Department of Trauma Surgery, Orthopedics and Plastic Surgery, University Medical Center Goettingen, Robert-Koch-Str. 40, 37099 Göttingen, Germany.
Department of Otorhinolaryngology, Head and Neck Surgery, University Medical Center Goettingen, Robert-Koch-Str. 40, 37099 Göttingen, Germany.
Cells. 2023 Aug 29;12(17):2167. doi: 10.3390/cells12172167.
Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization, subsequently increasing healthcare costs while limiting quality of life. In sarcopenic muscle fibers, the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32) is expressed at substantially higher levels, driving ubiquitin-proteasomal muscle protein degradation. As one of the key regulators of muscular equilibrium, the transcription factor Forkhead Box O3 (FOXO3) can increase the expression of Fbxo32, making it a possible target for the regulation of this detrimental pathway. To test this hypothesis, murine C2C12 myoblasts were transduced with AAVs carrying a plasmid for four specific siRNAs against Foxo3. Successfully transduced myoblasts were selected via FACS cell sorting to establish single clone cell lines. Sorted myoblasts were further differentiated into myotubes and stained for myosin heavy chain (MHC) by immunofluorescence. The resulting area was calculated. Myotube contractions were induced by electrical stimulation and quantified. We found an increased Foxo3 expression in satellite cells in human skeletal muscle and an age-related increase in Foxo3 expression in older mice in silico. We established an in vitro AAV-mediated FOXO3 knockdown on protein level. Surprisingly, the myotubes with FOXO3 knockdown displayed a smaller myotube size and a lower number of nuclei per myotube compared to the control myotubes (AAV-transduced with a functionless control plasmid). During differentiation, a lower level of FOXO3 reduced the expression Fbxo32 within the first three days. Moreover, the expression of Myod1 and Myog via ATM and Tp53 was reduced. Functionally, the Foxo3 knockdown myotubes showed a higher contraction duration and time to peak. Early Foxo3 knockdown seems to terminate the initiation of differentiation due to lack of Myod1 expression, and mediates the inhibition of Myog. Subsequently, the myotube size is reduced and the excitability to electrical stimulation is altered.
肌肉减少症在老年人群中患病率很高。肌肉减少症具有巨大的社会经济重要性，因为它会导致跌倒和住院，从而增加医疗保健成本，同时限制生活质量。在肌肉减少症的肌纤维中，E3 泛素连接酶 F-Box 蛋白 32（Fbxo32）的表达水平显著升高，导致泛素蛋白酶体肌蛋白降解。作为肌肉平衡的关键调节因子之一，叉头框蛋白 O3（FOXO3）可以增加 Fbxo32 的表达，使其成为调节这条有害途径的可能靶点。为了验证这一假设，用携带针对 Foxo3 的四个特定 siRNA 的质粒转染 C2C12 肌母细胞，通过流式细胞术分选成功转染的肌母细胞以建立单克隆细胞系。分选的肌母细胞进一步分化为肌管，并通过免疫荧光染色检测肌球蛋白重链（MHC）。计算出结果区域。通过电刺激诱导肌管收缩并进行量化。我们发现人类骨骼肌卫星细胞中的 Foxo3 表达增加，并且在衰老小鼠中 Foxo3 表达随着年龄的增长而增加。我们在体外建立了 AAV 介导的 FOXO3 敲低。令人惊讶的是，与对照肌管（转导了无功能对照质粒的 AAV）相比，FOXO3 敲低的肌管显示出更小的肌管大小和每肌管更少的核数。在分化过程中，FOXO3 水平降低会在最初的三天内降低 Fbxo32 的表达。此外，ATM 和 Tp53 途径的 Myod1 和 Myog 表达降低。功能上，Foxo3 敲低肌管的收缩持续时间和峰值时间更长。早期的 Foxo3 敲低似乎由于缺乏 Myod1 表达而终止分化的启动，并介导了 Myog 的抑制。随后，肌管大小减小，对电刺激的兴奋性发生改变。
