The Role of Structural Proteins in Normal and Abnormal Heart Development

Lara Bowell, Elias Diamantis, Jessica Gates and Lorna Neill

Aims:•Define a Structural Protein•Cardiomyopathy•Structural Proteins:

▫MYH6▫MYH7▫ACTC1▫TNNI3▫MYBP-C

•Summary

What is a structural Protein?•A protein involved in maintaining the

cytoskeleton, found all over the body.

•Sarcomeric proteins are a specific subset of structural proteins involved in heart contractility.

•Important in the heart as they ensure the

organised structure and maintain contractility.

Cardiomyopathy-‘Disease of the Sarcomere’•Geisterfer-Lowrance et al (1990), almost

all sarcomeric proteins are implicated in cardiomyopathy including: MYH7, MYH6, ACTC1, TNNI3, TNNT2, MYBP-C, MYL3, MYL2, TPM1

•Cardiomyopathy is a weakening of heart muscle.▫4 different types: Hypertrophic, Dilated,

Restrictive and Non-Compaction

Defects in structural proteins•Cardiomyopathy – Obviously!

•Congenital Heart Defects?

Myosin Heavy Chain Proteins

CARDIAC MYOSIN STRUCTURE•The heavy chains occur as two isoforms: •MYH6 alpha-MHC (fast)•MYH7 beta-MHC (slow)

The MYH7 gene is about 4 kilobases downstream from MYH6.

The same job…. Why have 2?

Normal development

Linear tube stage MYH6 mRNA - strongly biased towards the inflow tract.MYH7 mRNA - more homogenously expressed

Subsequent development Atria: 84.7% MYH6Ventricles: 99.3% MYH7

Somi et al 2006 (using the equivalents in chicks: Atrial-MHC and Ventricular-MHC)

AMHC(MYH6)

VMHC(MYH7)

HH30

HH30

So what’s the difference?Narolska et al (2004)•Samples from atria obtained

during heart bypass surgery

•Samples from ventricles obtained from donor hearts which could not be used for technical reasons

•Ratios of MYH7:MYH6 in ventricles and atria verified by electrophoresis

•Ventricles: 99.3% MYH7

•Atria: 84.7% MYH6

•ATPase activity (energy consumption) is coupled with NADH oxidation. The NAD+:NADH ratio can be measured photometrically.

“Tension cost” in ventricles is much lower; much more efficient than atria.

Maximum rate (speed) of force development of atria is much higher; more responsive than ventricles.This leads to the required efficiency

of the ventricles and required responsiveness of the atria.

Myosin Heavy Chain 6 - MYH6

ABNORMAL DEVELOPMENT•MYH6 has been linked to these specific types of cardiomyopathy:

Cardiomyopathy familial hypertrophic type 14 (CMH14)Cardiomyopathy dilated type 1EE (CMD1EE)

•However more recently, and more interestingly, it has now been linked to Dominantly inherited Atrial Septal Defects (ASD3 or Ostium Secundum)

•Also mutations in transcription factors (TBX5 and NKX2-5) disrupt regulation of MYH6, leading to other cardiac defects.

Ching et al (2005)• Study looks at a large family with dominantly inherited ASD and no other cardiac abnormalities.

• Screened the 39 exons of MYH6 in all members of the family identified a single nucleotide change, a I8429T A transversion

• Missence substitution I820N in the MYH6 neck region at the start of the QI domain.

• Destabilised structure.

•Reduced binding affinity and reduced ability to pull down the regulatory light chains.

• MYH6 knock-down using Morpholinos.

• MYH is expressed in the chick atrium from(stage 9) and knock-down was induced at (stage 13/14).

• Of the seven “knock-down” chickembryo’s:

four = no evidence of septum formation three = septum formation initiated but not complete

•No obvious other defects were seen.

NORMAL

Untreated “Wild Type” chick heart

One of the 3 embryo’s

where septation initiated – stump like protuberan

ce

MYH6 mismatch

morpholinos

No evidence of atrial

septation.

Rutland, et al. (2009)• The role of alpha MYH during early cardiogenesis.• Morpholino-induced knockdown of alpha MYH in chicks. • Three different phenotypes of the atrial septum which

were observed:

1. The atrial septum failed to initiate2. The septum was initiated but was growth restricted3. Incorrect specification occurred resulting in

multiple septa forming.

• Abnormally looped heart or an enlarged heart when there was a lower frequency of alpha MYH

• Actin filaments often appeared immature.

Summary of Phenotype Observed after alpha MYH Knockdown:

Granados-Riveron. J.T, et al (2010)•Analysed MYH6 mutations in 470 cases of isolated CHD, and detected one non-sense mutation, one splicing site mutation and seven non-synonymous coding mutations.

•Used plasmids to introduce mutant and non-mutant green fluorescent protein.

•MYH6 fusion proteins in mice myoblasts revealed that mutations in:

A230P and A1366D = significantly disrupt myofibril formationH252Q =significantly enhances myofibril assembly.

•Indicates functional variants of MYH6 are associated with cardiac malformations.

Myosin Heavy Chain 7 – MYH7

MYH7 - Cardiomyopathy mutations

• 194 hypertrophic cardiomyopathy mutations • 13 dilated cardiomyopathy mutations • 7 other mutations • 7 variants of uncertain effect • 15 polymorphisms

• These links are long-established and quite easy to understand: a defect in the protein structure will effect sarcomeric function and therefore muscle contraction.

MYH7 – Congenital Heart Defects•Budde et al 2007 - potential links

between MYH7 mutations and a range of defects.

•Postma et al 2010 - screened 141 patients with Ebstein’s anomaly for MYH7 mutations

Ebstein’s anomaly is a rare displacement of the tricuspid valve apically (“downwards”)

Tricuspid regurgitation

Type II (secundum) ASD present in 1/3 of patients

Postma et al 2010• 141 patients with Ebstein’s anomaly

• 8 were found to have MYH7 mutations: seven missense and one 3b.p deletion.

• Indicates link as both Ebstein’s anomaly and MYH7 are rare.

(NONE of the mutation free patients had left ventricular non-compaction)

Alpha Cardiac Actin 1– ACTC1

ACTC1 -normal development Actin polymerization has a role in cardiac looping by

causing tension and enabling dextro-looping, especially in C looping (Itasaki et al. 1991 and Latacha et al 2005)

Heart looping is divided into 2 stages!

S- looping: Atrium moves superior to the ventricles

C- looping:Ventral bending and dextral (rightwards) rotation of the heart tube

Männer 2009

Männer 2009

Mutations in ACTC1 lead to abnormal development

•E101K mutation in ACTC gene are associated with dilated and apical hypertrophic cardiomyopathy (Olsen et al 2000, Arad et al 2005)

•Missense mutation (Glu101Lys) where lysine is coded for instead of glutamine at position 101

E101K mutation present in patients with left ventricular noncompaction

but also.....

In 4 out of 94 families, septal defects (8 ASD, 1 VSD) present in E101K carriers, but absent in relatives without mutation

Monserrat et al 2007

ACTC1 mutation - ASD without signs of cardiomyopathy•2 large Swedish families studied with large incidence of ASD•Missense mutation M123V (methioine for valine) found in all affected individuals

Matsoon et al 2008

In vitro tests... Mutant M123V actin

Does not affect polymerization ability

Reduced binding affinity for myosin

Does not affect the velocity at which the

actin is moved by cardiac myosin

M123V mutation in subdomain 1, which contains binding sites, but

mutation is deep, so polymerization not affected

Mutation causes change in shape/flexibility of subunit =

reduced affinity

No effect on actomyosin motor properties of actin, so no

cardiomyopathy

Hypothesis

17-base pair deletion•One patient with ostium secundum defect found with heterozygous 17 b-p deletion in ACTC1•Patient’s father had deletion on one allele and had deviated intraventricular septum - linked to spontaneously closed VSD

Matsoon et al 2008

Hypothesis... Deletion will cause ACTC1 to be truncated and therefore non functional

Morpholino knockdown of chick embryos lead to reduced atrial septal size and also delayed s looping of the heart

Reduced atrial septal size...Lack of ACTC1 induces apoptosis of septum primum (Jiang et al 2010)....

.....Excessive apoptosis is known to cause ostium secundum defect!

Jiang et al found lack of ACTC1 expression in mRNA of patients with ASD, VSD and TOF.

Actin polymerization

known to be important in c

looping

Reduced/impaired levels of

ACTC

Affect c looping

Disrupts actin

polymerisation

Delay s looping

Delayed s looping...

Knockdown of ACTC1 leads to reduced septation and delayed cardiac looping in the chick

Wild type

Wild type

MO treated

MO treated

Here the arrows show the reduced septal size in the treated embryos (C&D)

Here the dotted line shows a difference in the relation between the atrium (a) and the truncus artiosus (ta), indicative of a looping problem

Troponin I - cTnI

Troponin •Troponin and Tropomyosin regulates the

binding of myosin to actin.•Troponin is a trimeric protein composed

of I, C and T subunits.•Troponin I binds to actin•Troponin C binds Ca2+

•Troponin T binds to tropomyosin

http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/structural-proteins/troponin.html

Troponin I•There are 3 forms of Troponin I:

▫cTnI from TNNI3 – ch 19q13.42

•cTnI can has inhibitory abilities – can inhibit the actomyosin ATPase activity independently of the other subunits.

Case studies•Yang et al (2010)

▫Case of an infant, with a perimembranous VSD at birth – heterozygous missense mutation in TNNI3 gene (R204H)

▫Developed cardiomyopathy by 18 months – mutation in TNNT2 gene

Myosin Binding Protein C – MYBP-C

MYBP-C – Myosin Binding Protein C•Binds to titin and myosin heavy chain,

role in structural integrity of the sarcomere and regulates contractility.

•Conclusively leads to 20-30% of all HCM when there are heterozygous mutations.

•Associated with late onset, mild phenotype and a good prognosis before 40.

However...• In Ohio, it was noted that there were very high

incidences of cardiovascular problems in neonates and a high incidence of death in the Amish communities.

• Xin B. et al 2007, studied 20 Amish neonates with cardiomegaly and cardiomyopathy. Some had small muscular VSDs.

• Zahka et al 2008, studied 10 Amish neonates who presented with a mixture of symptoms, including LVH, murmurs, small muscular VSDs

•Found a splice site mutation in intron 30 (c3330 + 2T>G) in all affected children. This mutation skips the 140bp exon 30, leading to a frame shift and a premature stop codon.

•Both these studies found that unless the children had heart transplants within the first year of life, they would die.

Future?•So far, no conclusive evidence linking

MYBP-C to VSDs.•However many of these patients had

VSDs with this defect.•Either way this mutation causes a defect

in the heart which causes very premature death of the neonate.

SummaryProtein Structural DefectMYH6 ASD

MYH7 Subtype of Ebstein’s Anomaly

ACTC1 Ostium Secundum ASD

TNNI3 Perimembranous VSD

MYBP-C Neonatal cardiomyopathy

Further Reading• MYBP-C

▫ Zahka et al 2008 Homozygous mutation of MYBPC3 associated with severe infantile hypertrophic cardiomyopathy at high frequency among the Amish. Heart 94 1326-1330

▫ Xin et al 2007 Homozygosity for a Novel Splice Site Mutation in the Cardiac Myosin-Binding Protein C Gene Causes Severe Neonatal Hypertrophic Cardiomyopathy. American Journal of Medical Genetics Part A 143A 2662-2667

▫ Deprez et al 2006 Two cases of severe neonatal hypertrophic cardiomyopathy caused by compound heterozygous mutations in the MYBPC3 gene. Journal of Medical Genetics 43 829-832

TNNI3▫ Troponin I 2009 http://www.scitopics.com/Troponin_I.html▫ Yang et al 2010. Ventricular septal defect and restrictive cardiomyopathy

in a paediatric TNNI3 mutation carrier. 20(5) 574-576▫ Roles of Troponin in heart development and cardiac function.

http://gradworks.umi.com/33/38/3338722.html▫ Sugimoto et al 2010 Pulmonary Hypertension-induced Myocardial

Injury: Evaluation Using A Highly Sensitive Cardiac Troponin-I Assay In Children With Congenital Heart Disease. Circulation. 122.A16382

• MYH6▫ Ching . Y.H, et al. Mutation in myosin heavy chain 6 causes atrial septal

defect. nature genetics. 37, pp. 423-428▫ Rutland. C, et al. (2009) Knockdown of alpha myosin heavy chain disrupts

the cytoskeleton and leads to multiple defects during chick cardiogenesis. Journal of Anatomy. 214(6): pp.905-915.

▫ Granados-Riveron. J.T, et al (2010). α-Cardiac myosin heavy chain (MYH6) mutations affecting myofibril formation are associated with congenital heart defects. Human Molecular Genetics.

• MYH7▫ Somi, et al, Atrial and Ventricular Myosin Heavy-chain Expression in the

Developing Chicken Heart: Strengths and Limitations of Non-radioactive In Situ Hybridization, Journal of Histochemistry and Cytochemistry, 54(6):649-64, 2006

▫ Narolska et al, Myocardial contraction is 5-fold more economical in ventricular than in atrial human tissue. Cardiovasc Res 65: 221–229, 2005 

▫ Budde, et al, Noncompaction of the Ventricular Myocardium Is Associated with a De Novo Mutation in the b-Myosin Heavy Chain Gene. PLoS ONE 2(12): e1362. doi:10.1371/journal.pone.0001362, 2007

▫ Postma, et al, Mutations in the Sarcomere Protein Gene MYH7 in Ebstein´s Anomaly, Published online before print December 2, 2010,doi: 10.1161/CIRCGENETICS.110.95798

• ACTC1▫ Männer, J. The anatomy of cardiac looping: a step toward the understanding of the

morphogenesis of several forms of congenital cardiac malformations. Clinical Antatomy. January 2009. 22: 21-25.

▫ Itasaki, N et al. Actin bundles on the right side in the caudal part of the heart tube play a role in dextro-looping in the embryonic chick heart. Anatomy and Embryology. 1991. 183: 2 9-39

▫ Latacha, K.S et al. Role of actin polymerization in bending of the early heart tube. Developmental Dynamics. August 2005. 233: 1272-86

▫ Olsen, T.T, Doan, T.P, Kishimoto, N.Y, Whitby, F.G, Ackerman, M.J, and Fananapazir, L. Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy. Journal of Molecular Cell Cardiology. September 2000. 32; 1687-1694

▫ Arad M, et al. Gene Mutations in apical hypertrophic cardiomyopathy. Circulation. November 2005. 112: 2805-11.

▫ Monserrat, L et al. Mutations in the alpha-cardiac actin gene associated with apical hypertrophic cardiomyopathy, left ventricular non-compaction, and septal defects. European Heart Journal. August 2007. 16: 1953-61.

▫ Matsson, H et al. Alpha cardiac actin mutations produce atrial septal defects. Human Molecular Genetics. January 2008. 17: 256-65

▫ Jiang, HK et al. Reduced ACTC1 expression might play a role in the onset of congenital heart disease by inducing cardiomyocyte apoptosis. Circulation Journal Japan. November 2010. 74(11): 2410-8

QUESTIONS

•?

Cardiomyopathy Familial Hypertrophic type 14• Asymmetric Ventricular hypertrophy often

involving the interventricular septum.• Symptoms include: dyspnea, syncope,

collapse, palpitations and chest pain. • Provoked by exercise.• Inter- and intrafamilial variability from benign to malignant • High risk of cardiac failure and suddencardiac death

Genetics• Transcription Factor (TF) GATA-4 forms a complex with

(TF) TBX5, which interacts with and regulates gene expression of MYH6. Mutant TBX5 doesn’t regulate MYH6 expression.

• TBX5 is important in heart and upper limb development.• Defects on TBX5 leads to Holt-Oram Syndrome, which

induces electrical conduction defects and abnormalities in the upper limb.

• Homeobox NKX2-5 also interacts with MYH6, and is also associated with defects in electrical conduction of the heart.

• Mutations of these transcription factors are associated with both ASD and VSD.

Physical Interaction between TBX5 and MEF2C Is Required for Early Heart Development (Ghosh.T.K, et al 2009)

Atrial Septal Defect 3• Incomplete closure of the wall

between the atria• Blood flow from the left to the

right atria• Abnormal mixing of

oxygenated and deoxygenated blood.

• Variable phenotypic effects depending on the size of the atrial septal shunt.

• Breathlessness, fatigue, poor growth, palpitations..

• Treatment: Small defects may close on their own after birth, larger defects may need surgery.

Cardiomyopathy dilated type 1EE• Ventricular dilation and impaired systolic

function• Congestive heart failure and arrhythmia• Patients are at risk of premature death