Dahlstedt Anders J, Katz Abram, Tavi Pasi, Westerblad Håkan
Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm, Sweden.
J Physiol. 2003 Mar 1;547(Pt 2):395-403. doi: 10.1113/jphysiol.2002.034793. Epub 2003 Jan 17.
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.
有活力的基因工程动物通常会表现出对改变后的基因型的适应性，这可能会掩盖目标蛋白质的作用。我们现在描述一种新方法，通过该方法可以在分离的成年骨骼肌细胞中区分改变后的基因型的直接影响与继发性适应性变化。我们研究了在微注射纯化的肌酸激酶（CK；CK-/-）前后，完全缺乏肌酸激酶的单个骨骼肌纤维中的收缩功能和细胞内Ca2+处理情况（与荧光Ca2+指示剂indo-1一起注射）。注射CK后，平均总CK活性估计约为4 mM s-1，约为野生型肌纤维活性的5%。注射CK后，CK-/-纤维在几个方面接近野生型表型：（a）在高强度刺激期间（占空比，即强直收缩持续时间除以强直收缩间隔 = 0.67），游离肌浆[Ca2+]（[Ca2+]i）增加，力量变化不大；（b）在短暂（350 ms）强直收缩期间，[Ca2+]i没有下降；（c）在低强度疲劳刺激期间（占空比 = 0.14），强直收缩[Ca2+]i在前10次强直收缩中增加，此后下降；（d）在低强度疲劳刺激的第二次强直收缩中，强直收缩[Ca2+]i和力量没有出现短暂降低。相反，未疲劳状态下CK-/-纤维的强直收缩力低于野生型纤维，并且在注射CK后这种差异仍然存在。注射失活的CK对任何测量参数都没有明显影响。总之，将CK微注射到CK-/-纤维中可显著恢复野生型表型的许多方面，但不是全部。
