Role of myoplasmic phosphate in contractile function of skeletal muscle: studies on creatine kinase-deficient mice.

1. Increased myoplasmic inorganic phosphate (P(i)) has been suggested to have an important role in skeletal muscle fatigue, especially in the early phase. In the present study we used intact fast-twitch muscle cells from mice completely deficient in creatine kinase (CK(-/-)) to test this suggestion. These CK(-/-) muscle cells provide a good model since they display a higher P(i) concentration in the unfatigued state and fatigue without significant increase of P(i). 2. Tetanic contractions (350 ms duration) were produced in intact single muscle fibres. The free myoplasmic [Ca(2+)] ([Ca(2+)](i)) was measured with the fluorescent indicator indo-1. The force-[Ca(2+)](i) relationship was constructed from tetani at different frequencies. 3. Compared with wild-type fibres, CK(-/-) fibres displayed lower force in 100 Hz tetani and at saturating [Ca(2+)](i) (i.e. 100 Hz stimulation during caffeine exposure), higher tetanic [Ca(2+)](i) during the first 100 ms of tetanic stimulation, reduced myofibrillar Ca(2+) sensitivity when measurements were performed 100-200 ms into tetani, and slowed force relaxation that was due to altered cross-bridge kinetics rather than delayed Ca(2+) removal from the myoplasm. 4. In wild-type fibres, a series of 10 tetani resulted in reduced tetanic force, slowed force relaxation, and increased amplitude of [Ca(2+)](i) tails after tetani. None of these changes were observed in CK(-/-) fibres. 5. Complementary experiments on isolated fast-twitch extensor digitorum longus muscles showed a reduction of tetanic force and relaxation speed in CK(-/-) muscles similar to those observed in single fibres. 6. In conclusion, increased P(i) concentration can explain changes observed in the early phase of skeletal muscle fatigue. Increased P(i) appears to be involved in both fatigue-induced changes of cross-bridge function and SR Ca(2+) handling.

Inhibition of creatine kinase reduces the rate of fatigue-induced decrease in tetanic [Ca(2+)](i) in mouse skeletal muscle.

1. Ca(2+)-phosphate (P(i)) precipitation in the sarcoplasmic reticulum (SR) may cause reduced SR Ca(2+) release in skeletal muscle fatigue. To study this, we inhibited the creatine kinase (CK) reaction with 2,4-dinitro-1-fluorobenzene (DNFB). The hypothesis was that with inhibition of CK, phosphocreatine would not break down to creatine and P(i). Therefore P(i) transport into the SR would be limited and Ca(2+)-P(i) precipitation would not occur. 2. Intact single fibres from a mouse foot muscle were fatigued by repeated short tetani under control conditions or after exposure to DNFB (10 microM). The free myoplasmic concentrations of Ca(2+) ([Ca(2+)](i)) and Mg(2+) ([Mg(2+)](i)) were measured with indo-1 and mag-indo-1, respectively. Changes in [Mg(2+)](i) were assumed to reflect alterations in myoplasmic ATP concentration. 3. During the first 10 fatiguing tetani, tetanic [Ca(2+)](i) increased both in control and after DNFB exposure. Thereafter tetanic [Ca(2+)](i) fell and the rate of fall was about fourfold lower after DNFB exposure compared with control. 4. Under control conditions, there was a good relationship between declining tetanic [Ca(2+)](i) and increasing [Mg(2+)](i) during the final part of fatiguing stimulation. This correlation was lost after DNFB exposure. 5. In conclusion, the present data fit with a model where Ca(2+)-P(i) precipitation inhibits SR Ca(2+) release in fatigue produced by repeated short tetani. Furthermore, the results suggest that the rate of P(i) transport into the SR critically depends on the myoplasmic Mg(2+)/ATP concentration.

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.

Is creatine kinase responsible for fatigue? Studies of isolated skeletal muscle deficient in creatine kinase.

Creatine kinase (CK) is a key enzyme for maintaining a constant ATP/ADP ratio during rapid energy turnover. To investigate the role of CK in skeletal muscle fatigue, we used isolated whole muscles and intact single fibers from CK-deficient mice (CK(-/-)). With high-intensity electrical stimulation, single fibers from CK(-/-) mice displayed a transient decrease in both tetanic free myoplasmic [Ca(2+)] ([Ca(2+)](i), measured with the fluorescent dye indo-1) and force that was not observed in wild-type fibers. With less intense, repeated tetanic stimulation single fibers and EDL muscles, both of which are fast-twitch, fatigued more slowly in CK(-/-) than in wild-type mice; on the other hand, the slow-twitch soleus muscle fatigued more rapidly in CK(-/-) mice. In single wild-type fibers, tetanic force decreased and [Ca(2+)](i) increased during the first 10 fatiguing tetani, but this was not observed in CK(-/-) fibers. Fatigue was not accompanied by phosphocreatine breakdown and accumulation of inorganic phosphate in CK(-/-) muscles. In conclusion, CK is important for avoiding fatigue at the onset of high-intensity stimulation. However, during more prolonged stimulation, CK may contribute to the fatigue process by increasing the myoplasmic concentration of inorganic phosphate.

Mechanisms underlying reduced maximum shortening velocity during fatigue of intact, single fibres of mouse muscle.

1. The mechanism behind the reduction in shortening velocity in skeletal muscle fatigue is unclear. In the present study we have measured the maximum shortening velocity (V0) with slack tests during fatigue produced by repeated, 350 ms tetani in intact, single muscle fibres from the mouse. We have focused on two possible mechanisms behind the reduction in V0: reduced tetanic Ca2+ and accumulation of ADP. 2. During fatigue V0 initially declined slowly, reaching 90 % of the control after about forty tetani. The rate of decline then increased and V0 fell to 70 % of the control in an additional twenty tetani. The reduction in isometric force followed a similar pattern. 3. Exposing unfatigued fibres to 10 microM dantrolene, which reduces tetanic Ca2+, lowered force by about 35 % but had no effect on V0. 4. In order to see if ADP might increase rapidly during ongoing contractions, we used a protocol with a tetanus of longer duration bracketed by standard-duration tetani. V0 in these three tetani were not significantly different in control, whereas V0 was markedly lower in the longer tetanus during fatigue and in unfatigued fibres where the creatine kinase reaction was inhibited by 10 microM dinitrofluorobenzene. 5. We conclude that the reduction in V0 during fatigue is mainly due to a transient accumulation of ADP, which develops during contractions in fibres with impaired phosphocreatine energy buffering.