Mitohormesis during advanced stages of Duchenne muscular dystrophy reveals a redox-sensitive creatine pathway that can be enhanced by the mitochondrial-targeting peptide SBT-20

Abstract

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.

Keywords: Antioxidant; Creatine; Mitochondria; Muscle; Respiration; Small molecule therapy.

Fig. 1

Anthropometrics and functional testing in 12-month-old D2.mdx mice. Body weight (A, n = 8–10), lower limb muscle volume assessed by microCT (B, n = 5), cage hang time (C, n = 5–10), voluntary wheel running in 24 h (D, n = 5–9) and grip strength (E, n = 5–10). Data were analyzed by unpaired t-tests. Results represent mean ± SD; *p < 0.05 compared with wild type.

Fig. 2

Complex I-supported mitochondrial respiration and mitochondrial H₂O₂ emission (mH₂O₂) in left ventricles from 12-month-old D2.mdx mice. Creatine-dependent (A) and -independent (B) ADP-stimulated respiration (JO₂) and ADP-suppression of mH₂O₂ during the process of oxidative phosphorylation (mH₂O₂/O₂) with low [ADP] (25uM; C) and high [ADP] (500uM; D) were assessed in cardiac left ventricle permeabilized muscle fibre bundles by stimulation with pyruvate (5 mM respiration, 10 mM mH₂O₂) and malate (2 mM) to stimulate Complex I with NADH. Mitochondrial creatine kinase (mtCK) total lysate protein content were assessed in heart samples remaining after removal of left ventricles (E). mH₂O₂ represents forward electron transfer that was achieved with NADH generated by pyruvate and malate to stimulate complex I with and without creatine in the experimental media. mH₂O₂ arising from electron slip in the electron transport chain during the process of oxidative phosphorylation is positively associated with membrane potential is therefore suppressed by [ADP] [11]. For A and B, several decades of research has contributed to this theoretical model whereby the matrix-derived ATP is transported through the inner membrane transporter ANT (adenine nucleotide translocase) to the intermembrane space (see Ref. [19] for review). In the presence of creatine, a phosphate is transferred from ATP to creatine by mtCK to produce phosphocreatine (PCr). The ADP product cycles back to the matrix while PCr is exported to the cytoplasm (theorized through VDAC; voltage dependent anion carrier) where it is used by cytoplasmic creatine kinase (cCK) to re-phosphorylate ADP to ATP to support ATP-dependent proteins with creatine returning to the mitochondria. This PCr/creatine system cycles faster than ATP/ADP due to faster diffusion kinetics of both PCr and creatine relative to ADP and ADP, and is estimated to represent up to 80 % of phosphate exchange between mitochondria and cytoplasmic compartments vs 20 % for the direct ATP/ADP shuttle. mtCK, ANT and VDAC are thought to be bound to cardiolipin (see Ref. [9] for review). Both systems are thought to be active in vitro in the presence of creatine. Diffusion distances are not to scale. Data were analyzed by Two-way ANOVA for data in panels C and D, and unpaired t-test for panel E. Results represent means ± SD; n = 7–10. *p < 0.05; nd means ‘no difference’.

Fig. 3

Mitochondrial creatine kinase (mtCK) cysteine redox state and cellular glutathione redox state in the heart from 12-month-old D2.mdx mice. Measurements were made in frozen heart following the removal of left ventricles. Greater cysteine oxidation on immunoprecipitated mtCK from D2.mdx left ventricles is demonstrated by lower binding of the maleimide-tagged fluorescent IR-dye 800 CW probe compared to wild type (A). Glutathione was measured in left ventricle lysates using HPLC-UV for the detection of GSH (B) and HPLC-fluorescence for GSSG (C). The GSH:GSSG ratio (D) and total glutathione (GSH + 2x GSSG; E) were calculated from GSH and GSSG. Data were analyzed by unpaired t-tests between wild type and D2.mdx Results represent mean ± SD; n = 4–8. *p < 0.05 compared to wild type.

Fig. 4

The effects of SBT-20 on complex I-supported mitochondrial respiration and mitochondrial H₂O₂ emission (mH₂O₂) in left ventricles from D2.mdx mice. Mice received daily subcutaneous injections of SBT-20 from day 4–∼12.5 weeks of age. Creatine-dependent and -independent ADP-stimulated respiration (JO₂) and ADP-suppression of mH₂O₂ during the process of oxidative phosphorylation (mH₂O₂/O₂) with low [ADP] (25uM, A, B) and high [ADP] (500uM; C, D) were assessed in cardiac left ventricle permeabilized muscle fibre bundles by stimulation with pyruvate (5 mM respiration, 10 mM mH₂O₂) and malate (2 mM) to stimulate Complex I with NADH. Data were analyzed Two-way ANOVA for data in panels C and D Results represent mean ± SD; n = 8–12. *p < 0.05; nd means ‘no difference’.