Nonsense-mediated mRNA decay and ubiquitin-proteasome system regulate cardiac myosin-binding protein C mutant levels in cardiomyopathic mice.

TY - JOUR

T1 - Nonsense-mediated mRNA decay and ubiquitin-proteasome system regulate cardiac myosin-binding protein C mutant levels in cardiomyopathic mice.

AU - Vignier, Nicolas

AU - Schlossarek, Saskia

AU - Fraysse, Bodvael

AU - Mearini, Giulia

AU - Krämer, Elisabeth

AU - Pointu, Hervé

AU - Mougenot, Nathalie

AU - Guiard, Josiane

AU - Reimer, Rudolph

AU - Hohenberg, Heinrich

AU - Schwartz, Ketty

AU - Vernet, Muriel

AU - Eschenhagen, Thomas

AU - Carrier, Lucie

PY - 2009

Y1 - 2009

N2 - RATIONALE: 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. METHODS AND 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.

AB - RATIONALE: 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. METHODS AND 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.

M3 - SCORING: Zeitschriftenaufsatz

VL - 105

SP - 239

EP - 248

JO - CIRC RES

JF - CIRC RES

SN - 0009-7330

IS - 3

M1 - 3

ER -