Dr#Pauline#Parisot#Centre#de#référence#M3C#

Malforma6ons#Cardiaques#Congénitales#Complexes#Université#Paris#V#–#AP@HP#

Hôpital#Necker@Enfants#Malades##

Heart#development#Implica6ons#for#cardiac#disease#

Master#de#Géné6que##Novembre#2012#

Congenital heart diseases 1% of live births 5000 children/year (France)

- Ventricular septal defects 30 %

- Conotruncal malformations 20%

- Various CHDs 50% Frequent among pediatric population

- 350 new cases of Cystic Fibrosis / year

15% of CHDs have a genetic disorder (≈ 750 cases/year)

Heart#anatomy#

In humans the heart is the first organ to develop and to function The 3rd to the 10th weeks are critical

heart#morphogenesis#

The heart is formed from mesoderm

•  Migration: bilateral clusters of progenitor cells that coalesce to form a cardiac crescent

•  the crescent fuses at the midline to form the primitive

cardiac tube which rapidly begins to pump blood

E7.5 –E8.25 : From cardiac crescent to linear heart

tube

•  Looping = first asymmetrical process

•  Looping to the right: D-loop

E 8.5: Cardiac Looping

LOOPING BALLOONING

Convergence Wedging Septation

Second Heart Field CCN

EC

After looping…

Dividing the heart tube - septation

•  Atrioventricular septation (atria from ventricles) –  AV cushions

•  Atrial septation (L & R atriums) –  1o & 2o interatrial septums

•  Ventricular septation (L & R ventricles) –  Interventricular septum

•  Outflow tract septation (pulmonary artery from aorta) –  OFT cushions

AV septation

Atrioventricular Septal Defect

•  Common AV junction (not separate L & R) •  Partial (ostium primum) type has separate L & R orificies

–  Shunt at atrial level only –  Can be asymptomatic

•  Complete (common AV canal) type has single orifice with common (bridging) valve leaflets –  Shunt at atrial and ventricular levels –  Often leads to heart failure, requires surgery, usually <1yr

•  Causes: common (~40%) in Down’s syndrome

Outflow tract septation

All components of the heart from the “horseshoe” primordium?

OFT morphogenesis

VE"

VD"VG"

Different#actors#

Myocardium#

Endocardium#

Cardiac##cushions###Cardiac#Neural#Crest#Cell##

E7.5 E8.5 E10.5

Cardiac crescent Second heart field

RV LV

RA LA OFT

A new paradigm in heart formation Second#Heart#Field#

Buckingham#et#al.#Nat$Rev$Genet$2005#

Second Heart Field

-  Gene expression : Fgf10 Kelly et al., Dev Cell (2001)

-  Fate mapping : Islet1 (Cre-lox) Cai et al., Dev cell (2003)

-  Clonal analyses Meilhac et al., Dev cell (2004)

Mlc1v.24$$(Fgf10)$

Additions to the “horseshoe”

•  “Anterior (or secondary) heart field” –  Outflow (& right ventricle?)

•  Dorsal mesocardium –  Spina vestibuli (atrial & AV septums)

•  Neural crest cells –  OFT separation (no material contribution) –  Nerves, wall of arterial trunks

•  Proepicardial organ –  Epicardium (visceral serous pericardium) –  Coronary arteries and veins

Cardiac#Neural#Crest#Cell#

Kirby#and#Waldo,$Circula;on$1990#

Pulmonary stenosis

Ventricular Septal

Right ventricular

hypertrophy

Overriding Aor

Clinical importance of the second heart field contribution in congenital heart defects

Di Felice and Zummo, Trends Cardiovasc Med 2009

Second heart field

First heart field

Normal heart Tetralogy of Fallot

PT Ao

IVS LV

RV

Pulmonary stenosis

Ventricular septal defect

Right ventricular

hypertrophy

Overriding aorta 1888

There are many types of OFT defect

How to classify congenital heart diseases?

Segmental classification

Pathogenetic Classification of Congenital Cardiovascular malformations (Clark 1996)

•  Ectomesenchymal tissue migration abnormalities –  Conotruncal septation defects

•  Abnormal intracardiac blood flow –  Hypoplastic left heart syndrome

•  Abnormal targeted growth –  Anomalous pulmonary venous return

•  Cell death abnormalities –  Muscular VSD

•  Extracellular matrix defects –  Atrioventricular septal defect

•  Abnormal situs and looping –  Heterotaxia

Concordance#according#to#pathogene6c#classifica6on#

Tetralogy**of*Fallot*

IAA* TOF+PA*

CAT*

Phenotype spectrum

Hypoplas6c#leY#heart#syndrome#######Aor6c#Coarcta6on##############################Bicuspid#Aor6c#valve#

Holoproencephaly

Solitary incisor tooth

New#classifica6on#:#ACC@CHD#1.#Heterotaxy#2.#Anomalies#of#the#venous#return#3.#Anomalies#of#the#atria#and#interatrial#communica6ons#4.#Anomalies#of#the#atrioventricular#junc6ons#and#valves#5.#Complex#anomalies#of#atrioventricular#connec6ons#6.#Func6onally#univentricular#hearts#7.#Ventricular#septal#defects##8.#Anomalies#of#the#ventricular#oualow#tracts#(ventriculo@arterial#connec6ons)#9.#Anomalies#of#the#extrapericardial#arterial#trunks#10.#Congenital#anomalies#of#the#coronary#arteries#

Houyel#et#al.$Orphanet$Journal$of$Rare$Diseases#2011,#

Different genes involved in CHDs with and without genetic syndromes

Syndrome#de#Di#George#@ #IAA,#TAC,#T4F,#APSO,#AVP,#CIV#par#malalignement,#VDDI#@ #anomalie#des#arcs,#aorte#à#droite#@ #microdélé6on#22q11.2#@ #gène#Tbx1$

CIV T4F APSO TAC VDDI

IAA AVP

Basson. PNAS 1999

HOLT-ORAM Syndrome ASD and limb defects

TBX 5

ASD + Cardiac conduction disorders

NKX 2.5

Noonan syndrome

PTPN11$gene$

Williams#–#Beuren#Syndrome#

Del#7q11.23#(elas6n#gene#dele6on)##

Genetic heterogeneity

1 CHD = several genes

ASD#:#TBX5,$NKX2.5,$GATA4,$MYH6$VSD#:#NKX2.5,$TBX5,$GATA4$AVSD#:#CRELD,$HEY2,$EVC,$EVC2$TOF+/@PA,#IAA#:#TBX1,$NKX2.5,$FOG2,$JAGGED1$CAT:$NKX2.6$TGA#:#CFC1,$PROSIT$240,$ZIC3$HLHS#:#NOTCH1$Pulmonary#stenosis#:$PTPN11$

Genetic heterogeneity 1 CHD = several genes#

Genetic heterogeneity among conotruncal CHDs

•  Del 22q11 TBX1 •  Alagille JAGGED1 •  Wardenburg PAX 3 •  Down syndrome •  CHARGE syndrome •  NKX2.5 •  Fog2 mutations •  GATA, etc…..

Genetic Heterogeneity of AVSD

Down syndrome Smith-Lemli-Opitz

Noonan syndrome Ellis van Creveld syndrome

8p23 del Looping anomalies

Genetic heterogeneity

•  Di George syndrome : TBX1 •  Oram syndrome: TBX5 •  Alagille syndrome: JAGGED1, HEY2 •  Noonan syndrome, LEOPARD : PTPN11 •  CHARGE syndrome : CHD7 •  Williams-Beuren syndrome : 7q1.123 •  Ellis van Creveld syndrome: EVC, EVC2 •  Smith-Lemli-Opitz syndrome : DHCR7 •  Neurofibromatosis : NF1 •  Rubinstein-Taybi syndrome : CREBBP, EP300 •  Wardenburg syndrome : PAX 3 •  Mowat-Wilson syndrome: ZFHX1B

Phenotype-Genotype Correlation

Trisomy 21 Normal karyotype Normal karyotype Cardiac isomerism

Different#phenotype#according#to#the#gene6c#disorder#

Non del 22q11 del 22q11

Phentoypic#severity#

Of mice and men…

Remodeling of the outflow tract

Bajolle et al., 2006

Mlc1v-24 (Fgf10) 96-16 T55

Transgenic markers of OFT myocardium at E10.5

OFT OFT OFT

OFT (and RV) inferior wall of the OFT superior wall of the OFT

The 96-16 cardiosensor line provides a marker of myocardium at the base of the pulmonary trunk

E9.5 E10.5 E10.5

E16.5 E15.5 E12.5

The β-galactosidase product of the 96-16 transgene provides evidence for rotation of OFT myocardium

E9.5 E10.5 E11.5 E12.5

Outflow tract remodeling

(1) neural crest is disturbed (Splotch - Pax3 mutants)

(2) left/right signalling is disturbed (Pitx2δc mutants)

*

Analysis of OFT rotation in mouse mutants with cardiac defects

Rotation of the outflow tract myocardium is affected in Splotch -/- hearts with PTA

E13.5

E11.5

E13.5

E11.5

Rotation of the outflow tract myocardium is affected in Splotch -/- hearts with DORV

E14.5 E14.5

96-16 expression in Pitx2δc -/- heart with PTA

E15.5 E15.5

96-16 expression in Pitx2δc -/- heart with TGA

E15.5 E15.5

PTA

OFT

PT

Ao

PTA

Tbx1 +/-

Tbx1 -/-

Tbx1

96-16 +/-

Di George syndrome del22q11.2

SHF regulator Subpulmonary myocardium misplaced and reduced

Persistant truncus arteriosus

Tbx1 +/-

TAC Tbx1 -/-

96-16

E18.5

Cx40-eGFP

Ao

TP

E14.5

96-16

E10.5

Tbx1 is a regulator of subpulmonary myocardium

Théveniau-Ruissy et al. Circ Res. 2008

Ao

TP

96-16

X 96-16

Sema3C : Tbx1-dependant expression in subpulmonary myocardium

E10.5

Ao

TP

Sema3C Sema3C

96-16

E13.5 E12.5

Tbx1 +/-

Sema3C

Tbx1 -/-

Sema3C

Semaphorin 3C is required for outflow tract septation and aortic arch artery remodeling

• Sema3C is a neurovascular guidance molecule and a cue for cNCC migration

• Sema3C acts through heterodimeric receptors Neuropilin1 or 2 and PlexinD1

Feiner et al. Development 2001

CAT + IAA type B CAT + IAA type C Sema 3C+/+

• Sema3C -/- mice display a common arterial trunk with interrupted aortic arch"

Sema 3C-/- Sema 3C-/-

Gitler et al. Dev Cell 2004

E10.5 E12.5

Tbx1

-/-

Sem

a3c

-/-

WT

E18.5

Comparative study of Sema3C and Tbx1 common trunks

T55 Coronary A Valves

Looking closely at OFT morphogenesis and outlet valve development in these two mouse models, we can observe important morphological differences that reflect the different upstream etiologies (SHF vs NCC)converging on common trunk.

Russell et al. J Thorac Cardiovasc Surg 2010

Recent classification for common arterial trunks based on the nature of the systemic pathway

Aortic dominance Pulmonary dominance

Genetics..what else?

Drugs: Alcohol, lithium, thalidomide, non steroidal anti-inflammatory drugs, Retinoic acid Maternal diseases : diabetes, Lupus, phenylketonuria, rubella… Induced diseases: right ventricular outflow tract obstruction due to twin to twin transfusion syndrome

Can genetics explain it all?

Building links between bedside and bench….

Pax3nLacZ/+! Pax3nLacZ/nLacZ!96-16! Mlc1v-24!

Coronary*vessel*development#

Vasculogenesis*Angiogenesis*Arteriogenesis*

E9# E12#

Wessels$and$Perez.Pomares$$2004$

Differencia?on*pathways*of*epicardial*derived*cells*

Aortic arch remodelling

The conduction system

Development of conduction tissue

•  Conduction tissue is specialized myocardium, not nerves

•  Conduction system differentiates by progressive localized recruitment from heart tube myocardium –  Local signals, e.g. endothelin & neuregulin from

endocardium & coronary arteries

•  Insulation –  AV by fibro-fatty layer (site of AV cushions) –  Ventricular bundle branches: fibrous sheath

Lecture Outline

•  Heart morphogenesis – Multiple sources of cells

•  Separating the chambers – Congenital defects

•  Conduction system