Download Sarcomeric Protein Mutations in Dilated Cardiomyopathy DCM

Sarcomeric Protein Mutations
in Dilated Cardiomyopathy
DCM
• A heart disease characterized by dilatation and
impaired contraction of the left ventricle or both
ventricles.
• A relatively common disease, affecting about 36.5 out
of 100,000 people, of which, 25–30% are familial
DCM.
• Autosomal dominant familial DCM accounts for
56% of all familial DCMs, and the causes of this
form of DCM is strongly associated with mutations in
proteins of the cardiac myocyte.
Mutations associated with DCM
• Identified in the proteins of the sarcomere,
cytoskeleton, and the sarcolemma.
• In most diseased hearts, only single mutations are
identified
• All mutations lead to the same disease phenotype
characteristic of DCM.
• A progressive process starting from the initial effects
of the mutations on contractility.
• The convergence of protein dysfunction signals to a
common pathway leading to the manifestation and
progression of DCM
Mutated proteins
• Mutations have been identified in β-myosin
heavy chain (β-MHC), myosin binding
protein-C (MyBP-C), actin, α- tropomyosin
(Tm), troponin T (TnT), troponin I (TnI),
troponin C (TnC), of the sarcomere; and titin,
T-cap, desmin, vinculin, and muscle LIM
protein (MLP) of the cytoskeleton.
DCM Associated Mutations Found
in MYH7
• Of the total number of DCM associated
mutations identified, the most prevalent are
mutations in the MYH7 gene, which encodes
for the β-myosin heavy chain (β-MHC) protein.
• All of the myosin mutations were discovered
through genetic screening studies of patients
who have been diagnosed with idiopathic
DCM.
Mutation types
• β-myosin heavy chain mutations associated
with DCM can be grouped into those which
are found in the S1, those which are not found
in the S1, and those which transition to DCM
from HCM.
Cardiac myosin
• In cardiac muscle, two MHCs and two each of
regulatory and essential light chains form the
complete myosin molecule.
• The structure of myosin is comprised of head, neck,
and tail regions.
• The myosin head, also known as myosin
subfragment 1 (S1), functions as the catalytic portion
of the myosin structure.
• The essential and regulatory light chains wrap around
the base of the head mediate myosin function.
The crystral structure of myosin head
Crystal structure of myosin-S1
• The relative
positions of the
DCM mutations are
found
in the myosin head.
• The seven mutations
which are associated
with DCM, and the
three which are
associated with
DCM that
transitions from
HCM, are shown.
Mutation sites
• Myosin mutations listed in Table 1, 10 in the S1
region of myosin, and 6 in the tail region.
• The mutations which localize to the head motor
domain could affect the actin-myosin interaction,
thereby causing contractile dysfunction.
• The mutations found in the tail region, occur in
highly conserved residues, the substitution of which,
may affect the structure of the myosin molecule.
Ser532Pro
• Found in the highly conserved α-helical
structure of the 50 kDa domain in myosin,
which contributes to the tight binding of actin.
• The proline for serine substitution introduces a
kink in the α-helix structure, possibly
disrupting stereospecific interactions between
myosin and actin.
Phe764Leu
• Found in the center of the region known as the
converter region, which is responsible for the
transmittal of movement from the myosin head
to the neck.
Ala223Thr and Ser642Leu
• Ala223Thr mutation causes a charge change in
a location near the ATP binding site of myosin.
• Ser642Leu mutation is located in the actinmyosin interaction region.
• The effects of these mutations are speculative,
since functional studies of the mutations have
yet to be reported.
Mutations at myosin tail
• Two mutations which are found in the myosin
tail, Arg1053Gln and Arg1500Trp, are both
weakly associated with DCM.
• Arg1053Gln is associated with DCM which
had transitioned from HCM, and the
Arg1500Trp mutation has no evidence for
familial heritage.
Cardiac Myosin Binding Protein C
• cMyBP-C: a protein associated with the thick
filament, localizes to the cross-bridge containing
C zones of the sarcomere, through interactions
with specific regions of titin and myosin.
• Contains several phosphorylation sites, play a role
in regulating force generation
• Not essential for cardiac development and
function, but involved in determining efficiency of
muscle contraction.
Electron micrograph of cardiac muscle
stained with antibody to MyBP-C
Diagram of the cDNA encoding cardiac MyBPC with the binding sites for myosin and titin
Mutations
• cMyBP-C is second only to myosin, in the
number of HCM associated mutations.
• Mutations result in the expression of truncated
forms of cMyBP-C.
• Only three mutations in cMyBP-C associated
with DCM, Asn948Thr, Arg820Gln, and a
nonsense mutation Gln1012X.
DCM associated mutations found in actin
• Glu361Gly and Arg312His.
• Any alteration to its amino acid sequence may
have profound effects on the structure and its
interaction with myosin, Tm, and possibly
even itself (actin polymerization).
G-actin showing the location of the two DCM
associated mutations.
DCM Associated Mutations in
Tropomyosin
• Through Ca2+ dependent movement
transmitted via the Tn complex, Tm sterically
blocks or opens the myosin binding sites on
actin. This regulatory mechanism is essential
for muscle contraction and relaxation.
• Only three known DCM associated mutations
in Tm, Glu40Lys, Glu54Lys, and Glu180Val.
DCM associated mutations in the
regulatory proteins of the thin filament.
Tn complex: TnT, TnI, and TnC.
• TnT has important structural and regulatory roles in
the thin filament, as the arm holding the Tn complex
to Tm. Through its N-terminal end, TnT strongly
binds to Tm, while the C-terminal end interacts with
TnI and TnC.
• TnI is the inhibitor of actin and myosin interaction.
The binding of actin in low Ca2+ and the alternative
binding of TnC in the presence of high Ca2+, by the
inhibitory region of TnI, provides a Ca2+ sensitive
mechanism for inhibition of muscle
• TnC is the main Ca2+ buffer in the sarcomere.
Calcium switch
• As intracellular Ca2+ concentration increases, the
binding of Ca2+ to the regulatory site of TnC causes
a conformational change in TnC and TnI.
• TnC’s affinity for TnI increases, while TnI’s affinity
for actin decreases.
• The decrease in TnI interaction with actin, caused by
Ca2+ binding to TnC, and the subsequent
conformational changes in the Tn subunits, allow
movement of Tm along the actin filament.
• The movement of Tm facilitates the interaction
between myosin and actin, leading to muscle
contraction.
TnT activates muscle contraction
• In the presence of TnI and TnC, there is a
Ca2+ dependent activation of actomyosin
ATPase rates, which results from the direct
interaction of TnT with TnC.
• The direct interaction of TnT with TnC in
mediating activation, suggests a dual role for
Ca2+ binding to TnC, the release of the
ATPase inhibition by TnI, and the activation of
the ATPase through interaction with TnT
DCM Associated Mutations in Tn
• DCM related mutations have been found in all three
subunits of Tn.
• All display functional characteristics distinct from
those caused by HCM associated mutations.
• In contrast to the mutations associated with familial
HCM, the DCM associated mutations are all
presented with slightly decreased Ca2+ sensitivities
and decreased actomyosin ATPase rates.
DCM associated mutations in Troponin
TnT mutations I
• Two mutations, Arg92Trp and Lys273Glu,
start off as HCM.
• The arginine to tryptophan mutation in
position 92, occurs in the Tm binding region of
TnT.
• The lysine to glutamic acid mutation in
position 273, associated with the transition
from HCM, results in a charge change in an
important binding site for TnI or TnC.
TnT mutations II
• Six mutations occur in the C-terminal half
of TnT, where it interacts with TnI and TnC,
Arg131Trp, Arg141Trp, Ala171Ser,
Arg205Leu, Lys210del, and Asp270Asn.
TnI mutation
• The mutation in TnI, Ala2Val, found in the
TNNI3 gene, is associated with recessive
DCM.
• Significant impairment of TnI and TnT
interaction when compared with wild-type
control.
TnC mutations
• The TnC mutation associated with DCM,
Gly159Gln, is localized in a domain
constitutively occupied by Ca2+ .
• A mammalian 2-hybrid assay demonstrated an
impaired interaction between TnT and TnC
when compared with wild-type.
Titin
• Known as connectin
• A giant muscle protein expressed in cardiac
and skeletal muscles.
• Referred to as the third myofilament of the
cardiac muscle sarcomere
• Spans the entire half of the sarcomere from Zline to M-line, and two titins form a continuous
myofilament along the whole length of the
sarcomere by overlapping at the M-line.
Structure and arrangement of titin
(connectin) in the muscle sarcomere
DCM Associated Mutations in Titin
DCM Associated Mutations in NonContractile Proteins
• MLP is a member of the cysteine-rich protein family,
whose function is implicated in muscle differentiation
and sarcomere assembly, through its interactions with
zyxin and α-actinin.
• Mice with deficiencies in MLP are reported to exhibit
chamber dilation and contractile dysfunction,
characteristic of DCM
• W4R in MLP causes defects in its interaction with
telethonin cap(T-cap)
• Gln9Arg and Lys69Arg affect its interaction withαactinin-2.
DCM Associated Mutations in T-cap
• T-cap is a 19 kD protein, whose role as a titin
binding protein at the Z-disc is required for
normal sarcomere development
• The interaction betweenT-cap and titin
requires the presence of MLP.
• T-cap R87Q, E132Q mutations cause an
impairment of the MLP binding affinity.
DCM Associated Mutations in Desmin
• Desmin is a cytoskeletal protein which forms muscle specific
intermediate filaments, connecting the nuclear and plasma
membranes of cardiomyocytes.
• Desmin is involved in the regulation of intermediate filament
organization and structure during the cell cycle, through the
post-translational modifications ofphosphorylation and
glycosylation, in its tail domain.
• It is found at the Z-lines and intercalated disks, and because of
the connections it forms between the nuclear and plasma
membranes, is believed to also play a role the stabilization of
the sarcomere.
• Ile451Met mutation identified sits in the carboxyl end of
human desmin
DCM associated mutations in
metavinculin
• Vinculin isoform larger than by 68 amino acids
• Expressed only in cardiac and smooth muscle
• In cardiac myocytes, localizes to intercalated disks and
costameres with vinculin.
• The head portion interacts with membrane-associated ligand
proteins, while the other end interacts with actin filaments.
• Have a unique role of anchoring actin filaments to intercalated
discs in the heart.
• Three mutations linked to DCM, Ala934Val, Arg975Trp, and
• a Leu954del.