Div. of Cellular Pharmacology, Dept. of Pharmacology, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de maio, 100, São Paulo, SP, Brazil.
J Appl Physiol (1985). 2011 Dec;111(6):1710-8. doi: 10.1152/japplphysiol.00586.2011. Epub 2011 Sep 15.
The molecular regulation of skeletal muscle proteolysis and the pharmacological screening of anticatabolic drugs have been addressed by measuring tyrosine release from prepubertal rat skeletal muscles, which are thin enough to allow adequate in vitro diffusion of oxygen and substrates. However, the use of muscle at accelerated prepubertal growth has limited the analysis of adult muscle proteolysis or that associated with aging and neurodegenerative diseases. Here we established the adult rat lumbrical muscle (4/hindpaw; 8/rat) as a new in situ experimental model for dynamic measurement of skeletal muscle proteolysis. By incubating lumbrical muscles attached to their individual metatarsal bones in Tyrode solution, we showed that the muscle proteolysis rate of adult and aged rats (3-4 to 24 mo old) is 45-25% of that in prepubertal animals (1 mo old), which makes questionable the usual extrapolation of proteolysis from prepubertal to adult/senile muscles. While acute mechanical injury or 1- to 7-day denervation increased tyrosine release from adult lumbrical muscle by up to 60%, it was reduced by 20-28% after 2-h incubation with β-adrenoceptor agonists, forskolin or phosphodiesterase inhibitor IBMX. Using inhibitors of 26S-proteasome (MG132), lysosome (methylamine), or calpain (E64/leupeptin) systems, we showed that ubiquitin-proteasome is accountable for 40-50% of total lumbrical proteolysis of adult, middle-aged, and aged rats. In conclusion, the lumbrical model allows the analysis of muscle proteolysis rate from prepubertal to senile rats. By permitting eight simultaneous matched measurements per rat, the new model improves similar protocols performed in paired extensor digitorum longus (EDL) muscles from prepubertal rats, optimizing the pharmacological screening of drugs for anticatabolic purposes.
骨骼肌蛋白分解的分子调控以及抗分解代谢药物的药理学筛选已经通过测量来自青春期前大鼠骨骼肌的酪氨酸释放来进行，这些肌肉足够薄，可以允许氧气和底物充分体外扩散。然而，使用处于加速青春期生长的肌肉限制了成年肌肉蛋白分解或与衰老和神经退行性疾病相关的蛋白分解的分析。在这里，我们建立了成年大鼠蚓状肌（每只后爪 4 块肌肉/每只大鼠 8 块肌肉）作为一种新的原位实验模型，用于动态测量骨骼肌蛋白分解。通过将附着在各自跖骨上的蚓状肌在 Tyrode 溶液中孵育，我们表明成年和老年大鼠（3-4 至 24 月龄）的肌肉蛋白分解率为青春期前动物（1 月龄）的 45-25%，这使得通常从青春期前到成年/老年肌肉的蛋白分解外推变得值得怀疑。虽然急性机械损伤或 1-7 天去神经支配会使成年蚓状肌的酪氨酸释放增加高达 60%，但在与β-肾上腺素受体激动剂、forskolin 或磷酸二酯酶抑制剂 IBMX 孵育 2 小时后，其释放量会减少 20-28%。使用 26S-蛋白酶体（MG132）、溶酶体（甲胺）或钙蛋白酶（E64/亮肽素）系统抑制剂，我们表明泛素-蛋白酶体占成年、中年和老年大鼠蚓状肌总蛋白分解的 40-50%。总之，蚓状肌模型允许分析从青春期前到老年大鼠的肌肉蛋白分解率。通过允许每只大鼠同时进行八次匹配测量，该新模型改进了在青春期前大鼠的配对伸趾长肌（EDL）肌肉中进行的类似方案，优化了抗分解代谢药物的药理学筛选。
