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

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... ozygotes 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. (Circ Res. 2009;105:239-248.) Key Words: cardiomyopathy Ⅲ hypertrophic cardiomyopathy Ⅲ mRNA stability Ⅲ transgenic mice Ⅲ ubiquitin C ardiac myosin-binding protein (cMyBP)-C is a major component of the A-band of the sarcomere, where it interacts with myosin, actin and titin (see elsewhere 1,2 and reviewed previously 3 ). It is exclusively expressed in the heart in humans and mice. 4,5 Its role has been enigmatic for long, but accumulating recent evidence suggests that cMyBP-C is essential for normal diastolic relaxation by inhibiting actin-myosin interactions at low intracellular Ca 2ϩ concentrations. 6 -10 Mutations in MYBPC3 encoding cMyBP-C cause hypertrophic cardiomyopathy (HCM) (reviewed previously 3,11 ). HCM is an autosomal-dominant disease characterized by left ventricular (LV) hypertrophy, which predominantly involves the interventricular septum and is associated with myocardial disarray and interstitial fibrosis. 12 HCM involves more than 450 mutations in at least 13 genes encoding sarcomeric proteins. 11,13 Out of them, mutations in MYBPC3 are frequent. 14 In contrast to other disease genes, in which the majority of the mutations are missense, Ϸ70% of MYBPC3 mutations result in a frameshift creating a premature termination codon (PTC) and should produce C-terminal truncated proteins. 11, 14, 15 The mechanism by which MYBPC3 mutations Original