We have blocked creatine kinase (CK)-mediated phosphocreatine (PCr) -->/<-- ATP transphosphorylation in skeletal muscle by combining targeted mutations in the genes encoding mitochondrial and cytosolic CK in mice. Contrary to expectation, the PCr level was only marginally affected, but the compound was rendered metabolically inert. Mutant muscles in vivo showed significantly impaired tetanic force output, increased relaxation times, altered mitochondrial volume and location, and conspicuous tubular aggregates of sarcoplasmic reticulum membranes, as seen in myopathies with electrolyte disturbances. In depolarized myotubes cultured in vitro, CK absence influenced both the release and sequestration of Ca2+. Our data point to a direct link between the CK-PCr system and Ca2+-flux regulation during the excitation and relaxation phases of muscle contraction.
Ectopic expression of telomerase blocks both telomeric attrition and senescence, suggesting that telomeric attrition is a mitotic counting mechanism that culminates in replicative senescence. By holding human ®broblast cultures con¯uent for up to 12 weeks at a time, we con®rmed previous observations and showed that telomeric attrition requires cell division and also, that senescence occurs at a constant average telomere length, not at a constant time point. However, on resuming cell division, these long-term con¯uent (LTC) cultures completed 15 ± 25 fewer mean population doublings (MPDs) than the controls prior to senescence. These lost divisions were mainly accounted for by slow cell turnover of the LTC cultures and by permanent cell cycle exit of 94% of the LTC cells, which resulted in many cell divisions being unmeasured by the MPD method. In the LTC cultures, p27 KIP1 accumulated and pRb became under-phosphorylated and under-expressed. Also, coincident with permanent cell cycle exit and before 1 MPD was completed, the LTC cultures upregulated the cell cycle inhibitors p21 WAF and p16 INK4A but not p14 ARF and developed other markers of senescence. We then tested the relationship between cell cycle re-entry and the cell cycle-inhibitory proteins following subculture of the LTC cultures. In these cultures, the downregulation of p27 KIP1 and the phosphorylation of pRb preceded the complete resumption of normal proliferation rate, which was accompanied by the down-regulation of p16 INK4A . Our results show that most normal human ®broblasts can accumulate p16 INK4A , p21 WAF and p27 KIP1 and senesce by cell division-independent mechanism(s). Furthermore, this form of senescence likely requires p16 INK4A and perhaps p27 KIP1 . Oncogene (2001) 20, 3541 ± 3552.
Continuous cycles of muscle fiber necrosis and regeneration are characteristic of the muscular dystrophies, and in some cases this leads to premature replicative senescence of myoblasts in vitro. The molecular mechanism of senescence in human myoblasts is poorly understood but there is evidence to suggest that telomeric attrition may be one of the ways by which this is achieved. We report here, for the first time, the extension of normal human skeletal muscle cell replicative life span by the reconstitution of telomerase activity. The telomerase-expressing cells show no features of transformation in vitro and have stable genomes with diploid karyotypes, do not express exceptionally high levels of c-myc and have wild-type, unmethylated CDKN2A genes. In vivo, they regenerate to repair muscle injury in immunosuppressed RAG-1 mice. This work suggests that telomerase expression to repair short telomeres may aid the expansion of diploid human muscle cells and consequently attempts at gene therapy for muscle diseases.
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