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Neonatal Cardiology
Third Edition
Michael Artman, MD
Joyce C. Hall Eminent Scholar in Pediatrics
Senior Vice President, Pediatrician-in-Chief
Children’s Mercy Hospital
Chair, Department of Pediatrics
University of Missouri-Kansas City School of Medicine
University of Kansas School of Medicine
Kansas City, Missouri
Lynn Mahony, MD
Professor, Department of Pediatrics
University of Texas Southwestern Medical Center
Dallas, Texas
David F. Teitel, MD
Professor, Department of Pediatrics and the Cardiovascular Research Institute
University of California, San Francisco
San Francisco, California
New York Chicago San Francisco Athens London Madrid Mexico City
Milan New Delhi Singapore Sydney Toronto
Artman_FM_i_xii.indd 1 2/10/17 6:25 PM

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This book is dedicated to our families for their unwavering support, understanding, and sacrifices.
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so well for our lives, careers, and academic endeavors.

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Contents
Contributors ....................................................................vii Chapter 8
Foreword to the Second Edition.......................................ix Approach to the Infant With Inadequate
Foreword to the Third Edition.........................................xi Systemic Perfusion........................................ 137
Chapter 1 Chapter 9
Cardiac Morphogenesis: Implications for Cardiomyopathy and Other Causes of
Congenital Cardiovascular Diseases................... 1 Ventricular Dysfunction.................................. 155
Adriana C. Gittenberger-de Groot, PhD, Chapter 10
Robert E. Poelmann, PhD, Arrhythmias.................................................. 185
Margot M. Bartelings MD, PhD,
Marco C. DeRuiter, MD, PhD, Chapter 11
and Monique R. M. Jongbloed, MD, PhD Principles of Medical Management................. 217
Chapter 2 Chapter 12
Myocyte Contraction and Relaxation................. 19 Cardiovascular Drug Therapy.......................... 235
Perinatal Cardiovascular Physiology.................. 39 Care of the Postoperative Patient................... 255
Prenatal Evaluation and Management............... 55 Neurology of Congenital Cardiovascular Disease:
Brain Development, Acquired Injury,
Chapter 5 and Neurodevelopmental Outcome................. 267
Initial Evaluation of the Newborn With
Shabnam Peyvandi, MD
Suspected Cardiovascular Disease................... 67
and Patrick McQuillen, MD
Chapter 6
Approach to the Cyanotic Infant........................ 81
Chapter 15
Epidemiology, Etiology, and Genetics
Chapter 7 of Congenital Cardiovascular Disease............. 279
Approach to the Infant With Excessive
Pulmonary Blood Flow................................... 111 Index ..............................................................................291


Contributors
Margot M. Bartelings MD, PhD Patrick McQuillen, MD
Department of Anatomy and Embryology Departments of Pediatrics and Neurology
Leiden University Medical Center University of California, San Francisco
Leiden, Netherlands San Francisco, California
Marco C. DeRuiter, MD, PhD Shabnam Peyvandi, MD

Department of Anatomy and Embryology Department of Pediatrics
Leiden University Medical Center University of California, San Francisco
Leiden, Netherlands San Francisco, California
Adriana C. Gittenberger-de Groot, PhD Robert E. Poelmann, PhD

Department of Anatomy and Embryology Department of Anatomy and Embryology
Leiden University Medical Center Leiden University Medical Center
Leiden, Netherlands Leiden, Netherlands
Monique R. M. Jongbloed, MD, PhD

Department of Anatomy and Embryology
Leiden University Medical Center
Leiden, Netherlands
vii


Foreword to the Second Edition
During the 8 years since the publication of the first edi- Their importance in development of congenital cardio-
tion of Neonatal Cardiology in 2002, many remarkable vascular malformations is now being explored.
advances in our understanding of normal cardiovascular The association of neurological abnormalities with
development and of mechanisms, resulting in congenital congenital cardiovascular malformations has been recog-
cardiovascular malformations, have been achieved. nized for many years; in many genetic syndromes, both
The concept that these malformations had little impact developmental delay and cardiac defects are encountered.
on normal fetal development was widely accepted; the In recent years, concern has been raised regarding intel-
influence of congenital cardiovascular malformations lectual and behavioral impairment in children with several
on fetal blood flow patterns and oxygenation, as well as congenital cardiac anomalies. The possibility that this was
their effect on fetal cardiac and vascular development, is related to surgical procedures during infancy was consid-
increasingly being recognized. Furthermore, the potential ered, but recent evidence suggests interference with brain
effects of these defects on other organ systems, particu- development occurs during fetal life. In a most interesting
larly the brain, has engendered great interest and study. new chapter, authored by Dr. Patrick McQuillen, neuro-
In this second edition, Drs. Michael Artman, Lynn logical development and mechanisms by which congenital
Mahony, and David Teitel have continued the general phi- cardiovascular malformations could affect it are presented.
losophy of the first edition; they have presented clinical and This topic is of great importance, because it is suggested
hemodynamic manifestations of congenital and acquired that in some infants with these cardiac lesions, correction
cardiovascular disturbances, based on biological informa- during the neonatal period may not improve the neuro-
tion regarding development and function. All chapters have logical deficit. This would support the concept of interven-
been significantly modified to address the increased under- tion to correct the circulatory disturbance during fetal life.
standing of basic biology and factors affecting normal devel- The use of nonsurgical approaches to cardiac lesions
opment and performance. As in the first edition, the graphic has become standard practice over the past decade. Many
material largely presents diagrams that help to explain basic of these procedures are not yet applicable to neonates, but
physiological concepts and pathophysiology. The quality of as discussed in this edition, interventions to relieve aortic
the diagrams has been greatly improved, making the infor- and pulmonary stenoses by balloon valvuloplasty have
mation presented more readily assimilable. now become standard practice, thus avoiding the high
The first chapter, on cardiac embryology, contrib- risks of surgery in critically ill infants.
uted by Dr. Kathleen Ruppel, reviews the advances in the This edition of Neonatal Cardiology continues to provide
understanding of genetic pathways involved in cardiac pediatric cardiologists, neonatologists, and obstetricians
morphogenesis. Also, the revolutionary changes in our with an invaluable resource for the differential diagnosis
appreciation of the embryology of the heart and great ves- of cardiovascular disturbances and their management in
sels are presented. It was widely accepted that all struc- newborn infants. The symptom-complex approach is par-
tures developed from the primitive heart tube; it is now ticularly helpful to students and residents in understanding
recognized that other primitive cells from the anterior the mechanisms responsible for clinical manifestations.
or secondary heart field, as well as neural crest cells, are
major contributors to cardiac and great vessel formation. Abraham M. Rudolph, MD
ix


Foreword to the Third Edition
Congenital heart disease is the most common serious con- genetics. In addition, they have enlisted Dr. Gittenberger
genital defect. It occurs in ~1% of all live births, and annually de Groot and her colleagues to discuss current concepts of
about 1.5 million children are born with congenital heart cardiac embryology. Understanding embryology not only
disease. (A similar number have bicuspid aortic valves, but helps us understand how the anomaly formed but some-
these seldom cause problems in childhood.) Given the high day also will be integrated with genetics and perhaps lead
potential early mortality and morbidity of these diseases, to prevention of an anomaly. Furthermore, knowing how
any book such as this one that improves the effectiveness of anomalies such as aortic atresia develop gives a guide to the
treatment will make a major contribution to world health. best time for intrauterine treatment. In another chapter,
Neonatal cardiology is concerned mainly with congeni- Drs. McQuillen and Peyvandi discuss the relation between
tal heart disease. The period of transition from fetus to neo- cardiac malformations and neurodevelopment. Now that
nate is often difficult, and congenital or acquired disease at most forms of congenital heart disease are treatable with
this age may, for many reasons, be more difficult to manage low mortality, our concentration must be on the quality of
than at older ages. It is not surprising, therefore, that the life that results. Chief among these is neurological function,
highest mortality in children with congenital heart disease and recent studies have shown that fetuses with congenital
who are not treated occurs in the neonatal period. What is heart disease often have neurological abnormalities before
surprising is that so few books have concentrated on this birth. Whether these changes are due to abnormal brain
critical period. This third edition of Neonatal Cardiology has blood flow secondary to the congenital heart disease (and
gone a long way to correcting the deficiency. thus potentially amenable to treatment) or due to the same
With the advances in imaging methods, diagnos- disturbance that has altered cardiac development remains
tic cardiac catheterization has been replaced by echocar- to be determined.
diography, CT, and MRI. As a result, the emphasis today Included in this book are discussions of arrhythmias
is placed on anatomic abnormalities rather than on their and pharmacological treatment as they relate to congenital
physiological consequences, even though it is these conse- heart disease. Because pharmacology is used to manipulate
quences that often determine the outcome. The authors of physiology for the patient’s benefit, knowing the basis of the
this book, basing their work on the groundbreaking studies physiological disturbance leads to more effective therapy.
by Dr. Abraham Rudolph of abnormal fetal development Although knowledge of pathological anatomy is impor-
and the physiological changes due to congenital heart dis- tant in understanding congenital heart disease, knowledge
ease, have shown how understanding the physiology as well of the associated pathophysiology is mandatory if we wish
as the anatomy of these lesions improves our ability to treat to provide optimal care. This book is one of the few to com-
these patients. bine both aspects and represents a hallmark in the treatment
In this third edition, Drs. Teitel, Mahony, and Artman of congenital heart disease.
have updated and expanded what was in the second edition
with added information about myocytes, arrhythmias, and Julien I.E. Hoffman, MD
xi


CH APT ER
Cardiac Morphogenesis:
1
Implications for Congenital
Cardiovascular Diseases
■■ INTRODUCTION Incorporation of the Sinus Venosus, Atrial
■■ CARDIAC PROGENITORS AND THE CONCEPTS OF Septation, and Pulmonary Vein Development
FIRST AND SECOND HEART FIELD Ventricular Inflow and Outflow Tract Septation
Pharyngeal Arch Development
■■ THE NEURAL CREST
Valvulogenesis
■■ THE EPICARDIUM Conduction System Development
■■ BREAKING SYMMETRY Development of the Epicardium, Myocardium,
■■ CRITICAL DEVELOPMENTAL TIME WINDOWS and Coronary Arteries
■■ CARDIAC MORPHOGENESIS AND ■■ SUGGESTED READINGS
DYSMORPHOGENESIS
■■ INTRODUCTION understanding of mutations in humans that lead to con-

genital cardiovascular disease.
Knowledge of the role of cardiac-specific genes and their
This chapter summarizes the initial phases of cardiac
modulating factors has increased tremendously over the
development. We then describe in more detail how cardiac
last decade, although 85% of human congenital cardio-
morphogenesis leads to formation of the four-chambered
vascular disease is still considered to be multifactorial in
heart and how abnormal cardiogenesis contributes to
origin. Advances in the molecular biology of the develop-
congenital cardiovascular disease.
ing heart have greatly contributed to our understanding
of cardiac morphogenesis. Manipulation of conserved
■■ CARDIAC PROGENITORS AND THE
genes from a variety of model organisms has increased
CONCEPTS OF FIRST AND SECOND
our understanding of how genetic factors and cellular
HEART FIELD
interactions contribute to cardiac development. Trans-
genic mouse models have allowed time-specific tracing Heart development starts with two cardiogenic plates
of cells and their role in heart formation. The problem derived from the lateral splanchnic mesoderm. These
of embryo-lethality after manipulating “cardiac-specific” plates fuse in the midline in the anterior (cranial) region
genes has been overcome by inducible knockout strategies. of the embryo. The crescent of the cardiogenic plates is
Whole genome sequencing programs have also increased referred to as the first heart field (FHF) and is flanked
Artman-Ch01_p001_018.indd 1 2/10/17 6:27 PM

2 Chapter 1
medially by the second heart field (SHF) mesoderm toward various parts of the embryo, including the heart.
(Figure 1A). Upon fusion in the midline, the FHF forms Cardiac neural crest cells differentiate into cells of the
the two-layered primary heart tube with myocardium autonomic nervous system and into vascular smooth
on the outside lined by endocardium on the inside. muscle cells of the pharyngeal arch arteries and contrib-
The myocardium secretes a glycoprotein-rich layer, the ute to the arterial pole of the heart entering the endo-
cardiac jelly, toward the endocardium. The primary heart cardial outflow tract cushions (Figure 1-1E). Neural
tube connects to the arterial pole cranially and to the crest cells within the heart are involved in modulation
venous pole caudally (Figure 1-1B) but does not contain and induction of semilunar valve formation and genera-
all segments of the four-chambered heart. Venous tribu- tion of the myocardial component of the outflow tract
taries abut on the small atrial component, followed down- septum. At the venous pole, where their contribution is
stream by the future atrioventricular canal and a primitive less important, a similar effect is observed in the atrio-
left ventricle. Finally, the outflow tract connects to the ventricular cushions. More recently, cardiac neural crest
aortic sac at the arterial pole (Figure 1-1C). The various cells contributing smooth muscle cells to the coronary
components can be distinguished soon after, as both the arteries have been identified.
AV canal and the outflow tract contain an increasing
amount of cardiac jelly that forms the endocardial cush- ■■ THE EPICARDIUM
ions. The cushions become even more prominent as they
acquire mesenchymal cells, derived from the endocardial The epicardium (splanchnic mesodermal lining of the
lining as a result of endocardial-mesenchymal transition. pericardial cavity) is a secondary layer covering the
Subsequently, the primary heart tube starts the develop- myocardial tube and the intrapericardial part of the arte-
mentally determined rightward looping. rial pole. During embryonic development, the epicar-
At the same time, the SHF adds progenitor cells to dium originates from both the venous and the arterial
both the venous and the arterial poles, which ultimately poles (Figure 1-1E–G). The larger epicardial population
form the essential components of the right ventricle (RV) derives from a protrusion of the coelomic wall, covering
and at least a part of the right side of the interventricu- the sinus venosus and liver primordium (Figure 1-1F).
lar septum (Figure 1-1D). At the venous pole, the SHF The cells of the proepicardium spread along the outer
forms cardiomyocytes encapsulating the sinus venosus wall of the ventricles and atria to the border of the myo-
and its tributaries. The sinus venosus is incorporated cardium at the arterial pole. Here, they join the arte-
subsequently into the wall of the right and left atrium. rial epicardium, which is derived from a much smaller
Likewise, the walls of the great arteries, the embryonic arterial proepicardium exhibiting a slightly different
pharyngeal arch arteries that connect to the aortic sac, are phenotype (Figure 1-1G). On activation, the epithelial
partly built from SHF-originating cells. Neural crest cells epicardium undergoes endocardial-mesenchymal tran-
also contribute to formation of the great arteries, as will sition, and the resulting mesenchymal cells fill the sub-
be explained below in this chapter. epicardial space as epicardium-derived cells. These cells
Multiple specific transcription factors and signaling migrate between the myocardial cells of the heart tube,
molecules are essential to the early stages of cardiogenesis. the atrioventricular cushions, and the future fibrous
These include the earliest markers of the precardiac meso- annulus. These epicardium-derived cells, in contrast
derm, including the homeobox-containing gene Nkx2.5 to neural crest cells, differentiate into several cell lines
and the zinc-finger-containing GATA4/5/6 subfamily. including the majority of the cardiac fibroblasts and the
Members of the T-box family, TBx1/5/18/20, also have vascular smooth muscle cells of the coronary vascular
essential roles in SHF differentiation. Myocardial differ- system (Figure 1-2).The endothelial lining of the coro-
entiation is regulated by several myocyte-specific genes, nary vascular system is derived from the endothelium
including myosin light and heavy chain, alpha-cardiac of the sinus venosus/liver primordium adjacent to the
actin, and cardiac troponin I. proepicardium (Figures 1-1F, 1-2).
■■ THE NEURAL CREST ■■ BREAKING SYMMETRY

The neural crest cells are an ecto-mesodermal deriva- Starting with the developmentally determined rightward
tive arising from the crest of the neural tube, migrating looping, it is clear that the heart and its connections to the

CARDIAC MORPHOGENESIS: IMPLICATIONS FOR CONGENITAL CARDIOVASCULAR DISEASES 3
Arterial pole RVV LVV

First heart RCV PV LVC DA
field Second heart SVC
field AoS Ao
RVV
Cardiac jelly PT
A
LA
OFT
EC ∗ AVC EC RA
LV
RV LV
RV
Venous pole
IVC
A B C D
NH18 HH17
PAA
Anterior SHF
SV OFT
aPEO
NCC
PC
Posterior SHF
PC
vPEO
vPEO
CV L
F G
E
FIGURE 1-1. Cardiac development. A. Schematic depiction of the precardiac mesoderm in the primi-
tive plate. The brown area reflects the mesoderm of the FHF, whereas the yellow area corresponds
to the putative SHF mesoderm. B. Primary heart tube derived of FHF mesoderm. The tube consists of
myocardium, lined by cardiac jelly. C. Heart tube after looping. The yellow areas reflect SHF-derived
contributions. The SHF contributions to the outflow tract have not been depicted. Note that in this
stage the atria are still positioned entirely above the primitive left ventricle, whereas the outflow tract
is positioned above the primitive right ventricle. D. Advanced stage of heart development. Septation
has now occurred at the level of the atria, ventricles, and outflow tract. E. Sagittal view of an embryo.
The (anterior and posterior) SHF mesoderm and its derivatives are depicted in yellow. Contributions
from neural crest cells are depicted in blue. A proepicardial organ can be distinguished at the venous
pole as well as a smaller proepicardial organ at the arterial pole. F. Scanning electronic microscopic
section at the level of the venous pole in a chick embryo, showing the venous pole of the proepicardial
organ. G. Scanning electronic microscopic section at the level of the outflow tract in a chick embryo,
showing the arterial pole of the proepicardial organ (arrowheads, asterisk). Abbreviations: A, atrium;
Ao, aorta; AoS, aortic sac; aPEO, atrial pole of proepicardial organ; AVC, atrioventricular canal; EC,
endocardial cushions; FHF, first heart field; HH, Hamburger and Hamilton; IVC, inferior vena cava;
LA, left atrium; L, liver; LCV, left cardinal vein; LV, left ventricle; LVV, left venous valve; PAA, pharyn-
geal arch artery; PC, pericardial cavity; PT, pulmonary trunk; PV, pulmonary vein; RA, right atrium;
RCV, right cardinal vein; RV, right ventricle; RVV, right venous valve; SHF, second heart field; SV, sinus
venosus; SVC, superior vena cava; VPEO, venous pole of proepicardial organ. B–E: Adapted from:
Gittenberger-de Groot AC et al. Ann Med. 2014;46(8):640-652. F and G: Adapted from: Gittenberger-de
Groot AC et al. Differentiation. 2012;84(1):41-53.

4 Chapter 1
Development Congenital/disease Stem cells
Compact myocardium
Spongious
myocardium
Infarction
Myocardium
CMPCs
Cardiomyocyte
precursor
Conduction
anomalies
Cardiac jelly
Trabeculae
∗ Purkinje fibres
Endocardium Endocardial cushions Cardiac

valves
Interstitial fibroblats Annulus fibrosis
Meso-
derm Valvulopathies
∗ EPDCs
Fibroelastosis
PEO epithelium Epicardium EPDCs
Fistulae
Sinus VSMCs fibroblasts
ECs
venosus/
liver Pattern anomalies
Endothelial Coronary capillaries Coronary
precursor vessels
Myocyte line Endocardial line

∗
Reactivation Composite
EPDC line Endothelial line in adults structures
FIGURE 1-2. Cell lines contributing to the developing heart. Schematic representation of cardiac
cell lines (cardiomyocytes, endocardium, epicardium, and endothelium) that are derived from the first
and second heart field mesoderm and are the main contributors to the definitive heart and vessels.
Epicardium-derived cells (in yellow) as well as inadequate interaction with other cardiac cell types may
play a role in some cardiac malformations (green boxes). The second heart field and neural crest cell
contribution to the great vessels is not represented. Abbreviations: CMPCs, cardiomyocyte progenitor
cells; EPDCs, epicardium-derived cells; ECs, endothelial cells; VSMCs, vascular smooth muscle cells.
Adapted from: Gittenberger-de Groot AC et al. Differentiation. 2012;84(1):41-53.
lungs are not symmetric. Situs inversus and heterotaxy abnormal cardiac development and even early embryo-
occur in humans; several mouse models have increased lethality. This results in early spontaneous abortion in
our knowledge on essential signaling factors related to humans. In homozygous mouse strains, 50% of spontane-
determination of situs. It is remarkable that only the ous early embryo-lethality related to mutations is caused
atrial situs and its contributing posterior SHF seem to be by cardiovascular abnormalities. In the usually non-
influenced by these factors, while right ventricle and left homozygous human, the percent of spontaneous abor-
ventricle with their specific morphologies do not copy the tions caused by congenital cardiovascular disease cannot
atrial situs anomalies (eg, inversus or ambiguous). be determined unequivocally. The FHF lesions are consid-
ered to be the most critical for embryonic demise. Most
of the cardiac malformations that clinicians encounter
■■ CRITICAL DEVELOPMENTAL
in the perinatal period occur during looping and events
TIME WINDOWS
mediated by the SHF. Spontaneous abortion in the second
In the preceding paragraphs, the cellular building blocks trimester caused by congenital cardiovascular disease is
of the cardiovascular system have been presented. Serious less common, as most forms of cardiac malformations are
disturbances of one or more cell populations can lead to compatible with intrauterine survival.

■■ CARDIAC MORPHOGENESIS AND incorporated into the posterior wall of both the right and
DYSMORPHOGENESIS the left atria. The atrial appendages are probably related
to the FHF.
In the human embryo, the formation of the four-
The left and right cardinal veins (the embryonic supe-
chambered heart occurs by about 8 weeks’ gestation.
rior and inferior caval veins) are incorporated into the
Thereafter, maturation and remodeling of, eg, the pha-
right atrium and are flanked by the folds of the embry-
ryngeal arch arteries and valves are essential for ensuing
onic right and left venous valves (Figure 1-1C, D). The
proper functioning and postnatal survival. The most
left inferior cardinal vein (future coronary sinus) and the
important elements of cardiac morphogenesis (summa-
left superior cardinal vein (regressing in the human heart
rized in Figure 1-3), including septation, valve formation,
as the ligament of Marshall) all drain into the cavity of
conduction system maturation, and coronary vascular
the right atrium. A splanchnic vascular plexus surrounds
development, will be presented.
the developing lung buds. During early developmental
stages, the primary route of pulmonary drainage from this
Incorporation of the Sinus Venosus, Atrial plexus is toward the systemic veins; a direct connection of
Septation, and Pulmonary Vein Development the primitive pulmonary veins to the heart is achieved
The precardiac mesoderm of the SHF at the venous pole from different tissue later during development. The
develops uniquely from an Nkx2.5-negative but myo- anlage of the primitive pulmonary vein, the so-called
sin light chain positive cell population, surrounding the mid-pharyngeal endothelial strand, initially does not
lumen of the sinus venosus. This myocardial cell lineage is have a lumen and is connected to the sinus venosus.
Heart tube
Heart loop
Atrial septation
Ventricular septation
Conduction system
Valve formation
Pulmonary veins
Aortic arch/
pulmonary artery
Ductus arteriosus
Coronary vasculature
Ovulation in days 18 24 26 28 29 31 33 35 37 39 41 43
Crown-rump length in mm 1-5 2-3 3-5 4-5 6-7 7-8 9-10 11-1414-1617-20 22-2425-26
Horizons IX XI XII XIII XIV XV XVI XVII XVIII XIX XXI XXII
chicken/mouse: sequence overlap identical
FIGURE 1-3. Cardiac morphogenesis. Schematic representation focusing on the time line and the
major events during cardiac morphogenesis. Many processes essential to heart formation overlap dur-
ing the 7 to 8 weeks of development, making it difficult to determine the separate molecular pathways
or the primary insult that leads to congenital cardiovascular malformations, either isolated or complex.
Valvulogenesis occurs relatively late in heart formation, while completion of atrial septation (closure
of the foramen ovale) and ductus arteriosus differentiation (closure of the ductus arteriosus) naturally
occur after birth because of the unique requirements of the fetal circulation. Source: Jongbloed MRM,
et al. Development of the Cardiac Conduction System and the Possible Relation to Predilection Sites
of Arrhythmogenesis, TheScientificWorldJOURNAL, vol. 8, pp. 239-269, 2008.

6 Chapter 1
During atrial septation it connects to the dorsal wall of left atria. It partially disintegrates forming the ostium
the left atrium. The splanchnic pulmonary venous con- secundum, which is required for the formation of the
nections with the systemic cardinal (putative caval) veins embryonic foramen ovale (Figure 1-4A, B), the essential
gradually disappear during normal development. communication between the right and left atria during
During atrial septation, four components deserve fetal life. Fusion of a mesenchymal cap on the free rim of
attention: the primary septum, the dorsal mesenchymal the primary atrial septum with the atrioventricular endo-
protrusion, the septum secundum, and the endocar- cardial cushion mass and with the dorsal mesenchymal
dial cushions. The initially two-layered myocardial pri- protrusion closes the ostium primum. This structure is
mary atrial septum is positioned between the right and found at the right side of the primary atrial septum
SVC
SVC
DM OS DM PV
SAS PAS MC
DM
PV
RA RA
OP
LA LA
IVC
TO MO
AVC IVC
A B
FIGURE 1-4. Atrial septation. A. Early development of the dorsal and cranial wall of the right and
left atria. The gold indicates the contribution of the SHF to the incorporated sinus venosus myocar-
dium. The appendages are not depicted. Both the inferior and the superior vena caval veins enter in
the right atrium as well as the coronary sinus, which is derived from the left superior cardinal vein.
A mesenchymal cap (green) under the rim of the primary atrial septum borders the ostium primum,
which connects the right and left atria. The entrance of the primitive pulmonary vein is seen in the left
atrium. The infolding of the superior wall of the right atrium that will form the secundum atrial septum
is already seen merging with the dorsal mesenchymal protrusion. B. After completion of septation,
the ostium primum is closed by fusion of the mesenchymal cap with the atrioventricular cushions,
which have now divided the atrioventricular canal into a tricuspid and a mitral orifice. The perforations
in the primary atrial septum have enlarged to form an ostium secundum that in combination with the
free rim of the secundum atrial septum is part of the foramen ovale (arrow) that closes after birth.
Abbreviations: AVC, atrioventricular canal; DM, dorsal mesocardium; IVC, inferior vena cava; LA, left
atrium; MC, mesenchymal cap; MO, mitral orifice; OP, ostium primum; OS, ostium secundum; PAS,
primary atrial septum; PV, pulmonary vein; RA, right atrium; SAS, secundum atrial septum; SVC, supe-
rior vena cava; TO, tricuspid orifice. Used with permission from Gittenberger-de Groot AC, et al., (2011).
Normal and Abnormal Cardiac Development. In: Pediatric Cardiovascular Medicine, Second Edition
(eds JH Moller and JIE Hoffman), Wiley-Blackwell, Oxford, UK.

as a thick mesenchymal mass (Figure 1-4A, B) and caval vein. Mutations in genes involved in posterior SHF
will develop into the muscular base of the atrial sep- differentiation, such as TBx5 and NKx2.5, are linked to
tum. At the right side of the primary septum and usu- this relatively common group of malformations.
ally incorporating the left venous valve, a folding process
of the atrial myocardial wall forms the crescent ridge Atrioventricular septal defects. Data from animal mod-
of the atrial septum secundum (the limbus). Dur- els and humans suggest that most cases of atrioventricu-
ing development, the free edge of the primary atrial lar septal defect (atrioventricular canal) are caused by a
septum (the valve of the foramen ovale) and the rim of deficiency in the base of the atrial septum resulting from
the septum secundum (the limbus) will overlap, allowing underdevelopment of the dorsal mesenchymal protru-
blood to pass via the foramen ovale (Figure 1-4B). At a sion. Additionally, the posterior inlet ventricular septum
variable time after birth, these two rims often fuse, result- is shorter than normal; in combination, this results in
ing in the closure of the foramen ovale, although this partial fusion to absence of the atrioventricular cushions,
structure remains patent in up to 20% of normal adults. resulting in a common valve with either one- or two-valve
ostia (depending on the amount of fusion). Portions of
Implications for Congenital Cardiovascular Disease the atrioventricular cushions develop into the character-
istic atrioventricular valve leaflets observed in atrioven-
Abnormal pulmonary venous return. As described
tricular septal defects (ie, superior and inferior bridging
above, pulmonary drainage is initially via an extensive
leaflets and left and right-sided mural leaflets).
midsagittal splanchnic vascular network. Disturbance
of the SHF can lead to misalignment and faulty incorpo-
ration of the primitive pulmonary veins into the dorsal Ventricular Inflow and Outflow Tract Septation
left atrial wall. In case of absence or atresia of the mid- Viewed from the right, the ventricular septum is divided
pharyngeal endothelial strand, either the early pulmo- into several components, including the ventricular inlet
nary to systemic connections will persist or abnormal septum, an apical trabeculated component, and an ante-
connections will develop, leading to abnormal drainage rior (“infolding”) component (Figure 1-6A, B). The out-
of the pulmonary venous blood to three levels: subdia- flow tract septum (Figure 1-6C) develops as a separate
phragmatic (eg, scimitar syndrome; Figure 1-5A), car- structure. The different components with their specific
diac, and supracardiac (Figure 1-5B). The drainage of all developmental history, boundaries, and origin are asso-
the pulmonary veins can be abnormal (total anomalous ciated with varying congenital malformations of the ven-
pulmonary venous connection) or partial, with some pul- tricular septum.
monary veins entering in into left atrium and some into To understand the process of ventricular septation
either systemic veins (Figure 1-5B) or the right atrium. and the aberrations in development that lead to the most
The few known genetic causes are linked to abnormalities common septal defects, it is helpful to first review several
of left/right asymmetry (heterotaxy) caused by mutations novel findings concerning the contribution of both the
of the transcription factor PITx2 or by more downstream anterior SHF and the neural crest cells to the outflow tract
signaling abnormalities (eg, PDGFRα). septum and the trabecular portion of the right ventricle.
Several tracing studies in mouse embryos employing
Atrial septal defects. Several types of atrial septal surrogate markers for SHF-derived cells have shown an
defects occur. A primum atrial septal defect is caused by asymmetric contribution to both the myocardium and the
a deficient connection of the primary interatrial septum vascular wall of the right ventricular outflow tract and the
with the atrioventricular cushion complex. This anomaly pulmonary trunk. In this process, which we have termed
is often seen in conjunction with an atrioventricular sep- the “pulmonary push,” the embryonic left (pulmonary)
tal defect (see below) in which the dorsal mesenchymal side of the outflow tract is expanded by a relatively
protrusion is also underdeveloped. The most common large contribution of the SHF as compared to the right
anomaly of the atrial septum is a secundum atrial septal (aortic) side, eventually bringing the pulmonary orifice
defect, in which there is deficiency of an atrial septal com- to its normal anterior and cranial position with respect
ponent. More rarely, a sinus venosus type of atrial septal to the aorta. This pulmonary push is responsible for the
defect is seen, which is related to the superior or inferior so-called rotation of the outflow tract and great arteries

8 Chapter 1
BV
VV
RPA
SVC RPV LPV
LPA
RSPV SVC Ao
PT LSPV
LIPV
RA LA
RA LA
RAA
LV LAA
RV
RIPV
IVC IVC
A B
FIGURE 1-5. Anomalous pulmonary venous connection. A. Scimitar syndrome is characterized by a

partial or complete right-sided anomalous venous connection to the inferior vena cava. In this case,
the right inferior pulmonary vein has an anomalous connection to inferior vena cava. The other veins
depicted all drain normally to the left atrium. Other characteristics of Scimitar syndrome include
hypoplasia of the right pulmonary artery and lung, resulting in dextroposition of the heart. B. Total
anomalous pulmonary venous connection, extracardiac type. The right and left pulmonary veins drain
via a vertical vein into a systemic vein (eg, the left innominate vein, which drains into the superior
vena cava). The absence of pulmonary venous connections and incorporation into the left atrium lead
to a small left atrium that does not contain vessel wall tissue. Abbreviations: Ao, aorta; BV, brachio-
cephalic vein; IVC, inferior vena cava; LA, left atrium; LAA, left atrial appendage; LPA, left pulmonary
artery; LIPV, left inferior pulmonary vein; LPV, left pulmonary vein; LSPV, left superior pulmonary
vein; LV, left ventricle; PT, pulmonary trunk; RA, right atrium; RAA, right atrial appendage; RIPV, right
inferior pulmonary vein; RPA, right pulmonary artery; RPV, right pulmonary vein; RSPV, right superior
pulmonary vein; RV, right ventricle; SVC, superior vena cava; VV, vertical vein. Panel B is Adapted
from Douglas YL et al. Int J Cardiol. 2009;134:302-312.
and also explains the relatively deep position of the aortic left ventricular outflow tracts. This outflow tract separa-
orifice in the crux of the heart. tion complex (distinguishable only as an outflow tract
During this process, neural crest cells are incor- septum in specific cardiac anomalies) fuses by bringing
porated by ingression into the aortic sac, creating the together the septal and parietal cushion with the merged
aorto-pulmonary septum, which separates the aorta and atrioventricular cushions, thereby closing the embryonic
pulmonary artery. The aorto-pulmonary septum forms interventricular foramen and completing ventricular
the central condensed mesenchyme, as well as two prongs septation. During this process, an anterior folding sep-
extending into the septal and parietal endocardial cushions tum is formed resulting from the expansion of the right
present in the outflow tract (Figure 1-6C). The neural crest and left ventricles, pushing the two outer faces of both
cells have an induction effect on the outflow tract, recruit- ventricles together and trapping epicardium in between.
ing myocardial cells into the cushions that form the pos- Of note, the epicardium serves a similar function as the
terior wall of the subpulmonary infundibulum, which also endocardial cushions inside the heart, bringing two myo-
forms the septum between the right ventricular and the cardial faces together. The trapped epicardial cells will

PT Ao
APS
D
Ao PT
OTS SB MB P
RV
∗ IS TS
SB AFS
IS
MB TS
A B C
FIGURE 1-6. Ventricular septal components. A. Schematic representation showing the components
of the interventricular septum including the inlet septum, the anterior folding septum, and the tra-
becular (apical) septum. The septal band that continues into the moderator band is related devel-
opmentally to the inlet septum. The posterior wall of the subpulmonary infundibulum contains the
small outflow tract septum (asterisk). B. Postmortem specimen with the above-mentioned septal
components. C. Scanning electron microscopic picture of the outflow tract of a chicken embryo. The
aorto-pulmonary septum at the level between the aortic and pulmonary trunk orifices, which consists
at this stage of condensed mesenchyme of neural crest cell origin, will merge with the distal endo-
cardial outflow tract cushion and extend into the proximal outflow tract cushion. The distal level will
remodel into the semilunar valves of the great arteries, while the proximal endocardial cushion will,
by induction through the neural crest cell population, transform into myocardium and eventually form
the small outflow tract septum. Abbreviations: AFS, anterior folding septum; Ao, aorta; APS, aorto-
pulmonary septum; D, distal (endocardial outflow tract cushion); IS, inlet septum; MB, moderator
band; OTS, outflow tract septum; P, proximal (outflow tract cushion); PT, pulmonary trunk; SB, septal
band; TS, trabecular septum. A: Adapted from Gittenberger-de Groot, AC, et al., (2012). Normal and
Abnormal Cardiac Development, in Pediatric Cardiovascular Medicine, Second Edition (eds JH Moller and
JIE Hoffman), Wiley-Blackwell, Oxford, UK. B and C: Used with persimmon from Gittenberger-de Groot, AC,
et al., (2012). Normal and Abnormal Cardiac Development, in Pediatric Cardiovascular Medicine, Second
Edition (eds JH Moller and JIE Hoffman), Wiley-Blackwell, Oxford, UK.
differentiate into epicardium-derived cells, bringing these cushion mass. Since the fibrous connection between the
cells deep into the core of the septum. tricuspid and mitral orifice and valves derives mainly
from atrioventricular and outflow tract endocardial
Implications for Congenital Cardiovascular Disease cushions, the ventricular septal defect will in part be
Ventricular septal defects. Muscular ventricular septal flanked by fibrous tissue; hence, the term “perimembra-
defects can be found within the anterior folding septum, nous” is often used to describe these defects. The defect
within the inlet septum, and on the border of the septal can extend more posteriorly toward the inlet septum or,
band with the anterior folding septum, ie, central muscu- in case of a malaligned or shortened outflow tract sep-
lar ventricular septal defect. tum, toward the orifices of the great arteries. These are
generally referred to as subarterial, but more specifically
Perimembranous ventricular septal defects and they are subaortic in tetralogy of Fallot and subpul-
malalignment defects, tetralogy of Fallot, and double- monary in Taussig-Bing malformation. From a devel-
outlet right ventricle. These malformations are caused opmental point of view, a double muscular subarterial
primarily by an abnormal extension and malalignment of infundibulum or conus has been proposed to be essen-
the outflow tract septal complex with the atrioventricular tial for the anomaly called double-outlet right ventricle.

10 Chapter 1
Given that the “pulmonary push” is an essential element aortic sac with the bilateral dorsal aortae (Figure 1-7A–D).
of rotation and extension of the right ventricular outflow Stability of the vasculature is provided by a smooth muscle
tract, anomalies of this region are better understood as cell layer that is derived from several sources, including the
resulting from unbalanced contributions from SHF and SHF splanchnic mesoderm and cardiac neural crest cells.
neural crest cells. Remodeling of this system requires a balanced interaction
of the SHF and neural crest–derived cells (Figure 1-7E)
Pharyngeal Arch Development and differs among species. In fish, five or more branchial
The pharyngeal arches develop as a bilaterally symmetric arches persist and feed the pairs of gills. In reptiles, pairs of
system harboring skeletal, muscular, glandular, and vas- the third (carotid), fourth (aorta), and sixth (pulmonary)
cular elements of the face and neck region. The six pairs pharyngeal arch arteries persist, but in birds and mam-
of pharyngeal arch arteries begin their development as a mals, the arterial system becomes asymmetric. In birds, the
cranio-caudal series of endothelial tubes that connect the right fourth becomes the aortic arch, whereas the left one
RCA LCA RCA LCA

LSA
RSA DAo
III γ
IV
RSA β
VI AAo DA
LSA PT
AoSac PA DA
α LDAo
CoA Ao
RDAo PT
a
DesAo
A B C D E
Right arch Left arch
RDA
III
Chicken Mouse Human
F G H I
FIGURE 1-7. Remodeling of the pharyngeal arterial arch system. A–D. Schematic representation
of the remodeling of the pharyngeal arterial arch system during development. The left arch with
principal fourth arch segment becomes dominant, and right-sided structures disappear. The color
coding refers to the arterial segments derived from the pharyngeal arches (blue, third arch; pur-
ple, fourth arch; green, sixth arch). E. Relative contribution of neural crest cells (blue) in the wall
of the great arteries showing that there are mixed wall structures as well as sharp boundaries.
F and H. Species variation in pharyngeal arch remodeling in the chicken (right arch), and the mouse
and human (left arches). I. Development of a right-sided anomalous subclavian artery occurs when
the right fourth arch segment disappears (asterisk) and the right dorsal aortic segment persists.
Abbreviations: AoSac, aortic sac; Ao, ascending aorta; CoA, aortic coarctation; DesAo, descending
aorta; DA, ductus arteriosus; PA, pulmonary artery; PT, pulmonary trunk; RCA/LCA, right/left carotid
artery; RDA, right dorsal aorta; RDAo/LDAo, right/left descending aorta, RSA/LSA, right/left subclavian
artery. Adpated from: A–D: From Molin DG, et al. Cardiovasc Res. 2002; 56(2):312-22, by permission of
Oxford University Press. Reprinted with permission from Molin DG, et al. Birth Defects Res A Clin Mol
Teratol. 2004; 70(12):927-38.

degenerates during development (Figure 1-7F). In mam-

mals, including humans, the main part of the left fourth
develops into the B-segment of the aortic arch (between
the origins of the left carotid and left subclavian arteries;
Figure 1-7H), while the right fourth becomes part of the
right subclavian artery (Figure 1-7G, H). The remodeling
process involves apoptosis of the vascular segments that
disappear, probably combined with shear stress-invoked
signaling. The distal part of the left sixth pharyngeal arch DA
artery persists in mammals as the ductus arteriosus. The
proximal part, which can be very short, is connected to
the left pulmonary artery. On the right side, the distal part
of the sixth pharyngeal arch artery disappears together
with the right dorsal aortic segment, to which it joins cau- Ao
dally with the left dorsal aorta. The subclavian arteries are
thought to develop from the ipsilateral seventh interseg- PT
mental arteries, but how these arteries move superiorly dur-
ing development is poorly understood (Figure 1-7A–D). A
The contribution of SHF and neural crest cells to the
pharyngeal arch arterial system is mainly to the differen-
tiation of the smooth muscle layer, whereas the neural Vascular wall
crest cells also provide for the innervation of the various
segments. The contribution of SHF and neural crest cells Luman
differs within the aortic arch system, as there are precise
boundaries to the contributions of neural crest cells. The Ductal tissue
ascending aorta and pulmonary trunk, including their

roots, are of mixed origin. The ductus arteriosus is com- B
pletely derived from neural crest cells with a sharp bound-
ary to the descending part of the thoracic aorta that lacks FIGURE 1-8. Aortic coarctation. Schematic depiction of an
aortic coarctation involving muscle of the ductus arteriosus
neural crest cells (Figure 1-7E). The pulmonary and sub- A. Ductal tissue with thick intimal cushions (depicted in blue)
clavian arteries probably have a complete SHF origin. completely encircles the aortic arch. B. Histological section
showing the extension of ductal tissue covering most of the
Implications for Congenital Cardiovascular Disease endothelial surface of the aortic wall. Abbreviations: Ao,
aorta; DA, ductus arteriosus; PT, pulmonary trunk.
Coarctation and aortic arch malformations. A number
of abnormalities may occur during the extensive remodel-
ing of the aortic arch system. However, many are able to the aortic arch between the left subclavian and entrance
sustain the prenatal circulation. Based on variable SHF and of the ductus arteriosus or its ligament (Figure 1-7H), can
neural crest cell contribution and ensuing hemodynamic also be hypoplastic. The most common abnormality in this
alterations, some sites are especially vulnerable to develop segment is the localized aortic coarctation, in which the
abnormal anatomy. In humans, the most common site is contribution from the left-sided ductus arteriosus is obvi-
located in the fourth arch artery, leading to interruption ous. Normally, the ductus arteriosus closes after birth, but
or hypoplasia of the B-segment (Figure 1-7H) resulting if the ductal (muscular) tissue extends abnormally into the
in aortic interruption. The causal factor in this anomaly aortic arch, its constriction at birth can increase the sever-
is linked with the SHF and TBx1 disturbances, combined ity of the coarctation (Figure 1-8A, B).
with the resulting alterations in blood flow. Dominance
of the right-sided fourth arch artery may lead to a right Anomalous origin of the subclavian artery. A relatively
aortic arch with mirror image branching of the carotid and common abnormality is seen when the right subclavian
subclavian arteries. The A-segment, or isthmus, located in artery does not reach its proper cranial position before

12 Chapter 1
the right dorsal aortic segment disappears. Rather than (anterior, posterior, and septal), while the mitral valve
arising from the right innominate artery, it arises from has two leaflets (aortic and lateral leaflet). The atrioven-
the descending aorta, distal to the left subclavian artery. In tricular cushions initially adhere to the myocardium of
the presence of an abnormal right-sided aortic arch, the the atrioventricular canal (Figure 1-9A) but separate by a
left subclavian artery can arise anomalously (Figure 1-7I). process that is not well understood (Figure 1-9B). The free
rims of the developing leaflets remain connected by endo-
Valvulogenesis cardial cushion–derived chordae tendinae to the papillary
Both semilunar and atrioventricular valves derive from muscles of the respective ventricles. The mesenchymal
their respective endocardial cushions. This implies that content of the valves is derived by endocardial-mesenchy-
defective endocardial-mesenchymal transition, essential mal transformation from the underlying endocardium.
for proper valve differentiation, can be linked to abnormal Epicardium-derived cells also migrate from the surface
valve structure. However, both in humans and in animal of the heart to the area of the annulus fibrosis, separat-
models, only a few genes have been linked to deficient ing the atrial and ventricular myocardium, and into the
valve tissue (eg, NOTCH1, NFATC1). Since the origin endocardial valve tissue (Figure 1-9B). In the cushions,
of the semilunar and atrioventricular valves differs with epicardium-derived cells are found mainly in the develop-
respect to SHF and FHF and they receive different con- ing parietal (and not septal) leaflets, a pattern potentially
leading to the preferential distribution pattern of affected
tributions from neural crest cells and epicardium-derived
valve leaflets in anomalies like Ebstein malformation and
cells, the normal development of the respective valves is
mitral valve prolapse.
described below.
Atrioventricular Valves Semilunar Valves

The fibrous tissue of the atrioventricular valves derives Both the aortic and the pulmonary semilunar valves
from the endocardial cushions in the atrioventricular derive from the septal and parietal cushions of the out-
canal, including the superior and inferior cushion and a flow tract. Neural crest cells enter the endocardial cush-
small lateral atrioventricular cushion on each side. The ions at the border of the arterial wall and myocardium.
central portions of the superior and inferior atrioven- At the same time, endocardial-mesenchymal transforma-
tricular cushions fuse, separating the tricuspid and mitral tion of the endocardial lining is essential to bring in the
orifices. The tricuspid valve consists of three leaflets mesenchymal cells containing the cushion. This process
ES AVCu AL
AVCu
RV
A B C
FIGURE 1-9. Atrioventricular valve formation. A. The atrioventricular valves develop form the atrio-
ventricular endocardial cushions (depicted in light blue). B. With dissociation of the embryonic atrial
and ventricular myocardium, the epicardium-derived cells from the atrioventricular epicardial sulcus
(depicted in dark blue) migrate into the endocardial cushions providing the fibroblasts for the annu-
lus fibrosis and a population within the cushions. The cushions form the definitive valve leaflets by
delamination from the wall as well as the chordae tendinae that are attached to the papillary muscles.
C. Ebstein anomaly of the tricuspid valve. The anterior tricuspid valve leaflet is rather well developed,
whereas the septal and posterior leaflets have not delaminated from the ventricular wall. Abbreviations:
AL, anterior (tricuspid valve leaflet); AVCu, atrioventricular endocardial cushion; ES, epicardial sulcus;
RV, right ventricle.

is more prominent in the cusp side facing the aorta. Arte- these emerge from the main cushions or whether there
rial epicardial cells have recently been identified in the are differential contributions of SHF, neural crest cells,
developing valve leaflets. Additionally, two intercalated and endocardium to the respective cushions and leaflets.
cushions develop each in the aortic and pulmonary part of The latter is important for understanding malformations
the outflow tract (Figure 1-10A). It is not known whether like bicuspid aortic valve (Figure 1-10B).
Implications for Congenital Cardiovascular Disease

Overriding and straddling atrioventricular valves and
double-inlet left ventricle. The tricuspid orifice becomes
positioned above the right ventricle during remodeling of
the inner curvature of the heart, which is closely linked
to the repositioning of the outflow tract. Initially, there
is only a slit-like inlet portion of the right ventricle, and
the right side of the atrioventricular cushions is contained
within this area. If the remodeling or shift of the tricuspid
orifice is incomplete, a ventricular septal defect remains.
IC
The valve overrides the septal defect; occasionally, there is
B actual straddling in which the tricuspid valve has chordal
EC
attachments into both ventricles. The ventricular septal
defect is characteristically in a posterior or inlet position.
EC
If more than 50% of the tricuspid valve remains connected
to the left ventricle, the resulting anomaly is called double-
inlet left ventricle, which a common form of physiologic
single ventricle. It is much less common for the mitral
IC
valve to straddle the ventricular septum. This is usually
associated with transposition of the great arteries, in which
case the ventricular septal defect is located anteriorly.
Stenosis or atresia of an atrioventricular valve. The

complete atrioventricular canal is originally connected to
NCCs the primitive left ventricle (Figure 1-1C). In the absence
of a connection of the atrium to the developing right ven-
tricle, the tricuspid orifice is atretic. It is seen as a myo-
cardial dimple in the right atrium. In these cases, the right
A ventricle has only trabecular and outflow tract portions,
lacking the inlet portion.
FIGURE 1-10. Outflow tract valve development. A. Sche-
matic representation of the two main outflow tract endocar-
dial cushions (light blue) that line the myocardial outflow Ebstein anomaly of the tricuspid valve. This anomaly
tract, showing a saddle-shaped border with the vascular is caused by deficient delamination of the valve leaflets.
wall of the aortic sac. As a result of inward migration of The amount of nondelamination is highly variable, from
neural crest cells (indicated as dark blue dots), the cush-
minimal inferior displacement of the hinge point of the
ions are separated into future aortic and pulmonary valve
leaflets. In both orifices, the third leaflet arises from an leaflet to severe displacement with almost complete atri-
intercalated cushion (green). This process results in two alization of the right ventricle. The displacement is mainly
facing coronary aortic leaflets and a nonfacing or noncoro- of the origins of septal and posterior tricuspid leaflets
nary leaflet. The pulmonary valve leaflets have the same from the atrioventricular junction toward the right ven-
configuration but lack the coronary orifices. B. Postmortem
tricular apex. The anterior tricuspid leaflet is usually not
specimen showing a bicuspid aortic valve. Abbreviations:
EC, endocardial cushions; IC, intercalated cushion; NCCs, displaced. Redundant tissues and fenestrations of the
neural crest cells. anterior leaflet are frequently observed.

14 Chapter 1
Bicuspid and unicuspid semilunar valves. A bicuspid contributions or deficient epicardial-myocardial signal-
aortic valve (Figure 1-10) is the most common congenital ing during development.
cardiovascular malformation, encountered in about 1%
to 3% of the human population. The valve often functions evelopment of the Epicardium, Myocardium,
D
normally at younger ages. In some cases, this malforma- and Coronary Arteries
tion, as well as a unicuspid valve, may also present in the Initially, the ventricular myocardium is oxygenated by
neonatal period because of significant aortic valve dys- diffusion from the ventricular lumen. A separate structure
function (usually stenosis). for gas exchange becomes necessary as the myocardium
grows. In early development, an epicardial covering over
Conduction System Development the myocardium is essential for normal coronary vascu-
The atrioventricular conduction system differenti- lar development. However, the epicardium-derived cells
ates from a cardiomyocyte origin. The atrial part of the also fulfill an important role in driving compaction of the
conduction system is derived from the posterior SHF– outer myocardial layers of the ventricle, specifically the
derived myocardium, which is devoid of Nkx2.5 expres- anterior part of the folding septum. The coronary endo-
sion. This includes a transient left sino-atrial node and thelial cells arising from the sinus venosus spread in the
the definitive right sinus node. Expression of markers subepicardial space and invade the compacting myocar-
reflecting a pacemaker phenotype (eg, Tbx3, Podoplanin, dium. In the peritruncal area, they form an endothelial
and Shox2), as well as histological features, indicates that ring surrounding the aorta and pulmonary orifice. The
the developing cardiac conduction system covers the endothelial cells also invade the aortic wall and form
entire sinus venosus area during early embryonic stages, the main stems and orifices of the coronary arteries in
including the internodal myocardium as well as the the pulmonary artery-facing (left and right) sinuses of
myocardium surrounding the pulmonary veins. During Valsalva. The factors guiding this process as well as those
later development stages, expression is restricted to the that repel them from entry into the pulmonary wall and
definitive elements of the conduction system. The FHF from the noncoronary aortic cusp remain to be defined.
(atrioventricular canal myocardium) and the primitive
(left) ventricle appear to contribute to the compact part Implications for Congenital Cardiovascular Disease
of the atrioventricular node and the main parts of the Myocardial thinning and noncompaction. The myo-
ventricular conduction system, including the common cardial thinning based on deficient epicardial-myocardial
bundle of His and the left and right bundle branches. interaction resembles noncompaction cardiomyopathies.
Animal models have shown that proper development of Experiments in both avian and mouse models in which the
the fibrous annulus requires the presence of epicardium- epicardial cell population has been limited in its outgrowth
derived cells. If the epicardial outgrowth is inhibited, a and endocardial-mesenchymal transition lead to severe
phenotype with accessory pathways and pre-excitation thinning of the compact myocardium and a spongy inter-
occurs. ventricular septum. Whether the basis of these malforma-
tions is epicardial or myocardial in humans is unknown.
Implications for Arrhythmias
Embryonic conduction pathways that normally disap- Coronary vascular anomalies. In anomalies such as
pear may persist both in structure and in function. This transposition of the great arteries, the main coronary stems
leads to abnormal atrial automaticity at specific sites, take the shortest course to the facing sinus of Valsalva,
including the terminal crest (formerly the right venous explaining the observed variation in patterning. When
valve), the pulmonary venous myocardium, and the cor- epicardial outgrowth and spreading is inhibited, single
onary sinus. The occurrence of congenital cardiovascular or pinpoint arterial orifices are observed, suggesting that
disease with a combination of nondelamination of the epicardium-derived cells are necessary for proper con-
tricuspid valve, noncompaction cardiomyopathy, and nection of the coronary arteries to the aorta. Ventricular-
accessory pathways with pre-excitation, as is observed coronary artery connections (Figure 1-11A) or fistulae
in Ebstein anomaly and transposition-transposed develop if there is no connection of the coronary arteries
great arteries, could be related to abnormal epicardial to the aorta. In human congenital cardiovascular disease

M RV
RV
VCAC
VCAC CA
CA
SEP
SEP
B C
VCAC
FIGURE 1-11. Ventricular-coronary arterial communications. A. Schematic representation of a heart

with pulmonary atresia with intact ventricular septum. The right coronary artery has a pinpoint orifice
(arrowhead), and the main subepicardial coronary branches are severely thickened and in part obliter-
ated (arrows). There are ventricular-coronary arterial communications between the diseased coronary
arteries and the hypoplastic right ventricular lumen, which is common in this condition. B and C.
Sections of a human fetal heart with pulmonary atresia and intact ventricular septum showing that
the severe coronary arterial pathology in the subepicardial region is already present in the fetus. The
ventriculo-coronary arterial communications are primarily a coronary arterial (epicardium derived) dis-
turbance and not primarily of myocardial origin. Magnifications: Bars, B and C: 1 mm. Abbreviations:
CA, coronary artery; SEP, subepicardial region; VCAC, ventriculo-coronary arterial communications.
Adapted from Gittenberger-de Groot AC et al. Prog Pediatr Cardiol. 2010;29(1):3-9.
that includes coronary abnormalities, an epigenetic cause Wamstad JA, Alexander JM, Truty RM, et al. Dynamic and
of the supposed link between disturbed epicardium and coordinated epigenetic regulation of developmental transi-
ventricular-coronary arterial connection is still missing. It tions in the cardiac lineage. Cell. 2012;151(1):206-220.
has been suggested that the occurrence of connections and Neural Crest
coronary orifice pathology in a subgroup of the patients
Arima Y, Miyagawa-Tomita S, Maeda K, et al. Preotic neu-
with pulmonary atresia and intact ventricular septum is of
ral crest cells contribute to coronary artery smooth muscle
epicardial origin (Figure 1-11B, C). involving endothelin signalling. Nat Commun. 2012;3:1267.
Kirby ML, Hutson MR. Factors controlling cardiac neural
SUGGESTED READINGS crest cell migration. Cell Adh Migr. 2010;4(4):609-621.
Cardiac Progenitors and Heart Fields Poelmann RE, Mikawa T, Gittenberger-de Groot AC. Neu-
ral crest cells in outflow tract septation of the embryonic
Chen L, Fulcoli FG, Tang S, Baldini A. Tbx1 regulates pro-
chicken heart: differentiation and apoptosis. Dev Dyn.
liferation and differentiation of multipotent heart progeni-
1998;212(3):373-384.
tors. Circ Res. 2009;105(9):842-851.
Kathiriya IS, Nora EP, Bruneau BG. Investigating the Epicardium and Coronary Vasculature
transcriptional control of cardiovascular development. Gittenberger-de Groot AC, Vrancken Peeters MP, Mentink
Circ Res. 2015;116(4):700-714. MM, Gourdie RG, Poelmann RE. Epicardium-derived cells
Kelly RG. The second heart field. Curr Top Dev Biol. contribute a novel population to the myocardial wall and the
2012;100:33-65. atrioventricular cushions. Circ Res. 1998;82(10):1043-1052.

16 Chapter 1
Gittenberger-de Groot AC, Winter EM, Bartelings MM, Ebels T, Anderson RH, Devine WA, et al. Anomalies of the
et al. The arterial and cardiac epicardium in development, left atrioventricular valve and related ventricular septal
disease and repair. Differentiation. 2012;84(1):41-53. morphology in atrioventricular septal defects. J Thorac
Lie-Venema H, van den Akker NM, Bax NA, et al. Origin, Cardiovasc Surg. 1990;99(2):299-307.
fate, and function of epicardium-derived cells (EPDCs) Vida VL, Padalino MA, Boccuzzo G, et al. Scimitar syn-
in normal and abnormal cardiac development. Scientific drome: a European Congenital Heart Surgeons Association
World J. 2007;7:1777-1798. (ECHSA) multicentric study. Circulation. 2010;122(12):
Riley PR, Smart N. Vascularizing the heart. Cardiovasc Res. 1159-1166.
2011;91(2):260-268.
Tian X, Hu T, He L, et al. Peritruncal coronary endothe- Ventricular Septation and Outflow Tract
lial cells contribute to proximal coronary artery stems Development
and their aortic orifices in the mouse heart. PLoS One. Bajolle F, Zaffran S, Kelly RG, et al. Rotation of the
2013;8(11):e80857. myocardial wall of the outflow tract is implicated in
Tian X, Hu T, Zhang H, et al. Subepicardial endothelial the normal positioning of the great arteries. Circ Res.
cells invade the embryonic ventricle wall to form coronary 2006;98(3):421-428.
arteries. Cell Res. 2013;23(9):1075-1090. Poelmann RE, Gittenberger-de Groot AC, Vicente-Steijn
Winter EM, Grauss RW, Hogers B, et al. Preservation of R, et al. Evolution and development of ventricular septa-
left ventricular function and attenuation of remodeling tion in the amniote heart. PLoS One. 2014;9(9):e106569.
after transplantation of human epicardium-derived cells Scherptong RW, Jongbloed MR, Wisse LJ, et al. Mor-
into the infarcted mouse heart. Circulation. 2007;116(8): phogenesis of outflow tract rotation during cardiac
917-927. development: the pulmonary push concept. Dev Dyn.
2012;241(9):1413-1422.
Breaking Symmetry
Gittenberger-de Groot AC, Bartelings MM, Poelmann RE, Pharyngeal Arch Development
Haak MC, Jongbloed MR. Embryology of the heart and its Molin DG, Poelmann RE, DeRuiter MC, et al. Trans-
impact on understanding fetal and neonatal heart disease. forming growth factor beta-SMAD2 signaling regu-
Semin Fetal Neonatal Med. 2013;18(5):237-244. lates aortic arch innervation and development. Circ Res.
Srivastava D. Making or breaking the heart: from lineage deter- 2004;95(11):1109-1117.
mination to morphogenesis. Cell. 2006;126(6):1037-1048.
Bokenkamp R, DeRuiter MC, van Munsteren C, Gittenberger-
de Groot AC. Insights into the pathogenesis and genetic
Critical Developmental Windows
background of patency of the ductus arteriosus. Neonatology.
Gittenberger-de Groot AC, Calkoen EE, Poelmann RE, 2010;98(1):6-17.
Bartelings MM, Jongbloed MR. Morphogenesis and molec-
ular considerations on congenital cardiac septal defects.
Valvulogenesis
Ann Med. 2014;46(8):640-652.
Attenhofer Jost CH, Connolly HM, Dearani JA, Edwards
Atrial and Pulmonary Vein Development WD, Danielson GK. Ebstein’s anomaly. Circulation.
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Briggs LE, Kakarla J, Wessels A. The pathogenesis of atrial
and atrioventricular septal defects with special emphasis on Kruithof BP, Kruithof-De-Julio M, Poelmann RE, et al.
the role of the dorsal mesenchymal protrusion. Differentia- Remodeling of the myocardium in early trabeculation and
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Douglas YL, Jongbloed MR, den Hartog WC, et al. Pul- 2013;57(11-12):853-863.
monary vein and atrial wall pathology in human total Norris RA, Kern CB, Wessels A, et al. Identification and
anomalous pulmonary venous connection. Int J Cardiol. detection of the periostin gene in cardiac development. Anat
2009;134(3):302-312. Rec A Discov Mol Cell Evol Biol. 2004;281(2):1227-1233.
Douglas YL, Jongbloed MR, Deruiter MC, Gittenberger- Wessels A, van den Hoff MJ, Adamo RF, et al. Epicardially
de Groot AC. Normal and abnormal development of pul- derived fibroblasts preferentially contribute to the parietal
monary veins: state of the art and correlation with clinical leaflets of the atrioventricular valves in the murine heart.
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Conduction System Kolditz DP, Wijffels MC, Blom NA, et al. Epicardium-derived
Jensen B, Boukens BJ, Postma AV, et al. Identifying the cells in development of annulus fibrosis and persistence of
evolutionary building blocks of the cardiac conduction accessory pathways. Circulation. 2008;117(12):1508-1517.
system. PLoS One. 2012;7(9):e44231. van Duijvenboden K, Ruijter JM, Christoffels VM. Gene
Jongbloed MR, Vicente Steijn R, Hahurij ND, et al. regulatory elements of the cardiac conduction system. Brief
Normal and abnormal development of the cardiac Funct Genomics. 2014;13(1):28-38.
conduction system: implications for conduction and Van Weerd JH, Christoffels VM. The formation and
rhythm disorders in the child and adult. Differentiation. function of the cardiac conduction system. Development.
2012;84(1):131-148. 2016;143(2):197-210.


CH APT ER
Myocyte Contraction
2
and Relaxation
■■ INTRODUCTION Initiation of Contraction in the
■■ GENERAL OVERVIEW OF CELLULAR ASPECTS OF Embryonic Heart
CARDIAC FUNCTION Mechanisms of Contraction and Relaxation
Calcium Influx through Calcium Channels
■■ STRUCTURAL COMPONENTS INVOLVED IN
Calcium Fluxes through the Sodium-Calcium
CONTRACTION AND RELAXATION
Exchanger
Changes in Myocyte Size and Morphology a-Adrenergic Stimulation
Sarcolemma and Transverse Tubules Cyclic Nucleotide Phosphodiesterases
Sarcoplasmic Reticulum Calcium-Calmodulin–Dependent Protein Kinase
Contractile Elements Phosphatases
Mitochondria ■■ SUGGESTED READINGS
Cytoskeleton and Extracellular Matrix
■■ EXCITATION-CONTRACTION COUPLING
■■ INTRODUCTION relatively little known about fundamental mechanisms

As described in Chapter 1, the developing mammalian of excitation-contraction coupling and regulation of con-
heart undergoes a series of complex and tightly regulated tractile function in the immature human heart. A thor-
processes during structural organogenesis. Considerable ough understanding of the basic molecular and cellular
understanding of the genetic control of these pathways processes governing contractile function is essential for
has been gained in recent years. Of equal importance are development of rational and age-appropriate pharma-
the functional changes in cardiac contraction and relax- cological strategies for fetal and neonatal patients with
ation that accompany morphological development of the impaired cardiac contractile function.
cardiovascular system. However, compared to our under- It should be noted that current concepts of molecular
standing of structural organogenesis, much less is known and cellular aspects of myocyte function during cardiac
about the genetic, molecular, and cellular processes that development and maturation are derived mainly from
control cardiac contractile function during embryonic animal models. Limited information is available regard-
development and fetal maturation. ing these processes in the immature human heart. The list
Although age-related changes occur in the functional of suggested readings at the end of this chapter refers to
cardiac responses to pharmacological or physiological several monographs that provide a comprehensive and
interventions, understanding of the underlying mecha- detailed overview of contractile function in the mature
nisms is generally incomplete. In particular, there is heart. In addition, references are listed that provide
19

20 Chapter 2
additional information regarding developmental changes human myocardium would provide more relevant results,
in cardiac ultrastructure, metabolism, electrophysiology, but the availability of tissue for these types of research is
and responses to pathophysiological states. Integration limited, especially for fetal and neonatal human hearts.
of these myocellular changes into a larger perspective of
developmental physiology and cardiac mechanics is pre- ■■ STRUCTURAL COMPONENTS INVOLVED
sented in Chapter 3. IN CONTRACTION AND RELAXATION
■■ GENERAL OVERVIEW OF CELLULAR Changes in Myocyte Size and Morphology
ASPECTS OF CARDIAC FUNCTION In the human embryo, the elements required for rhyth-
mic contraction and relaxation of the heart become func-
Excitation, contraction, and relaxation of myocardial
tional by approximately 3 weeks after conception. The
cells are mediated by complex ion transport processes
and coordination of calcium delivery to and from the major functional units of the developing cardiac myocytes
contractile proteins (Figure 2-1). At rest, active transport are the sarcolemmal membrane, sarcoplasmic reticulum,
processes (mainly the sodium-potassium pump [Na/K- mitochondria, and contractile proteins. At the same time,
ATPase]) maintain electrochemical gradients across supporting structural components (fibroblasts and the
the sarcolemmal membrane. Consequently, a resting extracellular matrix) are also developing. Each of these
membrane potential is established with the cell interior elements undergoes progressive development and matura-
being negative relative to the extracellular space. Depo- tion throughout embryonic, fetal, and early postnatal life.
larization of the cardiac sarcolemmal membrane occurs In the early embryo and fetus, the ultrastructural appear-
largely due to opening of sodium channels, which results ance and spatial arrangement of cellular structures are
in a rapid influx of sodium and a brisk rise in membrane quite different compared to those of the fully mature heart.
potential from negative to positive values. As described Ventricular myocytes change considerably with regard
in more detail below, this change in membrane potential to size, shape, and overall appearance during the transition
is ultimately translated into an increase in intracellular from late fetus to the adult. In general, the perinatal matu-
cytosolic calcium, binding of calcium to the contractile ration phase is characterized largely by the addition of cel-
protein complex in the myofibrils, and cell shortening lular structures and more precise spatial organization of the
(contraction). Relaxation occurs as the resting sarcolem- elements involved in contraction and relaxation. Newborn
mal membrane potential is re-established, intracellular myocytes exhibit random orientation of myofibrils with
cytosolic calcium decreases, and calcium dissociates from incomplete sarcomeres. Myofibrils are frequently located
the contractile protein complex. in the subsarcolemmal region. Regularly scattered through-
These processes must be very tightly regulated to main- out the cell are ribosomes, rough endoplasmic reticulum,
tain calcium homeostasis and control of contraction and and mitochondria. Nuclei are round and centrally located.
relaxation. During the past several years, it has become Relative to cell volume, nuclear volume decreases steadily
clear that many of the pathways and proteins involved in after birth, coincident with progressive cellular hypertro-
these processes undergo developmental regulation. Con- phy. The clusters of ribosomes, rough endoplasmic reticu-
sequently, significant age-related differences exist in the lum, and extensive Golgi apparatus are all consistent with
fundamental mechanisms of cardiac contraction, relax- active protein synthesis during rapid cell growth.
ation, and regulation of contractile function. Although the general patterns are similar, the precise
As noted above, most of our knowledge regarding timing and temporal relationships of the ultrastructural
developmental aspects of cardiac contractile function is changes vary from species to species. Table 2-1 summa-
derived from animal models, such as chickens, rats, rab- rizes comparisons among various species for the timing
bits, mice, and zebra fish. A recurring problem in develop- of appearance and maturation of important elements
mental cardiology relates to extrapolating animal studies involved in cardiac contraction and relaxation. The infor-
to human physiology. Because cardiac development and mation presented in Table 2-1 is somewhat generalized
maturation are controlled by genetic and epigenetic fac- because maturation is not uniform throughout the same
tors, there may be species-specific events that are unique heart within a given species. Furthermore, considerable
for a given animal model. It is therefore useful to compare variation in morphologic appearance of myocytes can be
results from more than one animal model. Studies using found within the same heart.

MYOCYTE CONTRACTION AND RELAXATION 21
Na+ channel
K+ channel
L-type Ca2+ channel T-tubule Sarcolemma
SL Ca2+ pump
SR Ca2+ release channel
Na+ Ca2+
exchanger
Calsequestrin
Junctional SR
Na+ K+ pump
β receptor
Longitudinal SR α
GS protein
Phospholamban
Adenylyl
SR Ca2+ pump
cyclase
Myofilament
FIGURE 2-1. Schematic diagram of the major components involved in calcium transport, excitation-
contraction coupling, contraction, and relaxation in mature mammalian ventricular myocytes. At
rest, a negative membrane potential is maintained largely by the action of the sodium-potassium
pumps. Contraction in adult myocytes (see also Figure 2-5) is triggered by membrane depolarization
(opening of sodium channels), which then promotes opening of voltage-dependent L-type calcium
channels. The resulting influx of a relatively small amount of calcium causes the release of a large
amount of calcium from the junctional sarcoplasmic reticulum by triggering the opening of specific
sarcoplasmic reticulum calcium release channels. This process is termed “calcium-induced calcium
release.” The central role of T-tubules in providing the proper spatial orientation for close coupling
of L-type calcium channel calcium influx to sarcoplasmic reticulum calcium release is depicted. The
rise in cytosolic calcium results in calcium binding to troponin C, activation of the myofilaments,
and contraction. The predominant mechanism for lowering calcium to promote relaxation (see also
Figure 2-7) is the ATP-dependent reuptake of calcium into the longitudinal sarcoplasmic reticulum via
the actions of sarcoplasmic reticulum calcium pumps, which are in turn regulated by the phosphoryla-
tion state of phospholamban. During steady state, the same amount of calcium that enters the cell
is extruded, mainly by the sodium-calcium exchanger. The β-adrenergic receptor/G protein/adenylyl
cyclase complex is illustrated to signify the central role of this system in regulating cardiac contraction
and relaxation (see also Figure 2-8).
An important change during postnatal development to hypertrophy of existing myocytes, with relatively slow
is a progressive decrease in surface area–to–volume ratio. turnover and cell division. The mechanisms involved in
This is due largely to a progressive increase in myocyte this postnatal switch from hyperplastic to hypertrophic
volume during postnatal development. Myocyte division growth are incompletely defined at present. A number
(hyperplasia) is commonly observed during fetal growth of laboratories are working to unravel the basis for this
and in the early newborn period. However, shortly after fundamental change in myocyte biology since the impli-
birth, continued growth of the heart is attributable largely cations for cardiac repair and regeneration are profound.

22 Chapter 2
Sarcolemma and Transverse Tubules T-tubules limits the interaction between sarcolemmal
In cardiac myocytes, the sarcolemmal membrane is well calcium channels and sarcoplasmic reticulum calcium
defined throughout fetal and postnatal development, and release channels, inhibiting the sarcoplasmic reticulum
the glycocalyx can be visualized quite early in cardiac from participating in excitation-contraction coupling.
development. Transverse tubules (T-tubules) are invagi- Morphogenesis of T-tubules and the temporal relation-
nations of the sarcolemma into the cell that allow the ship between the postnatal acquisition of T-tubules in
extracellular environment to extend into the inner cellu- the human heart and the emergence of a mature exci-
lar structures. In species with small ventricular myocytes tation-contraction coupling phenotype remain to be
(eg, birds and fish), the T-tubular system is generally not determined.
present, presumably because it is not required for efficient
excitation-contraction coupling. However, in larger cells, Sarcoplasmic Reticulum
a T-tubular system is necessary to allow transarcolemmal Coincident with the progressive development and matu-
fluxes to occur deep within the cell interior. The presence ration of the T-tubule system is a change in the appear-
of T-tubules compensates for the increased cell volume by ance and function of the sarcoplasmic reticulum. The
providing a mechanism for overcoming diffusional restric- sarcoplasmic reticulum is a specialized form of endoplas-
tions by creating a much larger surface for ion fluxes from mic reticulum that is an essential component of contrac-
outside to inside the cell and vice versa. The T-tubular sys- tion and relaxation in the mammalian heart. In mature
tem is therefore an integral component of contraction and cardiac myocytes, the sarcoplasmic reticulum stores,
relaxation processes in mammalian myocytes. This is the releases, and reaccumulates the majority of the calcium
site of highest density of calcium channels (for the influx that is involved in contraction and relaxation.
of calcium) and the area where the sarcolemma is closest The sarcoplasmic reticulum is composed of junctional
to the sarcoplasmic reticulum (the source of activator cal- and longitudinal elements (the longitudinal sarcoplasmic
cium for myocyte contraction; Figure 2-1). reticulum is also referred to as the free sarcoplasmic retic-
In mammalian hearts, T-tubules are one of the last ulum). Important maturational changes in the amount,
organelles to develop and generally do not appear until appearance, and function of the sarcoplasmic reticulum
after birth. The time course of appearance of T-tubules are apparent during late fetal and early postnatal matura-
varies among species (Table 2-1). The relative paucity of tion. In the adult, sarcoplasmic reticulum membranes are
well organized and prominently related to the thick fila-
ments of myofibrils. The junctional sarcoplasmic reticu-
■■ TABLE 2-1. Species Differences in Maturation of lum is immediately adjacent to the T-tubules in structures
Ventricular Myocytes
termed “dyads.” It is this close physical relationship that
Maturation of is the central element in the transduction of the changes
Appearance of Sarcoplasmic in sarcolemmal membrane potential and calcium influx
Species T-Tubules Reticulum
to the sarcoplasmic reticulum that trigger calcium release
Rat 21 days of age 1 to 11 days of and contraction.
age In mature myocytes, the junctional sarcoplasmic retic-
Rabbit 8 to 10 days of 5 to 14 days of ulum contains the sarcoplasmic reticulum calcium release
age age channels. These channels (also known as ryanodine recep-
Hamster 16 days of age 1 to 30 days of tors because of their high-affinity binding to this neutral
age plant alkaloid), open during excitation-contraction cou-
Guinea pig Complete at birth Complete at birth pling to allow discharge of stored calcium into the cyto-
sol for contraction to occur. In addition, the junctional
Dog 60 days of age Postnatally
sarcoplasmic reticulum contains high concentrations of
Human 32 weeks’ First appearance calsequestrin, a calcium-binding protein. This is the site
gestation at 30 weeks’
of storage of most of the calcium that cycles through the
gestation; func-
tional maturation
sarcoplasmic reticulum to and from the contractile pro-
unknown teins with each sequence of contraction and relaxation.
Triadin and junctin are smaller proteins contained in the

junctional sarcoplasmic reticulum that are important in into a highly ordered network (see below).
maintaining the proper spatial relations between calse- The Z-discs contain projections toward the center of
questrin and the calcium release channel. Other structural the sarcomere that are termed “thin filaments” and con-
proteins include FK binding protein (also known as cal- tain an abundance of actin. Thick filaments are polymers
stabin), which helps to stabilize the calcium release chan- of myosin and titin, a very large structural protein. The
nels and promote coordinated calcium release. thick filaments are arranged along the same long axis of
Longitudinal sarcoplasmic reticulum contains the the sarcomere and are interspersed among the thin fila-
greatest density of the ATP-dependent calcium pumps ments (Figure 2-2). I-bands are composed of thin fila-
involved in calcium reuptake into the sarcoplasmic ments, troponin complexes, and tropomyosin. A-bands
reticulum. These proteins are termed “sarcoendoplas- are composed of overlapping thin and thick filaments.
mic reticulum calcium ATPases” (SERCA). The cardiac- The dark M-band in the center of the A-band consists of
specific isoform is SERCA2a. The reuptake of calcium thick filaments cross-linked to titin. Mutations in a vari-
into the sarcoplasmic reticulum by SERCA2a is modu- ety of sarcomeric and Z-disc proteins are associated with
lated by a regulatory protein, phospholamban, which is both hypertrophic and dilated forms of familial cardio-
in close physical relationship with SERCA2a. In the basal myopathy (Chapter 9).
unphosphorylated state, phospholamban acts as a “brake” In early fetal life, sarcomeres can be observed in
to inhibit SERCA2a activity and calcium reuptake into the cardiac myocytes. However, the contractile apparatus
sarcoplasmic reticulum. When phospholamban is phos- remains relatively disorganized in the fetal and early
phorylated (eg, in response to β-adrenergic stimulation), newborn heart. Myofibrils initially are irregular and scat-
the inhibition is removed, SERCA2a activity increases, tered about the interior of the cell. They are arranged in
and relaxation is facilitated due to enhanced sarcoplasmic the subsarcolemmal region around a large central mass of
reticulum calcium reuptake. nuclei and mitochondria in immature cells. As matura-
In the late fetus and early newborn, peripheral subsar- tion progresses, the myofibrils come to lie along the long
colemmal couplings between the junctional sarcoplasmic axis of the cell. Progressive organization of the sarcomeres
reticulum and sarcolemma can be observed prior to the occurs even as contraction and relaxation are occurring.
acquisition of a well-organized T-tubular system. How- A progressive increase in myofilament content and
ever, as the cells enlarge and the T-tubular system devel- maturation of the sarcomeres is consistent across mam-
ops, deeper internal couplings (dyads) are acquired. The malian species, although the timing may vary. A devel-
volume and distribution of the sarcoplasmic reticulum opmental increase in the myofibrillar population with a
increase during late fetal and early postnatal maturation. more orderly arrangement of myofilaments during matu-
Each of the components of the mature sarcoplasmic retic- ration is often cited as the primary factor responsible for
ulum undergoes developmental regulation and matura- the increase in force generation observed during the late
tion (ryanodine receptors, SERCA2a, phospholamban, fetal and early newborn period in the mammalian heart.
etc.). However, developmental changes in sarcomeric protein
isoform expression also play an important role in matu-
Contractile Elements rational changes in force generation (see below).
Sarcomeres Myofilament Proteins
The sarcomere is the primary contractile unit of striated Myofilaments are composed predominantly of myosin
muscle (Figure 2-2). Contraction and relaxation of cardiac and actin (approximately 80% of the total contractile
muscle depends on the structural organization of contrac- protein content). Other less abundant components, such
tile and modulatory proteins into filaments that repeat- as troponin and tropomyosin, provide regulatory control
edly move back and forth past one another. Anchoring of and structural support (Figure 2-2).
the structural filaments, combined with sliding of com-
plementary filaments, results in shortening and genera- Giant filament. An essential component of the struc-
tion of force. In mature cardiac cells, the myofibrils are tural foundation of the sarcomere is titin, the largest pro-
organized into sarcomeres, which are delineated at each tein known in humans. Titin spans half of the sarcomere
end by Z-discs. The Z-disc is a complex group of proteins from the Z-disc at the end to the M-line at the center. The
that links the myofilaments from contiguous sarcomeres N-terminal ends of titin overlap within the Z-disc, and the

24 Chapter 2
Sarcomere
Myofibril
Thick filament Thin filament

Titin M-line
Cypher/Zasp
α-Actinin
MLP
Telethonin
Z-disc A-band I-band
Sarcomere
α-Tropomyosin Troponin complex Actin
FIGURE 2-2. Schematic diagram showing arrangement of thick and thin filaments within the sar-
comere. Thick filaments contain myosin, myosin-binding protein C, and titin. The tails of the myosin
heavy chains are woven together to form the thick filament. The globular head projects outward to form
cross bridges. The thin filaments include actin (which forms the backbone of the thin filament), the
troponins, and tropomyosin. Tropomyosin binds to troponin T at multiple sites along the major groove
of the actin filament and inhibits actin-myosin interaction. Troponin T binds the troponin complex to
tropomyosin, troponin I inhibits interactions between actin and myosin, and troponin C binds calcium.
The sarcomere, which lies between two Z-discs, is anchored by interactions between titin and actin
with Z-disc proteins, including LIM domain-binding protein 3 (cipher/ZASP), α-actinin, cardiac LIM
domain protein (MLP), and telethonin. Adapted from Mahony L. Development of myocardial structure
and function. In: Allen HD, Driscoll DJ, Shaddy RE, Feltes TF, eds. Moss and Adams’ Heart Disease in
Infants, Children and Adolescents, 7th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2008:577.

C-terminal ends overlap at the M-line to create a continu- calcium-binding sites on TnC, attachment to the low-
ous filament system (so-called giant filament) that aligns affinity site II is thought to be the trigger for cross-bridge
the thick filaments within the myofibril. formation. Binding of calcium to this site removes the
Titin does more than simply connect the myofibrils. inhibition that the tropomyosin-troponin complex con-
The segment of titin at the I-band contains serial spring fers on the actin-myosin interaction and triggers force
elements that determine passive tension of the myocytes, generation and contraction. The number of cross bridges
thereby impacting late diastolic filling and resting sarco- formed contributes to the amount of force developed by
mere length. This likely contributes to the Frank Starling the contracting myocyte and depends on the amount
mechanism. In addition, this extensible region provides of calcium released by the sarcoplasmic reticulum and
elastic recoil that drives early diastolic filling. intrinsic properties of the contractile proteins, such as
calcium sensitivity. At any given calcium concentration,
Thick filaments. The predominant protein in thick fila- an increase in calcium sensitivity results in greater force
ments is myosin. Individual thick filaments contain sev- and the rate of force generation. Conversely, a decrease
eral different polypeptides comprising a globular head in calcium sensitivity reduces force and the rate of force
and a tail section (Figure 2-2). Each of the approximately generation. Relaxation occurs as calcium dissociates from
300 myosin molecules in each sarcomere is composed of TnC and the proteins return to their resting conforma-
two heavy-chain myosin monomers and a stalk of four tional states.
light-chain monomers. The heavy chains form a large, Regulation of contraction and relaxation is very com-
bilobed globular head and contain its ATPase activity, plex and cannot be explained simply by delivery of cal-
essential to release the energy necessary for cross-bridge cium to and removal from the contractile proteins. There
formation. The light chains (essential and regulatory) is compelling evidence that cytosolic calcium peaks well
form the long stalk of the thick filament, joined to the before maximum force production is achieved and that
head by a flexible joint. It is the globular head that forms calcium is nearly completely removed from the con-
the cross bridge with the actin molecule, the ultimate step tractile elements before the onset of relaxation. These
in sarcomere shortening. observations suggest that cooperative signaling and
An important protein associated with the thick fila- mechanical feedback through thick and thin filaments
ments is myosin-binding protein C. This protein helps to are critical determinants of contraction, force generation,
stabilize thick filaments by forming transverse fibers that and relaxation. In addition, a growing body of literature
connect adjacent filaments near the center of the sarco- implicates posttranslational modification of sarcomeric
mere. Other important structural proteins at the M-line proteins (phosphorylation, nitrosylation, and peroxida-
include myomesin, M-protein, and obscurin, all of which tion) via various signaling pathways as important regu-
appear to act with myosin-binding protein C to facilitate lators of calcium sensitivity and cross-bridge cycling
force transmission along the thick filaments. rates. The relationships among the various myofilament
proteins, cytoskeletal proteins, extracellular matrix, and
Thin filaments. Each thin filament contains three major
signal transduction pathways are complex and not fully
proteins: actin, tropomyosin, and troponin (Figure 2-2).
understood. However, mutations or abnormalities in the
Repetitive association and dissociation of actin with
function of many of these proteins clearly can have pro-
myosin results in contraction and relaxation. Troponin
found effects on cardiac contractile function. Mutations
is composed of three major subunits. The inhibitory sub-
in myofilament proteins are associated with cardiomyop-
unit is troponin I (TnI), the calcium-binding subunit is athy, which is more fully described in Chapter 9. As our
troponin C (TnC), and the tropomyosin-binding sub- understanding of intrinsic sarcomere function and regu-
unit is troponin T (TnT). These proteins are required lation continues to expand, it is likely that new therapeu-
in the final stages of activation of actin binding with tic approaches targeted at improving contractile function
myosin to form the actomyosin complex, which initiates will be developed, but the implications for newborns with
contraction. heart disease are not defined.
Calcium plays an integral role in relaxation and
contraction by binding to one of the troponin sub- Isoform switching. Developmental changes in thick and
units, troponin C (TnC). Although there are several thin protein isoforms impact the ability of the sarcomere

26 Chapter 2
to contract and relax. Important age-related changes in contrast, both TnI and TnT exist as multiple isoforms
regulatory processes include changes in myofibrillar with variable expression during development.
ATPase activity (determined by differential expression of Three isoforms of TnI have been identified that are
myosin isoforms), troponin regulatory proteins, and pro- the products of three separate genes. These isoforms are
teins involved in delivery of calcium to and removal from classified as the fast skeletal form (TnI-f), the slow skel-
the contractile complex. In addition to developmental etal form (TnI-s), and the cardiac muscle form (TnI-c).
regulation, other factors may affect isoform expression. A major difference of the cardiac isoform from the other
Hormonal changes, nutritional status, workload, and two forms is that TnI-c (but not TnI-s) has a long inter-
innervation all play an integrated role in regulating pro- nal sequence that becomes phosphorylated in response to
tein isoform expression in the developing heart. adrenergic stimulation. Phosphorylation of TnI-c plays
an important role in modulating contractile performance
Titin isoforms. Titin is encoded by a single gene, but by effects on cooperative activation and mechanical feed-
differential splicing generates isoforms with different back at the level of the sarcomeres.
degrees of extensibility. Titin isoform expression is devel- Developmental changes in TnI isoform expression are
opmentally regulated. In general, fetal hearts express proposed to at least partly explain changes in contractile
more compliant titin isoforms than those expressed in function during perinatal maturation of the heart. In the
mature hearts; the physiologic impact in humans is not human fetal heart, the predominant isoform is TnI-s, but
known. TnI-c is detectable. The transition to TnI-c alone with
the disappearance of TnI-s occurs by approximately 9
Myosin isoforms. The cardiac myosin heavy chain months of age in the human heart. This extended postna-
(MHC) exists in two isoforms, α-MHC and β-MHC. tal time course of TnI isoform switching is likely to affect
Myosin containing two α chains (V1) has the highest inotropic responsiveness.
ATPase activity. V2 contains an α-chain and a β-chain TnT plays a role in regulating myofibrillar ATPase
and exhibits intermediate ATPase activity. Myosin con- activity and the responsiveness to calcium. Troponin T
taining two β-chains (V3) has the lowest ATPase activity. exists as multiple isoforms that appear to be products of
ATPase activity of the heavy-chain correlates with veloc- a single gene that undergoes developmentally regulated
ity of shortening of the myofibril. Myosin heavy-chain alternative splicing. Expression patterns during develop-
isoform expression changes during development in some ment vary among species. Fetal human and rabbit hearts
species, but in the human ventricle, the V3 isoform pre- express four cardiac TnT isoforms, TnT1-4. Expression of
dominates during fetal, neonatal, and adult life. TnT1 is highest in the fetal human heart and neonatal rab-
bit heart. The dominant isoform in adult rabbit myocar-
Actin isoforms. The backbone of the thin filaments is
dium is TnT4, and in contrast, normal adult human hearts
formed by polymerization of two strands of actin mono-
express only TnT3. Isoform expression is also affected by
mers. Actin is a relatively small globular protein encoded
pathophysiological conditions. Cardiac cTnT1 and cTnT4
by a multigene family. Two isoforms, skeletal α-actin and
are up-regulated in failing human hearts harvested from
cardiac α-actin, are present in striated muscle. These iso-
forms differ by only four amino acids, two of which are transplant patients and in hearts from children with con-
in the myosin-binding region. During development, both gestive heart failure. The level of expression of cTnT4 is
actin isoforms are expressed in the human ventricle. In correlated with the severity of heart failure before surgery
fetal and neonatal hearts, skeletal α-actin mRNA consti- and with the duration of recovery. Changes in cardiac
tutes more than 80% of the total actin, but in the mature TnT isoform expression (both during development and
heart, the cardiac α-actin isoform constitutes more than in response to pathophysiological states) may contribute
60% of the total actin. The functional consequence of this to changes in force development and to differences of
isoform shift in the human heart is unknown. myocardial sensitivity to acidosis.
Troponin isoforms. The troponin complex of the thin Mitochondria

filament confers calcium sensitivity to the actin-myosin In immature myocardium, mitochondria are irregularly
complex. TnC, the calcium-binding subunit, remains scattered about the cell. As the cells mature, mitochondrial
constant and does not exhibit isoform switching. In size becomes more regular, and mitochondria become

centrally located and surrounded by myofilaments. To resting sarcomere length is shorter, and sarcomere short-
meet the high energy requirements of active muscle, the ening is slower. Thus, the relative disorganization of the
mitochondria become distributed in a highly regular fash- cellular structures due to cytoskeletal immaturity may
ion along the myofilaments as myofibrillar organization have an important impact on the rate and amount of ten-
advances. sion that an immature myocyte can generate.
In all mammalian species, a large increase in mito- The cytoskeleton also fosters the spatial arrange-
chondrial volume occurs during the postnatal period. In ment of subcellular protein complexes that is required
addition, the ultrastructural appearance of myocardial for proper intracellular signaling. Cytoskeletal struc-
mitochondria changes during maturation. In the fetal tural proteins allow for communication between internal
heart, cristae are sparse and widely spaced. With progres- and external environments of the cell and are integrally
sive maturation of the heart during the postnatal period, involved in signal transduction and cell-to-cell signaling.
the cristae become more densely packed. Cytoskeletal proteins organize the colocalization of ion
These changes in mitochondrial appearance, position, channels, signaling molecules, and messengers required
and number reflect the increasing energy requirements for transduction of extracellular signals and mechanical
following birth and parallel age-related changes in sub- stress. Mutations in several cytoskeletal proteins cause
strate utilization by the heart. Long-chain free fatty acids various form of cardiomyopathy (Chapter 9).
are the primary energy substrate in adult hearts. Activated The cytoskeleton is a much more dynamic struc-
free fatty acids are transported into the mitochondria and ture than what was originally thought. The cytoskeleton
then are metabolized by β-oxidation, producing ATP. remodels during cell growth and in response to patho-
The enzyme carnitine palmitoyl coenzyme A transferase physiologic signals, such as excessive systolic stress or
transports activated free fatty acids from the cytosol into diastolic stretch. Normal development and maturation
the mitochondria. In immature hearts, the activity of this of the heart and vasculature are critically dependent on
enzyme is decreased. As a result of these and other factors, proteins within the cytoskeleton and extracellular matrix.
the primary energy substrates in the immature heart are Developmental changes in the organization and location
lactate and carbohydrates. It should be noted that these of intracellular organelles reflect changes in the composi-
data are derived from animal studies and comparable tion and organization of the cytoskeleton.
information from fetal and neonatal human hearts is lack-
ing. Abnormalities in mitochondrial substrate metabo- Z-Discs
lism can result in cardiomyopathy (Chapter 9). At each end of the sarcomere is the Z-disc, which links
the myofilaments from opposing sarcomeres into a tightly
Cytoskeleton and Extracellular Matrix arranged compact lattice. A variety of proteins are local-
ized to Z-discs, including α-actinin, telethonin (T-cap),
The cytoskeleton and extracellular matrix supports the nebulette, muscle LIM protein, filamin, and myotilin.
various components of the cardiac myocyte during con- Z-discs are crucial elements in the transmission of tension
traction and relaxation. The cytoskeleton determines generated by individual sarcomeres along the length of
cell size and organization and allows tension developed the myofibril. In addition, Z-disc proteins serve as dock-
by the contractile proteins to be transmitted through- ing sites for transcription factors, calcium-signaling pro-
out the myocyte, to adjacent cells, and to the extracellu- teins, and a variety of kinases and phosphatases involved
lar matrix. The ultrastructural appearance of immature in regulation of contractile function. Increasing evidence
myocytes suggests that the cytoskeletal structure is much indicates that the Z-disc and its associated components
less organized compared with adult myocytes. Adult cells sense cellular mechanical signals and transduce them
are compartmentalized by intermediate filaments that into signals for cell growth, development, and remodel-
connect Z-discs to one another. The linking of adjacent ing (both electrical and mechanical).
sarcomeres organizes the cell and compartmentalizes
sarcomeres with longitudinal sarcoplasmic reticulum, Extramyofibrillar Cytoskeleton
mitochondria, and microtubules. In contrast, immature Microfilaments, intermediate filaments, and microtubules
myocytes contain central aggregations of nuclei and mito- make up the three major extramyofibrillar cytoskeletal
chondria. This results in an internal load against which components (Figure 2-3). Microfilaments are composed
the immature myofibrils contract. Consequently, the predominantly of actin and are found in the thin filaments

28 Chapter 2
Collagen
Cell
adhesion
Proteoglycan Cadherin
Laminin-2 Ion molecule
channel
Fibronectin
β α
β-Dystroglycan α-dystroglycan Ion pump
Caveolin Sacrospan
α γ β δ
nNOS Sarcoglycan
Ankyrin
β-Syntrophin Integrins
Dystrobrevin
α -Syntrophin Plakoglobin
Paxillin
Dystrophin
Vinculin Vinculin
Desmin Spectrin β-catenin
Talin
intermediate
filaments α-actinin α-catenin
α-actinin
Cytoplasmic
actin
Z-discs
FIGURE 2-3. Cytoskeletal architecture of the cardiac myocyte. The dystrophin-sarcoglycan complex
links the plasma membrane to cytoplasmic actin filaments. Integrins bind the cell to the extracellular
matrix and are composed of α- and β-subunits that attach through various proteins (fibronectin, laminin,
and proteoglycans) to collagen. The intracellular part of the β-subunit binds to cytoplasmic actin fila-
ments through another protein complex that includes α-actinin, talin, vinculin, and paxillin. Ankyrin links
membrane proteins such as ion pumps and channels together, thereby organizing interactions among
proteins with related functions. Spectrin links ankyrin to the cytoplasmic actin filaments and is also
associated with both costameres and intercalated discs at the sarcolemmal membrane. Cadherins
mediate connection of cell-to-cell contacts in both desmosomes and fascia adherens. In the fascia
adherens, cadherins link the cytoplasmic actin filaments of adjacent cells through vinculin, catenins,
plakoglobin, and α-actinin. In addition to contributing to the structural integrity of the cell, many of these
proteins are critically involved in cell signaling. Adapted from Mahony L. Development of myocardial struc-
ture and function. In: Allen HD, Driscoll DJ, Shaddy RE, Feltes TF, eds. Moss and Adams’ Heart Disease
in Infants, Children and Adolescents, 7th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2008:580.
of the sarcomere (sarcomeric actin) and in the cytosol (non- link Z-discs together and to costameres, the sarcoplasmic
sarcomeric or cytoplasmic actin). Cytosolic microfilaments reticulum, and sarcolemmal and nuclear membranes.
are localized mainly in the subsarcolemmal space and play Stress-induced alterations of this network of intermedi-
an important role in linking the intracellular cytoskeleton ate fibers may mediate changes in gene expression.
with the extracellular matrix and adjacent myocytes. Microtubules are formed from polymerized subunits
Intermediate fibers form an intracellular network that of α and β tubulin that undergo continuous depolymeriza-
helps to maintain the structural integrity of myocytes. tion and repolymerization. Stiffness of the cytoskeleton is
These fibers are formed from polymers of desmin and moderated by the total amount of tubulin and the relative

proportions in the polymerized state. Microtubules sur- cell signaling and gene expression that can result in cell
round the nucleus and spread longitudinally throughout growth or hypertrophy.
the cell. They serve to stabilize cell structure by anchoring Integrins are another essential component of the pro-
subcellular organelles and are involved in the transmis- tein network responsible for transmission of force between
sion of signals within and between cells. myocytes and the extracellular matrix. Integrin molecules
are heterodimers of various α- and β-subunits and found
Connections to Adjacent Cells and the within the sarcolemmal membrane adjacent to costameres.
Extracellular Matrix The intracellular domain of integrin β-subunits binds to
The extracellular matrix surrounds myocardial cells and actin microfilaments through costamere proteins, and
serves both structural and regulatory functions. The the extracellular portion of integrin interacts with various
extracellular matrix is complex and contains a variety of extracellular matrix proteins. Integrins are involved in sig-
components, including (1) connective tissue, mainly elas- naling pathways mediated by several G-proteins and pro-
tin and various types of collagen; (2) a gel-like substance tein kinases that modify interactions between the integrins
consisting of proteoglycans; (3) basement membrane and the extracellular matrix. These pathways are involved
proteins, such as collagen, laminin, and fibronectin; and in both cell growth and apoptosis and play an essential role
(4) other molecules, such as cytokines, growth factors, in pathophysiological conditions and in normal embry-
and proteases. The extracellular matrix modulates cell onic and fetal cardiovascular development.
migration, proliferation, adhesion, and cell-to-cell signal- The dystrophin glycoprotein complex also plays an
ing, thereby playing a critical role in normal growth and important role in linking the intracellular cytoskeleton to
development as well as in pathological remodeling of the the extracellular matrix. This complex binds actin micro-
ventricles. filaments to the extracellular matrix, thereby transmitting
Intercalated discs connect myocytes to adjacent myo- force to the extracellular matrix and providing another
cytes and serve as important sites of force transmission mechanism for signal transduction. Mutations in dys-
between myocytes. Intercalated discs are complex struc- trophin cause muscular dystrophy, and mutations in the
tures that contain fascia adherens (analogous to adher- components of the dystrophin glycoprotein complex are
ens junctions in nonmuscle cells), desmosomes, and gap present in some patients with dilated cardiomyopathy.
junctions. A variety of proteins are involved in connect- Spectrin is another cytoskeletal protein that binds to
ing cells both structurally and functionally by weaving actin and helps to coordinate membrane signaling sys-
together the various cytoskeletal elements. tems with the contractile filaments. Spectrin is found in
The many protein complexes that link the myofibrils costameres and intercalated discs and is a component of
and membrane systems of the myocyte to the extracellular protein complexes related to the sarcoplasmic reticulum
matrix are essential for stabilizing cell structure during at the Z-discs. Ankyrins are adaptor proteins that bind to
the stresses of contraction and relaxation. Additionally, spectrin and also to a structurally diverse group of mem-
these protein complexes are involved in organizing and brane proteins, such as ion channels and pumps, calcium-
coordinating membrane signaling processes with the release channels, and cell adhesion molecules. In this
contractile systems. The various cytoskeletal networks manner, various proteins are physically linked together
within the myocyte and the extracellular matrix are linked to foster interactions among proteins with related func-
through highly complex protein networks called costa- tions. Mutations in ankyrins are known to cause cardiac
meres (Figure 2-4). These structures, which contain pro- arrhythmias in humans and in murine models.
teins including vinculin, talin, tensin, paxillin, and zyxin,
encircle the lateral aspects of the myocyte perpendicular ■■ EXCITATION-CONTRACTION COUPLING
to its long axis, forming a transmembrane physical attach-
ment between the peripheral Z-discs and the extracellu- Initiation of Contraction in the
lar matrix. Costameres thus anchor the myofibrils to the Embryonic Heart
sarcolemma and transmit lateral contractile force from A functional circulation must be established for the
the sarcomere to the extracellular matrix and ultimately embryo to survive. This requires the developing heart
to neighboring myocytes. It appears that costameres play to begin to function as a rhythmic pump. Mature ven-
a role in converting mechanical stimuli to alterations in tricular myocytes are not spontaneously active and rely

30 Chapter 2
ECM
ion channels Dystroglycan

complex
Spectrin
Ankyrin Dystrophin integrin
Titin
Talin Kindlin
FAK Filamin
Kindlin ILK PINCH
Parvin
Paxillin
Vinculin
F-actin
nin
ti
Ac
α-
Myotilin Titin
Telethonin/
Cypher L Myopaliadin T-cap
MLP
ENH Titin
CapZ Cypher S
Calsarcin Nebulette
FIGURE 2-4. Costameric proteins associated with Z-lines. Dystrophin glycoprotein complex and
integrin–vinculin–talin complex are two major costameric protein complexes. Members of the dys-
trophin glycoprotein complex and integrin were reported to directly interact with protein components
at the Z-line, such as filamin-C. Filamin-C (γ -filamin and filamin2) physically links the costamere and
the sarcomere by interacting with two major costameric protein complexes: the sarcoglycans in the
dystrophin glycoprotein complex and integrin. In addition, filamin-C interacts with the Z-line proteins
calsarcin-1 and myotilin. Vinculin, the founding member of the costamere, interacts with multiple
proteins, including talin, paxillin, and α-actinin. Integrin interacts with the ILK-pinch-parvin complex
and FAK. Dystrophin binds to ankyrin B and G, which are essential to organize the dystrophin and
dystroglycan complex. Titin is the largest known protein and crosses longitudinally from the Z-line to
the M-line. The N-terminus of titin at the Z-line binds to ankyrin. Not all costameric proteins or Z-line
proteins are illustrated in this figure for simplicity. Key proteins are shown to highlight the bridges
between the sarcomere and the costamere. Reprinted with permission from Peter AK, et al. The costa-
mere bridges sarcomeres to the sarcolemma in striated muscle. Prog Pediatr Cardiol 2011;31:83-88.
Copyright © 2011 with permission from Elsevier.

on transmission of electrical signals from the pacemaker in the T-tubules) in close physical relation to junctional
cells in the sinoatrial node. sarcoplasmic reticulum ryanodine receptors in the dyadic
The mechanisms involved in establishing the beating junction. These structural components are known to
heart have been clarified in recent years. Two distinct undergo developmental changes that consequently impact
mechanisms were proposed in the past. Originally, it was significantly on the mechanisms of excitation-contraction
thought that because the sarcoplasmic reticulum is sparse coupling in the immature heart.
and underdeveloped in embryonic myocytes, spontane- Figure 2-5 illustrates the general features of excita-
ous depolarization of the sarcolemmal membrane trig- tion-contraction coupling and calcium transport during
gers calcium influx through voltage-activated calcium contraction in mature mammalian ventricular myocytes.
channels. Subsequently, it was proposed that spontane- Depolarization of the sarcolemmal membrane promotes
ous sarcoplasmic reticulum calcium oscillations drive opening of voltage-dependent L-type calcium channels.
contractions and electrical activity. Leaking of sarcoplas- This results in the influx of a relatively small amount of
mic reticulum calcium into the cytosol would activate the calcium, which alone is not sufficient to directly acti-
sodium-calcium exchanger to generate action potentials. vate the contractile proteins. It is clear that contraction
Recent experimental evidence suggests that both path- results from the much greater increase in intracellular
ways may be involved independently to generate action calcium that results from release of a large amount of cal-
potentials and global calcium transients. During sponta- cium from sarcoplasmic reticulum stores. The opening
neous sarcoplasmic reticulum calcium oscillations, the of specific sarcoplasmic reticulum calcium release chan-
sodium-calcium exchanger extrudes calcium and triggers nels (also known as ryanodine receptors) is triggered by
an action potential that then promotes calcium influx and the influx of calcium across the sarcolemma through the
restoration of the sarcoplasmic reticulum calcium store L-type calcium channels. This process is known as calcium-
so that the process can be repeated. Inositol 1,4,5 trispho- induced calcium release.
sphate may play an important role in this process. Con- The structural basis for calcium-induced calcium
versely, if the action potential occurs spontaneously, it release is the colocalization of sarcolemmal L-type cal-
can result in a global whole-cell calcium transient as well. cium channels (predominantly in the T-tubules) and
Having both mechanisms available allows embryonic car- junctional sarcoplasmic reticulum ryanodine receptors
diomyocytes to contract without requiring physical con- in the dyadic junction (Figure 2-5). The close physical
nections to other myocytes, enabling cell migration. relationship of L-type calcium channels and ryanodine
Although we have a better understanding of how the receptors (~10 nm) in this region allows a single or a
embryonic heart begins to contract, the question of why small cluster of ryanodine receptors to open in response
the embryonic heart begins to beat rhythmically is per- to a local, spatially restricted microdomain of elevated
haps less clear. The mammalian embryonic heart begins calcium concentration that results from the nearby influx
to beat before tissues need to be perfused with blood (and, of calcium through an L-type calcium channel. Localized,
indeed, before the blood cells have formed). Recently, nonpropagating sarcoplasmic reticulum calcium release
clues have been provided from studies of zebrafish and events (either evoked or spontaneously occurring) have
mice that indicate that a beating heart is necessary for been commonly termed “calcium sparks.” It is the sum-
normal vascular development and for development of mation of these local release events that results in a global
the hematopoietic system. Shear stress from fluid flow- rise in intracellular calcium and activation of contrac-
ing through the developing vascular bed stimulates the tion. Modulation of the force of contraction is achieved
expression of hematopoietic stem cells. Nitric oxide may primarily by regulation of the magnitude of the L-type
be a critical signal for this process. The mechanisms calcium current. Evidence suggests that calcium-induced
responsible for signaling the embryo that it is time to inactivation of ryanodine receptors is the primary mecha-
establish the circulation remain elusive. nism for the beat-to-beat termination of calcium-induced
calcium release. Contraction is terminated and relaxation
Mechanisms of Contraction and Relaxation occurs because of (1) a decrease in sarcoplasmic reticulum
The main components of the mature excitation-contraction calcium release channel openings in conjunction with
coupling phenotype of calcium-induced calcium release (2) the reuptake of cytosolic calcium back into the sarco-
are sarcolemmal L-type calcium channels (predominantly plasmic reticulum through calcium pumps (and, to a much

32 Chapter 2
L-type Ca2+ channel
SR Ca2+ release channel
Calsequestrin FK-Binding protein
FIGURE 2-5. Schematic diagram of excitation-contraction coupling in mature mammalian ventricu-

lar myocytes. The close relationship between L-type calcium channels and sarcoplasmic reticulum
calcium release channels is illustrated. Calcium (blue circles) entering the cell through L-type calcium
channels (concentrated in the T-tubules) triggers the opening of specific calcium release channels in
the junctional sarcoplasmic reticulum. When this occurs, a large amount of calcium is released into
the cytosol, resulting in contraction of the myofilaments. Sarcoplasmic reticulum calcium release
channels are also known as ryanodine receptors. The major calcium binding protein in the sarcoplas-
mic reticulum is calsequestrin. FK binding proteins maintain stability of the sarcoplasmic reticulum
calcium release channels and promote coordinated interaction among neighboring channels. See text
for additional details.
lesser extent, calcium extrusion from the cell via sodium- In addition to providing the source of calcium for con-
calcium exchange and sarcolemmal calcium pumps). traction, the sarcoplasmic reticulum plays a primary role
Morphological considerations in immature myo- in relaxation in the adult heart. After the rise in intracel-
cytes support the feasibility of transsarcolemmal calcium lular calcium that occurs after calcium release from the
influx as a direct source of activator calcium for contrac- junctional sarcoplasmic reticulum, specific calcium pumps
tions (Figure 2-6). Despite a lack of T-tubules, fetal and in the longitudinal sarcoplasmic reticulum sequester cal-
newborn myocytes are relatively small and have a much cium back into the sarcoplasmic reticulum (Figure 2-7).
higher cell surface area–to–volume ratio than adult myo- As cytosolic calcium declines, calcium dissociates from
cytes. Ryanodine receptors are not in close proximity to the contractile protein complex, and relaxation occurs.
the sites of calcium entry across the sarcolemma in imma- Sarcoplasmic reticulum calcium pump activity is in turn
ture myocytes. This feature and the subsarcolemmal posi- regulated by the phosphorylation state of phospholam-
tion of myofibrils in fetal and newborn myocytes favor ban, which provides another key mechanism for modulat-
direct transsarcolemmal calcium delivery to (and from) ing contractile function.
the contractile proteins for contraction and relaxation in During steady-state conditions, the amount of calcium
immature ventricular myocytes (Figure 2-6). that enters the myocyte from the extracellular space must

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■■ INTRODUCTION understanding of mutations in humans that lead to con-

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■■ THE NEURAL CREST ■■ BREAKING SYMMETRY

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Arterial pole RVV LVV

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Development Congenital/disease Stem cells

Endocardium Endocardial cushions Cardiac

Myocyte line Endocardial line

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FIGURE 1-5. Anomalous pulmonary venous connection. A. Scimitar syndrome is characterized by a

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RCA LCA RCA LCA

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degenerates during development (Figure 1-7F). In mam-

ascending aorta and pulmonary trunk, including their

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Atrioventricular Valves Semilunar Valves

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Implications for Congenital Cardiovascular Disease

Stenosis or atresia of an atrioventricular valve. The

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FIGURE 1-11. Ventricular-coronary arterial communications. A. Schematic representation of a heart

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■■ INTRODUCTION relatively little known about fundamental mechanisms

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