Download Accessory pathways during normal and abnormal cardiac

Accessory pathways during normal and abnormal
cardiac development: relevance for WPW syndrome
Nico A. Blom
Leiden University Medical Center, Leiden, The Netherlands
Physiology and experimental medicine symposium: cardiovascular performance
in fetal and neonatal disease
Hospital for Sick Children, Toronto, May 19, 2009
Development of the cardiac conduction
system: relation with clinical arrhythmias
•
Research project of the departments of Anatomy and Embryology,
Cardiology and Pediatric Cardiology of the LUMC
•
Focus: understanding of developmental background of clinical
arrhythmias and conduction abnormalities
•
Morphological studies and embryonic EP studies (since 2006)
•
Ectopic atrial tachycardias, “internodal pathways”(Circ 1999, JCE 2003),
Mahaim tachycardia (Circ Res 2005), AVN in CHD (Ped Res 2005),
Sinus node disease (Circ 2007, Dev Dyn 2009), WPW syndrome (Circ
2007, Circ 2008, Circ 2008)
1. Introduction on cardiac development
2. Embryonic electrophysiology/
development of the CCS
3. Accessory pathways during normal
and abnormal heart development
Cardiac Development
• Primary heart tube >
mature heart
• Embryonic “base to apex”
> mature “ apex to base”
impulse propagation
Arterial pole
• Development of the
specialized conduction
system (CCS) and
formation of the annulus
fibrosis (AF)
De Jong et al, 1992; Chuck et al, 1997
Venous pole
Development of the primary cardiac tube:
contribution of the first heart field
2nd
arterial pole
1st
venous pole
Human 23 days; mouse E 8.5
Looping and addition of the
second heart field
SV
AoS
SAR
LA
2nd
RA
VAR
AoS
AVR
RV
“LV”
PR
human 25 days;mouse E11.5
Differential gene and protein
expression in the SHF
Tbx1
Mef 2c
Tbx2
Isl1
Tbx3
Id2
Tbx5
Nkx2.5
Tbx18
HCN4
Shox2
Podoplanin
Migrating neural crest cells and epicardium
derived cells (EPDCs):
Embryonic electrophysiology
pacemaking and conduction
Pacemaker activity
- First activity at inflow of tubular heart
Activation/contraction pattern
- Tubular heart: slow and peristaltic
(ICa2+)
- Looped heart: sequential atrial and
ventricular activation (areas of
slow/fast conduction)
DeJong et al. 1992
Surface ECG in tubular/looping
heart
“Mature” ECG at HH16 despite absences AVN & HPS
12
13
14+
15
QRS
14
16
p
Embryonic electrophysiology
“Mature heart” (chick HH>35, mouse >13.5 days):
⇒ rapid atrial and ventricular activation (INa+)
⇒ development of compact AV-node
⇒ maturation of His-Purkinje system
⇒ Coincides with ventricular septation
Change in ventricular activation pattern:
- base to apex (chicken <HH 33-35, mouse <13.5 days)
- apex to base ( chick >HH35, mouse >13.5 days)
Chuck et al. 1997
Development of the specialized
CCS
What is the origin of cells of the CCS ?
- myocardial heart tube (first heart field)?
- second heart field ?
- non-myocardial cell populations (NC-cells) ?
⇒ Retroviral lineage tracing: myocardial origin of CCS
Visualization of the developing
CCS
• Antigen expression
- HNK-1/Leu 7
- Id2
- NCAM (anti-PSA)
- etc..
• Transgenic animals
- CCS-LacZ reporter mouse
- MinK-LacZ reporter mouse
- etc……..
The developing CCS
7 weeks
SS
SAN
LBB
LVV
RVV
His
RBB
RAVR
CS
SAN,“internodal pathways”, right AV ring bundle
AVN, His and bundle branches
Blom et al. 1999
Accessory AV pathways during normal
and abnormal cardiac development
relevance for the pathogenesis of WPW
syndrome
Wolff-Parkinson-White syndrome
• Incidence 0.15% (0.07 % in children)
• Accessory atrioventricular pathway (AP):
substrate for ventricular preexcitation and
atrioventricular reentrant tachycardia
• Concealed WPW (retrograde conduction
only): 25% adults, 75% of the neonates
• Majority no familial involvement,
• Pathogenesis unknown
• Minority of cases are inherited as single
gene disorder: PRKAG2 gene
mutation,others
Öhnell 1943
Accessory pathways in the fetus
and neonate
•
Supraventricular tachycardias relatively common in fetuses
and neonates
• >90% are AVRT
• Fetal/neonatal AVRT can be life-threatening (fetal hydrops,
severe forward failure)
• Two-third remain free of symptoms without medication after
one year
Accessory pathways during cardiac
development
Formation of the annulus fibrosis:
- Contribution of endocardial atrioventricular cushions on the luminal
side and inward migration of AV sulcus tissue
- Bone morphogenetic protein (BMP) signaling, Epicardium derived
cells (EPDCs), Periostin
Summary of three studies
1. Normal human heart development: (immuno) histochemical studies
2. Normal quail and mouse heart development: electrophysiological
experiments, immunohistochemical studies
3. Effect of EPDC inhibition in quail hearts: electrophysiological
experiments ,immunohistochemical studies
Human embryos/fetuses
•44 human hearts: 4 weeks of development – 2 days postnatally
•Myocardial markers (HHF-35, MLC2a), Fibrous tissue markers
(fibronectin, collagen VI, periostin)
•4-6 weeks: All myocardium continuous at primitive AV canal
•6-10 weeks: Mainly broad myocardial continuities (post-septated
hearts)
7,1 weeks
LA LA
35% 20
VS
LV
RV
RV
45%
%
VS
LV
MLC-2a
Hahurij, Circulation 2009
Results
10-20 weeks of development:
¾ Most accessory connections comprise a single strand of
myocardium crossing the annulus fibrosis
16 weeks
RA
RA
AO
RV
67% 16
RV
rAVR
17%
%
RF+
20 weeks-birth-2 days postnatal:
¾ No accessory connections were found
Hahurij, Circulation 2009
Accessory connections related to the AVN
14,2 weeks
RA
HIS
*
LBB
LA
TV
MV
VS
RV
AVN
LV
VS
RF+
Hahurij, Circulation 2009
¾ accessory AV connections
with nodal phenotype?
Conclusion (1)
• AF isolation is an ongoing process after
the first trimester and APs remain
present in normal human fetuses until
late fetal stages
• Clinical relevance?
• Conducting properties?
Accessory pathways in normal
embryonic quail and mouse hearts
RA
™ Temperature 35,5 – 37 0C LVB
™ Perfusion with oxygenated
RVB
Tyrode solution
LVA
™EP recordings in normal quail (n= 80) and mouse embryos (n=42) during SR
and RA pacing
™Mouse embryos: 13.5 –birth (postseptation early and late)
™Quail embryos: preseptational (HH 30-34) and postseptational (HH 35-44)
Kolditz, Circulation 2007, Hahurij 2009
Methods
Mean HR, AV conduction times
Ventricular activation sequence
• “base-to-apex”
(base ≥ 1 ms earlier than apex)
• “apex-to-base”
(base ≥ 1 ms earlier than apex)
• “concurrent”
(base and apex <1ms)
•Immunohistochemistry: α-MLC-2a α-Periostin, α-Cx43, αNkx2.5
•Location and size of APs at the developing Annulus Fibrosis
LV base to apex activation
Pre-septational heart
HH 31 quail heart; base-to-apex LV-activation
2 μσ
200 mm/s
Sinus Rhythm
1600 mm/s
Apex to base activation pattern
(post-septated heart)
ΗΗ 39 quail heart; apex-to-base LV-activation
LA
LVB
9 μσ
LVA
200 mm/s
RA pacing 120/min
1600 mm/s
Example of premature base activation in a
late post-septational heart
HRA pacing
HH 39; LV-apex-to-base, RVB first
Ω
atrial capture
8 ms
1 ms
Quails
Electrophysiological recordings
Apex-first
Group A
HH 30-44
Group B
HH 35-44
Base-first
Concurrent
RVB-first
LVB-first
7 % (n = 1)
20 % (n =
3)
40 % (n =
6)
33 % (n =
5)
49% (n =
32)
17% (n =
11)
8 % (n = 5)
26% (n =
17)
51% (n = 33) of post-septated hearts
shows premature base activation !
Quail heart
Right posteroseptal
AV-ring (HH 38)
ibr
osi
s
Immunohistochemistry – MLC2a
an
n
ulu
sf
RA
RA
LV
RV
MLC2a
4x
s
u
l
nu
n
a
sis
o
r
fib
RV
AV myocardial continuity
MLC2a
40x
Quail hearts
Immunohistochemistry – MLC2a & periostin
MLC2a
periostin
RA
LV
+
RV
4x
periostin
myocardial cell
fibroblast cell
Mouse hearts
MLCMorphological analysis
13,5dpc
RA
RV
LA
LV
MLC-2a
Nkx2.5 & periostin
LA
LV
MLC-2a
periostin
Mouse hearts
Morphological analysis
Mean AP width (μm)
Early post-septated
Late post-septated
early vs late
Total
347
214
P=0,035*
Left side
238
138
P=0,000*
Right side
109
76
P=ns
Hahurij et al 2009
Conclusions (2)
• Transient APs are present during
normal avian and mouse heart
development and have antegrade
conducting properties
• Periostin may have an inductive role in
formation of fibrous tissue of the AF
Formation of the annulus fibrosis:
Role of Epicardium Derived Cells (EPDCs)
-EPDCs: outgrowth of cells from the
proepicardial organ (PEO) to the naked heart
and form the epicardium (HH >16)
- Migration to myocardium and
subendocardium
-Epithelial-to-Mesenchymal
Transformation (EMT)
Tubular heart
- Differentiation to fibroblasts, smooth
muscle cells of coronary arteries
PEO
Epicardial Outgrowth
SV
cytokeratin
HH17
proepicardial organ
HH20
epicardium
EPDCs: invasion of sulcus and
AV-cushion
Gittenberger-de Groot 1998
RA
•Chimera-studies: quail PEO transplanted to chick-embryo with resected PEO
•EPDCs identified by staining with a specific anti-quail nuclear antibody
(QCPN)
Role of EPDCs in formation of the annulus
fibrosis?
Mechanically inhibition of PEO outgrowth
HH16
OT
Ao
RV
inserted egg
shell
membrane
Atria
Pro-Epicardial Organ
Wild type versus EPDC-inhibited
hearts
Ex ovo electrogram
Kolditz , Circulation 2008
EPDC-inhibited heart: in ovo ECG:
Ventricular preexcitation
HH40
Wild type
HH41
EPDC-inhibited
Wild type vs EPDCinhibition
AP volumes
Large APs left and
right lateral
EPDCs and Periostin expression
normal
EPDC inhibited
RA
TV
AV-isolation
RA
TV
RV
AV-connection
RV
Conclusions (3)
• Functional APs are present during normal heart
development in avians and mammals
• “physiological” APs may serve as substrate for
transient AVRT in the fetus and neonate
• EPDCs play an important role in the formation
of the annulus fibrosis
• Periostin plays an inductive role in fibrous tissue
formation
• Abnormal migration of EPDCs may play a role
in the pathogenesis of WPW syndrome ?
Acknowledgments
• Dept of Pediatric Cardiology, LUMC, The Netherlands
Nathan Hahurij MD
Regina Bökenkamp MD
• Dept of Cardiology, LUMC, The Netherlands
Denise P. Kolditz MD, PhD
Martin J. Schalij MD PhD
• Dept of Anatomy & Embryology, LUMC, The Netherlands
Adriana C. Gittenberger-de Groot PhD
Robert E. Poelmann PhD
• Medical University of South Carolina, Charleston SC, USA
Roger R. Markwald PhD
Thank you for your attention
Mouse hearts
Cx43 expression
15.5dpc
LA
LA
RA
LV
RV
MLC-2a
LV
MLC-2a
15.5dpc
RA
LA
LA
LV
RV
Cx43
LV
Cx43
AP myocardium: Cx43 negative (AV canal myocardium
Results
Electrophysiology
Apex > Base
™ 85BPM
Base > Apex
™ 77BPM
Results
Electrophysiology data
Activation pattern
Early post-septated
HR 115±4BPM
AV 80±17msec
Late post-septated
HR 95±27BPM
AV 81±18msec
Apex > Base
38% (n=11)
46% (n=6)
Base > Apex
62% (n=18)
54% (n=7)
LVB > Apex
7% (n=2)
31% (n=4)
RVB > Apex
7% (n=2)
8% (n=1)
Concurrent
48% (n=14)
15% (n=2)
Results
Morphological analysis
13,5dpc
LA
RA
RV
LA
LV
MLC-2a
LV
MLC-2a
periostin
Nkx2.5 & periostin
Mean AP width (μm)
Early post-septated
Late post-septated
early vs late
Total
347
214
P=0,035*
Left side
238
138
P=0,000*
Right side
109
76
P=ns
left vs right
P=0,000*
P=0,028*
* Statistical test XXX
Mouse hearts
electrophysiology data
Activation pattern
Early post-septated
HR 115±4BPM
AV 80±17msec
Late post-septated
HR 95±27BPM
AV 81±18msec
Apex > Base
38% (n=11)
46% (n=6)
Base > Apex
62% (n=18)
54% (n=7)
Concurrent
48% (n=14)
15% (n=2)
Stages of Heart Development
Primary
Heart Fields
Day 1 (HH6)
Tubular
Heart
Day 2 (HH8-10)
Looped
Heart
Day 3-8 (HH10-34)
Hatching Day 18-20 (HH 45-46)
Septated
Heart
Day >9 (HH>35)
Srivastana et al.
Electrophysiological recordings
•
8 embryo's gestational age 15.5 d.p.c
•
Isolation of RA/RV from the LA/LV
(including the AV-node)
•
Both preparations in the same tissue bath
•
Simultaneous EGM recordings from the
RA/RV and LA/LV
Jongbloed et al. 2004
2 out of 8 embryo’s
SR with 1:1 conduction RA 稀 RV
2:1 VA conduction LV 稀 LA
Right Lateral AV-connection
Jongbloed et al. 2004
“CCS-LacZ reporter mouse”
Rentschler et al. 2001
CCS-lacZ Expression in Pulmonary Veins
Jongbloed et al. 2004
Results: E 10.5
LacZ+ myocardium: AV-ring, Primary fold, VA-ring
VA
AV
Prim. Fold
Jongbloed et al. 2004
Results: E 11.5
Formation of myocardial groove and
outgrowth of LacZ- myocardium at inflow-tract
Jongbloed et al. 2004
Jongbloed et al. 2004
Results: E 13.5
Division of the primary fold tissue:
稀 Medial part: trabecula septomarginalis
稀 Lateral part: moderator band
MB
Jongbloed et al. 2004
Segments/Rings of the Tubular Heart
ARTERIAL POL
Arterial Pole
Outflow Tract
RV
VA-Ring
OUTFLOW TRA
Primary Ring
LV
AV-canal
Atrium
Sinus Venosis
AV-Ring
PRIMITIVE ATR
SA-Ring
SINUS VENOSU
HOMING AND ROLES OF NEURAL CREST CELLS
outflow tract (OFT)
migration
OFT
inflow tract (IFT)
SMC
differentiation
IFT
PNS
RA
myocardialisation
induction
conduction system (CCS)
RV
•
•
High expression of both Tbx5 and Nkx2.5 establishes a regionally
restricted program of ventricular CS gene expression, including the Id2,
Cx 40 and ANF promotors
This program recruits cells into CS lineage, including cell cycle exit,
adoption of fast conduction, expression of other CS markers
Moskowitz,Cell, 2007
Development of the primary cardiac tube:
contribution of the first heart field
arterial pole
2nd
OT
“LV”
A
1st
SV
venous pole
Human 23 days; mouse E 8.5
Alternating zones of “slow” and “fast”
conduction (chick)
HH 23
DeJong et al. 1992
HH 27
Development of CCS: Ring
Theory
Hematoxylin/Eosin staining
Wenink, 1976