Mitigation of calcium-dependent destruction of skeletal muscle mitochondria is considered as a promising adjunctive therapy in Duchenne muscular dystrophy (DMD). In this work, we study the effect of intraperitoneal administration of a non-immunosuppressive inhibitor of calcium-dependent mitochondrial permeability transition (MPT) pore alisporivir on the state of skeletal muscles and the functioning of mitochondria in dystrophin-deficient mdx mice. We show that treatment with alisporivir reduces inflammation and improves muscle function in mdx mice. These effects of alisporivir were associated with an improvement in the ultrastructure of mitochondria, normalization of respiration and oxidative phosphorylation, and a decrease in lipid peroxidation, due to suppression of MPT pore opening and an improvement in calcium homeostasis. The action of alisporivir was associated with suppression of the activity of cyclophilin D and a decrease in its expression in skeletal muscles. This was observed in both mdx mice and wild-type animals. At the same time, alisporivir suppressed mitochondrial biogenesis, assessed by the expression of Ppargc1a, and altered the dynamics of organelles, inhibiting both DRP1-mediated fission and MFN2-associated fusion of mitochondria. The article discusses the effects of alisporivir administration and cyclophilin D inhibition on mitochondrial reprogramming and networking in DMD and the consequences of this therapy on skeletal muscle health.

Alisporivir Improves Mitochondrial Function in Skeletal Muscle of mdx Mice but Suppresses Mitochondrial Dynamics and Biogenesis.

Duchenne muscular dystrophy (DMD) is a progressive hereditary disease caused by the absence of the dystrophin protein. This is secondarily accompanied by a dysregulation of ion homeostasis, in which mitochondria play an important role. In the present work, we show that mitochondrial dysfunction in the skeletal muscles of dystrophin-deficient mdx mice is accompanied by a reduction in K+ transport and a decrease in its content in the matrix. This is associated with a decrease in the expression of the mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) in the muscles of mdx mice, which play an important role in cytoprotection. We observed that the BKCa activator NS1619 caused a normalization of mitoBKCa expression and potassium homeostasis in the muscle mitochondria of these animals, which was accompanied by an increase in the calcium retention capacity, mitigation of oxidative stress, and improvement in mitochondrial ultrastructure. This effect of NS1619 contributed to the reduction of degeneration/regeneration cycles and fibrosis in the skeletal muscles of mdx mice as well as a normalization of sarcomere size, but had no effect on the leakage of muscle enzymes and muscle strength loss. In the case of wild-type mice, we noted the negative effect of NS1619 manifested in the inhibition of the functional activity of mitochondria and disruption of their structure, which, however, did not significantly affect the state of the skeletal muscles of the animals. This article discusses the role of mitoBKCa in the development of DMD and the prospects of the approach associated with the correction of its function in treatments of this secondary channelopathy.

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BKCa Activator NS1619 Improves the Structure and Function of Skeletal Muscle Mitochondria in Duchenne Dystrophy

Duchenne muscular dystrophy (DMD) is a rare genetic disease leading to progressive muscle wasting, respiratory failure, and cardiomyopathy. Although muscle fibrosis represents a DMD hallmark, the organisation of the extracellular matrix and the molecular changes in its turnover are still not fully understood. To define the architectural changes over time in muscle fibrosis, we used an mdx mouse model of DMD and analysed collagen and glycosaminoglycans/proteoglycans content in skeletal muscle sections at different time points during disease progression and in comparison with age-matched controls. Collagen significantly increased particularly in the diaphragm, quadriceps, and gastrocnemius in adult mdx, with fibrosis significantly correlating with muscle degeneration. We also analysed collagen turnover pathways underlying fibrosis development in cultured primary quadriceps-derived fibroblasts. Collagen secretion and matrix metalloproteinases (MMPs) remained unaffected in both young and adult mdx compared to wt fibroblasts, whereas collagen cross-linking and tissue inhibitors of MMP (TIMP) expression significantly increased. We conclude that, in the DMD model we used, fibrosis mostly affects diaphragm and quadriceps with a higher collagen cross-linking and inhibition of MMPs that contribute differently to progressive collagen accumulation during fibrotic remodelling. This study offers a comprehensive histological and molecular characterisation of DMD-associated muscle fibrosis; it may thus provide new targets for tailored therapeutic interventions.

/pdf/characterisation-of-progressive-skeletal-muscle-fibrosis-in-1c99dako.pdf

Characterisation of Progressive Skeletal Muscle Fibrosis in the Mdx Mouse Model of Duchenne Muscular Dystrophy: An In Vivo and In Vitro Study

Duchenne muscular dystrophy is caused by the loss of functional dystrophin that secondarily causes systemic metabolic impairment in skeletal muscles and cardiomyocytes. The nutraceutical approach is considered as a possible complementary therapy for this pathology. In this work, we have studied the effect of pyrimidine nucleoside uridine (30 mg/kg/day for 28 days, i.p.), which plays an important role in cellular metabolism, on the development of DMD in the skeletal muscles of dystrophin deficient mdx mice, as well as its effect on the mitochondrial dysfunction that accompanies this pathology. We found that chronic uridine administration reduced fibrosis in the skeletal muscles of mdx mice, but it had no effect on the intensity of degeneration/regeneration cycles and inflammation, pseudohypetrophy, and muscle strength of the animals. Analysis of TEM micrographs showed that uridine also had no effect on the impaired mitochondrial ultrastructure of mdx mouse skeletal muscle. The administration of uridine was found to lead to an increase in the expression of the Drp1 and Parkin genes, which may indicate an increase in the intensity of organelle fission and the normalization of mitophagy. Uridine had little effect on OXPHOS dysfunction in mdx mouse mitochondria, and moreover, it was suppressed in the mitochondria of wild type animals. At the same time, uridine restored the transport of potassium ions and reduced the production of reactive oxygen species; however, this had no effect on the impaired calcium retention capacity of mdx mouse mitochondria. The obtained results demonstrate that the used dose of uridine only partially prevents mitochondrial dysfunction in skeletal muscles during Duchenne dystrophy, though it mitigates the development of destructive processes in skeletal muscles.

/pdf/the-effect-of-uridine-on-the-state-of-skeletal-muscles-and-n3qy3cy7.pdf

The Effect of Uridine on the State of Skeletal Muscles and the Functioning of Mitochondria in Duchenne Dystrophy

Duchenne muscular dystrophy (DMD) is caused by the absence of the dystrophin protein and a properly functioning dystrophin-associated protein complex (DAPC) in muscle cells. DAPC components act as molecular scaffolds coordinating the assembly of various signaling molecules including ion channels. DMD shows a significant change in the functioning of the ion channels of the sarcolemma and intracellular organelles and, above all, the sarcoplasmic reticulum and mitochondria regulating ion homeostasis, which is necessary for the correct excitation and relaxation of muscles. This review is devoted to the analysis of current data on changes in the structure, functioning, and regulation of the activity of ion channels in striated muscles in DMD and their contribution to the disruption of muscle function and the development of pathology. We note the prospects of therapy based on targeting the channels of the sarcolemma and organelles for the correction and alleviation of pathology, and the problems that arise in the interpretation of data obtained on model dystrophin-deficient objects.

https://www.mdpi.com/1422-0067/24/3/2229/pdf?version=1675845342

Ion Channels of the Sarcolemma and Intracellular Organelles in Duchenne Muscular Dystrophy: A Role in the Dysregulation of Ion Homeostasis and a Possible Target for Therapy

Givinostat as metabolic enhancer reverting mitochondrial biogenesis deficit in Duchenne Muscular Dystrophy.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of perinuclear heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. However, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. We have performed a longitudinal genome-wide analysis of gene expression in primary HGPS fibroblasts from patients at two sequential stages of disease that revealed a progressive activation of Rho signaling and SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1). siRNA-mediated downregulation or pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by reduced levels of Progerin and correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector of HGPS pathogenesis and target for therapeutic interventions.

https://www.biorxiv.org/content/biorxiv/early/2021/11/05/2021.11.05.467259.full.pdf

Targeting SerpinE1 reverses cellular features of Hutchinson-Gilford progeria syndrome

Duchenne muscular dystrophy (DMD) is a lethal muscle degenerative disease caused by mutations in the dystrophin gene. Early in life, DMD patients can temporarily compensate for the continuous degenerative process imposed on contractile activity of dystrophin-deficient myofibers, through a compensatory regeneration mediated by adult muscle stem (satellite) cells. However, the regenerative potential of satellite cells eventually declines at later stages of DMD and muscle fibers are replaced by fibrotic tissue, calcium deposits, and fat infiltration. Treatment with corticosteroids results in short-term improvements at the cost of steroidrelated side effects, but there is no cure for DMD. Several strategies have been employed to prolong ambulation and to delay the onset of secondary complications. Among these are gene and cellular therapies and pharmacologic strategies resulting in the replacement of a modified dystrophin protein or aimed at reducing the inflammatory cascade and enhancing muscle regeneration. The mdx mouse and the golden retriever muscular dystrophy dog (GRMD) are the models most commonly used to study DMD, with the former displaying a milder phenotype compared with human patients. Phenotype variability at the individual and muscle level has been described in the GRMD, making this model suitable to study the pathophysiology of muscular dystrophy beyond the primary effects of dystrophin loss. On the other hand, the same variability may distort assessment of outcome measures in preclinical trials. The autophagic machinery has been recently identified among the secondary therapeutic targets. Indeed, autophagy has been shown to be involved in many cellular processes to protect cells in stress conditions, including: providing amino acids to sustain vitality in the face of nutrient stress; and removing non-functional organelles, damaged mitochondria, and pathogens after intense exercise, endoplasmic reticulum stress, hypoxia, oxidative stress, or infections. There is clear evidence that autophagy is required to maintain muscle mass and myofiber integrity. Muscle-specific deletion of the crucial autophagy genes autophagy related 7 (ATG7) and autophagy related 5 (ATG5) was shown to result in muscle atrophy and age-dependent decreases in force production. On the other hand, overexpressing forkhead box O3 (FOXO3)–BCL2 interacting protein 3 (BNIP3) as a key pathway regulating autophagy during muscle wasting leads to an atrophic phenotype. Hence, it is possible to outline a dual role of autophagy in muscle homeostasis: Defective autophagy compromises the clearance of damaged proteins, toxic compounds, and organelles, whereas excessive autophagy leads to muscle loss and atrophy, pointing to autophagy as a sensitive process that needs to be fine-tuned to guarantee proper muscle function. In muscular dystrophies, autophagy has been reported to be impaired in muscles lacking collagen VI that accumulate dysfunctional organelles, thus triggering apoptosis and muscle wasting. Reactivation of autophagic flux by either nutritional or pharmacologic and genetic tools has been shown to ameliorate the dystrophic phenotype by removing dysfunctional mitochondria. Recent findings highlighted a crucial role of autophagy in DMD. For example, a low-protein diet has been shown to rescue the muscular defects in mdx mice. In addition, treatment with the protein kinase AMP-activated catalytic subunit alpha 1 (AMPK) agonist 5Aminoimidazole-4-carboxamide ribonucleotide (AICAR induces autophagy and improves muscle structure and strength without inducing muscle fiber atrophy. Additional data demonstrate that increased reactive oxygen species (ROS) production by CYBB/ NOX2 leads to autophagic impairment in muscles from the mdx mouse. Treatment with simvastatin, an b-hydroxy b-methylglutaryl–coenzyme A (HMG-CoA) reductase inhibitor that inhibits Cytochrome b subunit beta (CYBB)/NADPH oxidase-2 (NOX2) and Funding: This work was supported by Research Project Grant Parent PA-13-302 R01 AR064873, AFM 20568, the Ministry of Health, and (to L.L.).

/pdf/bet-on-autophagy-in-the-race-against-muscular-dystrophies-2c41566i48.pdf

Bet on autophagy in the race against muscular dystrophies.

Abstract Hutchinson–Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of peripheral heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. Yet, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. A genome-wide analysis of gene expression in cultures of primary HGPS fibroblasts identified SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1), as central gene that propels a cell-autonomous pathogenic signaling from the altered nuclear lamina. Indeed, siRNA-mediated downregulation and pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by the correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector and target for therapeutic interventions in HGPS pathogenesis. 

/pdf/serpine1-drives-a-cell-autonomous-pathogenic-signaling-in-1oisr9cu.pdf

Giorgia Catarinella

Papers

Givinostat as metabolic enhancer reverting mitochondrial biogenesis deficit in Duchenne Muscular Dystrophy.

Targeting SerpinE1 reverses cellular features of Hutchinson-Gilford progeria syndrome

Bet on autophagy in the race against muscular dystrophies.

SerpinE1 drives a cell-autonomous pathogenic signaling in Hutchinson–Gilford progeria syndrome