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PURPOSE OF REVIEW: To highlight recent insights in different aspects of striated muscle laminopathies (SMLs) related to LMNA mutations. RECENT FINDINGS: Clinical and genetic studies allow better patient management and diagnosis, with confirmation of ventricular tachyarrhythmias (VTA) risk prediction score to help with ICD implantation and development of models to help with classification of LMNA variants of uncertain significance. From a pathophysiology perspective, characterization of lamin interactomes in different contexts revealed new lamin A/C partners. Expression or function modulation of these partners evidenced them as potential therapeutic targets. After a positive phase 2, the first phase 3 clinical trial, testing a p38 inhibitor targeting the life-threatening cardiac disease of SML, has been recently stopped, thus highlighting the need for new therapeutic approaches together with new animal and cell models. SUMMARY: Since the first LMNA mutation report in 1999, lamin A/C structure and functions have been actively explored to understand the SML pathophysiology. The latest discoveries of partners and altered pathways, highlight the importance of lamin A/C at the nuclear periphery and in the nucleoplasm. Modulation of altered pathways allowed some benefits, especially for cardiac involvement. However, additional studies are still needed to fully assess treatment efficacy and safety.
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Lamina-associated polypeptide 1 (LAP1), a ubiquitously expressed nuclear envelope protein, appears to be essential for the maintenance of cell homeostasis. Although rare, mutations in the human LAP1-encoding TOR1AIP1 gene cause severe diseases and can culminate in the premature death of affected individuals. Despite there is increasing evidence of the pathogenicity of TOR1AIP1 mutations, the current knowledge on LAP1's physiological roles in humans is limited; hence, investigation is required to elucidate the critical functions of this protein, which can be achieved by uncovering the molecular consequences of LAP1 depletion, a topic that remains largely unexplored. In this work, the proteome of patient-derived LAP1-deficient fibroblasts carrying a pathological TOR1AIP1 mutation (LAP1 E482A) was quantitatively analyzed to identify global changes in protein abundance levels relatively to control fibroblasts. An in silico functional enrichment analysis of the mass spectrometry-identified differentially expressed proteins was also performed, along with additional in vitro functional assays, to unveil the biological processes that are potentially dysfunctional in LAP1 E482A fibroblasts. Collectively, our findings suggest that LAP1 deficiency may induce significant alterations in various cellular activities, including DNA repair, messenger RNA degradation/translation, proteostasis and glutathione metabolism/antioxidant response. This study sheds light on possible new functions of human LAP1 and could set the basis for subsequent in-depth mechanistic investigations. Moreover, by identifying deregulated signaling pathways in LAP1-deficient cells, our work may offer valuable molecular targets for future disease-modifying therapies for TOR1AIP1-associated nuclear envelopathies.
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The ability to recapitulate muscle differentiation in vitro enables the exploration of mechanisms underlying myogenesis and muscle diseases. However, obtaining myoblasts from patients with neuromuscular diseases or from healthy subjects poses ethical and procedural challenges that limit such investigations. An alternative consists in converting skin fibroblasts into myogenic cells by forcing the expression of the myogenic regulator MYOD. Here, we directly compared cellular phenotype, transcriptome, and nuclear lamina-associated domains (LADs) in myo-converted human fibroblasts and myotubes differentiated from myoblasts. We used isogenic cells from a 16-year-old donor, ruling out, for the first time to our knowledge, genetic factors as a source of variations between the two myogenic models. We show that myo-conversion of fibroblasts upregulates genes controlling myogenic pathways leading to multinucleated cells expressing muscle cell markers. However, myotubes are more advanced in myogenesis than myo-converted fibroblasts at the phenotypic and transcriptomic levels. While most LADs are shared between the two cell types, each also displays unique domains of lamin A/C interactions. Furthermore, myotube-specific LADs are more gene-rich and less heterochromatic than shared LADs or LADs unique to myo-converted fibroblasts, and they uniquely sequester developmental genes. Thus, myo-converted fibroblasts and myotubes retain cell type-specific features of radial and functional genome organization. Our results favor a view of myo-converted fibroblasts as a practical model to investigate the phenotypic and genomic properties of muscle cell differentiation in normal and pathological contexts, but also highlight current limitations in using fibroblasts as a source of myogenic cells.
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Fibroblastos , Fibras Musculares Esqueléticas , Humanos , Adolescente , Diferenciación Celular/genética , Mioblastos/metabolismo , GenómicaRESUMEN
Laminopathies are a group of rare disorders due to mutation in LMNA gene. Depending on the mutation, they may affect striated muscles, adipose tissues, nerves or are multisystemic with various accelerated ageing syndromes. Although the diverse pathomechanisms responsible for laminopathies are not fully understood, several therapeutic approaches have been evaluated in patient cells or animal models, ranging from gene therapies to cell and drug therapies. This review is focused on these therapies with a strong focus on striated muscle laminopathies and premature ageing syndromes.
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Striated muscle laminopathies are cardiac and skeletal muscle conditions caused by mutations in the lamin A/C gene (LMNA). LMNA codes for the A-type lamins, which are nuclear intermediate filaments that maintain the nuclear structure and nuclear processes such as gene expression. Protein kinase C alpha (PKC-α) interacts with lamin A/C and with several lamin A/C partners involved in striated muscle laminopathies. To determine PKC-α's involvement in muscular laminopathies, PKC-α's localization, activation, and interactions with the A-type lamins were examined in various cell types expressing pathogenic lamin A/C mutations. The results showed aberrant nuclear PKC-α cellular distribution in mutant cells compared to WT. PKC-α activation (phos-PKC-α) was decreased or unchanged in the studied cells expressing LMNA mutations, and the activation of its downstream targets, ERK 1/2, paralleled PKC-α activation alteration. Furthermore, the phos-PKC-α-lamin A/C proximity was altered. Overall, the data showed that PKC-α localization, activation, and proximity with lamin A/C were affected by certain pathogenic LMNA mutations, suggesting PKC-α involvement in striated muscle laminopathies.
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Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Laminopatías/genética , Laminopatías/metabolismo , Proteína Quinasa C-alfa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Músculo Estriado/patología , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Mutación , Mioblastos/metabolismo , Ratas , Transducción de SeñalRESUMEN
Laminopathies are causally associated with mutations on the Lamin A/C gene (LMNA). To date, more than 400 mutations in LMNA have been reported in patients. These mutations are widely distributed throughout the entire gene and are associated with a wide range of phenotypes. Unfortunately, little is known about the mechanisms underlying the effect of the majority of these mutations. This is the case of more than 40 mutations that are located at exon 4. Using CRISPR/Cas9 technology, we generated a collection of Lmna exon 4 mutants in mouse C2C12 myoblasts. These cell models included different types of exon 4 deletions and the presence of R249W mutation, one of the human variants associated with a severe type of laminopathy, LMNA-associated congenital muscular dystrophy (L-CMD). We characterized these clones by measuring their nuclear circularity, myogenic differentiation capacity in 2D and 3D conditions, DNA damage, and levels of p-ERK and p-AKT (phosphorylated Mitogen-Activated Protein Kinase 1/3 and AKT serine/threonine kinase 1). Our results indicated that Lmna exon 4 mutants showed abnormal nuclear morphology. In addition, levels and/or subcellular localization of different members of the lamin and LINC (LInker of Nucleoskeleton and Cytoskeleton) complex were altered in all these mutants. Whereas no significant differences were observed for ERK and AKT activities, the accumulation of DNA damage was associated to the Lmna p.R249W mutant myoblasts. Finally, significant myogenic differentiation defects were detected in the Lmna exon 4 mutants. These results have key implications in the development of future therapeutic strategies for the treatment of laminopathies.
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Exones/genética , Lamina Tipo A/genética , Mutación/genética , Mioblastos/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Núcleo Celular/metabolismo , Forma del Núcleo Celular , Células Clonales , Daño del ADN , Femenino , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Desarrollo de Músculos , Fracciones Subcelulares/metabolismo , Proteínas de Unión a Telómeros/metabolismoRESUMEN
LMNA encodes for Lamin A/C, type V intermediate filaments that polymerize under the inner nuclear membrane to form the nuclear lamina. A small fraction of Lamin A/C, less polymerized, is also found in the nucleoplasm. Lamin A/C functions include roles in nuclear resistance to mechanical stress and gene regulation. LMNA mutations are responsible for a wide variety of pathologies, including Emery-Dreifuss (EDMD) and LMNA-related congenital muscular dystrophies (L-CMD) without clear genotype-phenotype correlations. Both diseases presented with striated muscle disorders although L-CMD symptoms appear much earlier and are more severe. Seeking for pathomechanical differences to explain the severity of L-CMD mutations, we performed an in silico analysis of the UMD-LMNA database and found that L-CMD mutations mainly affect residues involved in Lamin dimer and tetramer stability. In line with this, we found increased nucleoplasmic Lamin A/C in L-CMD patient fibroblasts and mouse myoblasts compared to the control and EDMD. L-CMD myoblasts show differentiation defects linked to their inability to upregulate muscle specific nuclear envelope (NE) proteins expression. NE proteins were mislocalized, leading to misshapen nuclei. We conclude that these defects are due to both the absence of Lamin A/C from the nuclear lamina and its maintenance in the nucleoplasm of myotubes.
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Lamina Tipo A/deficiencia , Lamina Tipo A/metabolismo , Distrofias Musculares/patología , Distrofia Muscular de Emery-Dreifuss/patología , Índice de Severidad de la Enfermedad , Animales , Células Cultivadas , Simulación por Computador , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Humanos , Lamina Tipo A/genética , Ratones , Fibras Musculares Esqueléticas/metabolismo , Distrofias Musculares/genética , Distrofia Muscular de Emery-Dreifuss/genética , Mutación/genética , Mioblastos/metabolismo , FenotipoRESUMEN
Gene therapy is a promising strategy to cure rare diseases. The lack of regulatory sequences ensuring specific and robust expression in skeletal and cardiac muscle is a substantial limitation of gene therapy efficiency targeting the muscle tissue. Here we describe a novel muscle hybrid (MH) promoter that is highly active in both skeletal and cardiac muscle cells. It has an easily exchangeable modular structure, including an intronic module that highly enhances the expression of the gene driven by it. In cultured myoblasts, myotubes, and cardiomyocytes, the MH promoter gives relatively stable expression as well as higher activity and protein levels than the standard CMV and desmin gene promoters or the previously developed synthetic or CKM-based promoters. Combined with AAV2/9, the MH promoter also provides a high in vivo expression level in skeletal muscle and the heart after both intramuscular and systemic delivery. It is much more efficient than the desmin-encoding gene promoter, and it maintains the same specificity. This novel promoter has potential for gene therapy in muscle cells. It can provide stable transgene expression, ensuring high levels of therapeutic protein, and limited side effects because of its specificity. This constitutes an improvement in the efficiency of genetic disease therapy.
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Emery-Dreifuss muscular dystrophy (EDMD) is a genetic condition characterized by early contractures, skeletal muscle weakness, and cardiomyopathy. During the last 20 years, various genetic approaches led to the identification of causal genes of EDMD and related disorders, all encoding nuclear envelope proteins. By their respective localization either at the inner nuclear membrane or the outer nuclear membrane, these proteins interact with each other and establish a connection between the nucleus and the cytoskeleton. Beside this physical link, these proteins are also involved in mechanotransduction, responding to environmental cues, such as increased tension of the cytoskeleton, by the activation or repression of specific sets of genes. This ability of cells to adapt to environmental conditions is altered in EDMD. Increased knowledge on the pathophysiology of EDMD has led to the development of drug or gene therapies that have been tested on mouse models. This review proposed an overview of the functions played by the different proteins involved in EDMD and related disorders and the current therapeutic approaches tested so far.
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We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild-type (WT) transcripts. We developed 5'-RNA pre-trans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among nine pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, three induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from an LMNA-related congenital muscular dystrophy (L-CMD) mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV) delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery, although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.
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BACKGROUND: Emery-Dreifuss muscular dystrophy (EDMD) is associated with mutations in EMD and LMNA genes, encoding for the nuclear envelope proteins emerin and lamin A/C, indicating that EDMD is a nuclear envelope disease. We recently reported mutations in FHL1 gene in X-linked EDMD. FHL1 encodes FHL1A, and the two minor isoforms FHL1B and FHL1C. So far, none have been described at the nuclear envelope. OBJECTIVE: To gain insight into the pathophysiology of EDMD, we focused our attention on the poorly characterized FHL1B isoform. METHODS: The amount and the localisation of FHL1B were evaluated in control and diseased human primary myoblasts using immunofluorescence and western blotting. RESULTS: We found that in addition to a cytoplasmic localization, this isoform strongly accumulated at the nuclear envelope of primary human myoblasts, like but independently of lamin A/C and emerin. During myoblast differentiation, we observed a major reduction of FHL1B protein expression, especially in the nucleus. Interestingly, we found elevated FHL1B expression level in myoblasts from an FHL1-related EDMD patient where the FHL1 mutation only affects FHL1A, as well as in myoblasts from an LMNA-related EDMD patient. CONCLUSIONS: Altogether, the specific localization of FHL1B and its modulation in disease-patient's myoblasts confirmed FHL1-related EDMD as a nuclear envelope disease.
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Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/genética , Lamina Tipo A/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/genética , Distrofia Muscular de Emery-Dreifuss/genética , Mioblastos/metabolismo , Proteínas Nucleares/metabolismo , Western Blotting , Estudios de Casos y Controles , Técnica del Anticuerpo Fluorescente , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas Musculares/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mutación , Lámina Nuclear , Isoformas de ProteínasRESUMEN
LMNA gene encodes lamin A/C, ubiquitous proteins of the nuclear envelope. They play crucial role in maintaining nuclear shape and stiffness. When mutated, they essentially lead to dilated cardiomyopathy with conduction defects, associated or not with muscular diseases. Excessive mechanical stress sensitivity has been involved in the pathophysiology. We have previously reported the phenotype of Lmna(delK32) mice, reproducing a mutation found in LMNA-related congenital muscular dystrophy patients. Heterozygous Lmna(delK32/+) (Het) mice develop a progressive dilated cardiomyopathy leading to death between 35 and 70 weeks of age. To investigate the sensitivity of the skeletal muscles and myocardium to chronic exercise-induced stress, Het and wild-type (Wt) mice were subjected to strenuous running treadmill exercise for 5 weeks. Before exercise, the cardiac function of Het mice was similar to Wt-littermates. After the exercise-period, Het mice showed cardiac dysfunction and dilation without visible changes in cardiac morphology, molecular remodelling or nuclear structure compared to Wt exercised and Het sedentary mice. Contrary to myocardium, skeletal muscle ex vivo contractile function remained unaffected in Het exercised mice. In conclusion, the expression of the Lmna(delK32) mutation increased the susceptibility of the myocardium to cardiac stress and led to an earlier onset of the cardiac phenotype in Het mice.
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Cardiomiopatía Dilatada/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Carrera/fisiología , Animales , Cardiomiopatía Dilatada/patología , Técnicas de Sustitución del Gen , Heterocigoto , Contracción Isométrica/fisiología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Esquelético/patología , Mutación , Miocardio/patología , Fenotipo , ARN Mensajero/metabolismo , Distribución Aleatoria , Estrés Fisiológico/fisiologíaRESUMEN
BACKGROUND: Dystonia, cerebellar atrophy, and cardiomyopathy constitute a rare association. METHODS: We used homozygosity mapping and whole exome sequencing to determine the mutation, western blot and immunolabelling on cultured fibroblasts to demonstrate the lower expression and the mislocalization of the protein. RESULTS: We report on a boy born from consanguineous healthy parents, who presented at three years of age with rapidly progressing dystonia, progressive cerebellar atrophy, and dilated cardiomyopathy. We identified regions of homozygosity and performed whole exome sequencing that revealed a homozygous missense mutation in TOR1AIP1. The mutation, absent in controls, results in a change of a highly conserved glutamic acid to alanine. TOR1AIP1 encodes lamina-associated polypeptide 1 (LAP1), a transmembrane protein ubiquitously expressed in the inner nuclear membrane. LAP1 interacts with torsinA, the protein mutated in DYT1-dystonia. In vitro studies in fibroblasts of the patient revealed reduced expression of LAP1 and its mislocalization and aggregation in the endoplasmic reticulum as underlying pathogenic mechanisms. CONCLUSIONS AND RELEVANCE: The pathogenic role of TOR1AIP1 mutation is supported by a) the involvement of a highly conserved amino acid, b) the absence of the mutation in controls, c) the functional interaction of LAP1 with torsinA, and d) mislocalization of LAP1 in patient cells. Of note, cardiomyopathy has been reported in LAP1-null mice and in patients with the TOR1AIP1 nonsense mutation. Other cases will help delineate the clinical spectrum of LAP1-related mutations.
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Cardiomiopatías/genética , Enfermedades Cerebelosas/genética , Distonía/genética , Fibroblastos/metabolismo , Proteínas del Choque Térmico HSC70/genética , Chaperonas Moleculares/genética , Mutación Missense , Western Blotting , Cardiomiopatías/etiología , Técnicas de Cultivo de Célula , Enfermedades Cerebelosas/etiología , Preescolar , Distonía/etiología , Exoma , Proteínas del Choque Térmico HSC70/metabolismo , Humanos , Inmunohistoquímica , Masculino , Chaperonas Moleculares/metabolismo , LinajeRESUMEN
The mechanisms underlying the cell response to mechanical forces are crucial for muscle development and functionality. We aim to determine whether mutations of the LMNA gene (which encodes lamin A/C) causing congenital muscular dystrophy impair the ability of muscle precursors to sense tissue stiffness and to respond to mechanical challenge. We found that LMNA-mutated myoblasts embedded in soft matrix did not align along the gel axis, whereas control myoblasts did. LMNA-mutated myoblasts were unable to tune their cytoskeletal tension to the tissue stiffness as attested by inappropriate cell-matrix adhesion sites and cytoskeletal tension in soft versus rigid substrates or after mechanical challenge. Importantly, in soft two-dimensional (2D) and/or static three-dimensional (3D) conditions, LMNA-mutated myoblasts showed enhanced activation of the yes-associated protein (YAP) signaling pathway that was paradoxically reduced after cyclic stretch. siRNA-mediated downregulation of YAP reduced adhesion and actin stress fibers in LMNA myoblasts. This is the first demonstration that human myoblasts with LMNA mutations have mechanosensing defects through a YAP-dependent pathway. In addition, our data emphasize the crucial role of biophysical attributes of cellular microenvironment to the response of mechanosensing pathways in LMNA-mutated myoblasts.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Lamina Tipo A/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Microambiente Celular/fisiología , Humanos , Lamina Tipo A/genética , Microscopía Confocal , Mutación , Fosfoproteínas/genética , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAPAsunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM/genética , Proteínas Musculares/genética , Enfermedades Musculares/genética , Enfermedades Musculares/fisiopatología , Animales , Progresión de la Enfermedad , Corazón/efectos de los fármacos , Corazón/fisiopatología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas Musculares/metabolismo , Músculos/patología , Músculos/fisiopatología , Enfermedades Musculares/tratamiento farmacológico , Enfermedades Musculares/patología , Fenotipo , Isoformas de ProteínasRESUMEN
Lamins A and C, encoded by LMNA, are constituent of the nuclear lamina, a meshwork of proteins underneath the nuclear envelope first described as scaffolding proteins of the nucleus. Since the discovery of LMNA mutations in highly heterogeneous human disorders (including cardiac and muscular dystrophies, lipodystrophies and progeria), the number of functions described for lamin A/C has expanded. Lamin A/C is notably involved in the regulation of chromatin structure and gene transcription, and in the resistance of cells to mechanical stress. This review focuses on studies performed on knock-out and knock-in Lmna mouse models, which have led to decipher some of the lamin A/C functions in striated muscles and to the first preclinical trials of pharmaceutical therapies.
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Cardiomiopatías/genética , Lamina Tipo A/genética , Músculo Estriado/patología , Distrofias Musculares/genética , Lámina Nuclear/genética , Citoesqueleto de Actina/patología , Animales , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/patología , Desmina/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Técnicas de Sustitución del Gen , Humanos , Ratones , Ratones Noqueados , Músculo Estriado/citología , Membrana Nuclear , Vimentina/genéticaRESUMEN
AIMS: Dilated cardiomyopathy (DCM) is characterized by ventricular dilation associated with systolic dysfunction, which could be caused by mutations in lamina/C gene (LMNA). LMNA-linked DCM is severe in males in both human patients and a knock-in mouse model carrying a homozygous p.H222P mutation (LmnaH222P/H222P). The aim of this study was to investigate the molecular mechanisms underlying the gender difference of LMNA-linked DCM. METHODS AND RESULTS: A whole-exome analysis of a multiplex family with DCM exhibiting the gender difference revealed a DCM-linked LMNA mutation, p.R225X. Immunohistochemical analyses of neonatal rat cardiomyocytes expressing mutant LMNA constructs and heart samples from the LMNA-linked DCM patients and LmnaH222P/H222P mice demonstrated a nuclear accumulation of androgen receptor (AR) and its co-activators, serum response factor, and four-and-a-half LIM protein-2. Role of sex hormones in the gender difference was investigated in vivo using the LmnaH222P/H222P mice, where male and female mice were castrated and ovariectomized, respectively, or treated with testosterone or an antagonist of AR. Examination of the mice by echocardiography, followed by the analyses of histological changes and gene/protein expression profiles in the hearts, confirmed the involvement of testicular hormone in the disease progression and enhanced cardiac remodelling in the LmnaH222P/H222P mice. CONCLUSION: These observations indicated that nuclear accumulation of AR was associated with the gender difference in LMNA-linked DCM.
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Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Lamina Tipo A/genética , Mutación , Receptores Androgénicos/metabolismo , Transporte Activo de Núcleo Celular , Animales , Cardiomiopatía Dilatada/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Mutantes , Miocitos Cardíacos/metabolismo , Orquiectomía , Ovariectomía , Linaje , Ratas , Caracteres Sexuales , TransfecciónRESUMEN
Dilated cardiomyopathy (DCM) associates left ventricular (LV) dilatation and systolic dysfunction and is a major cause of heart failure and cardiac transplantation. LMNA gene encodes lamins A/C, proteins of the nuclear envelope. LMNA mutations cause DCM with conduction and/or rhythm defects. The pathomechanisms linking mutations to DCM remain to be elucidated. We investigated the phenotype and associated pathomechanisms of heterozygous Lmna(ΔK32/+) (Het) knock-in mice, which carry a human mutation. Het mice developed a cardiac-specific phenotype. Two phases, with two different pathomechanisms, could be observed that lead to the development of cardiac dysfunction, DCM and death between 35 and 70 weeks of age. In young Het hearts, there was a clear reduction in lamin A/C level, mainly due to the degradation of toxic ΔK32-lamin. As a side effect, lamin A/C haploinsufficiency probably triggers the cardiac remodelling. In older hearts, when DCM has developed, the lamin A/C level was normalized and associated with increased toxic ΔK32-lamin expression. Crossing our mice with the Ub(G76V)-GFP ubiquitin-proteasome system (UPS) reporter mice revealed a heart-specific UPS impairment in Het. While UPS impairment itself has a clear deleterious effect on engineered heart tissue's force of contraction, it also leads to the nuclear aggregation of viral-mediated expression of ΔK32-lamin. In conclusion, Het mice are the first knock-in Lmna model with cardiac-specific phenotype at the heterozygous state. Altogether, our data provide evidence that Het cardiomyocytes have to deal with major dilemma: mutant lamin A/C degradation or normalization of lamin level to fight the deleterious effect of lamin haploinsufficiency, both leading to DCM.
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Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Haploinsuficiencia , Heterocigoto , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patología , Núcleo Celular/ultraestructura , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Lamina Tipo A/química , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Mutación , Contracción Miocárdica/genética , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismoRESUMEN
A-type lamins are components of the nuclear lamina, a filamentous network of the nuclear envelope in metazoans that supports nuclear architecture. In addition, lamin A/C can also be found in the interior of the nucleus. This nucleoplasmic lamin pool is soluble in physiological buffer, depends on the presence of the lamin-binding protein, lamina-associated polypeptide 2α (LAP2α) and regulates cell cycle progression in tissue progenitor cells. ΔK32 mutations in A-type lamins cause severe congenital muscle disease in humans and a muscle maturation defect in Lmna(ΔK32/ΔK32) knock-in mice. Mutant ΔK32 lamin A/C protein levels were reduced and all mutant lamin A/C was soluble and mislocalized to the nucleoplasm. To test the role of LAP2α in nucleoplasmic ΔK32 lamin A/C regulation and functions, we deleted LAP2α in Lmna(ΔK32/ΔK32) knock-in mice. In double mutant mice the Lmna(ΔK32/ΔK32)-linked muscle defect was unaffected. LAP2α interacted with mutant lamin A/C, but unlike wild-type lamin A/C, the intranuclear localization of ΔK32 lamin A/C was not affected by loss of LAP2α. In contrast, loss of LAP2α in Lmna(ΔK32/ΔK32) mice impaired the regulation of tissue progenitor cells as in lamin A/C wild-type animals. These data indicate that a LAP2α-independent assembly defect of ΔK32 lamin A/C is the predominant cause of the mouse pathology, whereas the LAP2α-linked functions of nucleoplasmic lamin A/C in the regulation of tissue progenitor cells are not affected in Lmna(ΔK32/ΔK32) mice.
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Proteínas de Unión al ADN/metabolismo , Lamina Tipo A/metabolismo , Proteínas de la Membrana/metabolismo , Distrofias Musculares/metabolismo , Membrana Nuclear/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Lamina Tipo A/genética , Proteínas de la Membrana/genética , Ratones , Ratones Mutantes , Distrofias Musculares/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
A-type lamins A and C are nuclear intermediate filament proteins in which mutations have been implicated in multiple disease phenotypes commonly known as laminopathies. A few studies have implicated sumoylation in the regulation of A-type lamins. Sumoylation is a post-translational protein modification that regulates a wide range of cellular processes through the attachment of small ubiquitin-related modifier (sumo) to various substrates. Here we showed that laminopathy mutants result in the mislocalization of sumo1 both in vitro (C2C12 cells overexpressing mutant lamins A and C) and in vivo (primary myoblasts and myopathic muscle tissue from the Lmna(H222P/H222P) mouse model). In C2C12 cells, we showed that the trapping of sumo1 in p.Asp192Gly, p.Gln353Lys, and p.Arg386Lys aggregates of lamin A/C correlated with an increased steady-state level of sumoylation. However, lamin A and C did not appear to be modified by sumo1. Our results suggest that mutant lamin A/C alters the dynamics of sumo1 and thus misregulation of sumoylation may be contributing to disease progression in laminopathies.