Abstract
Laminopathies are a clinically heterogeneous group of disorders caused by mutations in LMNA. The main proteins encoded by LMNA are Lamin A and C, which together with Lamin B1 and B2, form the nuclear lamina: a mesh-like structure located underneath the inner nuclear membrane. Laminopathies show striking tissue specificity, with subtypes affecting striated muscle, peripheral nerve, and adipose tissue, while others cause multisystem disease with accelerated aging. Although several pathogenic mechanisms have been proposed, the exact pathophysiology of laminopathies remains unclear, compounded by the rarity of these disorders and lack of easily accessible cell types to study. To overcome this limitation, we used induced pluripotent stem cells (iPSCs) from patients with skeletal muscle laminopathies such as LMNA-related congenital muscular dystrophy and limb-girdle muscular dystrophy 1B, to model disease phenotypes in vitro. iPSCs can be derived from readily accessible cell types, have unlimited proliferation potential and can be differentiated into cell types that would otherwise be difficult and invasive to obtain. iPSC lines from three skeletal muscle laminopathy patients were differentiated into inducible myogenic cells and myotubes. Disease-associated phenotypes were observed in these cells, including abnormal nuclear shape and mislocalization of nuclear lamina proteins. Nuclear abnormalities were less pronounced in monolayer cultures of terminally differentiated skeletal myotubes than in proliferating myogenic cells. Notably, skeletal myogenic differentiation of LMNA-mutant iPSCs in artificial muscle constructs improved detection of myonuclear abnormalities compared to conventional monolayer cultures across multiple pathogenic genotypes, providing a high-fidelity modeling platform for skeletal muscle laminopathies. Our results lay the foundation for future iPSC-based therapy development and screening platforms for skeletal muscle laminopathies.
Highlights
The LMNA gene encodes two major protein isoforms: Lamin A and C; these nuclear intermediate filament proteins are expressed in most somatic cells, but absent from undifferentiated cells such as embryonic, germ and pluripotent cells (Dechat et al, 2010a; Worman, 2012)
Three distinct heterozygous dominant LMNA mutations were represented by these induced pluripotent stem cells (iPSCs): p.K32del and p.L35P and p.R249W (Table 1)
LMNA-iPSC were differentiated into inducible myogenic cells using our protocol that generates an expandable population of mesodermal cells similar to mesoangioblasts (i.e., HIDEMs: Human iPSC Derived Mesoangioblast-like cells), which can be efficiently induced to terminal skeletal myogenic differentiation by transient expression of the myogenesis regulator MyoD (Tedesco et al, 2012; Gerli et al, 2014; Maffioletti et al, 2015)
Summary
The LMNA gene encodes two major protein isoforms: Lamin A and C; these nuclear intermediate filament proteins are expressed in most somatic cells, but absent from undifferentiated cells such as embryonic, germ and pluripotent cells (Dechat et al, 2010a; Worman, 2012). The nuclear lamina provides structural support to the nucleus, and participates in mechanotransduction, heterochromatin tethering and regulation of transcription (Azibani et al, 2014; Gruenbaum and Foisner, 2015). Mutations in LMNA cause at least 16 rare disorders, collectively known as laminopathies (Scharner et al, 2010; Worman, 2012). While all four have cardiac involvement, EDMD, LGMD1B and LCMD affect skeletal muscle (Worman, 2012; Bonne and Quijano-Roy, 2013; Azibani et al, 2014). The gene expression and stem cell differentiation hypothesis suggests that mutant Lamin A/C deregulates expression of certain genes, which causes defective cell differentiation and function (Azibani et al, 2014; Gruenbaum and Foisner, 2015)
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