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\begin{document}

\title{A Reassessment of the anatomical features of multiple ventricular septal
defects}



\author[1]{Diane E. Spicer}%
\author[2]{Robert Anderson}%
\author[3]{Ujjwal Chowdhury}%
\author[3]{Lakshmi  Sankhyan}%
\author[4]{Niwin George}%
\author[3]{Niraj Pandey}%
\author[3]{Saurabh Gupta}%
\author[3]{Shikha Goja}%
\affil[1]{All Children's Hospital Foundation}%
\affil[2]{1Institute of Genetic Medicine, Newcastle University, Newcastle-upon-Tyne, United Kingdom Birmingham Children’s Hospital, Birmingham, United Kingdom}%
\affil[3]{All India Institute of Medical Sciences}%
\affil[4]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 19, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Over the course of time, new developments associated with embryogenesis
of the murine heart have served to clarify the developmental processes
observed in the human heart. This evidence allows for creation of a
developmental framework for many congenital cardiac defects. Here, we
aim to solidify the framework related to the categorization of both
solitary and multiple ventricular septal defects. Mice having genetic
perturbation of the Furin enzyme have demonstrated perimembranous and
juxta-arterial ventricular septal defects, permitting the inference to
be made that these defects can co-exist with defects occurring within
the apical muscular septum. Based on developmental evidence,
furthermore, all interventricular communications can be placed into one
of three groups, namely, those which are perimembranous, juxta-arterial,
and muscular. All of the defects are described based on their borders as
seen from the morphologically right ventricle. Our focus here will be on
those defects within the muscular ventricular septum, recognizing that
such defects can co-exist with those that are perimembranous. We discuss
the differentiation of multiple discrete defects from those referred to
as the `Swiss cheese' variant. As we show, appropriate surgical
management requires understanding of the specific terminology, as the
surgical approach may differ depending on the combination of the
individual defects. Data from the Society for Thoracic Surgeons revealed
that both mortality and morbidity were increased in the setting of
multiple as opposed to solitary ventricular septal defects.%
\end{abstract}%



\sloppy


\textbf{A Reassessment of the anatomical features of multiple
ventricular septal defects}

\textbf{Running title: Multiple ventricular septal defects}

\textbf{Diane E. Spicer, BSc\textsuperscript{1}}

\textbf{Robert H. Anderson, BSc, MD, PhD (Hon), FRCPath, FRCS Ed
(Hon)\textsuperscript{2}}

\textbf{Ujjwal Kumar Chowdhury, MCh, Diplomate NB\textsuperscript{3}}

\textbf{Lakshmi Kumari Sankhyan, MCh\textsuperscript{4}}

\textbf{Niwin George, MCh\textsuperscript{3}}

\textbf{Niraj Nirmal Pandey, DM\textsuperscript{3}}

\textbf{Saurabh Kumar Gupta, DM\textsuperscript{3}}

\textbf{Shikha Goja, MS\textsuperscript{3}}

\textsuperscript{1}Heart Institute, Johns Hopkins All Children's
Hospital, St. Petersburg, Florida, USA

and Department of Pediatric Cardiology, University of Florida,
Gainesville, Florida, USA

\textsuperscript{2}Institute of Biomedical Sciences, Newcastle
University, Newcastle-upon-Tyne, United Kingdom

\textsuperscript{3}Cardiothoracic Centre, All India Institute of Medical
Sciences, New Delhi, India

\textsuperscript{4}Department of Cardiothoracic Surgery, All India
Institute of Medical Sciences, Bilaspur, Himachal Pradesh, India

\emph{Corresponding author:}

\textbf{Dr. Ujjwal Kumar Chowdhury, M.Ch., Diplomate NB}

Professor

Department of Cardiothoracic and Vascular Surgery

AIIMS, New Delhi-110029, INDIA

Tel.: 91-11-26594835

Fax: 91-11-26588641

Email: ujjwalchowdhury@gmail.com

Orcid ID: http: //orcid.org/0000-0002-1672-1502

\textbf{Word Count:} Abstract (229), Text (2747), References (595),
Figure Legends (528)

\textbf{Statement of human rights/ethical approval:} The authors assert
that all procedures contributing to this study comply with the ethical
standards of the relevant national guidelines on human experimentation
and with the Helsinki declaration of 1975, as revised in 2008.

\textbf{Conflict of interest} : No potential conflicts exist

\textbf{Source of funding} : No funding was provided

\textbf{Availability of Data:} Data available in the articles cited in
references.

\emph{}

\textbf{Abstract}

Over the course of time, new developments associated with embryogenesis
of the murine heart have served to clarify the developmental processes
observed in the human heart. This evidence allows for creation of a
developmental framework for many congenital cardiac defects. Here, we
aim to solidify the framework related to the categorization of both
solitary and multiple ventricular septal defects. Mice having genetic
perturbation of the Furin enzyme have demonstrated perimembranous and
juxta-arterial ventricular septal defects, permitting the inference to
be made that these defects can co-exist with defects occurring within
the apical muscular septum. Based on developmental evidence,
furthermore, all interventricular communications can be placed into one
of three groups, namely, those which are perimembranous, juxta-arterial,
and muscular. All of the defects are described based on their borders as
seen from the morphologically right ventricle. Our focus here will be on
those defects within the muscular ventricular septum, recognizing that
such defects can co-exist with those that are perimembranous. We discuss
the differentiation of multiple discrete defects from those referred to
as the `Swiss cheese' variant. As we show, appropriate surgical
management requires understanding of the specific terminology, as the
surgical approach may differ depending on the combination of the
individual defects. Data from the Society for Thoracic Surgeons revealed
that both mortality and morbidity were increased in the setting of
multiple as opposed to solitary ventricular septal defects.

\textbf{Keywords:} Congenital heart disease, Muscular ventricular septal
defects, Swiss-cheese ventricular septal defects



\textbf{Introduction}

Deficient ventricular septation is the commonest abnormality found when
the heart is congenitally malformed. It can be found in isolation, or as
an integral part of multiple lesions. When encountered as the major
problem, the arrangement is appropriately described in terms of
ventricular septal defects. When deficient septation is part of lesions
such as double outlet right ventricle, or common arterial trunk, the
channel between the ventricles is more accurately considered as an
interventricular communication.\textsuperscript{1} Such distinctions,
however, are largely of semantic interest. The understanding of the
differences in the terms, nonetheless, is key to appropriate surgical
management. This is also the case when the defects are multiple. It is
now agreed that, when considered as an overall group, and taking account
of the fact that the communications can also be part of more complex
lesions, the defects themselves, when assessed in isolation, exhibit one
of three phenotypic patterns. There are the muscular, perimembranous,
and juxta-arterial variants.\textsuperscript{2} When approached on this
basis, it should be no surprise that the perimembranous, or
juxta-arterial, variants can co-exist with additional channels within
the muscular ventricular septum. Several discrete channels can also be
found within the confines of the muscular septum itself. On occasion,
furthermore, the multi-fenestrated septum is justifiably compared to
Swiss cheese. Yet another subtly different arrangement is found when a
solitary channel within the apical part of the muscular septum is
crossed, on the right ventricular side, by multiple
trabeculations.\textsuperscript{3} This gives the spurious impression of
multiple defects.\textsuperscript{4} The recent analysis of the trawl of
surgical results made by the Society of Thoracic Surgeons revealed that
both mortality and morbidity were increased in the setting of multiple
as opposed to solitary ventricular septal
defects.\textsuperscript{5-9}The surgical approach in presence of
multiple defects, however, is likely to be different depending on the
combinations of individual defects. It is unlikely that the same
operative intervention will be optimal for each of the different
patterns. To the best of our knowledge, the significance of these
different associations between multiple defects has yet to be explored
in the context of therapeutic management. In this, the introduction to a
review of such management, we have described the developmental
background to the existence of multiple defects. We demonstrate the
phenotypic combinations, and discuss their anatomical features. In our
subsequent review, we will discuss the significance of the anatomical
variations to diagnosis and options for treatment.

\textbf{Developmental considerations}

Towards the latter part of the twentieth century, one of us wrote an
inflammatory chapter in which we suggested that knowledge of cardiac
development was a hindrance rather than a help.\textsuperscript{10} At
the time, there was some justification for that statement, since the
alleged knowledge of the developmental events was mostly speculative,
and tended to be based on comparisons made between the anatomy of the
malformations and the presumed fashion of development of the normal
heart. All of that has now changed. We now have detailed knowledge of
the stages of anatomical development of the murine heart, and we are
able to compare those changes to abnormal arrangements found in mice
with deficient ventricular septation.\textsuperscript{11} Evidence is
now also becoming available to show that the changes observed in the
murine heart provide an accurate framework for understanding human
cardiac development.\textsuperscript{12} These changes now permit us to
provide an equally solid framework to underpin the categorisation of
both solitary and multiple ventricular septal defects.

In terms of cardiac development, the first evidence of ventricular
septation is seen subsequent to so-called ``looping'' of the primary
heart tube. This occurs on the tenth day of development in the mouse
heart (Figure 1A). This is equivalent to around 5 weeks of development
in the human, representing stage 13 in the system developed by the
Carnegie Institute.\textsuperscript{12} At this stage, the apical
components of the developing ventricles are beginning to expand from the
inlet and outlet parts of the heart tube by a process known as
``ballooning''.\textsuperscript{13} As the apical components extend
centrifugally, so the muscular part of the ventricular septum develops
between them. Significantly, the septum first becomes evident when it is
a meshwork of trabeculations. Such trabculations, at this early stage,
form the greater part of the developing ventricular
walls.\textsuperscript{14} Another significant feature of this early
stage of development is that, initially, the developing atrioventricular
canal is supported exclusively by the developing left ventricle, while
the outflow tract arises entirely above the cavity of the developing
right ventricle (Figure 1). The consequence of this arrangement is that,
initially, all the blood entering the heart through the venous
tributaries must pass through the interventricular communication so as
to reach the outflow tract and the arterial pole. The channel between
the ventricles as seen at this stage, therefore, can be described as the
primary interventricular communication.

With ongoing development, there is significant remodelling of this
initial interventricular communication.\textsuperscript{11} This can be
described in terms of two phases. In the first phase, there is expansion
of the atrioventricular canal such that the cavity of the right
ventricle achieves its own direct communication with the cavity of the
right atrium. It is this expansion that produces the right
atrioventricular junction. Once the right ventricle has its own
connection with the right atrium, the communication between the
ventricles can be considered to represent a secondary interventricular
communication (Figure 2A). Subsequent to expansion of the
atrioventricular canal, however, the entirety of the outflow tract
remains supported above the cavity of the right ventricle, which now
possesses its own inlet. This, of course, is comparable to the situation
found in the setting of double outlet right
ventricle.\textsuperscript{1} The outflow tract, at this stage, is
itself being divided into aortic and pulmonary channels by the formation
and fusion of endothelial components described as cushions (Figure 2A).
The role of these cushions is directly comparable to the steps taken by
the cardiac surgeon in correcting double outlet right ventricle. The
fusion of the cushions produces a shelf in the roof of the right
ventricle, which tunnels the blood passing through the secondary
interventricular communication into the aortic root (Figure 2B). During
this process, additional endothelial cushions have been separating the
atrioventricular canal into discrete tricuspid and mitral orifices. It
is the formation of additional processes, known as tubercles, from the
rightward margins of these cushions that eventually closes the remaining
communication between the aortic root and the cavity of the right
ventricle (Figure 2C).\textsuperscript{11} The channel that is
eventually closed, therefore, is a tertiary interventricular
communication. This is because the process of completion of ventricular
septation will have converted the secondary interventricular
communication into the subaortic outflow tract. This tunnelling of the
aortic root to the left ventricle is the second phase of remodelling of
the initial primary communication. And, if development proceeds
normally, the proximal outflow cushions, which themselves have formed
the shelf in the roof of the right ventricle, become muscularised and
remodelled, thus forming the free-standing subpulmonary
infundibulum.\textsuperscript{15} Only if development is incomplete,
with the tertiary foramen persisting as a ventricular septal defect, do
these myocardialised cushions persist as a muscular outlet septum.

\textbf{Development relative to the variable patterns of multiple
ventricular septal defects}

It is the changes that take place during normal development that now
underscore our understanding of the phenotypic variations to be found
amongst isolated ventricular septal defects. The inferences made from
normal development, furthermore, have now been validated by the finding
of such defects in developing mice in which the Furin enzyme had been
genetically perturbed. We are unsure precisely why perturbation of the
Furin enzyme should interfere with ventricular septation, but the
interrogation of three-dimensional datasets from developing fetuses
sacrificed at the stage when, in normal development, the ventricular
septum is intact, show the phenotypic features in different fetuses of
the different types of ventricular septal defect. Analysis of normal
development has also shown that the muscular ventricular septum is
formed by the coalescence of the components of the initial trabecular
meshwork. It is often presumed that these trabeculations come together
to form the compact layer of the ventricular walls. This is not the
case.\textsuperscript{14} The trabeculations do coalesce, nonetheless,
to form the papillary muscles of the atrioventricular valves, and also
the muscular part of the ventricular septum. In the mice in which the
Furin enzyme was perturbed, the process of septal coalescence was
disturbed, producing the arrangement also known as the Swiss-cheese
septum (Figure 3A). The inference can be made that failure of such
coalescence might be less severe, leaving discrete but multiple defects
with muscular borders at any site within the septum. In a proportion of
the mice having undergone perturbation of the Furin enzyme, there was
persistence of the tertiary embryonic interventricular communication. In
these mice, there was fibrous continuity in the postero-inferior
quadrants of the defects between the leaflets of the tricuspid and
mitral valves. This, of course, is the phenotypic feature of the
perimembranous defect.\textsuperscript{16} In a small number of the mice
with perturbation of the Furin enzyme, furthermore, there was evidence
that the proximal outflow cushions had fused so as to separate the
aortic and pulmonary roots, but had failed to muscularise. This had left
an interventricular communication between the outflow tracts that was
bordered cranially by the fused proximal outflow cushions. It is this
feature that is diagnostic for the ventricular septal defects that are
juxta-arterial.\textsuperscript{15} The inference can also be made, on
the basis of the development observed in the normal mice, and those
suffering perturbation of the Furin enzyme, that either the
perimembranous or juxta-arterial defects could co-exist with defects in
the apical muscular part of the septum. It also follows that multiple
individual defects might be anticipated to exist within the muscular
septum, or that failure of coalescence could hav been sufficiently
severe to produce the Swiss-cheese arrangement. All of these
possibilities are borne out by examination of hearts with ventricular
septal defects as found in archival collections.

\textbf{The Phenotypic Features of Multiple Ventricular Septal Defects}

When taking note of our current knowledge of cardiac development, we can
now argue that all interventricular communications, depending on their
borders, can be placed into one of three
groups.\textsuperscript{11,15}The significant feature of the first group
is that the defects are within the substance of the ventricular septum.
These are the so-called muscular defects (Figure 4A). As can also be
inferred from the developmental evidence, they can be found anywhere
within the muscular septum. Multiple muscular defects, therefore, can be
found when opening through different parts of the septum. The most
obvious multiple muscular defects, nonetheless, are found when the
septum has not properly coalesced during development. Our analysis of
the hearts contained within historical archives shows that this problem
can manifest as two patterns, which we interpret as representing a
spectrum of incomplete coalescence. At the milder end of the spectrum,
the apical part of the septum is itself intact, but multiple discrete
defects, of variable size, are found at the borders of the apical
ventricular components with the ventricular inlets and outlets (Figure
5). In the heart shown in Figure 5, two of the defects are large, and
are well seen from both the right and left side of the septum. The
severe end of the spectrum is shown in Figure 6. In this heart, there is
persisting excessive trabeculation at the apex of the left ventricle
(Figure 6B). The entire apical part of the septum, furthermore, shows
evidence of inappropriate coalescence of the muscular septum. As such,
it is exceedingly difficult to recognise the multiple individual defects
that percolate through the substance of the septum. This feature is even
worse to recognise when assessed from the right ventricular aspect
(Figure 6A). This arrangement is the so-called ``Swiss cheese'' variant.
As is shown, it is impossible, on the basis of direct examination, to
establish the precise number of fenestrations within such a septum. This
is not the case when the spectrum of coalescence is less severe (Figure
5). As we will discuss in the our surgical review, this means that the
``Swiss-cheese'' variant can be difficult to repair, the more so since
it is usually the most apical part of the septum that has failed to
coalesce.

The group of defects reflect failure of closure of the tertiary
interventricular communication.\textsuperscript{15} Its phenotypic
feature is fibrous continuity between the leaflets of the mitral and
tricuspid valves (Figure 4B).\textsuperscript{16} The defect
incorporates within its borders the atrioventricular component of the
membranous septum. It often additionally has a flap in its
postero-inferior border formed by the interventricular part of the
membranous septum. It is because the myocardial margins of the defect
extend around these components of the membranous septum that the defect
is designated as being perimembranous.\textsuperscript{17} The defect,
which opens directly beneath the aortic root, can co-exist with muscular
defects existing anywhere within the muscular part of the septum. The
combination of particular importance is that which exists with a
muscular inlet defect (Figure 7A). This is because, in this setting, the
atrioventricular conduction axis extends through the myocardial bar
which separates the two individual defects (Figure 7B). Should the heart
be very small, as is the case in neonates and infants, the bar
separating the defects may be of insufficient size to permit sutures to
be placed so as to close each defect individually.\textsuperscript{18}In
this setting, therefore, it may be judicious to place a single patch
covering the right ventricular exits of both defects. The alternative is
to temporise until it is judged that the muscular bar is of sufficient
size to permit sutures to be placed so as to close each defect without
jeopardising the conduction axis.

The third group of defects is characterised by failure of formation of
the muscular subpulmonary infundibulum (Figure
3C).\textsuperscript{15}The phenotypic feature is fibrous continuity
between the leaflets of the arterial valves (Figure 4C). This is the
rarest type to be found in the setting of multiple defects, but must be
anticipated to co-exist with muscular defects opening either to the apex
or inlet of the right ventricle.

The final combination to be considered is not truly an example of
multiple defects. This is when there is a large defect in the apical
part of the muscular septum (Figure 8A). When viewed from the right
ventricle, however, the defect is seen to be crossed by apical
trabeculations, giving the impression of multiple defects (Figure
8B).\textsuperscript{4} The understanding of this defect has been
obfuscated by suggestions that it extends into the infundibulum of the
right ventricle.\textsuperscript{3} It represents a defect within the
apical part of the muscular septum. As is now evident from development,
formation of the infundibulum is a late event. It cannot be completed
until the secondary interventricular communication is tunnelled into the
aortic root. These processes, in themselves, show that an outlet defect
could not open into the apical part of the right ventricle. It is also
clear from the anatomical arrangement that the infundibular part of the
right ventricle is found cranial to the limbs of the septomarginal
trabeculation, or septal band.\textsuperscript{16}

\textbf{Discussion}

As we have shown, multiple, or seemingly multiple, ventricular septal
defects can be found in three discrete patterns. In the first
arrangement, separate and discrete defects co-exist within different
parts of the ventricular septum. Most usually this is because of a
combination of perimembranous and muscular defects. This variant is of
particular surgical significance when the muscular defect opens to the
inlet of the right ventricle. This is because the atrioventricular
conduction axis is particularly vulnerable in this setting (Figure 7).
The second arrangement is found when the muscular ventricular septum has
failed properly to coalesce during cardiac development. This then
produces two variants according to the extent of coalescence. When less
severe, multiple discrete holes can be observed within the muscular
septum, usually at the borders between the right ventricular inlet and
outlet and the apical part of the septum. It is the more severe end of
the spectrum of failure of coalescence that manifests as the so-called
Swiss-cheese septum. This variant poses perhaps the greatest challenge
for surgical correction. The final arrangement is not truly an example
of multiple defects. It is found when a large, but solitary, defect in
the apical part of the muscular septum is crossed in the right ventricle
by trabeculations, giving the spurious impression of multiple
defects.\textsuperscript{4} The surgical approach to each of these
variants will itself vary, although several options have been proposed
for each specific sub-set. It is those options that we will discuss in
the our surgical review.

\textbf{Acknowledgements}

We express our gratitude to Prof. Wouter Lamers and Dr. Jill Hikspoors,
from the Univerity of Maastricht in the Netherlands, who produced the
reconstruction from which we created the illustration in Figure 1B. We
are equally indebted to Dr. Timothy J. Mohun, from the Crick Institute
in London, who created the episcopic datasets from which we produced the
figures shown in Figures 1A, 2 and 3. It was also Dr Mohun who produced
the abnormal mice with perturbation of the Furin enzyme.



\textbf{Author's contribution}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\textbf{Author's name} & \textbf{Concept/ design} & \textbf{Data
analysis/ interpretation} & \textbf{Drafting article} & \textbf{Critical
revision} & \textbf{Approval of article} & \textbf{Statistics} &
\textbf{Funding} & \textbf{Data Collection}\tabularnewline
\midrule
\endhead
Diane E. Spicer & ? & ? & ? & ? & ? & - & - & ?\tabularnewline
Robert H. Anderson & ? & ? & ? & ? & ? & - & - & ?\tabularnewline
Ujjwal Kumar Chowdhury & ? & ? & ? & ? & ? & - & - & ?\tabularnewline
Lakshmi Kumari Sankhyan & - & ? & ? & ? & ? & - & - & ?\tabularnewline
Niwin George & - & ? & ? & ? & ? & - & - & ?\tabularnewline
Niraj Nirmal Pandey & - & ? & ? & ? & ? & - & - & ?\tabularnewline
Saurabh Kumar Gupta & - & ? & ? & ? & ? & - & - & ?\tabularnewline
Shikha Goja & - & ? & ? & ? & ? & - & - & ?\tabularnewline
\bottomrule
\end{longtable}



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\end{enumerate}



\textbf{Figure Legends}

Figure 1. The images show the arrangement of the developing heart at the
stage of the beginning of formation of the muscular ventricular septum.
The left hand panel shows a section from a murine embryo sacrificed on
embryonic day 10, while the right hand panel shows a reconstruction of
the cavities of the developing human heart at around 5 weeks of
development, which is graded as Carnegie stage 13. The star shows the
site of formation of the apical muscular septum, separating the
ballooning apical ventricular components.

Figure 2. The images show the stages of remodelling of the primary
interventricular communication to form the secondary and tertiary
communications in the developing murine heart. Panel A shows the stage
subsequent to expansion of the atrioventricular canal. Panel B, at a
later stage, shows how the proximal outflow cushions have fused (dotted
line) to form a shelf in the roof of the right ventricle. Panel C, from
the same heart, shows the persisting tertiary interventricular
communication between the aortic root and the cavity of the right
ventricle.

Figure 3. The images show the different types of defects produced in
developing mice in which the Furin enzyme has been perturbed. Panel A
shows the non-compacted apical muscular septum in a heart which also
shows an ostium primum defect. Panel B shows a perimembranous defect,
while panel C shows an outlet defect that is juxta-arterial because of
failure of formation of the subpulmonary infundibulum.

Figure 4. The images show the features of the different types of
ventricular septal defects as determined on the basis of their borders.
Panel A shows the features of a muscular inlet defect, with panel B
showing a perimembranous defect, both in cuts replicating the
echocardiographic four-chamber plane. Panel C shows a juxta-arterial
defect in a cut simulating the parasternal long axis plane.

Figure 5. The images show multiple muscular defects found at the border
between the inlet of the right ventricle and its apical component when
there has been a degree of inappropriate coalescence of the septum
during development. Panel A shows the view from the right ventricular
aspect. As can be seen when assessing the situation from the left
ventricular apex (Figure 5B). the most apical part of the septum is
intact.

Figure 6. The images show the situation produced when there has been
severe lack of coalescence of the muscular ventricular septum during
cardiac development. Panel A shows the right ventricular aspect, with
Panel B showing the view from the left side. Although the entire apical
part of the septum has failed to coalesce, it is difficult to identify
individual defects. Note also the excessive apical trabeculation in the
left ventricle.

Figure 7. The images show multiple defects produced by the combination
of perimembranous and muscular inlet defects. The red dashed line shows
the location of the atrioventricular conduction axis, which runs through
the myocardial bar separating the defects. The defects are shown from
the right (panel A) and left (panel B) ventricular aspects.

Figure 8. The images show how a solitary defect when viewed from the
left ventricle (panel A) can seem to represent multiple defects when
viewed from the right ventricle (panel B).\selectlanguage{english}
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