Exon Skipping in Cardiac Troponin T of Turkeys with Inherited Dilated Cardiomyopathy

Biesiadecki, Brandon J.; Jin, Jian Ping

doi:10.1074/jbc.m200788200

Cited by 49 publications

(95 citation statements)

References 53 publications

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“…In contrast to the fact that most adult avian and mammalian hearts express only a single cTnT isoform, we have found the expression of an additional cTnT splicing variant resulting from the exclusion of 12 amino acids encoded by the exon 8 in the heart of the dilated cardiomyopathy (DCM) turkey (13). This abnormal splicing pathway resulted in a low molecular weight cTnT exhibiting altered molecular conformation and binding affinity for troponin I (TnI) and tropomyosin (Tm).…”

Section: Troponin T (Tnt)mentioning

confidence: 88%

Cardiac Troponin T Variants Produced by Aberrant Splicing of Multiple Exons in Animals with High Instances of Dilated Cardiomyopathy

Biesiadecki

Elder

et al. 2002

Journal of Biological Chemistry

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Adult cardiac muscle normally expresses a single cardiac troponin T (cTnT). As a potential pathogenic mechanism for turkey dilated cardiomyopathy, the splice-out of a normally constitutive exon generates an additional low molecular weight cTnT with altered conformation and function. We further found that aberrant splicing of cTnT also occurs in several mammals correlating to dilated cardiomyopathy. Skipping of the same exon as that in the turkey was found in the canine cTnT. Spliceout of the adjacent exon 6 occurred in the guinea pig cTnT. Retention of the embryonic exon 5 was found in the cTnT of cat, dog, and guinea pig. These aberrant splicing variants significantly altered the structure of cTnT to sustain functional effects as that in the myopathic turkey cTnT. The genomic sequence of canine cTnT gene shows no specific alterations. However, the alternative splicing patterns of canine cTnT are different in developing cardiac and skeletal muscles, suggesting abnormality of trans-regulatory factors. Transgenic expression of the aberrant cTnT variants resulted in contractile changes in mouse cardiomyocytes. The findings support the hypothesis that thin filament heterogeneity due to the co-expression of alternatively spliced cTnT variants may desynchronize myocardial contraction and contribute to the pathogenesis and pathophysiology of cardiomyopathy and heart failure.

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Section: Troponin T (Tnt)mentioning

confidence: 88%

Cardiac Troponin T Variants Produced by Aberrant Splicing of Multiple Exons in Animals with High Instances of Dilated Cardiomyopathy

Biesiadecki

Elder

et al. 2002

Journal of Biological Chemistry

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100

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“…There was no difference between high and low molecular weight truncated variants. Because the solid phase enzyme-linked immunosorbent assay protein binding experiments are done with repeated washing, these results only reflect the loss of high affinity binding (31), although low affinity binding between truncated slow TnT and tropomyosin is expected (42).…”

Section: Loss Of High Affinity Binding Of Truncated Slowmentioning

confidence: 99%

“…The fractions containing human slow TnT-(1-179) fragment were dialyzed against 0.1 mM EDTA and concentrated by lyophilization. The truncated slow TnT was further purified by Sephadex G-75 gel filtration chromatography in 6 M urea, 0.5 M KCl, and 10 mM imidazole-HCl, pH 7.0, as described previously (31). Both high and low molecular weight variants of truncated human slow TnT were purified by this procedure.…”

mentioning

confidence: 99%

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

Wang

Huang

Breckenridge

et al. 2005

Journal of Biological Chemistry

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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.

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“…Therefore, the ineffective binding between TnI and TnC in bacterial cells might not be simply due to the solution environment but result from ineffective folding of the bacterially made troponin proteins (most likely cardiac TnI since the TnC protein was in excess and only a small portion needed to be correctly folded to saturate the co-expressed cardiac TnI). It has been demonstrated by us and many other laboratories that TnI and TnC purified from bacterial expression both have biochemical activities and can form functional TnI-TnC binary complex and tertiary troponin complex [3,7,10]. These results indicate that denaturing (urea buffer) and renaturing (final dialysis) steps during the purification of bacterial made TnI are necessary for the yield of biologically active proteins.…”

Section: Bi-cistronic Co-expression With Tnc Improves the Expression mentioning

confidence: 99%

“…The total protein extracts were clarified by room temperature centrifugation at top speed in a microcentrifuge and resolved by 12% Laemmli SDS-gel with an acrylamide:bisacrylamide ratio of 45:1. Bovine cardiac TnI purified from ventricular muscle [10] and mouse cardiac/slow TnC purified from bacterial culture [10] were used as controls. The resulting gels were stained with Coomassie Brilliant Blue R250 to reveal the resolved protein bands and duplicate gels were electrically blotted to nitrocellulose membranes as previously described [18].…”

mentioning

confidence: 99%

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

Jin

2007

Archives of Biochemistry and Biophysics

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Troponin I (TnI) is a muscle-specific protein and plays an allosteric function in the Ca 2+ regulation of cardiac and skeletal muscle contraction. Expression of cloned cDNA in E. coli is an essential approach to preparing human TnI and mutants for structural and functional studies. The expression level of cardiac TnI in E. coli is very low. To reduce the potential toxicity of cardiac TnI to the host cell, we constructed a bi-cistronic expression vector to co-express cardiac TnI and cardiac/slow troponin C (TnC), a natural binding partner of TnI and a protein that readily expresses in E. coli at high levels. The co-expression moderately increased the expression of cardiac TnI although a high amount of TnC protein was produced from the bi-cistronic mRNA. The use of an E. coli strain containing additional tRNAs for certain low bacterial usage eukaryotic codons improved the expression of cardiac TnI. Modifications of two 5'-regional codons that have predicted low usages in bacterial cells did not reproduce the improvement, indicating that not the 5' but the overall codon usage restricts the translational efficiency of cardiac TnI mRNA in E. coli. However, deletion of the cardiac TnI-specific N-terminal 28 amino acids significantly improved the protein expression independent of the host cell tRNA modifications. The results suggest that the regulatory N-terminal domain of cardiac TnI is a dominant factor for the incompatibility in bacterial cells, supporting its role in modulating the overall molecular conformation. KeywordsTroponin I; Protein conformation; Bi-cistronic expression; Codon usage Muscle contraction is regulated by intracellular Ca 2+ via the thin filament-based troponintropomyosin system. Troponin is a striated muscle-specific protein complex contains three subunits: the Ca 2+ -binding subunit troponin C (TnC), the tropomyosin binding subunit troponin T (TnT) and the inhibitory subunit troponin I (TnI) [1,2]. During muscle contraction, cytoplasmic Ca 2+ rises and binds to TnC to induce a series of conformational changes in the troponin complex that release the inhibition of actomyosin ATPase and activate force development [3]. Three homologous TnI genes (cardiac, fast skeletal muscle, and slow skeletal muscle) have evolved in vertebrates to encode muscle-type-specific TnI isoforms [4]. Primary structures of cardiac, fast and slow skeletal muscle TnI isoforms are highly conserved. The main structural difference is a cardiac TnI-specific ~30 amino acids NH 2 -terminal extension [5].* To whom correspondence should be addressed. Tel: (847) 570-1960. Fax: (847)570-1865. E-mail: jpjin@northwestern.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered whi...

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Exon Skipping in Cardiac Troponin T of Turkeys with Inherited Dilated Cardiomyopathy

Cited by 49 publications

References 53 publications

Cardiac Troponin T Variants Produced by Aberrant Splicing of Multiple Exons in Animals with High Instances of Dilated Cardiomyopathy

Cardiac Troponin T Variants Produced by Aberrant Splicing of Multiple Exons in Animals with High Instances of Dilated Cardiomyopathy

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

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

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