α-Skeletal muscle actin nemaline myopathy mutants cause cell death in cultured muscle cells

Abstract

Nemaline myopathy is a neuromuscular disorder, characterized by muscle weakness and hypotonia and is, in 20% of the cases, caused by mutations in the gene encoding alpha-skeletal muscle actin, ACTA1. It is a heterogeneous disease with various clinical phenotypes and severities. In patients the ultrastructure of muscle cells is often disturbed by nemaline rods and it is thought this is the cause for muscle weakness. To search for possible defects during muscle cell differentiation we expressed alpha-actin mutants in myoblasts and allowed these cells to differentiate into myotubes. Surprisingly, we observed two striking new phenotypes in differentiating myoblasts: rounding up of cells and bleb formation, two features reminiscent of apoptosis. Indeed expression of these mutants induced cell death with apoptotic features in muscle cell culture, using AIF and endonuclease G, in a caspase-independent but calpain-dependent pathway. This is the first report on a common cellular defect induced by NM causing actin mutants, independent of their biochemical phenotypes or rod and aggregate formation capacity. These data suggest that lack of type II fibers or atrophy observed in nemaline myopathy patients may be also due to an increased number of dying muscle cells.

Actin Nemaline Myopathy Mouse Reproduces Disease, Suggests Other Actin Disease Phenotypes and Provides Cautionary Note on Muscle Transgene Expression

“…[32], [33]). In our hands C2C12 cells transfected with an ACTA1 D286G-EGFP construct produce cytoplasmic EGFP-positive aggregates in myoblasts and differentiating myotubes that are not seen in the nuclei (unpublished data), although it has been suggested that the D286G mutation does produce intranuclear rods [34].…”

“…It has been demonstrated by in vitro studies that certain ACTA1 mutations induce cell death [34], [58], [59]. The mutations studied in Wallefeld et al .…”

Dynamic regulation of sarcomeric actin filaments in striated muscle

“…A number of myopathy‐causing mutations have been identified in skeletal muscle α‐actin, which are located throughout the molecule [Feng and Marston, 2009; Laing et al, 2009]. These actin mutations cause a wide range of molecular defects in protein folding, binding to actin‐binding proteins, polymerization, and cytoskeletal organization, and it has been difficult to correlate the molecular defect with severity of the disease [Costa et al, 2004; Bathe et al, 2007; Vandamme et al, 2009a, b]. A mutant form of actin with defective polymerization or depolymerization could impair normal actin filament turnover in muscle, but whether a defect in actin dynamics is a major cause of congenital myopathies remains to be determined.…”

“…Many of the myopathy‐causing actin mutants induce actin rods or aggregates when they are exogenously expressed in muscle or nonmuscle cells [Costa et al, 2004; Bathe et al, 2007; Domazetovska et al, 2007; Vandamme et al, 2009a, b, c]. Although these actin rods and aggregates morphologically resemble nemaline rods in patient muscle, the presence of α‐actinin, a component of in vivo nemaline rods, is variable in cell culture models.…”

Severe congenital actin related myopathy with myofibrillar myopathy features

“…Also, wild-type ACTA1 formed filamentous structures whereas the mutant protein formed large aggregates, thus mimicking the structural lesions observed in the patient's muscle. Low expression in cell culture of some missense ACTA1 mutants was previously attributed to increased cell death by the mutant ACTA1 [17][18][19]. Actin accumulation in our patient muscle was not directly demonstrated due to lack of EM specimen and of muscle tissue for actin immunostaining.…”