Diaphragm assessment in mice overexpressing phospholamban in slow-twitch type I muscle fibers.
Diaphragm assessment in mice overexpressing phospholamban in slow-twitch type I muscle fibers.
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DOI：
10.1002/brb3.470
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发表时间：
2016-06
影响因子：
3.1
通讯作者：
Tupling AR
中科院分区：
文献类型：
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作者：
Fajardo VA;Smith IC;Bombardier E;Chambers PJ;Quadrilatero J;Tupling AR
磷蛋白（PLN）和肌磷脂（SLN）是调节肌内质网Ca2+ - atp酶（SERCA）泵的小抑制蛋白。我们实验室之前的研究表明，在过表达PLN （PLN OE）的小鼠比罗鱼肌和臀小肌中，SERCA功能受损，动力蛋白2（3-5倍）和SLN（7-9倍）上调，并观察到人类核中心肌病（CNM）的特征。在这里，我们通过结构和功能实验来评估Pln OE小鼠膈肌是否会出现CNM病理和肌肉无力。对来自Pln OE和WT小鼠的膈肌进行组织学/组织化学/免疫荧光染色、Ca2+‐atp酶和Ca2+摄取测定、Western blotting和体外电刺激。我们的研究结果表明，PLN过表达降低了SERCA对Ca2+的明显亲和力，但没有降低最大SERCA活性或Ca2+摄取率。SLN上调2.5倍，而dynamin 2的表达没有变化。关于CNM，我们没有观察到I型纤维优势、中央核或膈膜氧化活性的中央聚集，尽管存在I型纤维萎缩。此外，Pln OE膜片的体外收缩性评估显示，产生力的能力、最大松弛率或力的发展没有减少，但确实表明半松弛时间延长了。因此，PLN过表达对骨骼肌表型的影响在膈肌和体位比目鱼肌和臀小肌之间存在差异。我们的研究结果指出，SLN表达和I型纤维分布的差异是潜在的影响因素。
Phospholamban (PLN) and sarcolipin (SLN) are small inhibitory proteins that regulate the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump. Previous work from our laboratory revealed that in the soleus and gluteus minimus muscles of mice overexpressing PLN (Pln OE), SERCA function was impaired, dynamin 2 (3–5 fold) and SLN (7–9 fold) were upregulated, and features of human centronuclear myopathy (CNM) were observed. Here, we performed structural and functional experiments to evaluate whether the diaphragm muscles of the Pln OE mouse would exhibit CNM pathology and muscle weakness. Diaphragm muscles from Pln OE and WT mice were subjected to histological/histochemical/immunofluorescent staining, Ca2+‐ATPase and Ca2+ uptake assays, Western blotting, and in vitro electrical stimulation. Our results demonstrate that PLN overexpression reduced SERCA's apparent affinity for Ca2+ but did not reduce maximal SERCA activity or rates of Ca2+ uptake. SLN was upregulated 2.5‐fold, whereas no changes in dynamin 2 expression were found. With respect to CNM, we did not observe type I fiber predominance, central nuclei, or central aggregation of oxidative activity in diaphragm, although type I fiber hypotrophy was present. Furthermore, in vitro contractility assessment of Pln OE diaphragm strips revealed no reductions in force‐generating capacity, maximal rates of relaxation or force development, but did indicate that ½ relaxation time was prolonged. Therefore, the effects of PLN overexpression on skeletal muscle phenotype differ between diaphragm and the postural soleus and gluteus minimus muscles. Our findings here point to differences in SLN expression and type I fiber distribution as potential contributing factors.