Nuclear envelope alterations in fibroblasts from LGMD1B patients carrying nonsense Y259X heterozygous or homozygous mutation in lamin A/C gene

Mutations in the LMNA gene encoding nuclear lamins A and C are responsible for seven inherited disorders affecting specific tissues. We have analyzed skin fibroblasts from a patient with type 1B limb-girdle muscular dystrophy and from her deceased newborn grandchild carrying, respectively, a heterozygous (+/mut) and a homozygous (mut/mut) nonsense Y259X mutation. In fibroblasts(+/mut), the presence of only 50% lamins A and C promotes no detectable abnormality, whereas in fibroblasts(mut/mut) the complete absence of lamins A and C leads to abnormally shaped nuclei with lobules in which none of the analyzed nuclear proteins were detected, i.e., B-type lamins, emerin, nesprin-1alpha, LAP2beta, and Nup153. These lobules perturb cell division as fibroblast(mut/mut) cultures with large proportions of cells with dysmorphic nuclei grow more slowly than controls and the cell proliferation normalizes when the number of these abnormally shaped nuclei declines. In all fibroblasts(mut/mut), nesprin-1alpha-like emerin exhibited aberrant localization in the endoplasmic reticulum. Transfection of wild-type lamin A or C cDNAs restored the correct localization of both emerin and nesprin-1alpha. These data demonstrate that lamin C, like lamin A, interacts in vivo directly with nesprin-1alpha and with emerin and that lamin A or C is sufficient for the correct anchorage of emerin and nesprin-1alpha at the nuclear envelope in human cells.     

Both lamin A and lamin C mutations cause lamina instability as well as loss of internal nuclear lamin organization

We have applied the fluorescence loss of intensity after photobleaching (FLIP) technique to study the molecular dynamics and organization of nuclear lamin proteins in cell lines stably transfected with green fluorescent protein (GFP)-tagged A-type lamin cDNA. Normal lamin A and C proteins show abundant decoration of the inner layer of the nuclear membrane, the nuclear lamina, and a generally diffuse localization in the nuclear interior. Bleaching studies revealed that, while the GFP-tagged lamins in the lamina were virtually immobile, the intranuclear fraction of these molecules was partially mobile. Intranuclear lamin C was significantly more mobile than intranuclear lamina A. In search of a structural cause for the variety of inherited diseases caused by A-type lamin mutations, we have studied the molecular organization of GFP-tagged lamin A and lamin C mutants R453W and R386K, found in Emery-Dreifuss muscular dystrophy (EDMD), and lamin A and lamin C mutant R482W, found in patients with Dunnigan-type familial partial lipodystrophy (FPLD). In all mutants, a prominent increase in lamin mobility was observed, indicating loss of structural stability of lamin polymers, both at the perinuclear lamina and in the intranuclear lamin organization. While the lamin rod domain mutant showed overall increased mobility, the tail domain mutants showed mainly intranuclear destabilization, possibly as a result of loss of interaction with chromatin. Decreased stability of lamin mutant polymers was confirmed by flow cytometric analyses and immunoblotting of nuclear extracts. Our findings suggest a loss of function of A-type lamin mutant proteins in the organization of intranuclear chromatin and predict the loss of gene regulatory function in laminopathies.     

APOBEC2 negatively regulates myoblast differentiation in muscle regeneration

Recently we found that the deficiency of APOBEC2, a member of apoB mRNA editing enzyme, catalytic polypeptide-like family, leads to a diminished muscle mass and increased myofiber with centrally-located nuclei known as dystrophic phenotypes. APOBEC2 expression is predominant in skeletal and cardiac muscles and elevated exclusively at the early-differentiation phase of wild-type (WT) myoblast cultures; however the physiological significance is still un-known. Here we show that APOBEC2 is a key negative regulator of myoblast differentiation in muscle regeneration. APOBEC2-knockout (A2KO) mice myoblast cultures displayed a normal morphology of primary myotubes along with earlier increase in fusion index and higher expression levels of myosin heavy chain (MyHC), myogenin and its cooperating factor MEF2C than WT myoblasts. Similar response was observable in APOBEC2-knockdown cultures of WT myoblasts that were transfected with the specific siRNA at the differentiation phase (not proliferation phase). Importantly, cardiotoxin-injured A2KO gastrocnemius muscle provided in vivo evidence by showing larger up-regulation of neonatal MyHC and myogenin and hence earlier regeneration of myofiber structures with diminished cross-sectional areas and minimal Feret diameters. Therefore, the findings highlight a promising role for APOBEC2 in normal progression of regenerative myogenesis at the early-differentiation phase upon muscle injury.     

Immunolocalization of detergent-susceptible nucleoplasmic lamin A/C foci by a novel monoclonal antibody

The A-type lamins are localized in the interior of the nucleus as well as on the nuclear periphery. In this study, we have characterized a monoclonal antibody LA-2F9 produced against recombinant rat lamin A which stains a subpopulation of various cell types in a pattern of small nucleoplasmic foci that are unusually susceptible to mild detergent/salt extraction. The specific reactivity of mAb LA-2F9 towards lamins was confirmed by immunoblotting of HeLa and C3H10T(1/2) whole cell lysates and nuclear lysates. The epitope for LA-2F9 was narrowed down to amino acid residues 268-278 (SAKLDNARQSA). To check whether the appearance of lamin foci was cell-cycle-dependent, C3H10T(1/2) cells were serum-starved and then refed to trigger cells to enter the G(1) phase of the cell-cycle. The intensity of staining increased 3.5-fold within 6 h of refeeding, when the maximum number of cells were labeled with LA-2F9. We also checked whether the LA-2F9 foci colocalized with nuclear proteins known to be distributed in small foci such as hnRNPs, snRNPs, SC-35, and p80 coilin, but did not find evidence of colocalization. Our studies suggest that LA-2F9 has a novel and specific reactivity towards detergent-susceptible lower order lamin structures that are likely to be assembly intermediates.     

Effects of expressing lamin A mutant protein causing Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy in HeLa cells

Patients with the autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD) or familial partial lipodystrophy (FPLD) have specific mutations in the lamin A gene. Three such point mutations, G465D (FPLD), R482L, (FPLD), or R527P (EDMD), were introduced by site-specific mutagenesis in the C-terminal tail domain of a FLAG-tagged full-length lamin A construct. HeLa cells were transfected with mutant and wild-type constructs. Lamin A accumulated in nuclear aggregates and the number of cells with aggregates increased with time after transfection. At 72 h post transfection 60-80% of cells transfected with the mutant lamin A constructs had aggregates, while only 35% of the cells transfected with wild-type lamin A revealed aggregates. Mutant transfected cells expressed 10-24x, and wild-type transfected cells 20x, the normal levels of lamin A. Lamins C, B1 and B2, Nup153, LAP2, and emerin were recruited into aggregates, resulting in a decrease of these proteins at the nuclear rim. Aggregates were also characterized by electron microscopy and found to be preferentially associated with the inner nuclear membrane. Aggregates from mutant constructs were larger than those formed by the wild-type constructs, both in immunofluorescence and electron microscopy. The combined results suggest that aggregate formation is in part due to overexpression, but that there are also mutant-specific effects.     

Heat stress-induced localization of small heat shock proteins in mouse myoblasts: intranuclear lamin A/C speckles as target for alphaB-crystallin and Hsp25

We examined the effect of heat stress on localization of two sHsps, alphaB-crystallin and Hsp25, and of Hsc70, a member of a different class of heat shock proteins (Hsps), in both undifferentiated and differentiated mouse C2C12 cells. Under normal conditions, alphaB-crystallin and Hsp25 are found in the cytoplasm; only alphaB-crystallin is also found in the nucleus, distributed in a speckled pattern. Hsc70 is found to be homogeneously distributed throughout the cell. On heat stress, all these proteins translocate almost entirely into the nucleus and upon recovery relocate to the cytoplasm. Dual staining experiments using C2C12 myoblasts show that alphaB-crystallin and Hsp25, but not Hsc70, colocalize with the intranuclear lamin A/C and the splicing factor SC-35, suggesting interactions of sHsps and intranuclear lamin A/C. Interestingly, none of these proteins are found in the myotube nuclei. Upon heat stress, only Hsc70 translocates into the myotube nuclei. This differential entry of alphaB-crystallin and Hsp25 into the nuclei of myoblasts and myotubes upon heat stress may have functional role in the development and/or in the maintenance of muscle cells. Our study therefore suggests that these sHsps may be a part of the intranuclear lamin A/C network or stabilizing this specific network.     

Specific contribution of lamin A and lamin C in the development of laminopathies

Mutations in the lamin A/C gene are involved in multiple human disorders for which the pathophysiological mechanisms are partially understood. Conflicting results prevail regarding the organization of lamin A and C mutants within the nuclear envelope (NE) and on the interactions of each lamin to its counterpart. We over-expressed various lamin A and C mutants both independently and together in COS7 cells. When expressed alone, lamin A with cardiac/muscular disorder mutations forms abnormal aggregates inside the NE and not inside the nucleoplasm. Conversely, the equivalent lamin C organizes as intranucleoplasmic aggregates that never connect to the NE as opposed to wild type lamin C. Interestingly, the lamin C molecules present within these aggregates exhibit an abnormal increased mobility. When co-expressed, the complex formed by lamin A/C aggregates in the NE. Lamin A and C mutants for lipodystrophy behave similarly to the wild type. These findings reveal that lamins A and C may be differentially affected depending on the mutation. This results in multiple possible physiological consequences which likely contribute in the phenotypic variability of laminopathies. The inability of lamin C mutants to join the nuclear rim in the absence of lamin A is a potential pathophysiological mechanism for laminopathies.     

Increased solubility of lamins and redistribution of lamin C in X-linked Emery–Dreifuss muscular dystrophy fibroblasts

Emery–Dreifuss muscular dystrophy (EDMD) is caused by mutations in the gene encoding the nuclear membrane protein emerin (X-linked EDMD) or in the gene encoding lamins A/C (autosomal dominant EDMD). One hypothesis explaining the disease suggests that the mutations lead to weakness of the nuclear lamina. To test this hypothesis we investigated lamin solubility and distribution in skin fibroblasts from X-EDMD patients. Using in situ extraction of cells and immunofluorescence microscopy or biochemical fractionation and immunoblotting, we found that all lamin subtypes displayed increased solubility properties in fibroblasts from X-EDMD patients compared to normal individuals. Lamin and emerin solubility was mildly increased in fibroblasts from an X-EDMD carrier. Biochemical fractionation and immunoblotting also indicated that lamin C but no other lamin became redistributed from the nuclear lamina to the nucleoplasm in X-EDMD fibroblasts. Indirect immunofluorescence and confocal microscopy studies using lamin A- and lamin C-specific antibodies confirmed that lamin C but not lamin A became redistributed to the nucleoplasm. Interestingly, the lamin A/C binding protein LAP2α was also mislocalized in X-EDMD fibroblasts.     

Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.     

Generation and characterization of a conditional deletion allele for Lmna in mice

Extensive efforts have been devoted to study A-type lamins because mutations in their gene, LMNA in humans, are associated with a number of diseases. The mouse germline mutations in the A-type lamins (encoded by Lmna) exhibit postnatal lethality at either 4–8 postnatal (P) weeks or P16–18 days, depending on the deletion alleles. These mice exhibit defects in several tissues including hearts and skeletal muscles. Despite numerous studies, how the germline mutation of Lmna, which is expressed in many postnatal tissues, affects only selected tissues remains poorly understood. Addressing the tissue specific functions of Lmna requires the generation and careful characterization of conditional Lmna null alleles. Here we report the creation of a conditional Lmna knockout allele in mice by introducing loxP sites flanking the second exon of Lmna. The Lmna^flox/flox mice are phenotypically normal and fertile. We show that Lmna homozygous mutants (Lmna^Δ/Δ) generated by germline Cre expression display postnatal lethality at P16–18 days with defects similar to a recently reported germline Lmna knockout mouse that exhibits the earliest lethality compared to other germline knockout alleles. This conditional knockout mouse strain should serve as an important genetic tool to study the tissue specific roles of Lmna, which would contribute toward the understanding of various human diseases associated with A-type lamins.     

Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

Background

Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna^H222P/H222P mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna^H222P/H222P mice and assessed if adding a MEK1/2 inhibitor would provide added benefit.

Methods

Male Lmna^H222P/H222P mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated.

Results

Treatment of Lmna^H222P/H222P mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left ventricular fractional shortening at 20 weeks of age.

Conclusions

Both ACE inhibition and MEK1/2 inhibition have beneficial effects on left ventricular function in Lmna^H222P/H222P mice and both drugs together have a synergistic benefit when initiated after the onset of left ventricular dysfunction. These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor in addition to standard of care in patients with dilated cardiomyopathy caused by LMNA mutations.     

Role of nuclear Lamin A/C in cardiomyocyte functions

Lamin A/C is a structural protein of the nuclear envelope (NE) and cardiac involvement in Lamin A/C mutations was one of the first phenotypes to be reported in humans, suggesting a crucial role of this protein in the cardiomyocytes function. Mutations in LMNA gene cause a class of pathologies generically named ‘Lamanopathies’ mainly involving heart and skeletal muscles. Moreover, the well‐known disease called Hutchinson–Gilford Progeria Syndrome due to extensive mutations in LMNA gene, in addition to the systemic phenotype of premature aging, is characterised by the death of patients at around 13 typically for a heart attack or stroke, suggesting again the heart as the main site sensitive to Lamin A/C disfunction. Indeed, the identification of the roles of the Lamin A/C in cardiomyocytes function is a key area of exploration. One of the primary biological roles recently conferred to Lamin A/C is to affect contractile cells lineage determination and senescence. Then, in differentiated adult cardiomyocytes both the ‘structural’ and ‘gene expression hypothesis’ could explain the role of Lamin A in the function of cardiomyocytes. In fact, recent advances in the field propose that the structural weakness/stiffness of the NE, regulated by Lamin A/C amount in NE, can ‘consequently’ alter gene expression.     

Association of emerin with nuclear and cytoplasmic actin is regulated in differentiating myoblasts

Emerin is a nuclear envelope protein whose biological function remains to be elucidated. Mutations of emerin gene cause the Emery-Dreifuss muscular dystrophy, a neuromuscular disorder also linked to mutations of lamin A/C. In this paper, we analyze the interaction between emerin and actin in differentiating mouse myoblasts. We demonstrate that emerin and lamin A/C are bound to actin at the late stages of myotube differentiation and in mature muscle. The interaction involves both nuclear alpha and beta actins and cytoplasmic actin. A serine-threonine phosphatase activity markedly increases emerin-actin binding even in cycling myoblasts. This effect is also observed with purified nuclear fractions in pull-down assay. On the other hand, active protein phosphatase 1, a serine-threonine phosphatase known to associate with lamin A/C, inhibits emerin-actin interaction in myotube extracts. These data provide evidence of a modulation of emerin-actin interaction in muscle cells, possibly through differentiation-related stimuli.     

Enrichment and terminal differentiation of striated muscle progenitors in vitro

Enrichment and terminal differentiation of mammalian striated muscle cells is severely hampered by fibroblast overgrowth, de-differentiation and/or lack of functional differentiation. Herein we report a new, reproducible and simple method to enrich and terminally differentiate muscle stem cells and progenitors from mice and humans. We show that a single gamma irradiation of muscle cells induces their massive differentiation into structurally and functionally intact myotubes and cardiomyocytes and that these cells can be kept in culture for many weeks. Similar results are also obtained when treating skeletal muscle-derived stem cells and progenitors with Mitomycin C.