ABSTRACT
Pathogenic variants in ACTA1, which encodes skeletal muscle-specific α-actin (SKA), cause a
range of congenital myopathies; at the severe end of the disease spectrum are patients
diagnosed with nemaline myopathy with intranuclear rods, or intranuclear rod myopathy.
Intranuclear rod myopathy is characterized by the formation of SKA-containing rod-like
structures within the nucleus of myocytes, and is associated with infantile-onset muscle
weakness and death in early childhood due to respiratory failure, for which there are no current
targeted therapies. Recurrent variants affecting Val163 in SKA are associated with intranuclear
rod formation, but the pathogenic mechanisms driving the severity of disease in these patients
are unknown. We were the first to show that the smooth muscle-specific α-actin has a critical
function in the nucleus: loss of nuclear smooth muscle-specific α-actin prevents complete
differentiation of smooth muscle cells. Muscle biopsies from patients with intranuclear rod
myopathy show immature muscle fibers and increased numbers of progenitor-like satellite cells,
suggesting that incomplete differentiation of skeletal myocytes may be a pathologic feature of
this disease. We therefore hypothesize that SKA functions in the nucleus to promote skeletal
myocyte differentiation and that disruption of the nuclear functions of SKA in patients with
intranuclear rods causes incomplete skeletal myocyte differentiation, which underlies the severe
and early-onset disease. We will test our hypothesis by the following specific aims: 1) In the
C2C12 mouse myoblast cell line, we will identify intranuclear localization, protein binding
partners, and target genomic loci for nuclear SKA with and without p.Val163 variants. 2) We will
assess whether the intranuclear rod-associated p.Val163Leu variant prevents complete
differentiation in vitro using a model of human induced pluripotent stem cell (iPSCs)
differentiation to skeletal myocytes. We will differentiate Crispr/Cas9-edited ACTA1 p.Val163Leu
iPSCs alongside isogenic controls. 3) We will generate a conditional knock-in mouse model for
the Acta1 p.Val163Leu variant and induce knock-in in skeletal myocytes either during
development with a constitutive Acta1-Cre or after development with an inducible Acta1-
MerCreMer. We will assess whether introduction of the variant during development results in a
more severe phenotype of incompletely differentiated muscle. Taken together, these data will
identify a novel role for SKA in the nucleus during healthy skeletal myocyte development and
will identify a key pathogenic mechanism for intranuclear rod myopathy. Identifying a disease
mechanism may lead to development of therapeutic strategies for these patients.