Mitochondrial‐Targeted Peptide SBT‐20 Improves Mitochondrial Bioenergetics In Duchenne Muscular Dystrophy In a Mitochondrial Creatine Kinase Dependent Manner

Abstract

RATIONALEDuchenne Muscular Dystrophy is a progressive muscle wasting disease in males resulting from mutations in the X‐linked gene dystrophin. The loss of dystrophin in the dystroglycan complex causes severe muscle pathology including cardiomyopathy which can lead to early death. While the cardiac dysfunction in mouse models of this disease has been well characterized, the precise signaling events contributing to this myopathy have yet to be determined. Given that this disease is associated with markers of oxidative stress, and that other models of cardiac dysfunction are associated with impaired mitochondrial oxidative phosphorylation (respiration) and elevated oxidant emission (H2O2), we hypothesized that similar dysfunctions would exist in D2.B10‐DMDmdx/2J mice (DMD) that could be corrected with the mitochondrial targeted peptide SBT‐20.METHODSLeft ventricular mitochondrial H2O2 emission (spectrofluorometry; Amplex UltraRed fluorophore) and respiration (high‐resolution respirometry; Oroboros Oxygraph) were assessed in permeabilized muscle fibre bundles (PmFB) at 4 wk of age in DMD and DBA.2J wildtype (CONT), and again at 13–14 wk in DMD following daily subcutaneous injections of SBT‐20 (5mg/kg) or saline (0.9%) from 4 d for 12 weeks. H2O2 emission was measured in the presence of physiological ADP (25μM) given ADP is a critical suppressor of H2O2 emission during respiration that can be influenced by the phosphate‐shuttling activity of mitochondrial creatine kinase (mtCK). The contribution of mtCK was assessed by saturating [creatine] (Cr) in vitro.RESULTS AND INTERPRETATIONAt 4 wk, ADP‐suppression (25μM) of Complex I‐supported H2O2 (5mM Pyruvate/2mM Malate) was impaired in DMD in the presence of Cr (+Cr; 36+/−4% of emission at 0μM ADP in DMD vs 12+/−1% of emission at 0μM ADP in CONT when expressed per O2 consumed; p<0.01). This was also observed in the absence of Cr (−Cr; 36+/−4 vs 17+/−2% of 0μM ADP/O2 consumed, p<0.01) suggesting H2O2 emission is elevated in DMD independent of mtCK‐dependent phosphate shuttling. At this same [ADP], complex I‐supported respiration was lower in DMD vs CONT in both +Cr: (30.8+/−5.0 vs 54.2+/−10.2 pmol•s−1•mg wet wt−1, p<0.05) and ‐ Cr (35.0+/−1.2 vs 52.0+/−5.4 pmol•s−1•mg wet wt−1, p<0.01). After ~12 wk injections, SBT‐20‐treated DMD demonstrated greater ADP‐suppression of Complex I‐supported H2O2 vs CONT in +Cr (12+/−1% vs 15+/−0 % of emission at 0μM ADP/O2 consumed, p<0.01). However, there were no differences in ‐Cr (40+/−11 vs 42+/−13 % of 0μM ADP/O2 consumed), suggesting SBT‐20 specifically improved mtCK‐dependent ADP suppression of H2O2 emission. SBT‐20 did not change respiration in DMD vs CONT in +Cr (111.4 +/− 12.0 vs 82.1 +/− 11.2 pmol•s−1•mg wet wt−1, p=0.10) or –Cr condition (59.6 +/− 7.4 vs 65.3 +/− 6.8 pmol•s−1•mg wet wt−1). Collectively, these findings demonstrate that SBT‐20 is an effective therapeutic approach for improving the ability of mtCK to mediate ADP‐suppression of H2O2 in DMD heart.Support or Funding InformationFunding was provided to C.G.R.P. by National Science and Engineering Research Council (#436138‐2013) with infrastructure supported by Canada Foundation for Innovation, Ontario Research Fund and the James H. Cummings Foundation. M.C.H. was supported by an NSERC CGS‐D scholarship.