Elevated TGF \u3b22 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

Among rare diseases caused by mutations in LMNA gene, Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B are characterized by muscle weakness and wasting, joint contractures, cardiomyopathy with conduction system disorders. Circulating biomarkers for these pathologies have not been identified. Here, we analyzed the secretome of a cohort of patients affected by these muscular laminopathies in the attempt to identify a common signature. Multiplex cytokine assay showed that transforming growth factor beta 2 (TGF \u3b22) and interleukin 17 serum levels are consistently elevated in the vast majority of examined patients, while interleukin 6 and basic fibroblast growth factor are altered in subgroups of patients. Levels of TGF \u3b22 are also increased in fibroblast and myoblast cultures established from patient biopsies as well as in serum from mice bearing the H222P Lmna mutation causing Emery-Dreifuss Muscular Dystrophy in humans. Both patient serum and fibroblast conditioned media activated a TGF \u3b22-dependent fibrogenic program in normal human myoblasts and tenocytes and inhibited myoblast differentiation. Consistent with these results, a TGF \u3b22 neutralizing antibody avoided fibrogenic marker activation and myogenesis impairment. Cell intrinsic TGF \u3b22-dependent mechanisms were also determined in laminopathic cells, where TGF \u3b22 activated AKT/mTOR phosphorylation. These data show that TGF \u3b22 contributes to the pathogenesis of Emery-Dreifuss Muscular Dystrophy type 2 and Limb-Girdle muscular Dystrophy 1B and can be considered a potential biomarker of those diseases. Further, the evidence of TGF \u3b22 pathogenetic effects in tenocytes provides the first mechanistic insight into occurrence of joint contractures in muscular laminopathies

Failure of lamin A/C to functionally assemble in R482L mutated familial partial lipodystrophy fibroblasts: altered intermolecular interaction with emerin and implications for gene transcription

Familial partial lipodystrophy is an autosomal dominant disease caused by mutations of the LMNA gene encoding alternatively spliced lamins A and C. Abnormal distribution of body fat and insulin resistance characterize the clinical phenotype. In this study, we analyzed primary fibroblast cultures from a patient carrying an R482L lamin A/C mutation by a morphological and biochemical approach. Abnormalities were observed consisting of nuclear lamin A/C aggregates mostly localized close to the nuclear lamina. These aggregates were not bound to either DNA-containing structures or RNA splicing intranuclear compartments. In addition, emerin did not colocalize with nuclear lamin A/C aggregates. Interestingly, emerin failed to interact with lamin A in R482L mutated fibroblasts in vivo, while the interaction with lamin C was preserved in vitro, as determined by coimmunoprecipitation experiments. The presence of lamin A/C nuclear aggregates was restricted to actively transcribing cells, and it was increased in insulin-treated fibroblasts. In fibroblasts carrying lamin A/C nuclear aggregates, a reduced incorporation of bromouridine was observed, demonstrating that mutated lamin A/C in FPLD cells interferes with RNA transcription

Lamin A/C Mechanotransduction in Laminopathies

Mechanotransduction translates forces into biological responses and regulates cell functionalities. It is implicated in several diseases, including laminopathies which are pathologies associated with mutations in lamins and lamin-associated proteins. These pathologies affect muscle, adipose, bone, nerve, and skin cells and range from muscular dystrophies to accelerated aging. Although the exact mechanisms governing laminopathies and gene expression are still not clear, a strong correlation has been found between cell functionality and nuclear behavior. New theories base on the direct effect of external force on the genome, which is indeed sensitive to the force transduced by the nuclear lamina. Nuclear lamina performs two essential functions in mechanotransduction pathway modulating the nuclear stiffness and governing the chromatin remodeling. Indeed, A-type lamin mutation and deregulation has been found to affect the nuclear response, altering several downstream cellular processes such as mitosis, chromatin organization, DNA replication-transcription, and nuclear structural integrity. In this review, we summarize the recent findings on the molecular composition and architecture of the nuclear lamina, its role in healthy cells and disease regulation. We focus on A-type lamins since this protein family is the most involved in mechanotransduction and laminopathies

Pathogenesis of LMNA-related dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a progressive myocardial disease that leads to dilatation of cardiac ventricles, reduced contractile force, and a high risk of sudden cardiac death. The second most common gene mutated in the familial form of DCM is LMNA, and the founder mutation p.S143P is the most frequently reported mutation among Finnish DCM patients. LMNA gene encodes nuclear proteins lamin A/C that constitute the nuclear lamina and regulate several cellular functions, e.g., nuclear stability and gene expression. However, the mechanisms of how lamin mutations cause DCM are poorly understood, and there are no effective treatments available. The aim of this thesis project was to study how the p.S143P LMNA mutation affects cell function and viability and how these alterations contribute to DCM development. Using primary patient fibroblasts and transfection models, we showed that the p.S143P mutant lamin A/C is more nucleoplasmic, soluble, and dynamic compared to wild-type (WT) lamin A/C. Furthermore, in vitro assembly experiments showed that the p.S143P lamin A is not able to form typical filaments but forms disorganized aggregates. The consequences of the p.S143P mutation on induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were investigated in the second substudy, where mutant cells showed similar nucleoplasmic lamin A/C distribution. CMs were challenged with ischemic stress, which caused significant sarcomere damage and apoptosis in mutant CMs. In the third substudy, we analyzed the function of lamin A/C under heat shock (HS). We demonstrated that lamin A/C are hyperphosphorylated under HS in control and patient cells. However, the patient cells were more sensitive to heat stress than control cells, affecting cell survival during HS recovery. In the fourth substudy, we determined the mechanical stress response of the patient cells. Unlike the control cells, the mutant cells showed nuclear rupture, cytoskeletal damage, and cellular disarray under mechanical strain. To conclude, these results show that the p.S143P mutation changes the intrinsic properties of lamin A/C affecting its incorporation into the lamina and mislocalization to the nucleoplasm. Mislocalization of lamins deteriorates the lamina structure, leading to nuclear rupture and cytoskeletal damage under stress. These alterations inevitably contribute to the development of DCM.LMNA-geenimutaatioiden aiheuttaman laajentavan kardiomyopatian syntymekanismit Laajentava kardiomyopatia (DCM) on etenevä sydänlihassairaus, jolle on ominaista sydämen kammioiden laajeneminen, sydämen vajaatoiminta sekä lisääntynyt äkkikuoleman riski. LMNA-geenimutaatiot ovat yksi perinnöllisen DCM:n aiheuttaja ja suomalaisilla DCM potilailla p.S143P on yleisin tunnistettu LMNA-geenimutaatio. Lamiinit muodostavat tumakalvon sisäpinnalle säieverkoston (lamina), joka tukee tuman muotoa sekä säätelee geenien ilmentymistä. LMNA-geenin aiheuttaman DCM:n syntymekanismit ovat edelleen huonosti ymmärrettyjä eikä tautiin ole saatavilla tehokasta hoitomuotoa. Tämän väitöskirjatutkimuksen tavoitteena oli selvittää, miten p.S143P LMNAmutaatio vaikuttaa solujen toimintaan ja elinkelpoisuuteen sekä miten nämä muutokset vaikuttavat DCM syntyyn. Ensimmäisessä osatyössä analysoimme DCM potilailta eristettyjä fibroblasteja sekä transfektoituja soluja ja havaitsimme p.S143P mutaation estävän lamiini A:ta muodostamasta välikokoisia säikeitä, mikä johti proteiinin poikkeavaan sijoittumiseen tuman sisäosaan ja osassa soluista lamiinien aggregoitumiseen. Toisessa osatyössä tutkimme p.S143P mutaation vaikutuksia potilaiden fibroblasteista kantasolutekniikalla erilaistetuissa sydänsoluissa. Mutaatiota kantavissa sydänsoluissa lamiini A oli vastaavasti sijoittunut enemmän tuman sisäosaan. Iskeemisen stressin aikana, mutanteilla sydänsoluilla havaittiin selvästi enemmän sarkomeerirakenteen vaurioita sekä solukuolemia. Kolmannessa osatyössä tutkimme lamiini A/C:n vastetta lämpösokkiin. Havaitsimme, että lamiini A/C fosforyloituu lämpösokin aikana sekä potilas- että verrokki soluissa. Potilassolut osoittautuivat kuitenkin herkemmäksi lämpösokille kuin verrokkisolut, mikä heikensi potilassolujen elinkelpoisuutta lämpösokin jälkeen. Neljännessä osatyössä tutkimme potilassolujen vastetta mekaaniseen rasitukseen, mikä aiheutti potilassoluille tuman hajoamisen sekä tukirakenteen vaurioita. Tutkimustulosten perusteella voidaan päätellä, että p.S143P mutaatio estää lamin A/C:ta muodostamasta normaaleita välikokoisia säikeitä, mikä johtaa proteiinin poikkeavaan sijaintiin tumassa. Lamiini A/C sijoittuminen tuman sisäosiin heikentää laminan rakennetta ja aiheuttaa tuman hajoamisen sekä tukirangan vaurioita stressin aikana. Nämä tekijät vaikuttavat yhdessä DCM:n kehittymiseen

Prelamin A Influences a Program of Gene Expression In Regulation of Cell Cycle Control

The A-type lamins are intermediate filament proteins that constitute a major part of the eukaryotic nuclear lamina—a tough, polymerized, mesh lining of the inner nuclear membrane, providing shape and structural integrity to the nucleus. Lamin A (LA) filaments also permeate the nucleoplasm, providing additional structural support, but also scaffolding numerous tethered molecules to stabilize, organize, and facilitate molecular interactions to accomplish critical functions of cellular metabolism. Over the past 2 decades, much attention has been focused on roles of LA in maintenance of nuclear structural integrity. Only since the late 1990s have scientists discovered the devastating effects of LA gene (LMNA) mutations, as they have associated hundreds of LMNA mutations to a large group of diseases, called laminopathies, with a broad spectrum of phenotypes, ranging from skeletal, muscular, and neurological defects, to defective lipid storage, to accelerated aging phenotypes in diseases called progerias. Recent advances demonstrate LA regulatory functions include cell signaling, cell cycle regulation, transcription, chromatin organization, viral egress, and DNA damage repair. Amidst the flurry of fascinating research, only recently have researchers begun to focus attention on the different isoforms that exist for LA, a precursor form among them. LA is initially synthesized as Prelamin A (PreA), and undergoes a series of modifications that truncate the protein to produce “mature” LA. Existence of the precursor form, and its complex maturation pathway, have puzzled researchers since their realization. With a pattern of expression related to cell cycle phase, we hypothesized a role for PreA in cell cycle control. To investigate, we have performed array studies to assess gene expression effects at the levels of transcript expression, protein expression, and phosphorylation modification status. Here, we present evidence for a PreA-mediated program of cell cycle regulatory gene and protein expression modulation. Implicated pathways include RB-E2F, p53, p27Kip1, FoxOs, p300, and the Cyclins, with additional evidence indicating a role for the Pin1 prolyl isomerase in mediating PreA regulation of the cell cycle