DataSheet1_Loss of Unconventional Myosin VI Affects cAMP/PKA Signaling in Hindlimb Skeletal Muscle in an Age-Dependent Manner.PDF

Myosin VI (MVI) is a unique unconventional myosin ubiquitously expressed in metazoans. Its diverse cellular functions are mediated by interactions with a number of binding partners present in multi-protein complexes. MVI is proposed to play important roles in muscle function and myogenesis. Previously, we showed that MVI is present in striated muscles and myogenic cells, and MVI interacts with A-kinase anchoring protein 9 (AKAP9), a scaffold for PKA and its regulatory proteins. Since PKA directly phosphorylates the MVI cargo binding domain, we hypothesized that the cellular effects of MVI are mediated by the cAMP/PKA signaling pathway, known to play important roles in skeletal muscle metabolism and myogenesis. To elucidate the potential role of MVI in PKA signaling in hindlimb muscle function, we used mice lacking MVI (Snell’s waltzer, SV), considered as natural MVI knockouts, and heterozygous littermates. We used muscles isolated from newborn (P0) as well as 3- and 12-month-old adult mice. We observed a significant increase in the muscle to body mass ratio, which was most evident for the soleus muscle, as well as changes in fiber size, indicating alterations in muscle metabolism. These observations were accompanied by age-dependent changes in the activity of PKA and cAMP/PKA-dependent transcriptional factor (CREB). Additionally, the levels of adenylate cyclase isoforms and phosphodiesterase (PDE4) were age-dependent. Also, cAMP levels were decreased in the muscle of P0 mice. Together, these observations indicate that lack of MVI impairs PKA signaling and results in the observed alterations in the SV muscle metabolism, in particular in newborn mice.

肌球蛋白VI（MVI）是一种独特的非传统肌球蛋白，在多细胞生物中普遍表达。其多样的细胞功能通过与其存在于多蛋白复合物中的多种结合伙伴的相互作用而实现。MVI被认为在肌肉功能和肌发生中发挥着重要作用。先前研究表明，MVI存在于横纹肌和肌生成细胞中，并与A激酶锚定蛋白9（AKAP9）相互作用，AKAP9是PKA及其调节蛋白的支架。由于PKA直接磷酸化MVI货物结合域，我们假设MVI的细胞效应是通过已知的在骨骼肌代谢和肌发生中发挥重要作用的cAMP/PKA信号通路介导的。为了阐明MVI在腓肠肌功能中的PKA信号通路中的潜在作用，我们使用了缺乏MVI（Snell's waltzer，SV）的小鼠，这些小鼠被视为天然的MVI敲除小鼠及其同窝的杂合子。我们使用了新生（P0）以及3个月和12个月大的成年小鼠的肌肉。我们观察到肌肉与体重的比例显著增加，其中比目鱼肌最为明显，以及纤维尺寸的变化，这表明肌肉代谢发生了改变。这些观察伴随着PKA和cAMP/PKA依赖性转录因子（CREB）活性的年龄依赖性变化。此外，腺苷酸环化酶同种异构体和磷酸二酯酶（PDE4）的水平也随年龄而变化。还有，P0小鼠的cAMP水平降低。综上所述，这些观察结果表明MVI的缺失损害了PKA信号通路，并导致了SV肌肉代谢中观察到的改变，尤其是在新生小鼠中。