Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany; Institute of Vegetative Physiology, Medical Faculty, University of Cologne, Cologne, Germany.
Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany.
Eur J Cell Biol. 2024 Jun;103(2):151399. doi: 10.1016/j.ejcb.2024.151399. Epub 2024 Feb 20.
Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.
结蛋白基因突变导致肌病和心肌病。我们之前描述的携带与常见人类结蛋白突变 R350P 同源物的 R349P 结蛋白病小鼠，其肌肉组织中线粒体形态和功能发生明显改变。通过从小鼠 R349P 结蛋白病和 p53 敲除鼠的后代中分离骨骼肌成肌细胞，我们建立了一种永生化的细胞疾病模型。杂合和纯合 R349P 结蛋白敲入和野生型成肌细胞可以很好地分化为自发收缩的多核肌管。结蛋白病成肌细胞表现出特征性的结蛋白细胞骨架破坏和结蛋白蛋白聚集，结蛋白病肌管表现出特征性的肌原纤维不规则。长期电脉冲刺激促进肌管分化，并显著增加其自发收缩率。在杂合和纯合 R349P 结蛋白病肌管中，这种处理恢复了野生型肌管中可见的规则的肌原纤维横纹图案。通过密度梯度超速离心从肌管中纯化的线粒体的高分辨率呼吸测定显示正常的氧化磷酸化能力，但来自纯合 R349P 结蛋白敲入细胞的线粒体质子泄漏显著减少。与线粒体内膜质子通量减少一致，我们对纯化线粒体的定量蛋白质组学分析显示，在纯合 R349P 结蛋白敲入基因型中 ADP/ATP 转位酶的水平显著降低。由于这种改变也在 R349P 结蛋白病小鼠的比目鱼肌中检测到，与从培养细胞中纯化的线粒体相比，其显示出多种其他失调的线粒体蛋白，因此我们认为这一发现是结蛋白病继发性线粒体病发病机制中的早期步骤。
