Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy

: SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired

Abnormal Cellular Phenotypes Induced by Three TMPO/LAP2 Variants Identified in Men with Cardiomyopathies

A single missense variant of the TMPO/LAP2α gene, encoding LAP2 proteins, has been associated with cardiomyopathy in two brothers. To further evaluate its role in cardiac muscle, we included TMPO in our cardiomyopathy diagnostic gene panel. A screening of ~5000 patients revealed three novel rare TMPO heterozygous variants in six males diagnosed with hypertrophic or dilated cardiomypathy. We identified in different cellular models that (1) the frameshift variant LAP2α p.(Gly395Glufs*11) induced haploinsufficiency, impeding cell proliferation and/or producing a truncated protein mislocalized in the cytoplasm; (2) the C-ter missense variant LAP2α p.(Ala240Thr) led to a reduced proximity events between LAP2α and the nucleosome binding protein HMGN5; and (3) the LEM-domain missense variant p.(Leu124Phe) decreased both associations of LAP2α/β with the chromatin-associated protein BAF and inhibition of the E2F1 transcription factor activity which is known to be dependent on Rb, partner of LAP2α. Additionally, the LAP2α expression was lower in the left ventricles of male mice compared to females. In conclusion, our study reveals distinct altered properties of LAP2 induced by these TMPO/LAP2 variants, leading to altered cell proliferation, chromatin structure or gene expression-regulation pathways, and suggests a potential sex-dependent role of LAP2 in myocardial function and disease

Mitochondrial CYP2E1 is sufficient to mediate oxidative stress and cytotoxicity induced by ethanol and acetaminophen.

International audienceSeveral cytochromes P450 (CYPs) are not only located in the endoplasmic reticulum but also within mitochondria. One such CYP is CYP2E1 which metabolizes numerous substrates and generates significant amount of reactive oxygen species. The presence of CYP2E1 in these organelles raises questions regarding its physiological role but also its possible deleterious effects in the context of drug-induced cytotoxicity. The aim of our study was to investigate the role of mitochondrial CYP2E1 in the toxicity of acetaminophen and ethanol. Hence the effects of these two compounds in cells expressing CYP2E1 in mitochondria only, or in both endoplasmic reticulum and mitochondria, were compared to those observed in mock-transfected cells. Our results indicated that when acetaminophen or ethanol were used as CYP2E1 substrates, the exclusive localization of CYP2E1 within mitochondria was sufficient to induce reactive oxygen species overproduction, depletion of reduced glutathione, increased expression of mitochondrial Hsp70, mitochondrial dysfunction and cytotoxicity. Importantly, these harmful events happened despite lower cellular level and activity of CYP2E1 when compared to cells expressing CYP2E1 in both endoplasmic reticulum and mitochondria, and this was particularly obvious with acetaminophen. Taken together, these data suggest that mitochondrial CYP2E1 could play a major role in drug-induced oxidative stress and cell demise

Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.

During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair

Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient

International audienceSpecific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD) and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A

Potent induction of apoptosis in human hepatoma cell lines by targeted cytotoxic somatostatin analogue AN-238

The efficacy of a targeted cytotoxic hybrid somatostatin analogue AN-238 and of its superactive radical 2-pyrrolinodoxorubicin (AN-201) to induce apoptosis of HepG2 and Hep3B human hepatoma cell lines were studied. AN-238 was designed to selectively target tumor cells expressing somatostatin receptor subtypes (sst s). Its effects on HepG2 or Hep3B cells displaying or lacking tumor suppressor p53, respectively, were compared. Normal rat isolated hepatocytes were also tested. sst s were characterized by binding assays and RT-PCR. Cytotoxicity was quantified by flow cytometry. DNA fragmentation was studied by gel electrophoresis, PARP cleavage by Western blot and ROS formation using fluorescent probes. Specific binding of iodinated RC-160 to HepG2 and Hep3B cells, and its displacement by AN-238 was characterized. mRNA for hsst 2A was found in both cell lines. Flow cytometry showed a stronger effect of AN-238 than AN-201 to induce sub-G1 phase. DNA fragmentation, nuclear bodies, and PARP cleavage were observed. In addition, AN-238 increased formation of ROS more potently than AN-201. However, no inductions of DNA fragmentation by AN-201 or AN-238 were observed on rat hepatocytes. Our results indicate that, in liver cancer, the cytotoxic somatostatin analogue AN-238 is a powerful agent that can induce apoptosis, through sst s and independently of p53

The beneficial effect of myostatin deficiency on maximal muscle force and power is attenuated with age

International audienceThe prolonged effect of myostatin deficiency on muscle performance in knockout mice has as yet been only poorly investigated. We have demonstrated that absolute maximal force is increased in 6-month old female and male knockout mice and 2-year old female knockout mice as compared to age- and sex-matched wildtype mice. Similarly, absolute maximal power is increased by myostatin deficiency in 6-month old female and male knockout mice but not in 2-year old female knockout mice. The increases we observed were greater in 6-month old female than in male knockout mice and can primarily result from muscle hypertrophy. In contrast, fatigue resistance was decreased in 6-month old knockout mice of both sexes as compared to age- and sex-matched wildtype mice. Moreover, in contrast to 2-year old female wildtype mice, aging in 2-year old knockout mice reduced absolute maximal force and power of both sexes as compared to their younger counterparts, although muscle weight did not change. These age- related decreases were lower in 2-year old female than in 2-year old male knockout mice. Together these results suggest that the beneficial effect of myostatin deficiency on absolute maximal force and power is greater in young (versus old) mice and female (versus male) mice. Most of these effects of myostatin deficiency are related neither to changes in the concentration of myofibrillar proteins nor to the slow to fast fiber type transition

Sex-Specific Patterns of Diaphragm Phospholipid Content and Remodeling during Aging and in a Model of SELENON-Related Myopathy

Growing evidence shows that the lipid bilayer is a key site for membrane interactions and signal transduction. Surprisingly, phospholipids have not been widely studied in skeletal muscles, although mutations in genes involved in their biosynthesis have been associated with muscular diseases. Using mass spectrometry, we performed a phospholipidomic profiling in the diaphragm of male and female, young and aged, wild type and SelenoN knock-out mice, the murine model of an early-onset inherited myopathy with severe diaphragmatic dysfunction. We identified 191 phospholipid (PL) species and revealed an important sexual dimorphism in PLs in the diaphragm, with almost 60% of them being significantly different between male and female animals. In addition, 40% of phospholipids presented significant age-related differences. Interestingly, SELENON protein absence was responsible for remodeling of 10% PL content, completely different in males and in females. Expression of genes encoding enzymes involved in PL remodeling was higher in males compared to females. These results establish the diaphragm PL map and highlight an important PL remodeling pattern depending on sex, aging and partly on genotype. These differences in PL profile may contribute to the identification of biomarkers associated with muscular diseases and muscle aging