ABSTRACT
Title
Effects of staurosporine in rodents skeletal muscle: a pharmacological model of muscle atrophy.
Authors
G.Cannone1, S.Calzolaro1, A. Mele1, D.Conte Camerino1 and D. Tricarico1.
1Dept. of Pharmacobiology, Section of Pharmacology, Faculty of Pharmacy - University of Bari, Italy.
1Dept. of Pharmacobiology, Section of Pharmacology, Faculty of Pharmacy - University of Bari, Italy.
Abstract
Atrophy is a regressive process under the control of different pathways, growth and transcription factors which modulate, at different levels, protein synthesis and/or degradation.This work evaluates the potential atrophyc effects of staurosporine, a known apoptotic agent, on rodent. The study was conducted on Soleus (SOL), Exstensor Digitorum Longus (EDL) and Flexor Digitorum Brevis (FDB) skeletal muscles fibers of rats and mice, male and female, incubated with control (DMEM+) and staurosporine (1µg/mL) for 1-72 hours.The effects of staurosporine (1µg/mL) were assessed through total proteins content muscle measurements, the evaluation of caspase-3 activity, organ vitality assay (cell counting kit-8) and fibers diameter by single-blind measurements of FDB fibers incubated at 1-6-24-48-72 hours with Stauroporine alone or in combination with diazoxide (100 µM), a known cytoprotective drug.
Total proteins content quantification, the evaluation of the caspase-3 activity and the organ vitality assay were evaluated in themuscle homogenates, while the culture mediumwas used for the evaluation of creatine kinase (CK) activity. In mice staurosporine showed a more powerful atrophyc effect in respect to rats with the following scale: SOL≈EDL>FDB suggesting a tissue-specific effects of the molecule in the different muscular phenotypes; in fact the drug showed a reduction of total proteins content/muscle of 49 %, 50% and 42% for SOL, EDL and FDB, respectively. No gender atrophyc effects of the drug on SOL, EDL and FDB skeletal muscles were observed. Furthermore, in mice, staurosporine did not show any effects on caspase-3 activity suggesting the existence of a possible atrophy caspase-3 indipendent mechanism, instead the same drug caused a significant increase of CK activity respect to controls, as cells damage index. Then, organ vitality test allowed to estimate the entity of damage caused by the substance through the evaluation of the enzymatic activity of mitochondrial succinate dehydrogenase. In rats, it was observed a significant increase of enzyme activity mainly in SOL muscle then EDL and FDB, possibly related with the oxidative metabolism of SOL. Finally, staurosporine produced a reduction of FDB fibers diameter of 11-13%, mainly at 6 and 24 hours of incubation, while a smaller decrease of 5-6% has been observed in fibers coincubated with the diazoxide; the incubation of the muscles with staurosporine+diazoxide, in part, prevented the reduction of the fibers diameters.
All these findings suggest that the staurosporine is a powerful atrophyc agent on skeletal muscle fibers and its atrophyc effect depends on the mechanism of action of the drug. Its target is the protein kinase C (PKC), which is involved in various cellular mechanisms: it inhibites FOXO transcription factors leading to activation of the ubiquitin-proteasome and lysosomal-autophagic pathways; PKC has also a role in regulating KATP channels, not directly, in a not fully known way. In fact, KATP channels are definied as molecular sensor of atrophy in skeletal muscle (Tricarico et al., 2010), and this may explane also the preventive effect of diazoxide on the reduction of fibers diameters when the fibers were coincubated with staurosporine and diazoxide respect to the cells incubated with staurosporine alone.
References:
Tricarico et al. (2010). J Physiol. 588.5, 773-784.
Total proteins content quantification, the evaluation of the caspase-3 activity and the organ vitality assay were evaluated in themuscle homogenates, while the culture mediumwas used for the evaluation of creatine kinase (CK) activity. In mice staurosporine showed a more powerful atrophyc effect in respect to rats with the following scale: SOL≈EDL>FDB suggesting a tissue-specific effects of the molecule in the different muscular phenotypes; in fact the drug showed a reduction of total proteins content/muscle of 49 %, 50% and 42% for SOL, EDL and FDB, respectively. No gender atrophyc effects of the drug on SOL, EDL and FDB skeletal muscles were observed. Furthermore, in mice, staurosporine did not show any effects on caspase-3 activity suggesting the existence of a possible atrophy caspase-3 indipendent mechanism, instead the same drug caused a significant increase of CK activity respect to controls, as cells damage index. Then, organ vitality test allowed to estimate the entity of damage caused by the substance through the evaluation of the enzymatic activity of mitochondrial succinate dehydrogenase. In rats, it was observed a significant increase of enzyme activity mainly in SOL muscle then EDL and FDB, possibly related with the oxidative metabolism of SOL. Finally, staurosporine produced a reduction of FDB fibers diameter of 11-13%, mainly at 6 and 24 hours of incubation, while a smaller decrease of 5-6% has been observed in fibers coincubated with the diazoxide; the incubation of the muscles with staurosporine+diazoxide, in part, prevented the reduction of the fibers diameters.
All these findings suggest that the staurosporine is a powerful atrophyc agent on skeletal muscle fibers and its atrophyc effect depends on the mechanism of action of the drug. Its target is the protein kinase C (PKC), which is involved in various cellular mechanisms: it inhibites FOXO transcription factors leading to activation of the ubiquitin-proteasome and lysosomal-autophagic pathways; PKC has also a role in regulating KATP channels, not directly, in a not fully known way. In fact, KATP channels are definied as molecular sensor of atrophy in skeletal muscle (Tricarico et al., 2010), and this may explane also the preventive effect of diazoxide on the reduction of fibers diameters when the fibers were coincubated with staurosporine and diazoxide respect to the cells incubated with staurosporine alone.
References:
Tricarico et al. (2010). J Physiol. 588.5, 773-784.