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Sarcoplasmic-reticulum biogenesis in contraction-inhibited skeletal-muscle cultures
J H Charuk1, C Guerin, P C Holland
1Department of Neurology and Neurosurgery, Montreal Neurological Institute, Quebec, Canada.
Abstract:
We have previously shown that inhibition of the spontaneous contractile activity of cultured embryonic-chick skeletal-muscle fibres with tetrodotoxin (TTX) leads to decreased sarcoplasmic-reticulum Ca(2+)-transport rates and steady-state concentrations of the high-energy Ca(2+)-ATPase phosphoenzyme intermediate [Charuk & Holland (1983) Exp. Cell Res. 144, 143-157]. In the present study we used a monoclonal antibody to the Ca(2+)-ATPase to show that there is a decreased amount of enzyme accumulated by contraction-inhibited myotubes. Indirect immunofluorescence microscopy using the monoclonal antibody to the Ca(2+)-ATPase also revealed a disordered subcellular organization of the sarcotubular system in contraction-inhibited myotubes. The biogenesis of sarcoplasmic-reticulum proteins in TTX-paralysed myofibres was studied by labelling cells with [35S]methionine before isolation of the active Ca(2+)-pump membrane fraction. Protein turnover was selectively increased in that fraction from TTX-treated muscle cultures. Electrophoretic analysis and quantitative fluorography confirmed that decreased accumulation of the Ca(2+)-ATPase enzyme in contraction-inhibited myotubes was associated with increased turnover of this protein. The present results demonstrate that biogenesis of the sarcoplasmic-reticulum Ca(2+)-ATPase is regulated by the contractile activity of skeletal-muscle fibres.
Insights
Inhibition of muscle fiber contraction reduces sarcoplasmic reticulum Ca(2+)-ATPase enzyme levels and disrupts cellular organization. This indicates that muscle activity regulates the biogenesis of this critical calcium pump.
Area of Science:
- Muscle physiology
- Cellular biology
- Biochemistry
Background:
- Previous work showed tetrodotoxin (TTX) inhibition of muscle contraction decreases sarcoplasmic reticulum Ca(2+) transport rates.
- This also reduced the high-energy Ca(2+)-ATPase phosphoenzyme intermediate concentration.
Purpose of the Study:
- To investigate the effect of inhibited contractile activity on the amount and organization of Ca(2+)-ATPase in skeletal muscle.
- To examine the biogenesis and turnover of sarcoplasmic reticulum proteins in TTX-paralyzed muscle fibers.
Main Methods:
- Used a monoclonal antibody to detect Ca(2+)-ATPase levels via indirect immunofluorescence microscopy.
- Studied protein biogenesis by labeling with [35S]methionine and isolating the Ca(2+)-pump membrane fraction.
- Analyzed protein turnover using electrophoresis and quantitative fluorography.
Main Results:
- Contraction-inhibited myotubes showed decreased accumulation of Ca(2+)-ATPase.
- Immunofluorescence revealed a disordered subcellular organization of the sarcotubular system in these myotubes.
- Increased protein turnover of Ca(2+)-ATPase was observed in TTX-treated muscle cultures.
Conclusions:
- Muscle contractile activity regulates the biogenesis of the sarcoplasmic reticulum Ca(2+)-ATPase.
- Disruption of contractile activity leads to reduced enzyme levels and altered cellular structure.