Hypertrophic cardiomyopathy (HCM) occurs in many of the affected patients due to heterozygous mutations in sarcomeric genes, such as myosin binding protein C (cMyBP-C, MYBPC3). Interestingly, the majority of mutations in MYBPC3 lead to premature termination codons (PTC). PTC containing mRNAs are targeted by nonsense mediated mRNA decay (NMD), a cellular quality control mechanism, which induces degradation of these mRNAs. This could lead to an overall reduced MYBPC3-mRNA content per cell, resulting in cMyBP-C haploinsufficiency, often observed in HCM-patients with MYBPC3-mutations. In the present study, we analyzed the involvement of NMD during MYBPC3-expression in HCM-patients with different MYBPC3 truncation mutations (MYBPC3_trunc) and the consequences on mRNA and protein stoichiometry.

To distinguish between effects caused by mutations or by hypertrophy, we compared cardiac tissue from HCM-patients with MYBPC3_trunc mutations to heart healthy donors and patients with hypertrophy due to aortic stenosis (AS). We analyzed the number of active transcription sites (aTS) by RNA-fluorescence in situ hybridization (RNA-FISH) for MYBPC3 and compared it to cardiac troponin I (cTnI, TNNI3) as a second sarcomeric gene. We found increased numbers of aTS per nucleus for both genes in HCM-patients and AS-patients but not in donors. This increased transcriptional activity could be explained by hypertrophic growth of the hearts and thereby reflect an increased demand on gene expression due to more sarcomeres. In patients with AS, this did not affect MYBPC3/TNNI3-mRNA ratios as shown by qPCR, indicating that stoichiometry of these sarcomeric genes is unchanged. In contrast, the ratio of MYBPC3 to TNNI3-mRNA was reduced in patients with MYBPC3_trunc mutations compared to donors and patients with AS. This reduction was further translated to protein level, where we detected a decreased cMyBP-C/cTnI ratio by western blot. These results indicate that HCM-patients with MYBPC3_trunc mutations show cMyBP-C haploinsufficiency, which most likely is the result of degradation of mutated MYBPC3-mRNAs.

To analyze if NMD could be involved in the reduction of mutated MYBPC3-mRNA in HCM-patients, we performed RNA-sequencing and subsequent gene set enrichment analysis. Indeed, we found enrichment of NMD in patients with MYBPC3_trunc mutations and specifically upregulation of NMD-associated factor UPF3B. We could confirm upregulation of UPF3B at mRNA and protein level in MYBPC3_trunc-patients compared to donors and patients with AS. By immunofluorescence staining we can show localization of UPF3B in the z-discs of sarcomeres, indicating that degradation of sarcomeric proteins by NMD might occur at the z-discs.

Together, our results demonstrate that HCM-patients with MYBPC3_trunc mutations show cMyBP-C haploinsufficiency presumably due to reduction of mutated MYBPC3-mRNA. Increased expression of NMD components and especially UPF3B indicates an often hypothesized involvement of NMD in degradation of mutated MYBPC3-mRNA.