
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00297-0
10.1016/j.redox.2024.103319
103319
Short Communication
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☆
Hughes Meghan C. hughesmeghanc@gmail.com
a1
Ramos Sofhia V. sofhia.ramos@adventhealth.com
a1
Brahmbhatt Aditya N. anbrahm4@yorku.ca
a
Turnbull Patrick C. patrick.c.turnbull@gmail.com
a
Polidovitch Nazari N. n.polidovitch@gmail.com
b
Garibotti Madison C. mgarib@yorku.ca
a
Schlattner Uwe uwe.schlattner@univ-grenoble-alpes.fr
c
Hawke Thomas J. hawke@mcmaster.ca
d
Simpson Jeremy A. jeremys@uoguelph.ca
ef
Backx Peter H. pbackx@yorku.ca
b
Perry Christopher GR. cperry@yorku.ca
a⁎
a School of Kinesiology and Health Science and the Muscle Health Research Centre, York University, Toronto, ON, Canada
b Department of Biology and the Muscle Health Research Centre, York University, Toronto, ON, Canada
c University Grenoble Alpes, Inserm U1055, Laboratory of Fundamental and Applied Bioenergetics (LBFA), and Institut Universitaire de France, Grenoble, France
d Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada
e Department of Human Health and Nutritional Sciences and Cardiovascular Research Group, University of Guelph, Guelph, ON, Canada
f IMPART Team Canada Investigator Network, Saint John, New Brunswick, Canada
⁎ Corresponding author. School of Kinesiology and Health Science, Muscle Health Research Centre, 341 Norman Bethune College York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada. cperry@yorku.ca
1 These authors contributed equally.

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© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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

Mitochondria
Muscle
Antioxidant
Respiration
Creatine
Small molecule therapy
==== Body
pmcAbbreviations

ADP adenosine diphosphate

ATP adenosine triphosphate

ANT adenine nucleotide translocase

cCK cytosolic creatine kinase

mH2O2 mitochondrial H2O2 emission

mtCK mitochondrial creatine kinase

PCr phosphocreatine

PDC pyruvate dehydrogenase complex

VDAC voltage dependent anion carrier

1 Introduction

Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease affecting 1 in 5000 boys [1]. DMD is caused by X-linked recessive mutation in the dystrophin gene that almost exclusively affects males, and the loss of this structural protein triggers many cellular dysfunctions including cell membrane fragility, impaired calcium homeostasis and redox and metabolic stress [2]. Conventional glucocorticoid therapy targeting inflammation partially delays the progression of cardiac, respiratory and locomotor muscle dysfunction. However, there is no cure which underscores the need for new therapies [3]. Furthermore, recent exon-skipping therapies, for example, have limited effects in skeletal muscle with no appreciable benefits being identified in the heart [4]. Identifying specific relationships between redox stress and metabolic dysfunction could provide foundational knowledge for pursuing new paradigms of therapy development.

A variety of mitochondrial stress responses have been reported in humans and mouse models, including elevated mitochondrial-induction of apoptosis through permeability transition pore activity, reduced oxidative phosphorylation, and elevated hydrogen peroxide emission (mH2O2) (reviewed in Ref. [5]). In cardiac left ventricle and skeletal muscle from young (4 weeks) D2.mdx dystrophin-deficient mice, we previously reported ADP-stimulated mitochondrial respiration was attenuated and mH2O2 was higher due specifically to a reduced ability of ADP to attenuate mH2O2 particularly when creatine was included in the experimental media compared with when creatine was absent [6,7]. These comparisons were designed to test phosphate shuttling from mitochondrial to cytoplasmic compartments through two theoretical systems comprised of ATP export/ADP import (slow diffusion) and a faster phosphocreatine export/creatine import (faster diffusion) regulated in part by mitochondrial creatine kinase (mtCK) in the intermembrane space (reviewed in Refs. [8,9]). Matrix ADP/ATP turnover is accelerated with creatine as mtCK activity reduces the diffusion distance for the slower diffusing ADP/ATP to the matrix-intermembrane space interface. This creatine-dependent enhancement of ADP-stimulated ATP synthesis also causes greater ADP-suppression of mH2O2 [10] by lowering membrane potential [11]. Therefore, the greater attenuations in creatine-dependent bioenergetics in 4-week-old D2.mdx mice suggests dystrophin deficiency impairs the more effective method of phosphate shuttling at an early stage of the disease.

These findings in 4-week-old dystrophin deficient mice provide insight into unique mitochondrial remodeling events that occur in the early stages of disease. However, the extent to which mitochondria respond in much later stages of the disease is unpredictable. While indices of mitochondrial dysfunction are to be expected, there remains the possibility of a hormetic response over time whereby mitochondria may adapt to chronic dysfunction in specific mitochondrial pathways whilst others continue to fail. Indeed, the concept of mitochondrial hormesis predicts that chronic stress can stimulate mitochondrial adaptations that lead to improved functioning [12]. Our previous findings that creatine-dependent bioenergetics are attenuated moreso than creatine independent system at 4 weeks of age in D2.mdx mice raises an intriguing uncertainty over whether both systems eventually fail, or one can compensate for the other. Furthermore, the findings in D2.mdx mice that the faster creatine-dependent system is attenuated to a greater degree than the creatine-independent system implicates mtCK – a protein known to be inhibited by reactive oxygen species (ROS) [9] - as being particularly sensitive to the redox stress of dystrophin deficiency in the disease process. However, it remains unknown if mtCK oxidation occurs during dystrophin deficiency to explain this specific remodeling of mitochondrial creatine-dependent bioenergetics at any stage of disease.

In this study, we sought to determine whether both mitochondrial creatine-dependent and -independent bioenergetics are attenuated in advanced states of disease in 12-month-old D2.mdx mice or if either system adapts through a form of mitohormesis [12]. Our findings highlight considerable metabolic plasticity in these systems, particularly in the left ventricle, whereby severe reductions in creatine-dependent respiration and elevations in creatine-dependent mH2O2 are seemingly countered by higher respiration and lower mH2O2 in the creatine-independent system of phosphate shuttling. This observation led us to hypothesize that elevated creatine-dependent mH2O2 oxidized mtCK to explain the apparent reduction in mitochondrial sensitivity to creatine which proved to be the case. This finding of greater cysteine oxidation of mtCK linked to a unique impairment in creatine-dependent bioenergetics led us to perform a separate investigation demonstrating proof-of-principle that cardiac creatine-dependent bioenergetics are enhanced with 12 weeks of treatment with the ROS-lowering mitochondrial-targeting tetrapeptide SBT-20 [13].

2 Methods

Male D2.mdx mice originated from breeding colonies maintained at York University (Toronto, Canada) and sourced from The Jackson Laboratory (Bar Harbor, United States). DBA/2J wild type were purchased from The Jackson Laboratory at 4–5 weeks of age and aged in-house.

In the first part of this study, D2.mdx and wild type mice were aged to 52 weeks (12 months). Two to three days prior to tissue removal, mice were assessed for 24-h voluntary wheel running, hang time using an inverted cage lid and forelimb grip strength. Mice also received a single micro computed tomography scan for measurement of lower limb muscle volume. In the second part of the study, beginning at 4-days of age, D2.mdx mice received subcutaneous injections of 5 mg/kg SBT-20 (Stealth Biotherapeutics; Newton, MA, USA) 7 days/week continuously for 12 weeks. Thereafter, ultrasound assessments of cardiac function were performed, and muscles were removed.

Detailed methodology, including information on permeabilized muscle fibre bundles, high resolution respirometry, mitochondrial H2O2 emission, calcium retention capacity assessments, glutathione, western blots, redox assessments of mtCK, and statistical methods, are described in detail in the Supplemental Information (Appendix A).

3 Results

3.1 Mitochondrial respiration and mH2O2: modeling metabolic demand and phosphate shuttling

At 12 months of age, D2.mdx mice showed reduced body weight, lower limb muscle volume and cage hang time compared with age matched DBA/2J wild type mice (Fig. 1A–E) as expected with this model [7].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. 1

Creatine-dependent versus creatine-independent regulation of ADP-stimulated respiration were assessed by placing separate permeabilized left ventricle fibre bundles in experimental media with 20 mM creatine to saturate mtCK [14,15] or in media without creatine to model both theoretical models of phosphate shuttling (Fig. 2A and B). We found that left ventricles from 12-month-old D2.mdx mice have lower creatine-dependent Complex I-supported respiration compared to wildtype when stimulated by both low and high [ADP] modeling a range of metabolic demands for mitochondrial ATP synthesis (Fig. 2C and D). D2.mdx surprisingly showed increased respiration compared to wildtype when assessed in the absence of creatine at both low and high [ADP] suggesting the slower but direct ADP/ATP cycling system was upregulated despite reductions in the faster creatine-dependent system. Furthermore, the higher respiration seen in the condition with creatine compared to the absence of creatine in wildtype (Fig. 2C and D) is consistent with the known stimulatory effects of creatine on enhancing ADP-stimulated respiration reflective of faster adenylate cycling (Fig. 2A). In this regard, a critical observation is that this stimulatory effect of creatine was lost in the 12-month D2.mdx mice as shown by similar respiration rates when creatine was either present or absent (Fig. 2C and D) at both low or high [ADP]. Collectively, these results demonstrate a loss of mitochondrial creatine sensitivity in 12-month-old D2.mdx mice as well as an apparent compensation in the slower creatine-independent model of adenylate cycling.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. 2

In the presence of creatine, mH2O2 driven by Complex I-supported mH2O2 (NADH) through forward electron transfer was not different between wildtype and D2.mdx at low and high [ADP] (Fig. 2C and D) but was higher in D2.mdx with reverse electron transfer with high [ADP] (Supplemental Figs. S1A and B). This was noted when expressing mH2O2 relative to oxygen consumption at the same ADP concentration made in parallel fibre bundles to gain insight into how mH2O2 is regulated during the process of oxidative phosphorylation. Separate post-hoc analyses examining the ability of creatine to attenuate mH2O2 [16] revealed that wildtype hearts showed the expected effect whereby mH2O2 was lower when creatine was present compared to its absence when stimulated with both forward electron transfer during high [ADP] (Fig. 2C and D) and reverse electron transfer during both low and high [ADP] (Supplemental Figs. S1A and B), but this effect was lost in the D2.mdx whereby mH2O2 was similar in the presence and absence of creatine (Fig. 2C and D, Supplemental Figs. S1A and B). Given creatine is known to accelerate ADP/ATP cycling which enhances the well-established effect of ADP in lowering membrane potential-dependent mH2O2 ([6,[16], [17], [18]]; see discussion), these findings suggest that creatine is less capable of stimulating respiration, as described above, and attenuating mH2O2 in the left ventricles of 12-month-old D2.mdx mice. Furthermore, similar to the findings with respiration, there was an apparent compensation in the creatine-independent system whereby mH2O2 in the absence of creatine was lower in D2.mdx than wildtype with high [ADP] when driven by forward electron transfer (Fig. 2D) or with both low and high [ADP] when driven by reverse electron transfer (Supplemental Figs. S1A and B).

There were no changes in mtCK protein content of the left ventricles (Fig. 2E) indicating that factors independent of mtCK content may mediate the loss of creatine sensitivity in D2.mdx hearts.

Collectively, these results demonstrate divergent remodeling of left ventricular mitochondrial-cytoplasmic ADP/ATP cycling in 12-month-old D2.mdx mice whereby impairments in the faster creatine-dependent regulation of ADP-stimulated respiration and suppression of mH2O2 are countered by apparent compensations in the slower creatine-independent system.

We also questioned whether the reductions in creatine-dependent ADP governance of respiration and mH2O2 were unique to the heart or if it occurred in other muscles in D2.mdx at 12 months of age. Unlike the dystrophic heart (Fig. 2C and D), creatine stimulated increases in respiration in the diaphragm of 12-month-old D2.mdx but, similar to the heart, did not attenuate mH2O2 (Supplemental Figs. S2B, C, E, and F). In the diaphragm, creatine-stimulated respiration appeared to be greater during low and high [ADP] in 12-month-old -matched wildtype vs D2.mdx suggesting a partial impairment in creatine sensitivity nonetheless occurs in dystrophic diaphragm. However, unlike the left ventricle, there were no apparent compensatory increases in respiration in the absence of creatine but lower mH2O2 in this condition, similar to the heart, were observed (Supplemental Figs. S2B, C, E, and F). Also, no effect of creatine was seen in quadriceps or white gastrocnemius in 12-month-old wildtype, in contrast to the effects seen in our previous work at 4 weeks ([17,18] see discussion), or in 12-month-old D2.mdx (Fig. 2C and D).

We next explored whether changes in bioenergetics in each muscle type were related to altered contents of electron transport chain and phosphate shuttling components that are stimulated in most of the bioenergetic protocols employed in this study. In the heart, no changes in specific subunits of the electron transport chain, ANT1 or VDAC2 occurred, similar to mtCK (Fig. 2E), suggesting changes in respiration and mH2O2 might be linked to altered intrinsic activities, but this would require further investigation. Pyruvate dehydrogenase contents or activities, which regulates NADH production by pyruvate in these protocols, were not assessed. In the diaphragm of 12-month-old D2.mdx mice, certain components of the electron transport chain as well as ANT1 and VDAC2 were lower (Supplemental Figs. S3B and C) which could explain the lower respiration. In the white gastrocnemius, increased subunits of Complex I and IV as well as VDAC2 were observed (Supplemental Figs. S3B and C). The lack of differences in respiration in the white gastrocnemius suggests the higher contents of these proteins (all of which are involved in these ADP-stimulated respiration protocols) may have offset reductions in their specific activities in order to ‘maintain’ normal respiration rates expressed per mg tissue (Supplemental Figs. S2B, C, E, and F). Increases in complex II in the quadriceps would not be expected to influence the Complex I-stimulated respiration or mH2O2 protocols used in this study. Collectively, there are heterogeneous mitochondrial alterations across muscle type in 12-month-old D2.mdx but the loss of creatine sensitivity seems to be predominant in the cardiac left ventricles despite no changes in contents of many proteins stimulated in the bioenergetic protocols used in this study.

In an effort to explain why mitochondrial creatine sensitivity was uniquely impaired in the dystrophic heart and considering there were no changes in mtCK protein content of the left ventricles (see Fig. 2E), we next questioned whether mtCK was modified through redox-linked post-translational modifications given mH2O2 was elevated.

3.2 Cellular redox state and mtCK thiol oxidation

The increased creatine-dependent mH2O2 during the process of oxidative phosphorylation in 12-month-old D2.mdx mice guided us to hypothesize that mtCK thiols would be more oxidized than in wild type given mtCK is a redox-sensitive protein (reviewed in Ref. [9]). We next immunoprecipitated mtCK from left ventricles (Supplemental Fig. S3D) and incubated the extract in a maleimide-tagged fluorophore that binds irreversibly to reduced cysteine thiols, with no affinity to oxidized thiols, as previously described ([17,20,21], Supplemental Fig. S3). This experiment demonstrated that mtCK is more oxidized in the left ventricle of 12-month-old D2.mdx mice (Fig. 3A). This was related to a more oxidized glutathione (H2O2 scavenger) redox state as reflected by a lower GSH:GSSG (reduced to oxidized glutathione redox buffer) in lysate from left ventricles due apparently to high variability in GSSG (Fig. 3B–E). This oxidized environment in the left ventricle (Fig. 3B–E) was more pronounced than other specific muscles, although the diaphragm also showed a lower GSH:GSSG despite increases in both GSH and GSSG (Supplemental Fig. S3A). No changes were observed in quadriceps and white gastrocnemius (Supplemental Fig. S3A).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. 3

There were no differences in ANT1, VDAC2 protein contents or subunits of the electron transport chain in the left ventricle (Supplemental Fig. S3B and SC). While we did not assess their post-translational modifications, these collective observations guided us to examine mtCK-linked creatine-dependent bioenergetics in the heart in more detail.

3.3 In vivo treatment with the mitochondrial ROS-lowering peptide SBT-20

Given we observed attenuated creatine-sensitive control of bioenergetics by ADP in the left ventricle, as the second part of the study we performed a pilot study with in vivo injections of the mitochondrial targeting ROS-lowering peptide SBT-20 in D2.mdx mice from 4 days of age to ∼12.5 weeks of age. Treatment at an earlier age was chosen given a 12-month protocol was not possible. This 12-week treatment protocol increased pyruvate-supported ADP-stimulated respiration in the presence of creatine compared to saline-treated D2.mdx mice and had no effect on creatine-independent respiration at low or high [ADP] (Fig. 4A and C). Unlike 12-month-old D2.mdx where creatine did not increase cardiac mitochondrial respiration compared to the absence of creatine (Fig. 2C and D), left ventricles from 12-week-old mice appeared to retain some creatine sensitivity given respiration was higher in D2.mdx saline in the presence of creatine vs in the absence of creatine (Fig. 4A and C). Nonetheless, the results demonstrate that SBT-20 has a specific action of enhancing creatine-dependent respiration in D2.mdx mice given no effect was seen in the creatine-independent condition.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’.

Fig. 4

mH2O2 in the presence of creatine was not different than in the absence of creatine in D2.mdx saline treated mice (Fig. 4B and D). Unlike the respiration data, this suggests that creatine insensitivity developed in D2.mdx at least in regard to regulating mH2O2. While a wildtype 12-week-old group was not included to verify this observation, it is well-established that creatine enhances the effect of ADP on attenuating mH2O2 (see discussion) as was seen in older 12-month wildtype mice (Fig. 2C and D). This impaired ability of creatine to attenuate mH2O2 was not seen in SBT-20 treated mice given mH2O2 was lower with creatine compared to in its absence in both low and high [ADP] conditions (Fig. 4B and D) thereby demonstrating that SBT-20 enhances creatine—dependent suppression of mH2O2 in line with the greater creatine-dependent respiration. Lastly, while it is not possible to determine if the compensatory increase in creatine-independent bioenergetics seen in 12-month D2.mdx mice (Fig. 2C and D) occurred in these 12-week-old mice (Fig. 4A–D) without a 12-week-old wildtype control group, the enhanced action of creatine on respiration and mH2O2 with SBT-20 nonetheless demonstrates its ability to improve mitochondrial creatine sensitivity, particularly given D2.mdx mice that did not receive SBT-20 were clearly insensitive to creatine.

Increased susceptibility to calcium-induced mitochondrial permeability transition pore activity was observed given calcium retention capacity was reduced in 12-month-old D2.mdx mouse left ventricles (Supplemental Fig. S1C). There was no effect of SBT-20 on this measure compared to saline-treated D2.mdx mice at ∼12.5 weeks of age (Supplemental Fig. S1D).

Select cardiac functional parameters were not altered by SBT-20 compared to saline-treated D2.mdx mice (Supplemental Figs. S4A–D).

Lastly, to determine if higher mH2O2 in the presence of creatine was unique to the ADP-sensitive regulation of ROS production, we also assessed Complex III- and pyruvate dehydrogenase complex (PDC)-supported mH2O2 in the absence of ADP. In 12-month-old D2.mdx, Complex III-supported mH2O2 was lower in white gastrocnemius whereas PDC-supported mH2O2 was lower in the left ventricle, diaphragm, and quadriceps (Supplemental Fig. S1E). SBT-20 had no effect on these pathways. While this lower mH2O2 in 12-month-old D2.mdx mice warrant further investigation into how the contents or post-translational regulation of these pathways are altered during dystrophin deficiency, these data suggest the loss of creatine-dependent bioenergetics during pyruvate oxidation (Fig. 2) is a unique mechanism contributing to higher mH2O2 during attenuated oxidative phosphorylation in DMD.

4 Discussion

Creatine enhances the ability of ADP to stimulate oxidative phosphorylation and attenuate H2O2 emission in mitochondria. Here, we show 12-month-old D2.mdx mice have lower creatine-dependent bioenergetics that are related to greater cysteine oxidation of mtCK. In contrast, creatine-independent bioenergetics were apparently enhanced which may represent a form of mitohormesis in response to chronic disease in dystrophin deficient mice. Moreover, 12 weeks of treatment with the ROS-lowering mitochondrial-targeting peptide SBT-20 (up to ∼12.5 weeks of age) increased creatine-dependent respiration and lowered mH2O2, particularly under states of high metabolic demand in the left ventricle. These results demonstrate a specific mechanism linking redox and metabolic stress in mitochondria arising from dystrophin mutations and serve as a direction for continued development of mitochondrial-targeted therapies designed to restore metabolic and redox balance in DMD.

At 12 months of age, the left ventricle of D2.mdx mice also demonstrated a surprising increase in creatine-independent respiration, which contrasts the decreases we previously reported in this pathway in 4-week-old mice [6,7]. This observation suggests the slower mitochondrial ADP-ATP system may compensate for impairments in the faster creatine-dependent phosphate shuttling mechanism [9]. The greater abundance of cysteine oxidation on mtCK was consistent with higher rates of creatine-sensitive mH2O2 during the process of oxidative phosphorylation and the more oxidized cellular environment (lower GSH:GSSG). mtCK oxidation may be unique to advanced stages of this disease, given we previously reported no differences in 4-week D2.mdx mice, at least in skeletal muscle [17]. Further studies could examine the precise form of thiol modification that occurs in 12-month-old D2.mdx mice, such as glutathionylation, considering its emerging role in linking redox signaling to metabolic control [22,23] and considering the shift in GSH:GSSG noted in the present study. Likewise, the degree to which mtCK thiol redox state was preserved by SBT-20 could be considered given the positive effects noted in this part of the study that was otherwise limited by tissue availability. Further studies could assess the specific cystines that were oxidized given cysteine 278, which regulates mtCK activity, and C358, which may regulate mtCK tethering to the inner mitochondrial membrane, were previously shown to be redox sensitive [24]. ANT and VDAC thiol oxidation could also be assessed to explain the apparent compensatory increases in creatine-independent bioenergetics at late stages of disease given their protein contents did not change, although reconciling these measures with the divergent response of creatine-dependent and -independent systems may be challenging given both proteins are thought to be primary regulators in either system.

We were unable to assess cardiac function in 12-month-old D2.mdx mice due to their qualitatively frail nature. Furthermore, while we did not see robust differences in grip strength compared to age-matched wild type mice, we did note that the absolute values of grip strength in the 12 month old wild type mice are ∼50 % of the values we have reported previously in this strain at 4 weeks of age [7,17] suggesting an aging effect occurred in the control group. Nonetheless, the other parameters demonstrate a severe myopathy in the 12-month-old D2.mdx mice. Also, the effect of age on mitochondrial reprogramming in locomotor and respiratory muscle pathology compared to muscle dysfunction could also be considered, particularly in relation to the earlier remodeling seen in 4-week-old D2.mdx [6,7,17,18].

SBT-20 is a small tetrapeptide with high cell-penetrating potential that accumulates on cardiolipin in the inner mitochondrial membrane similar to the mitochondrial-targeting peptide elamipretide (formerly SS-31) [[25], [26], [27]] that prevents cytochrome c peroxidase activity, preserves oxidative phosphorylation, and prevents increases in superoxide production in response to stressors [28,29]. SBT-20 also preserved mitochondrial respiration in H2O2-treated cells and partially prevents cardiac infarct size in response to ischaemia reperfusion injury [13]. To our knowledge, this is the first study to report a unique creatine-specific preservation of bioenergetics by cardiolipin-targeting peptides. As mtCK is thought to be bound to cardiolipin [9], future studies could consider whether the age-related impairment in creatine-dependent reductions in respiration seen in the present study was due to altered cardiolipin tethering to mtCK, and whether the preserved creatine-dependent bioenergetics by SBT-20 preserved such interactions. As ANT and VDAC are also thought to be bound to cardiolipin, the potential for SBT-20 to regulate the system as a whole could be considered.

As an aged-match wild type control group (∼12 weeks of age) was not included for the SBT-20-treated D2.mdx experiments, we are not able to prove if the creatine-stimulated increases in respiration seen in D2.mdx vehicle treated animals were blunted compared to wildtype. We have previously demonstrated that creatine-dependent respiration is lower in 4-week-old D2.mdx mouse left ventricles [6]. Therefore, as creatine-dependent respiration is lower at both 4 weeks and 12 months as seen in Fig. 2, it is possible that similar reductions would exist at ∼12 weeks as well, but this would require the inclusion of an age-matched wildtype control group to be certain. However, the results clearly demonstrate that creatine does not lower cardiac mH2O2 in 12-week-old D2.mdx vehicle-treated mice which is consistent with the creatine insensitivity seen in 4-week-old [6,17] and 12-month-old D2.mdx hearts (Fig. 2C and D) depending on the [ADP]. SBT-20 treatment lowered mH2O2 only in the presence of creatine which demonstrates a remarkable ability to convert mitochondria from a creatine-insensitive to creatine-sensitive phenotype in D2.mdx mice. Likewise, SBT-20 increased creatine-dependent respiration in D2.mdx mice to levels higher than what was seen in vehicle treated mice. As such, this study provides first-time proof-of-principle evidence that mitochondrial creatine-dependent bioenergetics can be enhanced by this class of mitochondrial-targeted therapeutics.

Although select cardiac functional parameters were not altered by SBT-20 compared to saline-treated D2.mdx mice, we cannot determine if a dysfunction existed at this age in comparison to wild type. In fact, prior work at younger ages in the D2.mdx mouse have reported no overt cardiomyopathy [6] while much older ages are known to have a moderate left ventricular cardiomyopathy in this model [19], at least as assessed with non-invasive approaches.

SBT-20 did not alter calcium retention capacity suggesting that it did not have an effect on mitochondrial permeability transition – an event that links mitochondrial calcium overload to apoptosis [30]. This is in contrast to previous reports showing SBT-20 prevents cytochrome c release, a key trigger of mitochondrial-induced apoptosis, in response to ischaemic stress [29] that is known to trigger mitochondrial permeability transition [30]. As our pilot study did not include a wild type control group, we are unable to determine whether calcium retention capacity was reduced in the D2.mdx saline control group at this age in contrast to the clear reductions seen at 12 months of age (Supplemental Fig. S1).

Collectively, these findings suggest that a time-course design could be employed to determine whether SBT-20 prevents the unique time-dependent signatures of mitochondrial stress and delays the eventual onset of cardiomyopathy. To this end, we did not see an effect of SBT-20 on cardiac function assessed with echocardiography on ∼12.5-week-old mice. However, previous reports have shown an absence of overt cardiac dysfunction in D2.mdx mice at 4 weeks [6] or 7 weeks [19] of age but is apparent at 28 weeks and 52 weeks [19], with the latter age corresponding to our observation of oxidized mtCK. More in-depth analyses with invasive hemodynamics is also warranted given this approach could identify dysfunctions that are not detected with echocardiography.

4.1 Additional perspectives and limitations

This investigation was designed to determine whether creatine-dependent and -independent mechanisms of phosphate shuttling remodel in positive or negative manners during advanced stages of disease in dystrophin deficient mice. The elevated mH2O2 was linked to a greater degree of oxidized cysteines in mtCK in 12-month-old D2.mdx mice. The use of a thiol labeling technique in immunoprecipitated mtCK provides a unique insight into a potential target of mitochondrial ROS during dystrophin deficiency that is linked to a specific impairment in creatine-dependent respiration. Thiol labeling of enriched protein fractions enabled this discovery that would not be possible if the study relied solely on broader measures of redox conditions in the cell. Rather, the change in glutathione redox state provides insight into how changes in mH2O2 impacted this primary H2O2 scavenger of the cell. Additional measures of lipid peroxidation or broader protein redox states in the cell could be considered in future investigations but are tangential to the question of this investigation pertaining to creatine-dependent and -independent bioenergetics. In the 2nd investigation examining the effects of SBT-20, no redox measurements were performed to determine the impact of altered mH2O2 on cellular glutathione or other redox conditions or mtCK redox state. Such measurements should be considered in future investigations developing mitochondrial therapeutics for DMD in relation to measures of muscle function in order to more fully appreciate the impact of altered mitochondrial bioenergetics on redox-dependent processes regulating muscle contraction. Also, measurements of creatine, phosphocreatine, ADP and ATP (as examples) would add further insight into the degree to which the mitochondrial-cytoplasmic phosphate shuttling was altered in the cell. The extremely small muscle samples available from these 12-month-old frail dystrophin deficient mice were used in the most efficient way possible to obtain the dataset reported in this investigation such that future studies could consider adding these broader insights where possible.

5 Conclusions

These findings demonstrate that left ventricle mitochondrial creatine metabolism is attenuated in relation to oxidized mitochondrial creatine kinase in late stages of disease in 12-month-old D2.mdx mice. This impairment was linked to increases in creatine-independent bioenergetics which may represent a form of mitohormesis in response to chronic disease progression in dystrophin deficient mice. The ability of the ROS-lowering mitochondrial peptide SBT-20 to increase creatine-dependent pyruvate oxidation and lower creatine-dependent mitochondrial H2O2 emission demonstrates the potential for a mitochondrial-targeted therapeutic to enhance coupled respiration during dystrophin deficiency, particularly in regard to the regulation of mitochondrial creatine metabolism. This finding supports continued development of a new paradigm of mitochondrial-targeted redox and metabolic enhancing therapeutics that do not exist in the current standard of care of anti-inflammatory and other emerging treatments.

Funding

Funding was provided to C.G.R.P. and T.J.H. by the National Science and Engineering Research Council (no. 436138-2013 and no. 2018–06324 , respectively) and an Ontario Early Researcher Award (C.G.R.P., no. 2017-0351 ) with infrastructure supported by Canada Foundation for Innovation, the James H. 10.13039/100008176 Cummings Foundation , and the Ontario Research Fund. J.A.S was supported by the Heart and Stroke Foundation of Canada (HSFC; S13 SI 0592 ) and a new investigator award with the Heart and Stroke Foundation of Canada. P.B. was supported by 10.13039/501100000024 Canadian Institutes of Health Research , Project Grant (PJT 153159 ) and a Canada Research Chair in Cardiovascular Biology. M.C.H. and P.C.T. were supported by a NSERC CGS-PhD scholarship. S.V.R. was supported by an Ontario Graduate Scholarship.

CRediT authorship contribution statement

Meghan C. Hughes: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing. Sofhia V. Ramos: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. Aditya N. Brahmbhatt: Visualization, Writing – review & editing. Patrick C. Turnbull: Formal analysis, Methodology, Writing – review & editing. Nazari N. Polidovitch: Formal analysis, Methodology. Madison C. Garibotti: Data curation, Formal analysis, Writing – review & editing. Uwe Schlattner: Conceptualization, Formal analysis, Writing – original draft. Thomas J. Hawke: Formal analysis, Methodology, Writing – original draft, Writing – review & editing. Jeremy A. Simpson: Data curation, Formal analysis, Methodology, Writing – review & editing. Peter H. Backx: Data curation, Formal analysis, Methodology, Writing – review & editing. Christopher GR. Perry: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors have no conflict of interest to declare. Stealth Biotherapeutics supplied SBT-20 without funding.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Data availability

Data will be made available on request.

Acknowledgements

We thank Trevor Teich for providing technical assistance with certain experiments, and Dr Robert Tsushima for kindly providing access to the Vevo 2100 ultrasound imaging system for echocardiography.

☆ Present address for Sofhia Ramos: Translational Research Institute, AdventHealth, Orlando, FL, USA.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103319.
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References

1 Bladen C.L. Salgado D. Monges S. Foncuberta M.E. Kekou K. Kosma K. The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations Hum. Mutat. 36 2015 395 402 10.1002/humu.22758 25604253
2 Allen D.G. Whitehead N.P. Froehner S.C. Absence of dystrophin disrupts skeletal muscle signaling: roles of Ca2+, reactive oxygen species, and nitric oxide in the development of muscular dystrophy Physiol. Rev. 96 2016 253 305 10.1152/physrev.00007.2015 26676145
3 Kourakis S. Timpani C.A. Campelj D.G. Hafner P. Gueven N. Fischer D. Standard of care versus new-wave corticosteroids in the treatment of Duchenne muscular dystrophy: can we do better? Orphanet J. Rare Dis. 16 2021 117 10.1186/s13023-021-01758-9 33663533
4 Shah M.N.A. Yokota T. Cardiac therapies for Duchenne muscular dystrophy Ther. Adv. Neurol. Disord. 16 2023 17562864231182934 10.1177/17562864231182934
5 Bellissimo C.A. Garibotti M.C. Perry C.G.R. Mitochondrial stress responses in Duchenne muscular dystrophy: metabolic dysfunction or adaptive reprogramming? Am. J. Physiol. Cell Physiol. 323 2022 C718 C730 10.1152/ajpcell.00249.2022 35816642
6 Hughes M.C. Ramos S.V. Turnbull P.C. Edgett B.A. Huber J.S. Polidovitch N. Impairments in left ventricular mitochondrial bioenergetics precede overt cardiac dysfunction and remodelling in Duchenne muscular dystrophy J. Physiol. 598 2020 1377 1392 10.1113/JP277306 30674086
7 Hughes M.C. Ramos S.V. Turnbull P.C. Rebalka I.A. Cao A. Monaco C.M.F. Early myopathy in Duchenne muscular dystrophy is associated with elevated mitochondrial H2 O2 emission during impaired oxidative phosphorylation J. Cachexia Sarcopenia Muscle 10 2019 643 661 10.1002/jcsm.12405 30938481
8 Wallimann T. Tokarska-Schlattner M. Schlattner U. The creatine kinase system and pleiotropic effects of creatine Amino Acids 40 2011 1271 1296 10.1007/s00726-011-0877-3 21448658
9 Schlattner U. Kay L. Tokarska-Schlattner M. Mitochondrial proteolipid complexes of creatine kinase Subcell. Biochem. 87 2018 365 408 10.1007/978-981-10-7757-9_13 29464567
10 Meyer L.E. Machado L.B. Santiago A.P.S.A. Da-Silva W.S. De Felice F.G. Holub O. Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity J. Biol. Chem. 281 2006 37361 37371 10.1074/jbc.M604123200 17028195
11 Nicholls D.G. Ferguson S.J. Bioenergetics 4 2013 Elsevier
12 Ristow M. Zarse K. How increased oxidative stress promotes longevity and metabolic health: the concept of mitochondrial hormesis (mitohormesis) Exp. Gerontol. 45 2010 410 418 10.1016/j.exger.2010.03.014 20350594
13 Dai W. Cheung E. Alleman R.J. Perry J.B. Allen M.E. Brown D.A. Cardioprotective effects of mitochondria-targeted peptide SBT-20 in two different models of rat ischemia/reperfusion Cardiovasc. Drugs Ther. 30 2016 559 566 10.1007/s10557-016-6695-9 27747447
14 Walsh B. Tonkonogi M. Söderlund K. Hultman E. Saks V. Sahlin K. The role of phosphorylcreatine and creatine in the regulation of mitochondrial respiration in human skeletal muscle J. Physiol. 537 2001 971 978 10.1111/j.1469-7793.2001.00971.x 11744769
15 Perry C.G. Kane D.A. Lin C.T. Kozy R. Cathey B.L. Lark D.S. Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle Biochem. J. 437 2011 215 222 10.1042/BJ20110366 21554250
16 Meyer L.E. Machado L.B. Santiago A.P. da-Silva W.S. De Felice F.G. Holub O. Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity J. Biol. Chem. 281 2006 37361 37371 10.1074/jbc.M604123200 17028195
17 Bellissimo C.A. Delfinis L.J. Hughes M.C. Turnbull P.C. Gandhi S. DiBenedetto S.N. Mitochondrial creatine sensitivity is lost in the D2.mdx model of Duchenne muscular dystrophy and rescued by the mitochondrial-enhancing compound Olesoxime Am. J. Physiol. Cell Physiol. 324 2023 C1141 C1157 10.1152/ajpcell.00377.2022 36689672
18 Ramos S.V. Hughes M.C. Delfinis L.J. Bellissimo C.A. Perry C.G.R. Mitochondrial bioenergetic dysfunction in the D2.mdx model of Duchenne muscular dystrophy is associated with microtubule disorganization in skeletal muscle PLoS One 15 2020 e0237138 10.1371/journal.pone.0237138
19 Coley W.D. Bogdanik L. Vila M.C. Yu Q. Van Der Meulen J.H. Rayavarapu S. Effect of genetic background on the dystrophic phenotype in mdx mice Hum. Mol. Genet. 25 2016 130 145 10.1093/hmg/ddv460 26566673
20 Frasier C.R. Moukdar F. Patel H.D. Sloan R.C. Stewart L.M. Alleman R.J. Redox-dependent increases in glutathione reductase and exercise preconditioning: role of NADPH oxidase and mitochondria Cardiovasc. Res. 98 2013 47 55 10.1093/cvr/cvt009 23341578
21 Sloan R.C. Moukdar F. Frasier C.R. Patel H.D. Bostian P.A. Lust R.M. Mitochondrial permeability transition in the diabetic heart: contributions of thiol redox state and mitochondrial calcium to augmented reperfusion injury J. Mol. Cell. Cardiol. 52 2012 1009 1018 10.1016/j.yjmcc.2012.02.009 22406429
22 Young A. Gill R. Mailloux R.J. Protein S-glutathionylation: the linchpin for the transmission of regulatory information on redox buffering capacity in mitochondria Chem. Biol. Interact. 299 2019 151 162 10.1016/j.cbi.2018.12.003 30537466
23 Mailloux R.J. Protein S-glutathionylation reactions as a global inhibitor of cell metabolism for the desensitization of hydrogen peroxide signals Redox Biol. 32 2020 101472 10.1016/j.redox.2020.101472
24 Wendt S. Schlattner U. Wallimann T. Differential effects of peroxynitrite on human mitochondrial creatine kinase isoenzymes. Inactivation, octamer destabilization, and identification of involved residues J. Biol. Chem. 278 2003 1125 1130 10.1074/jbc.M208572200 12401781
25 Szeto H.H. Birk A.V. Serendipity and the discovery of novel compounds that restore mitochondrial plasticity Clin. Pharmacol. Ther. 96 2014 672 683 10.1038/clpt.2014.174 25188726
26 Zhao K. Luo G. Zhao G.-M. Schiller P.W. Szeto H.H. Transcellular transport of a highly polar 3+ net charge opioid tetrapeptide J. Pharmacol. Exp. Therapeut. 304 2003 425 432 10.1124/jpet.102.040147
27 Birk A.V. Liu S. Soong Y. Mills W. Singh P. Warren J.D. The mitochondrial-targeted compound SS-31 re-energizes ischemic mitochondria by interacting with cardiolipin J. Am. Soc. Nephrol. 24 2013 1250 1261 10.1681/asn.2012121216 23813215
28 Szeto H.H. Mitochondria-targeted cytoprotective peptides for ischemia-reperfusion injury Antioxidants Redox Signal. 10 2008 601 619 10.1089/ars.2007.1892
29 Birk A.V. Chao W.M. Liu S. Soong Y. Szeto H.H. Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia Biochim. Biophys. Acta 1847 2015 1075 1084 10.1016/j.bbabio.2015.06.006 26071084
30 Bernardi P. Gerle C. Halestrap A.P. Jonas E.A. Karch J. Mnatsakanyan N. Identity, structure, and function of the mitochondrial permeability transition pore: controversies, consensus, recent advances, and future directions Cell Death Differ. 30 2023 1869 1885 10.1038/s41418-023-01187-0 37460667
