Modeling Inheritable Myofibrillar Myopathy with Patient-Specific iPS Cells

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 DESCRIPTION (provided by applicant): Multiple mutations in the gene encoding aB-crystallin, CRYAB, have been identified in the literatures that result in skeletal myopathy. αB-Crystallin is small molecular-weight heat shock protein that has been shown to function as a chaperone for desmin, titin, and actin. Additionally, αB-crystallin impacts skeletal muscle differentiation throgh exhibiting effects on cell cycle exit and a muscle-specific, regulatory transcription factor, MyoD. A patient harboring the homozygous recessive, nonsense mutation, 343delT, in CRYAB develops symptoms of severe muscle stiffness and degeneration around age four months, following normal development. The patient's muscle biopsy shows dense, irregular staining of 343delT, possibly reminiscent of the aggregates visible with other mutant forms of aB-crystallin, such as R120G, which has been shown to also induce the aggregation of an intermediate filament protein, desmin, contributing to the disease pathology. The patient's symptoms also bear similarities to the CRYAB/HSPB2 double knockout mouse, though the mouse seems to have less severe and later onset muscle deterioration. Therefore, the question as to whether this disease pathology results from loss of αB-crystallin function or gain of toxic function of 343delT αB-crystallin remains and whether this impact is seen in myogenic progenitor cells, differentiated myotubes, or both. This proposal aims to examine this question using 343delT patient-specific induced pluripotent stem cell- (iPSC) derived myogenic progenitors and skeletal myotubes. iPSCs offer great utility in modeling cell autonomous diseases in vitro because of their continuous ability to self-renew and pluripotent differentiation potential; they are also amenable to genetic manipulation. iPSCs will be differentiated to myogenic progenitors and skeletal myotubes by an existing protocol. [Control iPSCs have been generated by gene correction in 343delT patient iPSCs] and will be used for phenotypic comparisons to examine the structure and function of differentiated myotubes as well as analyze cell cycle progression and gene expression throughout differentiation of myogenic progenitors to myotubes. Additionally, CRYAB null iPSCs have been generated from 343delT patient iPSCs for comparison to determine if phenotypes are due to loss or gain of function effects of the 343delT mutation. Successful completion of these aims will provide a human model system of 343delT-induced myopathy and a better understanding of how expression of the mutant and/or lack of expression contribute to myopathy, as well as the importance of a chaperone, aB-crystallin, in human skeletal muscle differentiation and maintenance.