Cardiac Myosin-Binding Protein C (MYBPC3) in Cardiac Pathophysiology

Authors, Journal, Affiliations, Type, DOI

• Authors: Lucie Carrier, Giulia Mearini, Konstantina Stathopoulou, Friederike Cuello
• Journal: Gene (2015), 573(2):188–197
• Affiliations: Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf; DZHK (German Centre for Cardiovascular Research)
• Type: Review article (Gene Wiki Review series)
• DOI: https://doi.org/10.1016/j.gene.2015.09.008
• Conflict of interest: University Medical Center Hamburg-Eppendorf has filed a patent for MYBPC3 gene therapy using AAV9 for HCM treatment.

Overview

This 2015 review from Hamburg provides a foundational overview of MYBPC3 biology covering three pillars: the mutation landscape in HCM and other cardiomyopathies; cMyBP-C protein structure, function, and posttranslational modifications; and therapeutic strategies for MYBPC3-associated disease. MYBPC3 mutations account for 40–50% of all HCM mutations (>350 variants), with >60% being truncating, and bi-allelic truncating mutations produce fatal neonatal cardiomyopathy within the first year of life. Haploinsufficiency — with absent truncated protein degraded via NMD/UPS/autophagy — is the dominant pathomechanism; reduced cMyBP-C increases myofilament Ca²⁺ sensitivity and impairs diastolic relaxation. Gene therapy approaches as of 2015 include AON-mediated exon skipping (proof-of-concept in mice), RNA trans-splicing, and AAV-mediated gene replacement (34-week prevention of HCM phenotype in homozygous knock-in mice).

Keywords

MYBPC3; cardiac myosin-binding protein C; cardiomyopathies; hypertrophy; posttranslational modifications; gene therapy

Key Takeaways

MYBPC3 Mutations: HCM

• More than 350 individual MYBPC3 mutations cause HCM, representing 40–50% of all HCM mutations — the most frequently mutated gene in HCM. More than 60% are truncating mutations (nonsense, insertions/deletions, splicing/branch-point), leading to COOH-terminally truncated cMyBP-C lacking major myosin- and/or titin-binding sites.
• Most mutations are heterozygous and associate with late disease onset and often benign progression. Patients with >1 mutation develop more severe phenotypes; 14% of childhood-onset HCM is caused by compound genetic variants.
• Bi-allelic truncating MYBPC3 mutations: 51 reported cases of homozygotes or compound heterozygotes; all 26 cases with double truncating mutations led to neonatal cardiomyopathy → heart failure and death within the first year of life. The only alternative is cardiac transplantation.

MYBPC3 Mutations: Other Cardiomyopathies

• DCM: MYBPC3 mutations represented the second highest number of disease-causing mutations in a European multi-centre DCM cohort of 639 patients (38/294, i.e., 13%; Haas et al. 2014).
• LVNC (Left Ventricular Non-Compaction): MYBPC3 mutations also cause LVNC, reflecting the cardiomyopathy phenotypic overlap with HCM; both missense and truncating mutations found in DCM and LVNC.
• South Asian founder variant: A 25-bp deletion in MYBPC3 intron 32 (Δ25, frameshift) — initially associated with HCM in 2 families — is present in 4% of the South Asian population and confers a 6.99-fold increased risk of heart failure (Dhandapany et al., 2009). Found in 13.8% of patients across HCM, DCM, and restrictive CMP.

Founder MYBPC3 Mutations by Population

• All founder mutations identified are truncating, lacking the phosphorylation M motif and/or major binding domains:
  • Iceland: c.927–2A>G (intron 11 splice); arose >550 years ago; accounts for 58% of Icelandic HCM
  • Veneto, Italy: c.912_913delTT (exon 11); 19.5% of HCM probands
  • Netherlands: c.2373_2374insG (17%), c.2864_2865delCT (2.6%), c.2827C>T (1.6%) — three different founder mutations
  • Finland: c.3183C>T / Gln1061X (exon 29); 17% of cases
  • Japan: c.1775delT / Val592fs/8 (exon 18); 16% of HCM probands
  • Tuscany, Italy: c.772G>A / Glu258Lys (exon 6); 14% of cases
  • Center-East France: c.1898–2A>G (intron 20); arose ~10 centuries ago; 8.4% of HCM patients
• High founder mutation prevalence in these regions increases the likelihood of homozygotes or compound heterozygotes — associated with severe pediatric cardiomyopathy.

cMyBP-C Protein Structure and Function

• MYBPC3 gene: >21 kbp, 35 exons (34 coding); located at chr11p11.2. cMyBP-C consists of 8 immunoglobulin-like (Ig) and 3 fibronectin-like (Fn3) domains. Cardiac-specific structural additions vs. skeletal isoforms: C0 domain (extra N-terminal Ig domain), multiple phosphorylation sites in the M motif between C1 and C2, and a 28-amino acid insertion in the C5 domain.
• cMyBP-C is located in the C-zone of the A-band (cross-bridge-bearing region), forming transverse stripes ~43 nm apart. It is not essential for sarcomere formation during embryogenesis but is crucial for sarcomere organization and normal cardiac function (lesson from Mybpc3⁻/⁻ knockout mice).
• Role in crossbridge cycling: cMyBP-C constrains actin-myosin interaction by tethering the myosin S1 heads (via N-terminal C1-M-C2 region binding to myosin S2), limiting loaded shortening velocity and power output. Loss of cMyBP-C accelerates crossbridge cycling rates (↑loaded shortening, ↑power output, ↑ktr at submaximal Ca²⁺).
• Control of filament states: N-terminus of cMyBP-C stabilises the ON state of thin filaments and OFF state of thick filaments, coordinating both filament systems (Kampourakis et al. 2014).
• Diastolic regulation: cMyBP-C prevents residual crossbridge cycling at low diastolic Ca²⁺, promoting diastolic relaxation. Reduced/absent cMyBP-C impairs diastolic relaxation → diastolic dysfunction as an early consequence of MYBPC3 mutation, independent of hypertrophy.

Pathomechanisms of MYBPC3 Mutations

• Truncating mutations → haploinsufficiency: Truncated cMyBP-Cs are not detectable in human patient myocardium (Western blot) or in heterozygous knock-in mice, implying the mutant protein is degraded — via nonsense-mediated mRNA decay (NMD), the ubiquitin-proteasome system (UPS), and autophagy-lysosome pathway.
• Missense mutations → poison polypeptide: Stable mutant cMyBP-Cs are (at least in part) incorporated into the sarcomere, potentially acting as poison polypeptides disrupting sarcomeric structure and/or function.
• Reduced cMyBP-C → increased myofilament Ca²⁺ sensitivity: Demonstrated across human samples, heterozygous and homozygous knock-in mice, and EHT models — the central functional consequence. Likely explains early diastolic dysfunction before overt hypertrophy (confirmed in MYBPC3 heterozygous patients and mice).
• UPS impairment: External stress (neurohumoral, aging) combined with Mybpc3 mutations impairs the UPS in mice; proteasomal activities are also depressed in human HCM patients (Predmore et al. 2010).
• Homozygous knock-in model: Born without phenotype; develop systolic dysfunction followed by compensatory hypertrophy soon after birth, mirroring neonatal cardiomyopathy.

Phosphorylation of cMyBP-C

• Three main phosphorylation sites in the M motif: Ser273, Ser282, Ser302 (mouse numbering). Ser282 acts as a hierarchical switch: its phosphorylation renders Ser273 and Ser302 more accessible.
• Kinases: PKA (beta-adrenergic stimulation, most important); CaMKII; RSK (p90 ribosomal S6 kinase); PKD (protein kinase D); PKC. GSK3β phosphorylates Ser131/133 outside the M-domain at the actin-binding site.
• Functional consequences: Dephosphorylated cMyBP-C binds myosin S2 → brakes crossbridge formation. When phosphorylated (by PKA via beta-adrenergic stimulation), cMyBP-C shifts to actin binding → accelerates crossbridge formation, enhances force development, promotes relaxation.
• Disease state: Overall cMyBP-C phosphorylation is reduced in human and experimental heart failure — loss of the beneficial phosphorylation state. Transgenic phosphomimetic cMyBP-C protected hearts from ischemia-reperfusion injury and reduced calpain-mediated degradation. Phosphorylation is therefore considered protective; preserving it combats disease progression.

Other Posttranslational Modifications of cMyBP-C

• Acetylation: 8 sites identified (5 in N-terminal C0-C1 region); confirmed in vivo during ischemia-reperfusion in mouse hearts. The 40-kDa calpain-degradation fragment is heavily acetylated — acetylation may promote proteolysis (contrast with phosphorylation which reduces calpain cleavage). Functional significance not fully understood.
• Citrullination: Detected at Arg696 in control and diseased tissue; not functionally characterised.
• S-glutathiolation: Three sites in murine cMyBP-C (Cys479, 627, 655); enhanced in hypertensive mouse models correlating with diastolic dysfunction. In vitro S-glutathiolation increases myofilament Ca²⁺ sensitivity — potentially deleterious. Mechanistic attribution to cMyBP-C specifically requires cell-level ablation experiments.
• S-nitrosylation: Cys1270 identified in mouse perfusion studies; functional role and in vivo occurrence not yet established.
• Carbonylation: Irreversible oxidative modification detected after doxorubicin exposure; potential contributor to chemotherapy-induced cardiac dysfunction.

Gene Therapy Approaches for MYBPC3 (as of 2015)

• Exon skipping (AON strategy): Antisense oligonucleotides (AONs) packaged under U7 promoter in tandem in AAV9 induce removal of exons 5 and 6 → in-frame deletion → expression of an alternatively spliced Mybpc3 variant. Proof-of-concept in heterozygous Mybpc3-knock-in newborn mice: prevented systolic dysfunction and left ventricular hypertrophy (Gedicke-Hornung et al. 2013). For human MYBPC3, skipping of 6 single or 5 double exons would preserve functionally important phosphorylation and protein interaction sites; ~half of missense or exonic/intronic truncating mutations could be addressed, including 35 mutations in exon 25.
• RNA trans-splicing: Two independently transcribed molecules (mutant pre-mRNA + therapeutic pre-trans-splicing molecule with WT sequence) are spliced together to produce a repaired full-length mRNA. Feasibility shown in isolated cardiac myocytes and in vivo in homozygous Mybpc3 knock-in mice, but efficiency was low and repaired protein insufficient to prevent disease phenotype (Mearini et al. 2013). Theoretically superior strategy — only 2 pre-trans-splicing molecules (targeting 5' and 3' of MYBPC3 pre-mRNA) would bypass all MYBPC3 mutations. Requires substantial efficiency improvement.
• Gene replacement (AAV-mediated): Full-length Mybpc3 gene transfer dose-dependently prevented development of cardiac hypertrophy and dysfunction over 34 weeks in homozygous knock-in mice (Mearini et al. 2014). Unexpectedly, exogenous Mybpc3 expression was associated with down-regulation of endogenous mutant Mybpc3 (mechanism unclear; likely sarcomeric incorporation competition). This result paved the way for testing in larger animal models (naturally occurring HCM cat model with MYBPC3 mutation) and potential translation for infants with bi-allelic mutations.
• CRISPR/Cas9: Not yet evaluated for MYBPC3 mutations as of 2015; considered under investigation in iPSC lines. Applicable for founder mutations. Efficiency, off-target effects, and long-term implications noted as unresolved issues.

Limitations of the Document

• 2015 publication: gene therapy sections are substantially outdated — CRISPR-based cardiac editing, SupRep, base editing, and prime editing have all been developed since; refer to more recent sources.
• Conflict of interest: Hamburg group holds AAV9-MYBPC3 gene therapy patent; gene replacement results should be interpreted in this context.
• Phosphorylation and PTM data predominantly from animal models; human clinical correlates limited.
• Gene Wiki Review series — educational format with space constraints acknowledged by authors; some important studies not cited.
• CRISPR iPSC experiments mentioned as "under investigation" — no data reported in this paper.

Key Concepts Mentioned

• concepts/Haploinsufficiency — central pathomechanism for truncating MYBPC3 mutations
• concepts/Sarcomere-Biology — cMyBP-C structural role in cardiac sarcomere
• concepts/AAV-Gene-Delivery — gene replacement and exon skipping vector platform
• concepts/CRISPR-Cas9-in-Channelopathies — CRISPR genome editing discussed as emerging approach

Key Entities Mentioned

• entities/MYBPC3 — primary subject of review
• entities/HCM — primary disease context
• entities/DCM — secondary disease context (MYBPC3 as 2nd most common DCM gene)
• entities/MYH7 — context as second most common HCM gene

Wiki Pages Updated

• wiki/sources/mybpc3-gene-2015.md — created (this file)
• wiki/sourceindex.md — new entry added
• wiki/wikiindex.md — no new concept/entity pages; existing MYBPC3 and HCM entries updated
• wiki/entities/MYBPC3.md — major update: mutation landscape, founder mutations, protein structure/function, pathomechanisms, phosphorylation, other PTMs, gene therapy; source_count 2→3
• wiki/entities/HCM.md — minor update: bi-allelic MYBPC3 neonatal phenotype, South Asian deletion, founder mutations; source_count 9→10
• wiki/entities/DCM.md — minor update: MYBPC3 as 2nd most common DCM gene; source_count 12→13