Degradation of MYBPC3 with Hypertrophic Cardiomyopathy-truncation mutations presumably takes place at Z-discs via nonsense mediated decay factor UPF3B

Mutations in myosin binding protein C (cMyBP-C, MYBPC3) belong to the most common mutations that lead to hypertrophic cardiomyopathy (HCM). Among the mutation positive patients 30-40% are heterozygous for these mutations. The majority of mutations in MYBPC3 lead to premature termination codons (PTC). PTC containing mRNAs are most often targeted by nonsense mediated mRNA decay (NMD), a cellular quality control mechanism, which induces degradation of these mRNAs. In case of MYBPC3_trunc mutations this leads to an overall reduced MYBPC3-mRNA content per cell, resulting in cMyBP-C haploinsufficiency and thereby altered biomechanical function, finally causing HCM. Depending on the fraction of mutant mRNA per cell, extend of haploinsufficiency also varies between cells within the myocardium, which could even aggravate cardiac dysfunction.

To date only few studies have shown direct involvement of NMD in cMyBP-C-haploinsufficiency. The aim of our study was therefore to identify potential NMD-pathways involved in HCM-development. We analyzed HCM-patients with different MYBPC3 truncation mutations (MYBPC3_trunc). To distinguish between effects caused by mutations or by hypertrophy, we compared cardiac tissue from HCM-patients with MYBPC3_trunc mutations not only to heart healthy donors and but also to patients with hypertrophy due to aortic stenosis (AS). We show that the HCM-patients show haploinsufficiency at protein level via Western Blot analysis, which is not caused by reduced transcriptional activity as shown by RNA-FISH and RT-qPCR. To identify potential candidates of NMD-associated genes which could underlie haploinsufficiency development, we performed RNA-sequencing and subsequent gene set enrichment analysis (GSEA) of the HCM-patients in comparison to donors and AS-patients. We found enrichment of NMD in patients with MYBPC3_trunc mutations and specifically upregulation of NMD-associated factor UPF3B. We confirmed this upregulation by RT-qPCR and Western Blot. Interestingly, NMD-factors UPF1 and UPF2, which are involved in alternative NMD-pathways, were not upregulated. This suggests that MYBPC3_trunc mRNAs are specifically targeted by UPF3B.

It has been shown that sarcomeric proteins are translated at the Z-discs, therefore we hypothesized that also NMD of MYBPC3_trunc mRNA could take place there. Therefore, we performed immunofluorescent staining of UPF1, UPF2, and UPF3B in cardiac tissue. Indeed, we could show that UPF3B is located at the Z-discs, whereas UPF1 is more pronounced in nuclei and cytoplasm and UPF2 was accumulated between the Z-discs.

In summary, we show that haploinsufficiency in HCM-patients with different truncation mutations in MYBPC3 mutations is likely established by NMD. Upregulation of NMD-factor UPF3B and its localization at the Z-discs strongly suggests that MYBPC3_trunc NMD is driven by the UPF2-independent UPF3B-pathway and takes place at the Z-discs.