Sarcomeric Protein Mutations in Dilated Cardiomyopathy

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Sarcomeric Protein Mutations in Dilated
Cardiomyopathy
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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, 2530 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.

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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

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Mutated proteins

• Mutations have been identified in ß-myosin heavy
  chain (ß-MHC), myosin binding protein-C (MyBP-C),
  actin, a- 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.

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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 ß-myosin
  heavy chain (ß-MHC) protein.
• All of the myosin mutations were discovered
  through genetic screening studies of patients who
  have been diagnosed with idiopathic DCM.

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Mutation types

• ß-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.

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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.

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The crystral structure of myosin head
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Crystal structure of myosin-S1

• The relative positions of the DCM mutations are
  foundin the myosin head.
• The seven mutations which are associated with
  DCM, and the three which are associated with DCM
  thattransitions from HCM, are shown.

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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.

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Ser532Pro

• Found in the highly conserved a-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 a-helix structure, possibly
  disrupting stereospecific interactions between
  myosin and actin.

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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.

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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 actin-myosin
  interaction region.
• The effects of these mutations are speculative,
  since functional studies of the mutations have
  yet to be reported.

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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.

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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.

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Electron micrograph of cardiac muscle stained
with antibody to MyBP-C
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Diagram of the cDNA encoding cardiac MyBP-C with
the binding sites for myosin and titin
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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.

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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).

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G-actin showing the location of the two DCM
associated mutations.
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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.

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DCM associated mutations in the regulatory
proteins of the thin filament.
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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.

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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.
• TnCs affinity for TnI increases, while TnIs
  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.

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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

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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.

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DCM associated mutations in Troponin
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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.

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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.

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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.

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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.

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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
  Z-line to M-line, and two titins form a
  continuous myofilament along the whole length of
  the sarcomere by overlapping at the M-line.

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Structure and arrangement of titin (connectin) in
the muscle sarcomere
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DCM Associated Mutations in Titin
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DCM Associated Mutations in Non-Contractile
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 a-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
  witha-actinin-2.

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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.

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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

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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.