Creatine kinase injection restores contractile function in creatine-kinase-deficient mouse skeletal muscle fibres.
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.
Dahlstedt
Anders J
AJ
Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm, Sweden.
Katz
Abram
A
Tavi
Pasi
P
Westerblad
Håkan
H
D000818:Animals; D002118:Calcium / Q000378:metabolism; D003402:Creatine Kinase / Q000008:administration & dosage / Q000172:deficiency* / Q000494:pharmacology*; D004558:Electric Stimulation; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D008845:Microinjections; D009119:Muscle Contraction / Q000187:drug effects*; D018763:Muscle Fatigue / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D009210:Myofibrils / Q000378:metabolism; D009994:Osmolar Concentration; D013997:Time Factors
D003402:Creatine Kinase; D002118:Calcium