Nonsense-Mediated mRNA Decay and Ubiquitin–Proteasome System Regulate Cardiac Myosin-Binding Protein C Mutant Levels in Cardiomyopathic Mice

Abstract

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. (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...

Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice

“…KI mice carry a G4A transition on the last nucleotide of exon 6, which results in three different mutant mRNAs (Fig. 1a) and a 480% reduction of total Mybpc3 mRNA and cMyBP-C protein levels 8 . We first evaluated the level of expression of FLAG-tagged Mybpc3 after gene transfer in cardiac myocytes isolated from KI neonatal mice.…”

“…Yet, we believe that prevention is the more realistic scenario in the patient group we would like to target with the gene therapy approach, namely infants with severe homozygous or compound heterozygous mutations who quickly develop a therapy-resistant form of systolic heart failure 14,[16][17][18][19][20] . Second, mouse models including our KI model clearly differ from human HCM in that they show a much milder phenotype, generally only at the homozygous state 8,40,41 . In fact, our KI mice, although likely the best murine HCM model available, have a normal life expectancy and, as shown also in this study, LVH and the decrease in LV contractile function remain stable over time.…”

“…More than 64% of MYBPC3 mutations are truncating, leading to unstable mutant polypeptides [2][3][4][5] . Findings in humans as well as in cat and mouse HCM models indicate that the most prevalent disease mechanism is haploinsufficiency, even for MYBPC3 missense mutations [6][7][8] . Yet, mutant proteins ('poison peptides') may play an additional role in the pathogenesis of the disease [9][10][11] .…”

Myomasp/LRRC39, a Heart- and Muscle-Specific Protein, Is a Novel Component of the Sarcomeric M-Band and Is Involved in Stretch Sensing

“…Likewise, A-band staining was investigated with a MYBPC3 antibody, because MYBPC3 has been shown to bind to the myosin subfragment-2 (S2) neck region with its C terminus 26 and to localize to the sarcomeric A-band. 27 Simultaneous staining of MYBPC3 and myomasp/ LRRC39 excluded an A-band localization of myomasp/ LRRC39 ( Figure 4A). In line with the subcellular localization in ARVCM, cryosections of intact rat heart and skeletal muscle tissues again revealed a sarcomeric M-band signal for myomasp ( Figure 4B) and colocalization with the M-bandspecific titin N2A domain ( Figure 4B).…”

The ubiquitin proteasome system in human cardiomyopathies and heart failure

“…In vitro studies have shown that the expression of truncated (77) or missense (4, 5) MYBPC3 can inhibit the proteasome. These findings were translated to a mouse knock-in model, MYBPC3 Glu264Lys (89). This mutation is in the last base of exon 6 in a conserved splice site and analogous to Glu258Lys in humans.…”