![Cardiac sarcomere structure](https://upload.wikimedia.org/wikipedia/commons/7/7f/Cardiac_sarcomere_structure.png)
The myosin-binding protein C, cardiac-type is a
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
that in humans is encoded by the ''MYBPC3''
gene
In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
.
This
isoform
A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some iso ...
is expressed exclusively in
heart muscle
Cardiac muscle (also called heart muscle, myocardium, cardiomyocytes and cardiac myocytes) is one of three types of vertebrate muscle tissues, with the other two being skeletal muscle and smooth muscle. It is an involuntary, striated muscle that ...
during
human
Humans (''Homo sapiens'') are the most abundant and widespread species of primate, characterized by bipedalism and exceptional cognitive skills due to a large and complex brain. This has enabled the development of advanced tools, cultu ...
and mouse development, and is distinct from those expressed in slow
skeletal muscle (
MYBPC1
Myosin-binding protein C, slow-type is a protein that in humans is encoded by the ''MYBPC1'' gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''gene ...
) and fast skeletal muscle (
MYBPC2
Myosin binding protein C, fast type is a protein that in humans is encoded by the MYBPC2 gene.
Function
This gene encodes a member of the myosin-binding protein C family. This family includes the fast-, slow- and cardiac-type isoforms, each of ...
).
Structure
cMyBP-C is a 140.5 kDa protein composed of 1273 amino acids.
cMyBP-C is a myosin-associated protein that binds at 43 nm intervals along the myosin thick filament backbone, stretching for 200 nm on either side of the
M-line M line may refer to:
* M-line (mittel line or middle line), a structure in a muscle sarcomere
* M Ocean View, a light rail and former streetcar line in San Francisco, California
* McKinney Avenue Transit Authority, a streetcar line in Dallas, Texas ...
within the crossbridge-bearing zone (C-region) of the
A band
The A Band are a British musical collective formed in Nottingham in the late 1980s. In 2005, The Wire compared the band's importance to that of AMM and SME, and noted their lack of musical training.
The line-up of the group has never been f ...
in striated muscle. The approximate stoichiometry of cMyBP-C along the thick filament is 1 per 9-10 myosin molecules, or 37 cMyBP-C molecules per thick filament. In addition to myosin, cMyBP-C also binds
titin
Titin (contraction for Titan protein) (also called connectin) is a protein that in humans is encoded by the ''TTN'' gene. Titin is a giant protein, greater than 1 µm in length, that functions as a molecular spring that is responsible for th ...
and
actin
Actin is a family of globular multi-functional proteins that form microfilaments in the cytoskeleton, and the thin filaments in muscle fibrils. It is found in essentially all eukaryotic cells, where it may be present at a concentration of ov ...
. The cMyBP-C isoform expressed in cardiac muscle differs from those expressed in slow and fast skeletal muscle (
MYBPC1
Myosin-binding protein C, slow-type is a protein that in humans is encoded by the ''MYBPC1'' gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''gene ...
and
MYBPC2
Myosin binding protein C, fast type is a protein that in humans is encoded by the MYBPC2 gene.
Function
This gene encodes a member of the myosin-binding protein C family. This family includes the fast-, slow- and cardiac-type isoforms, each of ...
, respectively) by three features: (1) an additional
immunoglobulin (Ig)-like domain on the N-terminus, (2) a linker region between the second and third
Ig domains, and (3) an additional loop in the sixth
Ig domain. cMyBP-C appears necessary for normal order, filament length and lattice spacing within the structure of the
sarcomere
A sarcomere (Greek σάρξ ''sarx'' "flesh", μέρος ''meros'' "part") is the smallest functional unit of striated muscle tissue. It is the repeating unit between two Z-lines. Skeletal muscles are composed of tubular muscle cells (called mus ...
.
Function
cMyBP-C is not essential for sarcomere formation during embryogenesis, but is crucial for sarcomere organization and maintenance of normal
cardiac function. Absence of cMyBP-C (''Mybpc3''-targeted knock-out mice) results in severe cardiac hypertrophy, increased heart-weight-to-body-weight-ratios, enlargement of ventricles, increased myofilament Ca2+ sensitivity and depressed diastolic and systolic function.
Histologically, ''Mybpc3''-targeted knock-out hearts display structural rearrangements with cardiac myocyte disarray and increased interstitial fibrosis similar to patients with
hypertrophic cardiomyopathy, without obvious alterations in shape or size of single cardiac myocytes. Ultrastructural examination revealed a loss of lateral alignment of adjacent myofibrils with their Z-lines misaligned.
cMyBP-C appears to act as a brake on cardiac contraction, as loaded shortening, power and cycling kinetics all increase in cMyBP-C knockout mice. Consistent with this notion, cMyBP-C knockout mice exhibit an abnormal systolic timecourse, with a shortened elastance timecourse and lower peak elastance in vivo, and an accelerated force development in isolated, skinned cardiac fibers suggesting that cMyBP-C is required to constrain the crossbridges in order to sustain a normal ejection.
cMyBP-C regulates the positioning of myosin and actin for interaction and acts as a tether to the myosin S1 heads, limiting their mobility. 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. Furthermore, cMyBP-C contributes to the regulation of cardiac contraction at short sarcomere length and is required for complete relaxation in diastole.
Interactions of cMyBP-C with its binding partners vary with its
posttranslational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribo ...
status. 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. In its dephosphorylated state, cMyBP-C binds predominantly to myosin S2 and brakes crossbridge formation, however, when phosphorylated in response to
β-adrenergic stimulation through activating
cAMP
Camp may refer to:
Outdoor accommodation and recreation
* Campsite or campground, a recreational outdoor sleeping and eating site
* a temporary settlement for nomads
* Camp, a term used in New England, Northern Ontario and New Brunswick to descri ...
-dependent protein kinase (
PKA), it favours binding to actin, then accelerating crossbridge formation, enhancing force development and promoting relaxation. Protein kinases identified thus far to phosphorylate cMyBP-C in the M motif are
PKA,
Ca
2+/calmodulin-dependent kinase II (
CaMKII
/calmodulin-dependent protein kinase II (CaM kinase II or CaMKII) is a serine/threonine-specific protein kinase that is regulated by the /calmodulin complex. CaMKII is involved in many signaling cascades and is thought to be an important mediator ...
),
ribosomal s6 kinase (RSK),
protein kinase D (PKD), and
protein kinase C (PKC).
Furthermore,
GSK3β
Glycogen synthase kinase-3 beta, (GSK-3 beta), is an enzyme that in humans is encoded by the ''GSK3B'' gene. In mice, the enzyme is encoded by the Gsk3b gene. Abnormal regulation and expression of GSK-3 beta is associated with an increased suscept ...
was described as another protein kinase to phosphorylate cMyBP-C outside the M-domain in the proline-alanine-rich actin-binding site at Ser133 in human myocardium (mouse Ser131). Phosphorylation is required for normal cardiac function and cMyBP-C stability, and overall phosphorylation levels of cMyBP-C are reduced in human and experimental heart failure. Other posttranslational modifications of cMyBP-C exist, which occur throughout the protein and are not thoroughly characterised yet, such as acetylation, citrullination, S-glutathiolation, S-nitrosylation and carbonylation.
Genetics
The cloning of the human ''MYBPC3'' cDNA and localization of the gene on human chromosome 11p11.2 has assisted the structure and function of cMyBP-C. ''MYBPC3'' became therefore the “best” candidate gene for the ''CMH4'' locus for
hypertrophic cardiomyopathy that was initially mapped by the group of Schwartz.
''MYBPC3'' mutations segregating in families with
hypertrophic cardiomyopathy have been identified. ''MYBPC3'' was thus the fourth gene for
hypertrophic cardiomyopathy, following
MYH7, encoding β-
myosin heavy chain
Myosins () are a superfamily of motor proteins best known for their roles in muscle contraction and in a wide range of other motility processes in eukaryotes. They are ATP-dependent and responsible for actin-based motility.
The first myosin (M2 ...
,
TNNT2
Cardiac muscle troponin T (cTnT) is a protein that in humans is encoded by the ''TNNT2'' gene. Cardiac TnT is the tropomyosin-binding subunit of the troponin complex, which is located on the thin filament of striated muscles and regulates muscle c ...
and
TPM1
Tropomyosin alpha-1 chain is a protein that in humans is encoded by the ''TPM1'' gene. This gene is a member of the tropomyosin (Tm) family of highly conserved, widely distributed actin-binding proteins involved in the contractile system of striate ...
, encoding cardiac
troponin T
Troponin T (shortened TnT or TropT) is a part of the troponin complex, which are proteins integral to the contraction of skeletal and heart muscles. They are expressed in skeletal and cardiac myocytes. Troponin T binds to tropomyosin and help ...
and α-
tropomyosin
Tropomyosin is a two-stranded alpha-helical, coiled coil protein found in actin-based cytoskeletons.
Tropomyosin and the actin skeleton
All organisms contain organelles that provide physical integrity to their cells. These type of organelles a ...
, respectively, earmarking
hypertrophic cardiomyopathy as a disease of the
sarcomere
A sarcomere (Greek σάρξ ''sarx'' "flesh", μέρος ''meros'' "part") is the smallest functional unit of striated muscle tissue. It is the repeating unit between two Z-lines. Skeletal muscles are composed of tubular muscle cells (called mus ...
.
To date, roughly 350 mutations in ''MYBPC3'' have been identified, and in large part, the mutations result in protein truncation, shifts in reading frames, and premature termination codons. Genetic studies have revealed significant overlap between genotypes and phenotypes as ''MYBPC3'' mutations can lead to various forms of cardiomyopathies, such as
dilated cardiomyopathy
Dilated cardiomyopathy (DCM) is a condition in which the heart becomes enlarged and cannot pump blood effectively. Symptoms vary from none to feeling tired, leg swelling, and shortness of breath. It may also result in chest pain or fainting. Co ...
and
left ventricular
A ventricle is one of two large chambers toward the bottom of the heart that collect and expel blood towards the peripheral beds within the body and lungs. The blood pumped by a ventricle is supplied by an atrium, an adjacent chamber in the upper ...
noncompaction cardiomyopathy. In patients with isolated or familial cases of dilated cardiomyoathy, ''MYBPC3'' mutations represented the second highest number of known mutations.
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. 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. All of them are truncating mutations, resulting in a shorter protein, lacking the regulatory phosphorylatable M motif and/or major binding domains to other sarcomeric proteins.
A body of evidence indicates that patients with more than 1 mutation often develop a more severe phenotype, and a significant fraction of childhood-onset
hypertrophic cardiomyopathy(14%) is caused by compound genetic variants. This suggests that a gene-dosage effect might be responsible for manifestations at a younger age. 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.
Pathomechanisms
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
Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka's lab in Kyoto, Japan, who showed in ...
(iPSC)-derived cardiac myocytes.
Although access to human myocardial samples is difficult, at least some studies provided evidence that truncated cMyBP-Cs, resulting from truncating ''MYBPC3'' mutations are not detectable in human patient samples by Western-immunoblot analysis.
This was supported in heterozygous ''Mybpc3''-targeted knock-in mice,
carrying the human c.772G>A transition (i.e. founder mutation in Tuscany
These data suggest
haploinsufficiency
Haploinsufficiency in genetics describes a model of dominant gene action in diploid organisms, in which a single copy of the wild-type allele at a locus in heterozygous combination with a variant allele is insufficient to produce the wild-type ...
as the main disease mechanism for heterozygous truncating mutations. A body of evidence exists that the mechanisms regulating the expression of mutant allele involve the
nonsense-mediated mRNA decay
Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that exists in all eukaryotes. Its main function is to reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons. Translation of these aberran ...
, the
ubiquitin-proteasome system
Proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases.
Proteasomes are part of a major mechanism by whi ...
(UPS) and the
autophagy-lysosomal pathway after gene transfer of mutant ''MYBPC3'' in cardiac myocytes or in mice ''in vivo''.
In contrast to truncating mutations, missense mutations lead, in most of the cases (although difficult to specifically detect), to stable mutant cMyBP-Cs that are, at least in part, incorporated into the sarcomere and could act as poison polypeptides on the structure and/or function of the
sarcomere
A sarcomere (Greek σάρξ ''sarx'' "flesh", μέρος ''meros'' "part") is the smallest functional unit of striated muscle tissue. It is the repeating unit between two Z-lines. Skeletal muscles are composed of tubular muscle cells (called mus ...
. Homozygous or compound heterozygous mutations are therefore likely subject to differential regulation depending on whether they are double missense, double truncating or mixed missense/truncating mutations. 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.
The human c.772G>A transition results in low levels of three different mutant ''Mybpc3'' mRNAs and cMyBP-Cs in homozygous mice, suggesting a combination of
haploinsufficiency
Haploinsufficiency in genetics describes a model of dominant gene action in diploid organisms, in which a single copy of the wild-type allele at a locus in heterozygous combination with a variant allele is insufficient to produce the wild-type ...
and polypeptide poisoning as disease mechanism in the homozygous state.
In addition, the combination of external stress (such as neurohumoral stress or aging) and ''Mybpc3'' mutations have been shown to impair the UPS in mice, and proteasomal activities were also depressed in patients with
hypertrophic cardiomyopathy or
dilated cardiomyopathy
Dilated cardiomyopathy (DCM) is a condition in which the heart becomes enlarged and cannot pump blood effectively. Symptoms vary from none to feeling tired, leg swelling, and shortness of breath. It may also result in chest pain or fainting. Co ...
.
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.
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. Therefore, EHTs are suitable to model the disease phenotype and recapitulate functional alterations found in mice with
hypertrophic cardiomyopathy. Another good system for modeling cardiomyopathies in the cell culture dish is the derivation of cardiac myocytes from iPSC. Reports of human iPSC models of sarcomeric cardiomyopathies showed cellular hypertrophy in most of the cases,
including one with the c.2995_3010del ''MYBPC3'' mutation that exhibited in addition to hypertrophy contractile variability in the presence of
endothelin-1.
Therapy
Because of their tissue selectivity and persistent expression recombinant
adeno-associated virus
Adeno-associated viruses (AAV) are small viruses that infect humans and some other primate species. They belong to the genus ''Dependoparvovirus'', which in turn belongs to the family ''Parvoviridae''. They are small (approximately 26 nm in di ...
es (AAV) have therapeutic potential in the treatment of inherited cardiomyopathy resulting from ''MYBPC3'' mutations- Several targeting approaches have been developed. The most recent is genome editing to correct a mutation by
CRISPR/Cas9 technology.
Naturally existing as part of the prokaryotic immune system, the
CRISPR/Cas9 system has been used for correction of mutations in the mammalian genome.
By inducing nicks in the double-stranded DNA and providing a template DNA sequence, it is possible to repair mutations by
homologous recombination
Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may ...
. This approach has not yet been evaluated for ''MYBPC3'' mutations, but it could be used for each single or clustered mutation, and therefore applied preferentially for frequent founder ''MYBPC3'' mutations.
Other strategies targeting the mutant pre-mRNA by
exon skipping
In molecular biology, exon skipping is a form of RNA splicing used to cause cells to “skip” over faulty or misaligned sections (exons) of genetic code, leading to a truncated but still functional protein despite the genetic mutation.
Mechanis ...
and/or
spliceosome
A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs ( snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to specif ...
-mediated RNA
trans-splicing
''Trans''-splicing is a special form of RNA processing where exons from two different primary RNA transcripts are joined end to end and ligated. It is usually found in eukaryotes and mediated by the spliceosome, although some bacteria and archa ...
(SMaRT) have been evaluated for ''MYBPC3''.
Exon skipping
In molecular biology, exon skipping is a form of RNA splicing used to cause cells to “skip” over faulty or misaligned sections (exons) of genetic code, leading to a truncated but still functional protein despite the genetic mutation.
Mechanis ...
can be achieved using
antisense oligonucleotide
Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids ...
(AON) masking exonic splicing enhancer sequences and therefore preventing binding of the splicing machinery and therefore resulting in exclusion of the exon from the mRNA. This approach can be applied when the resulting shorter, but in-frame translated protein maintains its function. Proof-of-concept of
exon skipping
In molecular biology, exon skipping is a form of RNA splicing used to cause cells to “skip” over faulty or misaligned sections (exons) of genetic code, leading to a truncated but still functional protein despite the genetic mutation.
Mechanis ...
was recently shown in homozygous ''Mybpc3''-targeted knock-in mice.
Systemic administration of
AAV-based AONs to ''Mybpc3''-targeted
knock-in In molecular cloning and biology, a gene knock-in (abbreviation: KI) refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found ...
newborn mice prevented both systolic dysfunction and left ventricular hypertrophy, at least for the duration of the investigated period.
For the human ''MYBPC3'' gene, skipping of 6 single exons or 5 double exons with specific AONs would result in shortened in-frame cMyBP-Cs, allowing the preservation of the functionally important phosphorylation and protein interaction sites. With this approach, about half of missense or exonic/intronic truncating mutations could be removed, including 35 mutations in exon 25. The other strategy targeting the mutant pre-mRNA is SMaRT. Hereby, two independently transcribed molecules, the mutant pre-mRNA and the therapeutic pre-trans-splicing molecule carrying the wild-type sequence are spliced together to give rise to a repaired full-length mRNA. Recently, the feasibility of this method was shown both in isolated cardiac myocytes and ''in vivo'' in the heart of homozygous ''Mybpc3''-targeted
knock-in In molecular cloning and biology, a gene knock-in (abbreviation: KI) refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found ...
mice, although the efficiency of the process was low and the amount of repaired protein was not sufficient to prevent the development of the cardiac disease phenotype.
In principle, however, this SmART strategy is superior to
exon skipping
In molecular biology, exon skipping is a form of RNA splicing used to cause cells to “skip” over faulty or misaligned sections (exons) of genetic code, leading to a truncated but still functional protein despite the genetic mutation.
Mechanis ...
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.
AAV-mediated gene transfer of the full-length ''Mybpc3'' (defined as “gene replacement”) dose-dependently prevents the development of cardiac hypertrophy and dysfunction in homozygous ''Mybpc3''-targeted
knock-in In molecular cloning and biology, a gene knock-in (abbreviation: KI) refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found ...
mice.
The dose-dependent expression of exogenous ''Mybpc3'' was associated with the down-regulation of endogenous mutant ''Mybpc3''. Additional expression of a sarcomeric protein is expected to replace partially or completely the endogenous protein level in the sarcomere, as it has been shown in transgenic mice expressing sarcomeric proteins.
Notes
References
Further reading
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External links
Mass spectrometry characterization of MYBPC3 at COPaKB GeneReviews/NIH/NCBI/UW entry on Familial Hypertrophic Cardiomyopathy Overview*
{{PDB Gallery, geneid=4607
Human proteins