[22] Consistent with this notion, cMyBP-C knockout mice exhibit an abnormal systolic timecourse, with a shortened elastance timecourse and lower peak elastance in vivo,[23] and an accelerated force development in isolated, skinned cardiac fibers[24] suggesting that cMyBP-C is required to constrain the crossbridges in order to sustain a normal ejection.

This results in a decreased number of crossbridges formed, which hinders force generation, due to its N-terminal C1-M-C2 region interacting with the myosin-S2 domain.

At least three extensively characterized phosphorylation sites (Ser273, 282 and 302; numbering refers to the mouse sequence) are localized in the M motif of cMyBP-C and are targeted by protein kinases in a hierarchical order of events.

[60] Furthermore, a 25-bp intronic MYBPC3 deletion leading to protein truncation is present in 4% of the population in South India and is associated with a higher risk to develop heart failure.

[62] Founder MYBPC3 mutations have been reported in Iceland, Italy, The Netherlands, Japan, France and Finland, where they represent a large percentage of cases with hypertrophic cardiomyopathy.

[63][64][65][66][67][68][69] A body of evidence indicates that patients with more than 1 mutation often develop a more severe phenotype,[70] and a significant fraction of childhood-onset hypertrophic cardiomyopathy (14%) is caused by compound genetic variants.

A total of 51 cases of homozygotes or compound heterozygotes have been reported, most of them with double truncating MYBPC3 mutations and associated with severe cardiomyopathy, leading to heart failure and death within the first year of life.

[72] A great understanding of how MYBPC3 mutations lead to the development of inherited cardiomyopathy came from the analyses of human myocardial samples, gene transfer in different cell lines, naturally-occurring or transgenic animal models and more recently disease modeling using induced pluripotent stem cells (iPSC)-derived cardiac myocytes.

The homozygous Mybpc3-targeted knock-in mice, which genetically mimic the situation of severe neonatal cardiomyopathy are born without phenotype and soon after birth develop systolic dysfunction followed by (compensatory) cardiac hypertrophy.

[91] Skinned trabeculae or cardiac myocytes obtained from human patients carrying a MYBPC3 mutation or from heterozygous and homozygous Mybpc3-targeted knock-in mice exhibited higher myofilament Ca2+ sensitivity than controls.

[92][78][93][94][95] Disease-modeling by engineered heart tissue (EHT) technology with cardiac cells from heterozygous or homozygous Mybpc3-targeted knock-in mice reproduced observations made in human and mouse studies displaying abbreviated contractions, greater sensitivity to external Ca2+ and smaller inotropic responses to various drugs (isoprenaline, EMD 57033 and verapamil) compared to wild-type control EHTs.

[100] Because of their tissue selectivity and persistent expression recombinant adeno-associated viruses (AAV) have therapeutic potential in the treatment of inherited cardiomyopathy resulting from MYBPC3 mutations-[101] Several targeting approaches have been developed.

[87] Systemic administration of AAV-based AONs to Mybpc3-targeted knock-in newborn mice prevented both systolic dysfunction and left ventricular hypertrophy, at least for the duration of the investigated period.

[88] In principle, however, this SmART strategy is superior to exon skipping or CRISPR/Cas9 genome editing and still attractive, because only two pre-trans-splicing molecules, targeting the 5’ and the 3’ of MYBPC3 pre-mRNA would be sufficient to bypass all MYBPC3 mutations associated with cardiomyopathies and therefore repair the mRNA.