Baqué S, Newgard C B, Gerard R D, Guinovart J J, Gómez-Foix A M
Department de Bioquímica i Fisiologia, Universitat de Barcelona, Spain.
Biochem J. 1994 Dec 15;304 ( Pt 3)(Pt 3):1009-14. doi: 10.1042/bj3041009.
The feasibility of using adenovirus as a vector for the introduction of glycogen phosphorylase activity into myocytes has been examined. We used the C2C12 myoblast cell line to assay the impact of phosphorylase gene transfer on myocyte glycogen metabolism and to reproduce in vitro the two strategies proposed for the treatment of muscle genetic diseases, myoblast transplantation and direct DNA delivery. In this study, a recombinant adenovirus containing the muscle glycogen phosphorylase cDNA transcribed from the cytomegalovirus promoter (AdCMV-MGP) was used to transduce both differentiating myoblasts and nondividing mature myotube cells. Muscle glycogen phosphorylase mRNA levels and total phosphorylase activity were increased in both cell types after viral treatment although more efficiently in the differentiated myotubes. The increase in phosphorylase activity was transient (15 days) in myoblasts whereas in myotubes higher levels of phosphorylase gene expression and activity were reached, which remained above control levels for the duration of the study (20 days). The introduction of muscle phosphorylase into myotubes enhanced their glycogenolytic capacity. AdCMV MGP-transduced myotubes had lower glycogen levels under basal conditions. In addition, these engineered cells showed more extensive glycogenolysis in response to both adrenaline, which stimulates glycogen phosphorylase phosphorylation, and carbonyl cyanide m-chlorophenylhydrazone, a metabolic uncoupler. In conclusion, transfer of the muscle glycogen phosphorylase cDNA into myotubes confers an enhanced and regulatable glycogenolytic capacity. Thus this system might be useful for delivery of muscle glycogen phosphorylase and restoration of glycogenolysis in muscle cells from patients with muscle phosphorylase deficiency (McArdle's disease).
已对使用腺病毒作为载体将糖原磷酸化酶活性导入心肌细胞的可行性进行了研究。我们使用C2C12成肌细胞系来测定磷酸化酶基因转移对心肌细胞糖原代谢的影响，并在体外重现为治疗肌肉遗传性疾病提出的两种策略，即成肌细胞移植和直接DNA递送。在本研究中，一种含有从巨细胞病毒启动子转录的肌肉糖原磷酸化酶cDNA的重组腺病毒（AdCMV-MGP）被用于转导分化中的成肌细胞和不分裂的成熟肌管细胞。病毒处理后，两种细胞类型中的肌肉糖原磷酸化酶mRNA水平和总磷酸化酶活性均有所增加，尽管在分化的肌管中效率更高。成肌细胞中磷酸化酶活性的增加是短暂的（15天），而在肌管中达到了更高水平的磷酸化酶基因表达和活性，在研究期间（20天）一直高于对照水平。将肌肉磷酸化酶引入肌管增强了它们的糖原分解能力。AdCMV MGP转导的肌管在基础条件下糖原水平较低。此外，这些工程细胞在受到刺激糖原磷酸化酶磷酸化的肾上腺素和代谢解偶联剂羰基氰化物间氯苯腙的作用下，均表现出更广泛的糖原分解。总之，将肌肉糖原磷酸化酶cDNA转移到肌管中可赋予增强的和可调节的糖原分解能力。因此，该系统可能有助于递送肌肉糖原磷酸化酶并恢复肌肉磷酸化酶缺乏症（麦克尔迪氏病）患者肌肉细胞中的糖原分解。
