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

Abstract

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.

Figure 2. The myofibrillar Ca²⁺ sensitivity is lower in CK^–/– fibres when measurements are performed early in the tetanus

Representative [Ca²⁺]_i and force records from 40 Hz tetani produced in a wild-type fibre (A) and a CK^–/– fibre (B). Period of stimulation is indicated by bar below force records. Observe the different tetanic [Ca²⁺]_i patterns, with a gradual increase in the wild-type fibre but not in the CK^–/– fibre. This has an impact on the force-[Ca²⁺]_i curves from the same fibres, which are shown in C (wild-type fibre) and D (CK^–/– fibre). ○ (connected by dashed line) and • (connected by continuous line) refer to measurements performed 100-200 ms and 250-350 ms into tetani, respectively. Note that while the two force-[Ca²⁺]_i curves were similar in the wild-type fibre, there was a clear shift to the right with early measurements in the CK^–/– fibre.

Figure 1. Tetanic force is markedly smaller in CK^–/– fibres than in wild-type fibres despite similar or even higher [Ca²⁺]_i

Typical records of [Ca²⁺]_i and force from 100 Hz tetani produced in a wild-type fibre (A) and a CK^–/– fibre (B).[Ca²⁺]_i records are shown both with (dashed line) and without (continuous line) kinetic correction of the indo-1 signal. Observe that kinetic correction reveals a [Ca²⁺]_i transient at the onset of contraction, and that the decrease of [Ca²⁺]_i at the end of stimulation is slightly delayed without correction. Horizontal bars below force records show period of stimulation.

Figure 3. Force relaxation is markedly slower in CK^–/– fibres than in wild-type fibres

A, real force records of the relaxation phase of 100 Hz tetani obtained in a wild-type fibre (continuous line) and a CK^–/– fibre (dashed line). B, calcium-derived force records from the same fibres produced from tetanic [Ca²⁺]_i records and their respective force-[Ca²⁺]_i curves. The time axis starts at the end of tetanic stimulation. Note that the markedly slower relaxation in the CK^–/– fibre is not accompanied by a slowed relaxation of calcium-derived force.

Figure 4. The amplitude of tails of elevated [Ca²⁺]_i is increased after 10 repeated tetani in wild-type but not in CK^–/– fibres

Mean [Ca²⁺]_i records from the relaxation phase of wild-type fibres (Wt; A) and CK^–/– fibres (B). Records were obtained in the first (thin line) and tenth (thick line) repeated tetanus.

Figure 5. Tetanic force is smaller and relaxation slower in CK^–/– EDL muscles

Typical force records from 100 Hz tetani produced in EDL muscles from wild-type (continuous line) and CK^–/– (dashed line) mice. The period of stimulation is indicated below the records.