produced by repeated, short tetanic contractions. Xenopus fibres displayed a marked vacuolation which started to develop about 2 min after fatiguing stimulation, reached a maximum after about 30 min, and then receded in about 2 h. Vacuoles were never seen during fatiguing stimulation. The vacuoles developed from localised swellings of t-tubules and were mostly located in rows of mitochondria. Mitochondrial staining, however, showed no obvious alterations of mitochondrial structure. There was no clear correlation between the presence of vacuoles and force depression; for instance, some fibres showed massive vacuole formation at a time when force had recovered almost fully. Vacuole formation was not reduced by cyclosporin A, which inhibits opening of the non-specific pore in the mitochondrial inner membrane. In mouse fibres there was no vacuole formation or obvious changes in mitochondrial structure after fatigue, but still these fibres showed a marked force depression at low stimulation frequencies ('low-frequency fatigue'). Vacuoles could be produced in mouse fibres by glycerol treatment and these vacuoles were not associated with any force decline. In conclusion, vacuoles originating from the t-tubular system develop after fatigue in Xenopus but not in mouse fibres. These vacuoles are not the cause of the delayed force recovery after fatigue.

Insulin increases near-membrane but not global Ca2+ in isolated skeletal muscle.

It has long been debated whether changes in Ca2+ are involved in insulin-stimulated glucose uptake in skeletal muscle. We have now investigated the effect of insulin on the global free myoplasmic Ca2+ concentration and the near-membrane free Ca2+ concentration ([Ca2+]mem) in intact, single skeletal muscle fibers from mice by using fluorescent Ca2+ indicators. Insulin has no effect on the global free myoplasmic Ca2+ concentration. However, insulin increases [Ca2+]mem by approximately 70% and the half-maximal increase in [Ca2+]mem occurs at an insulin concentration of 110 microunits per ml. The increase in [Ca2+]mem by insulin persists when sarcoplasmic reticulum Ca2+ release is inhibited but is lost by perfusing the fiber with a low Ca2+ medium or by addition of L-type Ca2+ channel inhibitors. Thus, insulin appears to stimulate Ca2+ entry into muscle cells via L-type Ca2+ channels. Wortmannin, which inhibits insulin-mediated activation of glucose transport in isolated skeletal muscle, also inhibits the insulin-mediated increase in [Ca2+]mem. These data demonstrate a new facet of insulin signaling and indicate that insulin-mediated increases in [Ca2+]mem in skeletal muscle may underlie important actions of the hormone.

Effects of CO2-induced acidification on the fatigue resistance of single mouse muscle fibers at 28 degrees C.

The role of reduced muscle pH in the development of skeletal muscle fatigue is unclear. This study investigated the effects of lowering skeletal muscle intracellular pH by exposure to 30% CO2 on the number of isometric tetani needed to induce significant fatigue. Isolated single mouse muscle fibers were stimulated repetitively at intervals of 4-2.5 s by using 80-Hz, 400-ms tetani at 28 degrees C in Tyrode solution bubbled with either 5 or 30% CO2. Stimulation continued until tetanic force had fallen to 40% of the initial value. Exposure to 30% CO2 caused a significant fall in intracellular pH of approximately 0.3 pH unit but did not cause any significant changes in initial peak tetanic force. During the course of repetitive stimulation, intracellular pH fell by approximately 0.3 pH unit in both normal and acidified fibers. The number of tetani needed to reduce force to 40% of the initial value was not significantly different in 5 and 30% CO2 Tyrode. The sole effect of acidosis was to reduce the rate of relaxation of force, especially in fatigued fibers. It is concluded that, at 28 degrees C, acidosis per se does not accelerate the development of fatigue during repeated tetanic stimulation of isolated mouse skeletal muscle fibers.

Mechanisms underlying the reduction of isometric force in skeletal muscle fatigue.

A decline of isometric force production is one characteristic of skeletal muscle fatigue. In fatigue produced by repeated short tetani, this force decline can be divided into two components: a reduction of the cross-bridges' ability to generate force, which comes early; and a reduction of the sarcoplasmic reticulum Ca2+ release, which develops late in fatigue. Acidification due to lactic acid accumulation has been considered as an important cause of the reduced cross-bridge force production. However, in mammalian muscle it has been shown that acidification has little effect on isometric force production at physiological temperatures. By exclusion, in mammalian muscle fatigue, the reduction of force due to impaired cross-bridge function would be caused by accumulation of inorganic phosphate ions, which results from phosphocreatine breakdown. The reduction of sarcoplasmic reticulum Ca2+ release in late fatigue correlates with a decline of ATP and we speculate that the reduced Ca2+ release is caused by a local increase of the ADP/ATP ratio in the triads.

Mechanisms underlying the slow recovery of force after fatigue: importance of intracellular calcium.

Recovery of force production after an intense bout of activity may sometimes take several days, especially at low activation frequencies ('low frequency fatigue'). This slow recovery can also be observed in isolated muscle and single muscle fibres. The origin of the force deficit is failure of excitation-contraction coupling at the level of the triads. The most likely cause of the failure is an elevated intracellular Ca2+ level, but the site of action of Ca2+ is unclear. Available evidence does not support the involvement of Ca2+-activated proteases. Ca2+-induced damage to mitochondria or swelling of t-tubules do not seem to be causative factors. Other mechanisms are discussed, including possible detrimental effects of Ca2+-activated lipases, calmodulin, and reactive oxygen species.

The effect of intracellular pH on contractile function of intact, single fibres of mouse muscle declines with increasing temperature.

1. The effect of altered intracellular pH (pHi) on isometric contractions and shortening velocity at 12, 22 and 32 degrees C was studied in intact, single fibres of mouse skeletal muscle. Changes in pHi were obtained by exposing fibres to solutions with different CO2 concentrations. 2. Under control conditions (5% CO2), pHi (measured with carboxy SNARF-1) was about 0.3 pH units more alkaline than neutral water at each temperature. An acidification of about 0.5 pH units was produced by 30% CO2 and an alkalinization of similar size by 0% CO2. 3. In acidified fibres tetanic force was reduced by 28% at 12 degrees C but only by 10% at 32 degrees C. The force increase with alkalinization showed a similar reduction with increasing temperature. Acidification caused a marked slowing of relaxation and this slowing became less with increasing temperature. 4. Acidification reduced the maximum shortening velocity (V0) by almost 20% at 12 degrees C, but had no significant effect at 32 degrees C. Alkalinization had no significant effect on V0 at any temperature. 5. In conclusion, the effect of pHi on contraction of mammalian muscle declines markedly with increasing temperature. Thus, the direct inhibition of force production by acidification is not a major factor in muscle fatigue at physiological temperatures.

Increased tetanic force and reduced myoplasmic [P(i)] following a brief series of tetani in mouse soleus muscle.

Muscle performance is improved after a brief period of exercise (warm-up). One factor that is known to strongly affect force production is the myoplasmic concentration of inorganic phosphate ([P(i)]). Improved performance after warm-up may therefore be due to a reduction of [P(i)]. Herein, we show that after a warm-up protocol (15 tetani at 2-s intervals), tetanic force is increased by approximately 6% (P < 0.05) and [P(i)] is almost halved (P < 0.05) in isolated mouse soleus muscle. A warm-up protocol with longer intervals (15 tetani at 5-s intervals) reduced tetanic force and did not alter [P(i)]. We conclude that a reduction of [P(i)] contributes to the force-potentiating effect of warm-up.

Effects of repetitive tetanic stimulation at long intervals on excitation-contraction coupling in frog skeletal muscle.

1. Single skeletal muscle fibres of Xenopus frogs were used to investigate the possibility that excitation-contraction (E-C) coupling can be impaired under conditions of elevated intracellular free Ca2+ ([Ca2+]i). 2. Fibres were stimulated with a train of up to 200 tetani at 10 or 20s intervals; this long-interval stimulation (LIS) scheme was chosen to minimize fatigue. After LIS, fibres were exposed to hypotonic Ringer solution for 5 min. At the end of LIS, force was about 90% of the original and the hypotonic challenge did not result in any force depression. 3. Caffeine, terbutaline and 2,5-di(tert-butyl)-1,4-benzohydroquinone increased both basal and tetanic [Ca2+]i. In ten out of thirteen fibres, the presence of any of these drugs during LIS resulted in a force reduction to about 10% of the control when fibres were returned to normal Ringer solution after the hypotonic challenge. Force production was severely depressed for at least 20 min and then recovered to control levels within 120 min. 4. Neither protease inhibitors nor a scavenger of reactive oxygen species prevented the impairment of E-C coupling. 5. It is concluded that after a period of elevated [Ca2+]i, E-C coupling in frog skeletal muscle becomes sensitive to the mechanical stress induced by exposure to hypotonic solution. The underlying molecular basis for this remains unclear.

Augmented force output in skeletal muscle fibres of Xenopus following a preceding bout of activity.

1. The effect of a brief period of activity on subsequent isometric tetanic force production was investigated in single muscle fibres of Xenopus laevis. 2. Following a train of ten tetani separated by 4 s intervals, tetanic force was significantly augmented by about 10%. The tetanic force augmentation persisted for at least 15 min and then slowly subsided. A similar potentiation was seen following trains of five and twenty tetani. 3. During the period of tetanic force potentiation, tetanic calcium was reduced by more than 30%, and intracellular pH was reduced from 7.15 +/- 0.07 to 7.03 +/- 0.11 (n = 4). 4. Fibre swelling was greatest at 1 min and then subsided over 15-20 min and possibly accounted for a small part of the observed force potentiation. 5. A reduction in the inorganic phosphate (P1) concentration of more than 40% was found in fibres frozen in liquid nitrogen at the peak of force potentiation compared with resting fibres. 6. It is concluded that the augmentation of tetanic force found after a brief preceding bout of activity is due to a reduction in inorganic phosphate. This mechanism may underlie the improved performance observed in athletes after warm-up.

Reversible depression of action potentials and force production in frog single muscle fibres by calmodulin-inhibitors.

The effects of the calmodulin-inhibitors trifluoperazine, thioridazine and zaldaride maleate on the responses to electrical stimulation in isolated frog skeletal muscle fibres were investigated. All three drugs initially reduced the amplitude of the action potentials but potentiated twitch force. This was followed by a total loss of action potentials and force production. However, the resting membrane potential was not changed. The effects were completely reversible upon removal of the drugs. These results suggest that an intact calmodulin system is required for normal function of the sarcolemmal sodium channels of frog skeletal muscle.

Slow recovery of force in single skeletal muscle fibres.

After a bout of intense exercise, especially in untrained persons recovery of muscle force is often slow. Force depression is much more marked at low frequencies of stimulation than at high frequencies ("low-frequency fatigue') and recovery can take more than 1 day. Delayed force recovery is also seen in single muscle fibres from frog and mouse after fatigue induced by repeated, brief contractions. Evidence from our own and other laboratories indicates that the impairment is unlikely to result from metabolic changes and points to a defect in excitation-contraction coupling. We demonstrate that the likely site of failure is in the coupling between t-tubule depolarization and release of Ca2+ from the SR. The causative agent appears to be a localized increase in cytoplasmic Ca2+ which initiates some disruptive process, which can, however, be fully reversed, albeit slowly. Our experimental evidence does not support the involvement of Ca(2+)-activated proteases. Attempts to clarify the possible role of Ca(2+)-activated lipases (phospholipase A2) and Ca2+/calmodulin have been hampered by side-effects of available inhibitors. Efforts to clarify how Ca2+ exerts its effects are continuing.