SPP1 genotype and glucocorticoid treatment modify osteopontin expression in Duchenne muscular dystrophy cells.

Glucocorticoids are beneficial in Duchenne muscular dystrophy (DMD). Osteopontin (OPN), the protein product of SPP1, plays a role in DMD pathology modulating muscle inflammation and regeneration. A polymorphism in the SPP1 promoter (rs28357094) has been recognized as a genetic modifier of DMD, and there is evidence suggesting that it modifies response to glucocorticoid treatment. The effect of the glucocorticoid deflazacort on SPP1 mRNA and protein expression was investigated in DMD primary human myoblasts and differentiated myotubes with defined rs28357094 genotype (TT versus TG). Both healthy and DMD myoblasts/myotubes abundantly express OPN. In immunoblot, OPN was detected as a doublet of 55 and 50 kDa bands, with a shift towards the lighter isoform in the transition from myoblasts to myotubes and to mature muscle. A significant increase in OPN expression was observed in DMD myotubes carrying the TG compared to the TT genotype at rs28357094. Deflazacort treatment led to a significant increase of OPN only in myotubes carrying the TG genotype, leading to OPN overexpression. Our study shows a strong effect of the rs28357094 G allele in increasing OPN expression in the presence of deflazacort, and adds to the evidence that rs28357094 polymorphism may predict response to glucocorticoids in DMD.

Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.

Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro.

Activation of mitochondrial ERK protects cancer cells from death through inhibition of the permeability transition.

We studied human cancer cell models in which we detected constitutive activation of ERK. A fraction of active ERK was found to be located in mitochondria in RWPE-2 cells, obtained by v-Ki-Ras transformation of the epithelial prostate RWPE-1 cell line; in metastatic prostate cancer DU145 cells; and in osteosarcoma SAOS-2 cells. All these tumor cells displayed marked resistance to death caused by apoptotic stimuli like arachidonic acid and the BH3 mimetic EM20-25, which cause cell death through the mitochondrial permeability transition pore (PTP). PTP desensitization and the ensuing resistance to cell death induced by arachidonic acid or EM20-25 could be ablated by inhibiting ERK with the drug PD98059 or with a selective ERK activation inhibitor peptide. ERK inhibition enhanced glycogen synthase kinase-3 (GSK-3)-dependent phosphorylation of the pore regulator cyclophilin D, whereas GSK-3 inhibition protected from PTP opening. Neither active ERK in mitochondria nor pore desensitization was observed in non-transformed RWPE-1 cells. Thus, in tumor cells mitochondrial ERK activation desensitizes the PTP through a signaling axis that involves GSK-3 and cyclophilin D, a finding that provides a mechanistic basis for increased resistance to apoptosis of neoplastic cells.

2-Deoxy-D-glucose couples mitochondrial DNA replication with mitochondrial fitness and promotes the selection of wild-type over mutant mitochondrial DNA.

Pathological variants of human mitochondrial DNA (mtDNA) typically co-exist with wild-type molecules, but the factors driving the selection of each are not understood. Because mitochondrial fitness does not favour the propagation of functional mtDNAs in disease states, we sought to create conditions where it would be advantageous. Glucose and glutamine consumption are increased in mtDNA dysfunction, and so we targeted the use of both in cells carrying the pathogenic m.3243A>G variant with 2-Deoxy-D-glucose (2DG), or the related 5-thioglucose. Here, we show that both compounds selected wild-type over mutant mtDNA, restoring mtDNA expression and respiration. Mechanistically, 2DG selectively inhibits the replication of mutant mtDNA; and glutamine is the key target metabolite, as its withdrawal, too, suppresses mtDNA synthesis in mutant cells. Additionally, by restricting glucose utilization, 2DG supports functional mtDNAs, as glucose-fuelled respiration is critical for mtDNA replication in control cells, when glucose and glutamine are scarce. Hence, we demonstrate that mitochondrial fitness dictates metabolite preference for mtDNA replication; consequently, interventions that restrict metabolite availability can suppress pathological mtDNAs, by coupling mitochondrial fitness and replication.

The clinical spectrum of CASQ1-related myopathy.

OBJECTIVE: To identify and characterize patients with calsequestrin 1 (CASQ1)-related myopathy. METHODS: Patients selected according to histopathologic features underwent CASQ1 genetic screening. CASQ1-mutated patients were clinically evaluated and underwent muscle MRI. Vacuole morphology and vacuolated fiber type were characterized. RESULTS: Twenty-two CASQ1-mutated patients (12 families) were identified, 21 sharing the previously described founder mutation (p.Asp244Gly) and 1 with the p.Gly103Asp mutation. Patients usually presented in the sixth decade with exercise intolerance and myalgias and later developed mild to moderate, slowly progressive proximal weakness with quadriceps atrophy and scapular winging. Muscle MRI (n = 11) showed a recurrent fibrofatty substitution pattern. Three patients presented subclinical cardiac abnormalities. Muscle histopathology in patients with p.Asp244Gly showed vacuoles in type II fibers appearing empty in hematoxylin-eosin, Gomori, and nicotinamide adenine dinucleotide (NADH) tetrazolium reductase stains but strongly positive for sarcoplasmic reticulum proteins. The muscle histopathology of p.Gly103Asp mutation was different, showing also NADH-positive accumulation consistent with tubular aggregates. CONCLUSIONS: We report the clinical and molecular details of the largest cohort of CASQ1-mutated patients. A possible heart involvement is presented, further expanding the phenotype of the disease. One mutation is common due to a founder effect, but other mutations are possible. Because of a paucity of symptoms, it is likely that CASQ1 mutations may remain undiagnosed if a muscle biopsy is not performed.

Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA.

Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.

Genetic Modifiers of Duchenne Muscular Dystrophy and Dilated Cardiomyopathy.

OBJECTIVE: Dilated cardiomyopathy (DCM) is a major complication and leading cause of death in Duchenne muscular dystrophy (DMD). DCM onset is variable, suggesting modifier effects of genetic or environmental factors. We aimed to determine if polymorphisms previously associated with age at loss of independent ambulation (LoA) in DMD (rs28357094 in the SPP1 promoter, rs10880 and the VTTT/IAAM haplotype in LTBP4) also modify DCM onset. METHODS: A multicentric cohort of 178 DMD patients was genotyped by TaqMan assays. We performed a time-to-event analysis of DCM onset, with age as time variable, and finding of left ventricular ejection fraction < 50% and/or end diastolic volume > 70 mL/m2 as event (confirmed by a previous normal exam < 12 months prior); DCM-free patients were censored at the age of last echocardiographic follow-up. RESULTS: Patients were followed up to an average age of 15.9 ± 6.7 years. Seventy-one/178 patients developed DCM, and median age at onset was 20.0 years. Glucocorticoid corticosteroid treatment (n = 88 untreated; n = 75 treated; n = 15 unknown) did not have a significant independent effect on DCM onset. Cardiological medications were not administered before DCM onset in this population. We observed trends towards a protective effect of the dominant G allele at SPP1 rs28357094 and recessive T allele at LTBP4 rs10880, which was statistically significant in steroid-treated patients for LTBP4 rs10880 (< 50% T/T patients developing DCM during follow-up [n = 13]; median DCM onset 17.6 years for C/C-C/T, log-rank p = 0.027). CONCLUSIONS: We report a putative protective effect of DMD genetic modifiers on the development of cardiac complications, that might aid in risk stratification if confirmed in independent cohorts.

Altered Ca2+ homeostasis and endoplasmic reticulum stress in myotonic dystrophy type 1 muscle cells.

The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.

Signal transduction to the permeability transition pore.

The permeability transition pore (PTP) is an inner mitochondrial membrane channel that has been thoroughly characterized functionally, yet remains an elusive molecular entity. The best characterized PTP-regulatory component, cyclophilin (CyP) D, is a matrix protein that favors pore opening. CyP inhibitors, CyP-D null animals, and in situ PTP readouts have established the role of PTP as an effector mechanism of cell death, and the growing definition of PTP signalling mechanisms. This review briefly covers the functional features of the PTP and the role played by its dysregulation in disease pathogenesis. Recent progress on PTP modulation by kinase/phosphatase signal transduction is discussed, with specific emphasis on hexokinase and on the Akt-ERK-GSK3 axis, which might modulate the PTP through CyP-D phosphorylation.