Mitochondrial creatine kinase (mtCK) regulates the “fast” export of phosphocreatine to support cytoplasmic phosphorylation of ADP to ATP which is more rapid than direct ATP export. Such “creatine-dependent” phosphate shuttling is attenuated in several muscles, including the heart, of the D2.mdx mouse model of Duchenne muscular dystrophy at only 4 weeks of age. However, the degree to which creatine-dependent and -independent systems of phosphate shuttling progressively worsen or potentially adapt in a hormetic manner throughout disease progression remains unknown. Here, we performed a series of proof-of-principle investigations designed to determine how phosphate shuttling pathways worsen or adapt in later disease stages in D2.mdx (12 months of age). We also determined whether changes in creatine-dependent phosphate shuttling are linked to alterations in mtCK thiol redox state. In permeabilized muscle fibres prepared from cardiac left ventricles, we found that 12-month-old male D2.mdx mice have reduced creatine-dependent pyruvate oxidation and elevated complex I-supported H2O2 emission (mH2O2). Surprisingly, creatine-independent ADP-stimulated respiration was increased and mH2O2 was lowered suggesting that impairments in the faster mtCK-mediated phosphocreatine export system resulted in compensation of the alternative slower pathway of ATP export. The apparent impairments in mtCK-dependent bioenergetics occurred independent of mtCK protein content but were related to greater thiol oxidation of mtCK and a more oxidized cellular environment (lower GSH:GSSG). Next, we performed a proof-of-principle study to determine whether creatine-dependent bioenergetics could be enhanced through chronic administration of the mitochondrial-targeting, ROS-lowering tetrapeptide, SBT-20. We found that 12 weeks of daily treatment with SBT-20 (from day 4–∼12 weeks of age) increased respiration and lowered mH2O2 only in the presence of creatine in D2.mdx mice without affecting calcium-induced mitochondrial permeability transition activity. In summary, creatine-dependent mitochondrial bioenergetics are attenuated in older D2.mdx mice in relation to mtCK thiol oxidation that seem to be countered by increased creatine-independent phosphate shuttling as a unique form of mitohormesis. Separate results demonstrate that creatine-dependent bioenergetics can also be enhanced with a ROS-lowering mitochondrial-targeting peptide. These results demonstrate a specific relationship between redox stress and mitochondrial hormetic reprogramming during dystrophin deficiency with proof-of-principle evidence that creatine-dependent bioenergetics could be modified with mitochondrial-targeting small peptide therapeutics.

中文翻译：

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杜氏肌营养不良症晚期阶段的线粒体兴奋作用揭示了氧化还原敏感的肌酸途径，线粒体靶向肽 SBT-20 可以增强该途径


线粒体肌酸激酶 (mtCK) 调节磷酸肌酸的“快速”输出，以支持 ADP 的细胞质磷酸化为 ATP，这比直接 ATP 输出更快。在仅 4 周龄的杜氏肌营养不良 D2.mdx 小鼠模型中，这种“肌酸依赖性”磷酸盐穿梭在包括心脏在内的多个肌肉中减弱。然而，在整个疾病进展过程中，肌酸依赖性和不依赖性磷酸盐穿梭系统逐渐恶化或可能以毒物兴奋效应方式适应的程度仍然未知。在这里，我们进行了一系列原理验证研究，旨在确定 D2.mdx（12 月龄）疾病后期阶段磷酸盐穿梭途径如何恶化或适应。我们还确定了肌酸依赖性磷酸盐穿梭的变化是否与 mtCK 硫醇氧化还原状态的变化有关。在从心脏左心室制备的透化肌纤维中，我们发现 12 个月大的雄性 D2.mdx 小鼠的肌酸依赖性丙酮酸氧化减少，复合物 I 支持的 H2O2 排放 (mH2O2) 增加。令人惊讶的是，不依赖肌酸的 ADP 刺激的呼吸增加，mH2O2 降低，这表明较快的 mtCK 介导的磷酸肌酸输出系统的损伤导致了 ATP 输出的替代较慢途径的补偿。 mtCK 依赖性生物能学的明显损害与 mtCK 蛋白含量无关，但与 mtCK 的硫醇氧化程度较高和氧化程度较高的细胞环境（较低的 GSH:GSSG）有关。接下来，我们进行了一项原理验证研究，以确定是否可以通过长期施用线粒体靶向、降低 ROS 的四肽 SBT-20 来增强肌酸依赖性生物能学。 我们发现，每天用 SBT-20 治疗 12 周（从第 4 天到~12 周龄），仅在肌酸存在的情况下，D2.mdx 小鼠的呼吸才会增加，mH2O2 才会降低，而不会影响钙诱导的线粒体通透性转变活性。总之，在老年 D2.mdx 小鼠中，与 mtCK 硫醇氧化相关的肌酸依赖性线粒体生物能减弱，这似乎可以通过增加不依赖于肌酸的磷酸盐穿梭作为一种独特的线粒体兴奋作用来抵消。单独的结果表明，肌酸依赖性生物能学也可以通过降低 ROS 的线粒体靶向肽来增强。这些结果证明了肌营养不良蛋白缺乏期间氧化还原应激与线粒体激效重编程之间的特定关系，并通过原理证明证明肌酸依赖性生物能学可以通过线粒体靶向小肽疗法进行修改。