Cellular Fate of Truncated Slow Skeletal Muscle Troponin T Produced by Glu180 Nonsense Mutation in Amish Nemaline Myopathy

Abstract

A nonsense mutation at codon Glu 180 in exon 11 of slow skeletal muscle troponin T (TnT) gene (TNNT1) causes an autosomal-recessive inherited nemaline myopathy. We previously reported the absence of intact or prematurely terminated slow TnT polypeptide in Amish nemaline myopathy (ANM) patient muscle. The present study further investigates the expression and fate of mutant slow TnT in muscle cells. Intact slow TnT mRNA was readily detected in patient muscle, indicating unaffected transcription and RNA splicing. Sequence of the cloned cDNAs revealed the single nucleotide mutation in two alternatively spliced isoforms of slow TnT mRNA. Mutant TNNT1 cDNA is translationally active in Escherichia coli and non-muscle eukaryotic cells, producing the expected truncated slow TnT protein. The mutant mRNA was expressed at significant levels in differentiated C 2 C 12 myotubes, but unlike intact exogenous TnT, truncated slow TnT protein was not detected. Transfective expression in undifferentiated myoblasts produced slow TnT mRNA but not a detectable amount of truncated or intact slow TnT proteins, indicating a muscle cell-specific proteolysis of TnT when it is not integrated into myofilaments. The slow TnT-(1-179) fragment has substantially lower affinity for binding to tropomyosin, in keeping with the loss of one of two tropomyosinbinding sites. Our findings suggest that inefficient incorporation into myofilament is responsible for the instability of mutant slow TnT in ANM muscle. Rapid degradation of the truncated slow TnT protein, rather than instability of the nonsense mRNA, provides the protective mechanism against the potential dominant negative effect of the mutant TnT fragment.

Deletion of a Genomic Segment Containing the Cardiac Troponin I Gene Knocks Down Expression of the Slow Troponin T Gene and Impairs Fatigue Tolerance of Diaphragm Muscle

“…This mutation prematurely terminates the ssTnT polypeptide, and the C-terminally truncated ssTnT-(1-179) has a much decreased binding affinity for tropomyosin and fails to incorporate into the myofilaments (4). As a result, the truncated ssTnT protein was degraded and undetectable in the muscle of Amish nemaline myopathy patients, consistent with the recessive phenotype of the disease (5).…”

Troponin T Core Structure and the Regulatory NH2-Terminal Variable Region

“…Transgenic mice overexpressing the cardiac TnT core lacking the entire NH 2 -terminal variable region in the heart exhibited a normal basal life activity without apparent disruption of cardiac muscle function. Previously, we demonstrated that a COOH-terminal truncated slow skeletal muscle TnT was unable to accumulate in the muscle cells because it did not incorporate into the myofibrils (52). The large amount of McTnT-ND 72 recovered in the Tn complex from the cardiac muscle thin filaments (the Guba-Straub solution extracted fraction) of the transgenic mice demonstrates that McTnT-ND 72 effectively incorporates into the Tn complex and renders a functional thin filament.…”

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain

“…5E, right graph). TnnT1 is the tropomyosin binding subunit of the troponin complex and as such plays an important role in Ca 2+ -induced striated muscle contraction [56,57]. Analysis of TnnT1 therefore delivers an important read-out for the functionality and maturity of skeletal muscle tissue [57].…”