School of Kinesiology and Health Science and the Muscle Health Research Centre, York University, Toronto, ON, Canada.
Department of Biology and the Muscle Health Research Centre, York University, Toronto, ON, Canada.
Redox Biol. 2024 Oct;76:103319. doi: 10.1016/j.redox.2024.103319. Epub 2024 Aug 20.
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 HO emission (mHO). Surprisingly, creatine-independent ADP-stimulated respiration was increased and mHO 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 mHO 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.
线粒体肌酸激酶（mtCK）调节“快速”输出磷酸肌酸，以支持细胞质中 ADP 向 ATP 的磷酸化，其速度比直接 ATP 输出更快。在只有 4 周大的 Duchenne 肌营养不良症 D2.mdx 小鼠模型的几种肌肉中，包括心脏，这种“依赖肌酸”的磷酸盐穿梭作用会减弱。然而，依赖肌酸和非依赖肌酸的磷酸盐穿梭系统在疾病进展过程中逐渐恶化或潜在适应的程度仍不清楚。在这里，我们进行了一系列原理验证研究，旨在确定 D2.mdx（12 个月大）中磷酸盐穿梭途径在疾病后期如何恶化或适应。我们还确定了依赖肌酸的磷酸盐穿梭变化是否与 mtCK 巯基氧化还原状态的改变有关。在从心脏左心室制备的通透性肌肉纤维中，我们发现 12 个月大的雄性 D2.mdx 小鼠的依赖肌酸的丙酮酸氧化减少，并且复合物 I 支持的 HO 发射（mHO）增加。令人惊讶的是，非依赖肌酸的 ADP 刺激呼吸增加，mHO 降低，这表明更快的 mtCK 介导的磷酸肌酸输出系统的损伤导致替代的较慢的 ATP 输出途径的补偿。依赖 mtCK 的生物能的明显损伤与 mtCK 蛋白含量无关，但与 mtCK 更大的巯基氧化和更氧化的细胞环境（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慢性给予靶向线粒体、降低 ROS 的四肽 SBT-20 来增强依赖肌酸的生物能。我们发现，在 12 周的每天治疗（从第 4 天到大约 12 周龄）中，在 D2.mdx 小鼠中，只有在存在肌酸的情况下，SBT-20 增加了呼吸并降低了 mHO，而不会影响钙诱导的线粒体通透性转换活性。总之，与 mtCK 巯基氧化有关，在年龄较大的 D2.mdx 小鼠中，依赖肌酸的线粒体生物能降低，似乎通过增加非依赖肌酸的磷酸盐穿梭来对抗，这是一种独特的线粒体应激适应形式。单独的结果表明，依赖肌酸的生物能也可以通过降低 ROS 的靶向线粒体肽来增强。这些结果表明，在肌营养不良症中，氧化应激与线粒体应激适应之间存在特定关系，并提供了原理验证证据，表明依赖肌酸的生物能可以通过靶向线粒体的小肽治疗来改变。
