International Journal of Molecular Sciences Article The Effect of Deflazacort Treatment on the Functioning of Skeletal Muscle Mitochondria in Duchenne Muscular Dystrophy Mikhail V. Dubinin 1,* , Eugeny Yu. Talanov 2, Kirill S. Tenkov 1, Vlada S. Starinets 1,2, Natalia V. Belosludtseva 2 and Konstantin N. Belosludtsev 1,3 1 Department of Biochemistry, Cell Biology and Microbiology, Mari State University, pl. Lenina 1, 424001 Yoshkar-Ola, Russia; [email protected] (K.S.T.); [email protected] (V.S.S.); [email protected] (K.N.B.) 2 Laboratory of Mitochondrial Transport, Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Institutskaya 3, 142290 Pushchino, Russia; [email protected] (E.Y.T.); [email protected] (N.V.B.) 3 Biophotonics Center, Prokhorov General Physics Institute, Russian Academy of Sciences, Vavilov st. 38, 119991 Moscow, Russia * Correspondence: [email protected]; Tel.: +7-987-701-0437 Received: 27 October 2020; Accepted: 17 November 2020; Published: 19 November 2020 Abstract: Duchenne muscular dystrophy (DMD) is a severe hereditary disease caused by a lack of dystrophin, a protein essential for myocyte integrity. Mitochondrial dysfunction is reportedly responsible for DMD. This study examines the effect of glucocorticoid deflazacort on the functioning of the skeletal-muscle mitochondria of dystrophin-deficient mdx mice and WT animals. Deflazacort administration was found to improve mitochondrial respiration of mdx mice due to an increase in the level of ETC complexes (complexes III and IV and ATP synthase), which may contribute to the normalization of ATP levels in the skeletal muscle of mdx animals. Deflazacort treatment improved the rate of Ca2+ uniport in the skeletal muscle mitochondria of mdx mice, presumably by affecting the subunit composition of the calcium uniporter of organelles. At the same time, deflazacort was found to reduce the resistance of skeletal mitochondria to MPT pore opening, which may be associated with a change in the level of ANT2 and CypD. In this case, deflazacort also affected the mitochondria of WT mice. The paper discusses the mechanisms underlying the effect of deflazacort on the functioning of mitochondria and contributing to the improvement of the muscular function of mdx mice. Keywords: Duchenne muscular dystrophy; skeletal muscle; deflazacort; mitochondria; Ca2+ uniporter; mitochondrial permeability transition 1. Introduction Duchenne muscular dystrophy (DMD) is the most prevalent muscular dystrophy afflicting ~1 in 3500–5000 live born males [1,2]. Regarded as a debilitating and fatal skeletal muscle disease, DMD is characterised by muscular weakness, exercise intolerance, and progressive deterioration of skeletal muscle. DMD is caused by large deletions and small forms of other mutations (deletions, duplications, insertions, and substitutions) in the dystrophin gene located on the short arm of the X-chromosome. The protein encoded by this gene is involved in the formation of a complex functional network (the so-called dystrophin-associated protein complex) which ensures the integrity and rigidity of muscle fibers. However, dystrophin deficiency and secondary reduction of these glycoproteins [3] render the myoctes and their fibers more susceptible to damage as they become structurally unstable and exceedingly porous to the extracellular environment. As a result, excessive Ca2+ influx, Int. J. Mol. Sci. 2020, 21, 8763; doi:10.3390/ijms21228763 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 18 Int. J. Mol. Sci. 2020, 21, 8763 2 of 18 Ca2+ influx, poor Ca2+ handling, activation of proteases/lipases, and mitochondrial Ca2+ overload poorprecede Ca2 muscle+ handling, degeneration activation [4–7]. of proteases While /thelipases, exact and underlying mitochondrial pathomechanisms Ca2+ overload still precede require muscle more degenerationresearch, over [4 time,–7]. While and as the regeneration exact underlying fails, pathomechanismsfatty and connective still tissue require replacement more research, culminates over time, in andnon-functional as regeneration muscle fails, tissue fatty [7]. and connective tissue replacement culminates in non-functional muscle tissueAccording [7]. to several reports, DMD is characterised by a systemic metabolic impairment, which is centralAccording to theto etiology several of reports, the disease. DMD isIndeed, characterised DMD is by accompanied a systemic metabolic by a significant impairment, deficiency which of is centralthe glycolysis to the etiology[8–10] and of thepurine disease. nucleotide Indeed, cycle DMD [11,12] is accompanied enzymes, as by well a significant as the enzymes deficiency involving of the glycolysisthe tricarboxylic [8–10] andacid purine (TCA) nucleotide cycle [10,13] cycle and [11 the,12] electron enzymes, transport as well as chain the enzymes (ETC) [14–16]. involving These the tricarboxylicchanges lead acid to reduced (TCA) cycle ATP [10 levels,13] and in skeletal the electron muscle transport in both chain DMD (ETC) patients [14–16 and]. These mdx changesmodels lead[17– to20]. reduced This, in ATP turn, levels results in skeletal in dysfunction muscle in of both the DMDcontractile patients apparatus, and mdx leadingmodels to [17 reduced–20]. This, muscular in turn, 2+ 2+ resultsstrength, in dysfunctiondysregulation of the of contractileintracellular apparatus, Ca buffering, leading to loss reduced of homeostasis, muscular strength, and dysregulationCa -induced ofdegeneration. intracellular Ca2+ buffering, loss of homeostasis, and Ca2+-induced degeneration. 2+ DMD-induced dysregulation of intracellularintracellular CaCa2+ homeostasis is hypothesized to associatedassociated with the dysfunction of of mitochondria mitochondria [14–16,21–23]. [14–16,21–23]. It Itwas was found found that that DMD DMD results results in ina decrease a decrease in 2+ inthe the efficiency efficiency of transport of transport and andaccumulation accumulation of Ca of in Ca mitochondria,2+ in mitochondria, which develops which develops in the skeletal in the skeletalmuscle already muscle in already the early in stages the early of postnatal stages of development postnatal development and is accompanied and is by accompanied a decrease in by the a rate of mitochondrial oxidative phosphorylation, as well as a decrease in resistance to the decrease in the rate of mitochondrial oxidative phosphorylation, as well as a decrease in resistance mitochondrial permeability transition (MPT) pore opening due to ROS overproduction and to the mitochondrial permeability transition (MPT) pore opening due to ROS overproduction and depolarization of organelles [14–16,23–25]. Recently, we showed that the dysregulation of calcium depolarization of organelles [14–16,23–25]. Recently, we showed that the dysregulation of calcium homeostasis in skeletal muscle mitochondria of mdx mice may be due to rearrangements in the homeostasis in skeletal muscle mitochondria of mdx mice may be due to rearrangements in the systems systems responsible for the transport of calcium ions and MPT pore opening [26]. responsible for the transport of calcium ions and MPT pore opening [26]. Currently, one of the amenable disease interventions available to DMD patients is through the Currently, one of the amenable disease interventions available to DMD patients is through the administration of a subclass of glucocorticoids, specifically prednisone and its oxazoline derivative administration of a subclass of glucocorticoids, specifically prednisone and its oxazoline derivative deflazacort (Dfz, Figure 1). Some other experimental trials are ongoing, such as myostatin inhibition deflazacort (Dfz, Figure1). Some other experimental trials are ongoing, such as myostatin inhibition [ 27] [27] and/or Sarepta’s micro-dystrophin gene therapy [28], which show promising results. However, and/or Sarepta’s micro-dystrophin gene therapy [28], which show promising results. However, currently currently Dfz is the first drug approved by the United States Food and Drug Administration (FDA) Dfz is the first drug approved by the United States Food and Drug Administration (FDA) for the for the management of DMD. It is known that Dfz in the form of an active metabolite, 21- management of DMD. It is known that Dfz in the form of an active metabolite, 21-desacetyldeflazacort, desacetyldeflazacort, is able to inhibit the release of cytokines by inhibiting the proliferation of CD4+ is able to inhibit the release of cytokines by inhibiting the proliferation of CD4+ lymphocytes and at lymphocytes and at the same time increasing the number of CD8+ cells, which helps to suppress the the same time increasing the number of CD8+ cells, which helps to suppress the immune response and immune response and inflammatory processes in muscle tissue [29,30]. Dfz-based therapy prolongs inflammatory processes in muscle tissue [29,30]. Dfz-based therapy prolongs ambulation by 2–5 years ambulation by 2–5 years and modestly improves muscle strength and cardiopulmonary function [30] and modestly improves muscle strength and cardiopulmonary function [30] but is often associated but is often associated with significant side effects such as a Cushingoid appearance, erythema, with significant side effects such as a Cushingoid appearance, erythema, hirsutism, increased weight, hirsutism, increased weight, nasopharyngitis, delayed puberty, cataracts, and vertebral fractures [31– nasopharyngitis, delayed puberty, cataracts, and vertebral fractures [31–33]. Glucocorticoids also 33]. Glucocorticoids