Nonsense-mediated mRNA decay and ubiquitin-proteasome system regulate cardiac myosin-binding protein C mutant levels in cardiomyopathic mice.
BACKGROUND
Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein (cMyBP)-C are frequent causes of hypertrophic cardiomyopathy, but the mechanisms leading from mutations to disease remain elusive.


OBJECTIVE
The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations.


RESULTS
We developed a cMyBP-C knock-in mouse carrying a point mutation. The level of total cMyBP-C mRNAs was 50% and 80% lower in heterozygotes and homozygotes, respectively. Surprisingly, the single G>A transition on the last nucleotide of exon 6 resulted in 3 different mutant mRNAs: missense (exchange of G for A), nonsense (exon skipping, frameshift, and premature stop codon) and deletion/insertion (as nonsense but with additional partial retention of downstream intron, restoring of the reading frame, and almost full-length protein). Inhibition of nonsense-mediated mRNA decay in cultured cardiac myocytes or in vivo with emetine or cycloheximide increased the level of nonsense mRNAs severalfold but not of the other mRNAs. By using sequential protein fractionation and a new antibody directed against novel amino acids produced by the frameshift, we showed that inhibition of the proteasome with epoxomicin via osmotic minipumps increased the level of (near) full-length mutants but not of truncated proteins. Homozygotes exhibited myocyte and left ventricular hypertrophy, reduced fractional shortening, and interstitial fibrosis; heterozygotes had no major phenotype.


CONCLUSIONS
These data reveal (1) an unanticipated complexity of the expression of a single point mutation in the whole animal and (2) the involvement of both nonsense-mediated mRNA decay and the ubiquitin-proteasome system in lowering the level of mutant proteins.
Vignier
Nicolas
N
Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany.
Schlossarek
Saskia
S
Fraysse
Bodvael
B
Mearini
Giulia
G
Krämer
Elisabeth
E
Pointu
Hervé
H
Mougenot
Nathalie
N
Guiard
Josiane
J
Reimer
Rudolph
R
Hohenberg
Heinrich
H
Schwartz
Ketty
K
Vernet
Muriel
M
Eschenhagen
Thomas
T
Carrier
Lucie
L
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D018389:Codon, Nonsense / Q000235:genetics*; D003513:Cycloheximide / Q000494:pharmacology; D004195:Disease Models, Animal; D004640:Emetine / Q000494:pharmacology; D005091:Exons / Q000235:genetics; D055879:Gene Knock-In Techniques; D006720:Homozygote; D017379:Hypertrophy, Left Ventricular / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017354:Point Mutation / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020871:RNA Stability / Q000235:genetics*; D025801:Ubiquitin / Q000378:metabolism*; D000099163:Myosin Binding Protein C
D002352:Carrier Proteins; D018389:Codon, Nonsense; D003513:Cycloheximide; D004640:Emetine; D046988:Proteasome Endopeptidase Complex; D011500:Protein Synthesis Inhibitors; D025801:Ubiquitin; D000099163:Myosin Binding Protein C