Creatine kinase injection restores contractile function in creatine-kinase-deficient mouse skeletal muscle fibres

Abstract

Viable genetically engineered animals generally exhibit adaptations to the altered genotype, which may mask the role of the protein of interest. We now describe a novel method by which the direct effects of the altered genotype can be distinguished from secondary adaptive changes in isolated adult skeletal muscle cells. We studied contractile function and intracellular Ca2+ handling in single skeletal muscle fibres that are completely deficient of creatine kinase (CK; CK-/-) before and after microinjection of purified CK (injected together with the fluorescent Ca2+ indicator indo-1). The mean total CK activity after CK injection was estimated to be approximately 4 mM s-1, which is approximately 5 % of the activity in wild-type muscle fibres. After CK injection, CK-/- fibres approached the wild-type phenotype in several aspects: (a) the free myoplasmic [Ca2+] ([Ca2+]i) increased and force showed little change during a period of high-intensity stimulation (duty cycle, i.e. tetanic duration divided by tetanic interval = 0.67); (b) [Ca2+]i did not decline during a brief (350 ms) tetanus; (c) during low-intensity fatiguing stimulation (duty cycle = 0.14), tetanic [Ca2+]i increased over the first 10 tetani, and thereafter it decreased; (d) tetanic [Ca2+]i and force did not display a transient reduction in the second tetanus of low-intensity fatiguing stimulation. Conversely, tetanic force in the unfatigued state was lower in CK-/- than in wild-type fibres, and this difference persisted after CK injection. Injection of inactivated CK had no obvious effect on any of the measured parameters. In conclusion, microinjection of CK into CK-/- fibres markedly restores many, but not all, aspects of the wild-type phenotype.

Figure 1. Injection of creatine kinase (CK) markedly improves the performance of CK^−/− skeletal muscle fibres during high-intensity stimulation

Typical records of [Ca²⁺]_i (upper) and force (lower) from a CK^−/− fibre injected with inactive CK (A) and subsequently with active CK (B). Note that the initial decline in tetanic [Ca²⁺]_i and force was clearly smaller after CK injection. For comparison, records from a wild-type fibre are shown in C. Fibres were activated by 20 cycles of 200 ms, 70 Hz stimulation and 100 ms rest.

Figure 2. CK injection results in a marked force restoration in CK^−/− fibres during high-intensity stimulation

Relative force restoration in the last (20th) stimulation train of high-intensity stimulation plotted against the estimated myoplasmic CK activity. Calculations of the relative force restoration were based on the assumption that with active CK, force remains constant during this type of stimulation, which was the result obtained in wild-type fibres (Dahlstedt et al. 2000). Thus, the percentage force restoration was calculated as (P_a - P_b)(100 - P_b)⁻¹, where P_a and P_b are the relative forces during the last stimulation train after and before injection of CK, respectively.

Figure 3. The performance of CK^−/− fibres injected with CK approaches the wild-type phenotype

Mean data (±s.e.m.) of tetanic [Ca²⁺]_i (upper) and force (lower) during high-intensity stimulation after injection of inactive CK (open triangles; n= 3) and active CK (filled circles; n= 5 for [Ca²⁺]_i and n= 7 for force). For comparison, mean tetanic [Ca²⁺]_i and force (dashed lines) from wild-type fibres are also shown (data from Dahlstedt et al. 2000).

Figure 4. The performance after pharmacological inhibition of CK is similar to that before CK injection

Original records of [Ca²⁺]_i (upper) and force (lower) from a CK^−/− fibre exposed to high-intensity stimulation (200 ms, 70 Hz given every 300 ms) before CK injection (A), after CK injection (B) and subsequently after application of 10 μm 2,4-dinitro-1-fluorobenzene (DNFB; C). Mean data (±s.e.m.; n= 3) of relative [Ca²⁺]_i (D) and force (E) obtained before CK injection (open triangles in E), after CK injection (filled circles) and finally after DNFB exposure (open circles). Data are expressed relative to [Ca²⁺]_i and force in a standard 350 ms, 70 Hz tetanus produced before the high-intensity stimulation protocol after CK injection.

Figure 5. [Ca²⁺]_i is better maintained in CK^−/− fibres after CK injection

Representative [Ca²⁺]_i records obtained from 350 ms, 70 Hz tetanic contractions elicited before (dashed line) and after (continuous line) injection of CK into a CK^−/− fibre. Note the fast [Ca²⁺]_i decline during the contraction before CK injection (rate of decline shown by straight lines).

Figure 6. CK-injected CK^−/− fibres approach the wild-type phenotype during low-intensity fatiguing stimulation

A, selected records of [Ca²⁺]_i and force from a CK-injected CK^−/− fibre fatigued by 350 ms, 70 Hz tetani given at 2.5 s intervals. Mean data of relative tetanic [Ca²⁺]_i (B) and force (C) are in each fibre expressed as a percentage of the first fatiguing tetanus. CK-injected CK^−/− fibres (filled circles, n= 5); wild-type fibres (open circles, n= 10); unmodified CK^−/− fibres (open triangles, n= 11).