Citation
Pijnappels, D. A. (2009, June 18). Electrophysiological deterioration and resurrection in the scarred heart. Retrieved from https://hdl.handle.net/1887/13851
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General Introduction
I
Chapter
General introduction and outline of the thesis
Background
T
he unique three dimensional architecture of the heart is essential for both elec- trical and mechanical activation of the myocardium. Any disruption of this ar- chitecture may have significant consequences for the pump function of the heart and may result in serious clinical conditions. The cardiovascular system has been subject of study for more than a century now. These studies provided us with novel insights, helped us to develop new therapeutic strategies and ultimately resulted in improved treatment options, and prognosis of patients with cardiovascular disorders. It beca- me possible to cure patients with supraventricular arrhythmias, protect them against sudden cardiac death and to alleviate the symptoms of coronary artery disease. Fur- thermore genetic disorders causing arrhythmias, for example, can be detected and affected persons can be treated.More recently, cell and gene therapy as potential therapeutic treatment modalities for patients with heart failure or ischemic heart disease were introduced. For cell and gene therapy to become successful it is not only necessary to select the most appro- priate cell types or the best target genes, but also to understand the way new cells will have to be incorporated in the functional cardiac syncytium to prevent rhythm and conduction disturbances and to improve cardiac function.
In order to understand the electrophysiological consequences of gene and cell the- rapy and to unravel the different processes playing a role in impulse conduction, it is important to consider normal electrophysiological characteristics of the heart first.
Electrophysiology of the Heart
Systemic and pulmonary circulation is maintained through rhythmic contractions of the heart, which are triggered by propagated electrical waves. Electrical activation of the heart starts with spontaneous impulse formation in the sinoatrial node. Next the impulse spreads rapidly across the atria followed by mechanical activation of the atria. Subsequently, the electrical impulse propagates slowly through the atrioven- tricular node, thereby allowing atrial activation to be completed prior to ventricular activation. The electrical impulse then enters the base of the ventricular septum at the bundle of His and rapidly propagated across the left and right bundle branches, towards the apex of the ventricles. These bundle branches diverge into an extensive network of Purkinje fibers from which the electrical activation front rapidly spreads from endocardium, across the ventricular walls, to the epicardium and base of the heart. Consequently, normal activation of working myocardium is fast and coordina-
ted, resulting in almost synchronous mechanical activation of the different ventricu- lar segments. Of note, relatively small, physiological conduction delays can be measu- red between different ventricular segments, which together contribute to a process of sequential force generation.1,2
This rapid, coordinated activation of the left ventricle is also referred to as synchro- nicity, and determines to a large extent the efficacy by which blood is extruded from the ventricles.3 Delayed activation of one or more ventricular segments may result in dyssynchronous activation of the ventricles resulting in a reduced ejection fraction and increase in energy demand.4,5
During electrical activation of the ventricular myocardium, calcium ions (Ca2+) enter the cardiomyocytes (CMCs). Once Ca2+ entered the cell, more Ca2+ is released from intracellular calcium stores. These calcium ions bind to troponin, allowing sarcomere interactions and mechanical contraction of the cells.6,7 This process is referred to as electro-mechanical coupling and is the key mechanism by which the regulation of contraction is managed in the heart.8
In fact, each CMC acts as a single excitable and contractile unit. However, in order to establish efficient electrical conduction, and to produce enough force to extrude the blood from the ventricles, CMCs need to form a three-dimensional multi-cellular structure with specific architectural characteristics.9,10 This three-dimensional myo- cardial syncytium is a well-regulated and organized structure that will remain active throughout the entire lifespan with only limited, or no, degree of self-renewal af- ter injury.11,12 Consequently, cardiomyocyte death will disrupt the three-dimensional syncytium resulting in conduction abnormalities, the occurrence of potentially lethal arrhythmias and ultimately in symptoms of heart failure.
Ventricular Architecture
The left ventricle has a typical ellipsoid shape, which is crucial in establishing and maintaining optimal transfer of blood from the ventricles into the systemic and pul- monary circulation by coordinated contractions.13 Underneath this basic principle of cardiac function lies the complex structure of the heart, which modifies both elec- trical and mechanical activation. Ventricular architecture has been described as a transmural spiral continuum between two helical fiber structures.3,14-16 In the long axis views of the ventricle, the fiber direction is mainly longitudinal in the endocar- dium (-60o) and gradually changes into a transverse (circumferential) direction in the midwall, after which it becomes longitudinal again in the epicardium (+60o). More- over, short axis views of the ventricle shows diverging myofibers sheets (myofibers consisting of adjacent CMC clustered together by collagen) separated by cleavage planes, associated with a change in orientation of less than 40o.9 Hence, the activation
wavefront propagates from the endocardium to the epicardium in a spiral-like fa- shion, guided by the orientation of myofibers in the working myocardium. As a result of this fiber arrangement and associated electrical activation pattern, the left ventri- cular wall shortens, thickens, and twists along the long axis during cardiac activation, extruding a maximal volume of blood from the ventricles.17 In addition, this typical cardiac architecture also influences diastolic function.3,18
Myocardial Tissue Structure and Anisotropic Propagation
During cardiac development, processes as cell differentiation, proliferation, migra- tion, and integration, contribute to the formation of myocardial tissue.19-23 Among these newly formed cells are CMCs, which are initially round-shaped, but become elongated through unidirectional growth and alignment in a specific direction, the- reby creating a short and long cell axis. How this process of elongation and alignment is governed, is still not completely understood, but it seems to involve processes as electrical activation24 and mechanical stretch.25,26 However, the role of the extracellu- lar matrix in cell alignment is more evident, as shown by certain cardiac pathologies associated with extracellular matrix malformations, giving rise to increased structu- ral heterogeneity.27 Later in cardiac development, intercalated disc components, such as gap junction proteins, become clustered at the longitudinal ends of CMCs.28 Con- sequently, these elongated rod-shaped cells are predominantly coupled in the longi- tudinal direction and organized in fiber bundles.29,30 Such CMCs are intertwined in an organized mesh of densely packed cells and therefore coupled to multiple neighbou- ring cells in different degrees of actual cell-cell contact. Importantly, this typical tissue structure of the healthy heart has functional implications for electrical conduction across the cardiac muscle.31 In particular, conduction of the electrical impulse parallel to the myocardial fiber axis (longitudinal) is about 3 times faster than perpendicular to the fiber axis (transverse), indicating anisotropic conduction.32
Anisotropy can be defined as heterogeneity of a physiological property for a certain material when measured along different axes, in contrast to isotropy, which referrers to homogeneity in each direction. Anisotropic conduction is determined by 3 fac- tors; cell geometry, cell size, and gap junction distribution patterns.33,34 In more detail, healthy myocardium consists of rod-shaped, ventricular CMCs mainly coupled by end-to-end connections, which results in gap junction resistances that are an order of magnitude smaller than those associated with side-to-side connections. Furthermore, longitudinal intracellular cytoplasmic resistance for a CMC (dimensions; ~100 μm x
~20 μm; resistance; ~0.5 MΩ) is comparable to that of gap junction resistance in the longitudinal direction.33 Therefore, conduction is mediated by relatively low-resistant cytoplasm and high-resistant cell membrane junctions. This heterogenic nature by
which conduction is mediated implies that by any given dimension, beyond the level of single cell, the number of junctional membranes to be crossed in longitudinal di- rection is less than in transverse direction. Consequently, conduction in longitudinal direction involves considerably more continuous low-resistant cytoplasm, allowing electrical conduction at significantly higher velocities, as compared to conduction in transverse direction. Of note, changes in the length-width ratio (geometry) of CMCs will therefore affect the anisotropic nature of electrical conduction in the heart.35 In addition, cell size and gap junction distribution patterns will also affect anisotropic electrical conduction, although to a lesser extent than cell geometry. Moreover, litera- ture provides evidence for the notion that the anisotropic nature of cardiac tissue also influences the excitability, and action potential characteristics in the heart.36
The degree of tissue anisotropy differs among the various regions in the heart. In par- ticular, large variations can be found in both the anisotropy ratio (CVLongitudinal/CVTrans-
versal) and CV in different cardiac regions. The maximal value for the anisotropy ratio is found in the crista terminalis of the right atrium, whereas the ventricles represent the lowest value, 10 versus 2-3, respectively.37 In addition, the fastest CV in longitudinal direction is measured in Purkinje fibers, while the slowest CV, in the same direction, is found in the ventricular mass, being ~2 m/s vs ~0.5 m/s, respectively.38,39 These differences in CV seem to contribute to the specific roles of the various cardiac struc- tures involved in electrical impulse propagation, together ensuring effective electrical and mechanical activation of the cardiac muscle.
In summary, both electrical activation and myocardial deformation are influenced by the anisotropic nature of the adult mammalian heart, giving rise to a highly integra- tive system. Therefore, any changes in the anisotropic characteristics of the myocar- dium may increase the risk of arrhythmias and decreased pumping efficiency.
Electrical Impulse Initiation and Propagation
Propagation of an electrical impulse at high velocity over large distances across the heart is ensured by a sensitive interplay between gap junctions, allowing cell-to-cell conduction, and excitable cell membranes, generating action potentials. Propagation of electrical impulses is therefore mainly determined by 3 factors, 1) the sarcolemmal electrical properties of CMCs to generate an action potential, 2) characteristics of the gap junctions that determine the syncytical behaviour of myocardial tissue, and 3) the anisotropic tissue strucuture.33 At a more cellular level, propagation is influenced by factors as cell shape and volume, and accumulation of ion channels and gap junc- tions.35
These gap junctions form intercellular channels that do not only allow low-resi- stance trafficking of electrical impulses but also the transfer of small molecules up
to 1 kD between cytoplasmic compartments. A single gap junction channel is as- sembled from 2 hemichannels or connexons, one from each cell. Such a connexon is formed by a cluster of 6 connexins, forming one part of the axial gap junction channel.40 These gap junctions are assembled by specific subtypes of connexins, fol- lowing a site-specific pattern, which allows distinct cardiac tissues to have different biophysical properties.41,42 In particular, tissue-specific connexin expression is quite similar among mammals, in which the ventricles express mainly Cx43 (>) and Cx45, while the atria and conduction system express mostly Cx40 (>), Cx43(>), and Cx45.43 Of note, each connexin subtype has unique biophysical properties, which together determine the gating and conduction characteristics of a gap junction channel. In the heart, unitary conductances of Cx channels vary, but in general the following values are found; >120 picosiemens (pS) for Cx40 channels, 50-90 pS for Cx43 channels, and 26-29 pS for Cx40 channels. Furthermore, Cx40 and Cx45 channels are relatively cation selective, from which the gating of Cx45 channels is voltage-dependent. In addition, Cx43 channels are relatively insensitive to changes in voltage and highly permeable to ions.44,45 The properties of gap junction channels can be modulated by a number of other mechanisms, including alterations in the phosphorylation state of specific connexin proteins, and extracellular fatty acid composition.40 Gap junction modulations are important to adapt effectively to physiological or pathophysiological changes, but cellular communication in the ventricles is controlled mainly by regula- tion of the number of functional gap junction channels. This type of regulation is me- diated through changes in channel protein trafficking, assembly, and degradation.46 All these processes can occur within a short time-frame of only hours, which is in part related to the high turnover-rate of connexin.47
Successful electrical impulse propagation depends not only on the presence of functi- onal gap junctions between adjacent CMCs, but also on the excitability of these cells.48 Excitability refers to the property of cells to generate an action potential by successive in- and outflow of ion current, and is traditionally divided in 5 phases, being phase (0) depolarization, phase (1) transient repolarization, phase (2) plateau, phase (3) repola- rization, and phase 4) resting membrane phase. This process of excitation is the main mechanism by which CMCs are able to maintain or strengthen the electrical charge generated by these cells. While the electrical impulse is propagated across the heart, excited cells are coupled to nonexcited cells and need to provide enough electrical charge, delivered through the gap junctions, to reach excitation threshold in these nonexcited cells. During this process of electrical loading, nonexcited cells change from an electrical sink into an electrical source and are now able to drive the next adjacent CMCs to reach excitation threshold, and thereby sustain rapid propagation of the electrical impulse.34 Successful propagation will continue as long as the charge
delivered by the source equals or exceeds the charge required to excite the membrane of the adjacent CMC.
Hodgkin and Huxley were the first to describe this concept of action potential initia- tion and propagation, and developed a model of electrical activation and propagation, in which these aspects were described in terms of the biophysical properties of cell membranes.49 To determine whether propagation will occur and how this is affected by alterations in cell properties, the so-called safety factor of conduction has been introduced. Safety factor equations comprise the influence of excitability, gap juncti- onal coupling, and cell geometry on conduction velocity (CV). Briefly, the equation as proposed by Rudy et al. involves parameters involved in source-sink interactions and predicts for a single strand of multiple cells whether propagation will fail (safety factor <1), due to impaired excitation or severely decreased or increased intercellular conductance, or succeed (safety factor >1).50 Computer simulations and equations, such as the safety factor of conduction, have been powerful tools in exploring com- plex phenomena such as electrical impulse propagation.
Biophysically, propagation of an electrical impulse can be considered as a continuous or discontinuous electrical phenomenon.33,51 In brief, propagation can be studied in a situation in which the impulse is propagated across a chain of excitable units, coupled to each other by high-conductance structures, without any significant resistant obsta- cles, creating a continuous cardiac syncytium. However, technical advances, allowing a more refined assessment, showed that, even in the normal heart, electrical conduc- tion is discontinuous and that gap junctions played a crucial role in this process.51 Further research supported these findings and demonstrated that discontinuous electrical behaviour can be attributed to a variety of factors, such as discontinuous distribution of intra- and intercellular resistance, structural obstacles, and aging.34,52,53 Fast and Kléber studied discontinuous conduction at a cellular level, using a high- resolution optical mapping technique and cultured synthetic chains of neonatal rat CMCs. In this study they showed significant differences between average conduction delays associated with either gap junctional (118 μs) or cytoplasmic impulse (38 μs) propagation, thereby proving electrical propagation to be discontinuous also at the level of cell-cell coupling.54 The concept of discontinuous cardiac propagation has greatly improved our understanding of the origin, maintenance, and termination of cardiac arrhythmias.
Disturbances in Impulse Propagation
Electrical propagation in the heart is maintained by a harmonious interplay between ion channels and gap junctions. However, electrical impulse propagation across car- diac tissue can be disturbed by different causes, which among others involve changes
in excitability and gap junction coupling.50,55-57 Action potential generation is a sensi- tive process as it involves multiple ion channels which could all be affected by diffe- rent circumstances, thereby decreasing the excitability of the myocardium. Secondly, diminished gap junction coupling will decrease intercellular conductance, and there- by further depress conduction of the electrical impulse. Of note, for electrical impulse propagation across healthy myocardial tissue, the safety factor of conduction is about 1.5, but this may drop below 1 in case of seriously disturbed electrical properties.50 For example, myocardial infarction may result in such serious disturbances as the CMCs may become less excitable and less coupled by gap junctions, a process also referred to as electrical remodelling.33,58 In this thesis, especially these infarct-related disturbances of electrical impulse initiation and propagation will be studied and dis- cussed.
Myocardial Ischemia and Infarction
Once the myocardium becomes ischemic, especially in the acute setting, the CMCs will rapidly uncouple by down-regulating their connexin expression.55 This process of uncoupling is probably initiated to reduce the flow of injury-related mediators towards adjacent cardiac tissue. Decreased intercellular coupling of CMCs also re- sults in conduction abnormalities that could eventually lead to decreased contractile function and increased arrhythmogenic risk. In addition, during acute ischemia, ac- tion potential characteristics will change (depolarized resting membrane potential, reduced upstroke velocity and amplitude, and changed action potential duration, and increased effective refractory period) due to electrical remodelling. However, after more than 30 minutes, CMCs will further depolarize, while necrosis is initiated, and conduction becomes blocked completely.59 At this point, an initially slow cascade of events starts transforming the endangered zone of excitable and well-coupled myo- cardium into a non-excitable and poorly coupled mesh of myocardial scar fibroblasts (also known as myofibroblasts), secreting large amounts of extracellular matrix. This process is also referred to as infarct healing and is usually completed within 6 weeks in humans. During this process the number of fibroblasts dramatically increases, there- by creating a fibrotic scar.60 The process of infarct scar formation is a complex, multi- stage process, regulated by different mechanisms, serving mainly to restore structural integrity of the damaged heart.61-63 However, the excessive presence of extracellular matrix secreted by scar fibroblasts can also contribute to the formation of insulating septa creating areas of nonuniform anisotropy, and extremely slow transverse CV as the impulse if forced to follow a zigzag course.64
The origin of these fibroblasts, populating the myocardial scar, is still a topic of discus- sion. However, regardless of their origin, these cells do form gap junctions between
other fibroblasts and CMCs and thereby are able to affect the syncytial behaviour of the surrounding cardiac tissue.65-67 Importantly, myofibroblasts are non-excitable cells with depolarized resting-membrane potentials (~-20mV), as compared to CMCs (~-80mV), and will therefore act as a depolarizing source on adjacent myocardial tis- sue. This fibroblast-mediated depolarization results in premature, partial inactivation of fast inward Na+ channels (SCN5A) in these CMCs and might therefore disturb glo- bal electrophysiological properties.68 Another consequence of this passive electrical behaviour is that impulse conduction across these fibroblasts is electrotonic, solely depending on gap junction-mediated ion flows, without any contribution of excitable membranes. Electrotonic conduction is decremental in nature and therefore associa- ted with conduction block in case of distances beyond the electrotonic range.69-71 This concept of passive impulse conduction was described by William Thomson in the Cable theory more than 150 years ago, and was later applied to the field of electrophy- siology.72 Of note, slow conduction and conduction block, due to electrophysiological properties of scar fibroblasts, is likely to contribute to increased activation delays between different ventricular segments, and thereby decrease cardiac performance.
As discussed earlier, the safety factor of conduction predicts whether successful propagation will succeed or fail. Changes in the safety factor of conduction are de- termined by either gap junction uncoupling or by reduced excitability (especially by inactivation of depolizaring Na+ current). Reduced activation of fast inward Na+ channels leads to a swift decrease in the safety factor of conduction and is associated with slow conduction and block. However, such a reduction in Na+-channel activity and associated conduction disturbances correspond to a much larger reduction in gap junction coupling.50,73
Loss of excitation and reduced gap junction coupling, as a result of myocardial in- farction, are not the only mechanisms by which the infarcted area affects cardiac function. Vital myocardial tissue is separated from the infarcted area by a border- zone, which is subjected to ongoing fibrosis. Infiltrating fibroblasts may cause he- terogeneity in orientation of these resident cells and thereby changing their degree of connectivity, which could affect their contribution to anisotropic conduction.56,74 Furthermore, in this borderzone, ion channel properties are changed, such as delayed recovery of the fast inward Na+ currents, and reduction in peak L-type inward Ca2+
currents (CACNA1C).75-77 These changes in ion channel properties do not only result in altered excitability, but also in altered refractoriness in these surviving CMCs.78 Hence, this causes serious disruptions in electrical conduction, thereby increasing the risk for ventricular arrhythmias to occur.
Traditionally, myocardial scar tissue was considered to be static and solely detrimen- tal. However, over the last decade this view has changed and now infarcted myocar-
dium is considered to be active and viable tissue, representing mainly scar fibroblasts and accumulating extracellular matrix, while still detrimental in nature.79 This new perspective of myocardial scar tissue also increased its value as therapeutic target, thereby raising new possibilities to revive the damaged areas in the infarcted heart.
Restoration of Electrophysiological Characteristics
As discussed previously, ischemic damage results in electrophysiological changes, leading to conduction abnormalities, and mechanical disturbances. These electrophy- siological changes involve decreased, or loss of, excitability in CMCs and diminished gap junction coupling between myocardial cells. Hence, in order to treat these con- duction abnormalities, either excitation in these electrically deteriorated cells should be restored or improved, or gap junction coupling should be improved within the da- maged myocardium, or both. This process of electrophysiological restoration can be achieved through gene transfer into infarcted myocardial tissue, which would modify the electrophysiological properties of target cells. Alternatively, cells with particular electrophysiological properties could be transplanted into the damaged myocardium to recover its electrophysiological status. Taken together, there is ample rationale for applying cell modification and cell transplantation techniques in the damaged myo- cardium, to improve the electrical properties of this damaged cardiac tissue.
Cell Modification
Molecular biology has provided us with an immense repertoire of novel research tools that contributed to the development of new screening, diagnostic, and treatment op- tions for a wide variety of diseases, including cardiovascular disorders. For example, it became clear that fully differentiated cells in adult organisms are still susceptible to genetic interventions. Genetic modification can take place through viral and non- viral methods, by which a synthetic strand of DNA is transferred into target cells.80-83 After DNA transfer, this genetic material can be used for protein synthesis, which may induce a phenotypic switch in these cells and modify their electrophysiological properties.
In the damaged myocardium, target cells can range from electrophysiologically dete- riorated CMCs to unexcitable, poorly coupled scar fibroblasts. In order to improve the electrophysiological properties of the infarcted myocardium by genetic manipulation, the excitability and gap junction coupling of the target cells should be modified. Such a modification would change conduction velocity across the working myocardium.
Several experimental in vivo studies have attempted to modify CV in damaged myo- cardium by genetic modification of resident cardiac cells with promising results.84-86 In addition, several in vitro studies revealed the underlying mechanisms by which
these therapeutic effects can be achieved, including modifications in the expression levels and functionality of both ion channels and connexins.87-89 However, most stu- dies on genetic modification investigated the effects on the onset and occurrence of ventricular arrhythmias, while the effects on cardiac dyssynchrony were not studied in much detail. Nevertheless, an increase in CV across damaged myocardial tissue may contribute to improved synchronicity of the ventricle by decreasing the activa- tion delay between different ventricular segments. Hence, a more synchronized acti- vation pattern of the cardiac muscle might improve cardiac function (Figure 1).
Besides the treatment of tachyarrhythmias, genetic modification of myocardial tis- sue has also been proposed for the treatment of bradyarrhythmias.90,91 Briefly, these disorders may arise from impaired impulse initiation in the sinus node, resulting in abnormally low heart rates. Therefore, modification of native pacemaker cells or con- trolled induction of pacemaker activity in other cardiac cells may improve cardiac function by restoring normal heart rate.92
The aforementioned studies have demonstrated the therapeutic potential of modi- fying electrophysiological properties of the damaged heart. However, knowledge concerning long-term effects and ongoing, non-electrophysiological, detrimental effects of these target cells is lacking. Modification of not only electrophysiological properties of the cell, but also modification of cell fate may therefore contribute to additional therapeutic effects related to such genetic interventions. In more de- tail, genetic studies have revealed that certain cardiac-specific transcription factors are essential for proper cardiac differentiation and development.93,94 Forced expres- sion of these cardiac transcription factors in non-cardiac cells might therefore lead to activation of cardiac genes and thereby induce a phenotypic switch in the tar- get cells or even directly reprogram these cells into fully excitable and contractile CMCs. This approach of reprogramming seems very attractive, as adult CMCs are not able to undergo mitosis to compensate for cell loss after myocardial infarction, for example.
Recently, an alternative approach for genetic reprogramming has been described by the group of Yamanaka, who reported that adult mouse and human somatic cells were reprogrammed into a pluripotent state by forced expression of only a small number of genetic factors.95,96 Soon, other research groups confirmed these findings,97-99 while the technical and genetic tools for this type of reprogramming were further refined.100-103 Such reprogrammed cells are now referred to as induced pluripotent stem (iPS) cells and appear to be very similar to embryonic stem (ES) cells in many aspects, including their potential to fully differentiate into functional excitable CMCs.96 This novel concept of reprogramming creates new perspectives with regard to patient-specific diagnosis and treatment.104 In theory, autologous
cardiac cells from diseased patients are easily available now for screening and trans- plantation purposes. However, in order to fulfill these future goals, the process of cardiomyogenic differentiation in iPS cells should be as least as efficient as in ES cells or other stem cells.
Figure 1. Overview of a normal, infarcted, and genetically modified heart, and magnified areas of interest, including isochronal lines. These lines connect points of equal local activation times, thereby providing insight into electrical homogeneity of specific regions. (A) Rapid and coordinated electrical activation of the myocardium, as shown by smooth, continuous isochronal lines in the left ventricle, results in a syn- chronous contraction, thereby maintaining optimal cardiac output (indicated by large-sized blue arrow displayed on the aorta) (upper panel). Homogenous electrical activation of the cardiac muscle is reflected by almost equally spaced isochronal lines over the entire surface of ventricular tissue at higher magni- fication. Such tissue consists mainly of CMCs, which are well-coupled by gap junctions (indicated in green) and able to generate action potentials, thereby contributing to rapid electrical impulse propagation (Lower panel). (B) Electrical activation is seriously disturbed in areas of fibrotic myocardium, hampering global cardiac function, illustrated by decreased cardiac output (small-sized blue arrow) (upper panel).
A higher magnified image of such fibrotic tissue (indicated in grey) shows curving and narrowing of the isochronal lines in this scar fibroblast-rich region (grey cells), indicating slow conduction pathways due to decreased gap junction coupling (red structures) and loss of excitation in these regions. Gene transfer into such regions by intramyocardial scar injection of viral particles (indicated in green) may present a novel option for the treatment of conduction abnormalities in the infarcted heart (lower panel). (C) Ge- netic modification of these fibrotic areas, aimed at improving electrical conduction, may enhance cardiac function by reducing the risk for ventricular arrhythmias, and improving pumping efficiency of the heart (medium-sized blue arrow) (upper panel). The isochronal map of such a genetically modified myocardium may show a homogeneous activation, while the isochronal lines might be slightly curved and increased in number, depending on how well electrical conduction is restored across these regions.
In addition, iPS cell-derived CMCs should maintain long-term phenotypic and ge- notypic stability. Today, only a few studies have tried to compare these aspects of cardiomyogenic differentiation in iPS and ES stem cells, but showed only limited me- chanistic insights in the differentiation processes of these cells.105-107
Cell Transplantation
Although genetic modification of cells residing in scarred areas of the infarcted heart might improve cardiac function, transplantation of cells into these damaged areas may achieve similar effects. Cell therapy for ischemic heart disease holds promise to regenerate infarcted myocardium, and thereby restore electrophysiological and con- tractile function.108 Adult CMCs are considered to be post-mitotic cells, and therefore stem or progenitor cells appear to be the ideal substrate to heal the infarcted myocar- dium. Although cell-based cardiac regeneration is still subject of intense, world-wide research, the initial enthusiasm is now partially replaced by caution. Formation of new CMCs from transplanted stem cells is now considered to be a very rare event and is probably not responsible for the therapeutic effects observed in clinical cell therapy trials. A more prominent role in the beneficial outcome is given to neovasculariza- tion, mediated through secretion of growth factors and cytokines by the engrafted cells. Still, the concept of cell-based therapy for ischemic heart disease has many in- teresting aspects worth to be further investigated.109,110
Transplantation or recruitment of new cells in the infarcted myocardium may lead to improved cardiac function by suppressing conduction abnormalities, mediated through restoration of gap junction coupling and excitation in these damaged are- as. However, in order to improve, and not to further worsen cardiac function, these transplanted cells should integrate functionally with surrounding cardiac tissue, and thereby contribute to the structural and functional homogeneity of the cardiac mus- cle. From a mechanical point of view, these transplanted cells, if differentiated into functional CMCs, should exhibit the same properties as host cells, in terms of rigidity and force generation. Concerning the electrophysiological aspects, these transplan- ted cells should couple to neighbouring cardiac cells, and, ideally, conduct the elec- trical impulses as fast as adjacent tissue. In addition, it is likely that cells implanted into damaged cardiac tissue should also align with native cardiac cells to restore tis- sue structure and contribute to anisotropic conduction. Moreover, if these implanted cells differentiate into functional, contractile CMCs, their alignment will also affect the amount of force that these cells generate in a specific direction. However, the alignment, or spatial integration, of transplanted cells with host cardiac tissue has not yet been studied in much detail.
Different cell types have been used for transplantation into the damaged heart, each
with their own electrophysiological properties.111-113 In addition, several studies have shown the beneficial effects of cell transplantation on conduction parameters in in- farcted regions of the heart, associated with improved cardiac performance.114,115 Interestingly, the beneficial effects of cell therapy appear to be mainly mediated by improved gap junction coupling in the damaged areas, leaving only a minor role for excitation. This was further demonstrated by transplantation of cells lacking Cx43, which significantly worsened cardiac function by formation of anatomic obstacles, thereby increasing electrical heterogeneity and the risk of reentrant arrhythmias.116,117 These experiments highlighted the importance of gap junction coupling of transplan- ted cells with native cells to gain therapeutic benefit from these interventions. Howe- ver, while gap junction coupling seems to be mandatory for a beneficial outcome of cell therapy, the extent of gap junction coupling between excitable and unexcitable cells, in terms of ratios, appeared to affect this outcome.118 In more detail, an increa- sing number of unexcitable mesenchymal stem cells (>10%) relative to the number of cultured, excitable CMCs, significantly increased the risk for reentrant arrhythmias in an in vitro model. However, co-cultures with a lower number of stem cells (<10%) did not result in these harmful conduction abnormalities. Interestingly, transplanta- tion of skeletal myoblasts into the post-infarction failing heart was associated with global downregulation of Cx43 expression in the host myocardium, an effect opposite to what cell therapy should achieve.119 This fall in gap junction coupling resulted most likely in decreased intercellular conductance, which was reflected by an increase in the incidence of conduction abnormalities, compared to control groups.
In summary, while currently available therapeutic options for the treatment of acute myocardial infarction are sufficient for the treatment of symptoms, the underlying causes usually remain unresolved, being loss of myocardial tissue. Recently, extensive research has been performed in the field of cell and gene therapy. The ultimate aim of cell and gene therapy is to “heal” the infarcted area on a more biological basis, by repopulating the damaged area with “new” cells that contribute to proper cardiac function.
However, further research is needed to gain insight into the integrative and functional aspects of these novel treatment strategies, with the purpose to improve outcome and reduce potential hazards.
Aim and Outline of the Thesis
In order to comprehend the potential therapeutic value and hazard of cell modifica- tion and transplantation for ischemic heart diseases, one should consider the heart as a highly integrative, electromechanical organ, see Chapter I. Therefore, the aim of this thesis was to explore, from a mechanistic and electrophysiological point of view, the integrative and functional aspects of cell modification and transplantation as the- rapeutic options to cure the damaged, ischemic heart.
Myocardial infarction results in the replacement of excitable and well-coupled myo- cardium into unexcitable and poorly coupled mesh of myocardial scar fibroblasts.
Genetic modification of these fibroblasts, by forced expression of transcription fac- tors involved in cardiac development, may induce differentiation or expression of va- rious cardiac proteins in these cells, and thereby improve their electrophysiological properties. Therefore it is tested, in Chapter II, whether transfer of the myocardin gene, a potent cardiac transcription factor, in cultured human ventricular scar fibro- blasts (hVSFs), results in a phenotypic switch, favouring electrical conduction across these genetically modified cells.
Improved electrical conduction across genetically modified hVSFs may restore ra- pid electrical activation of adjacent regions of cardiac tissue, thereby lowering the dyssynchronous nature of fibrotic myocardial tissue. This concept is explored in Chapter III, which studies the extent and underlying mechanisms, by which hVSFs contribute to dyssynchronous activation of cultured cardiac tissue. In addition, this chapter describes how genetic modification of scar fibroblasts can result in (1) re- synchronization of cardiac tissue by increased CV across these fibroblasts and (2) establishment of interconnecting tissue for electrical stimulation.
Although forced expression of myocardin in hVSFs improved electrical impulse con- duction across these cells, it did not generate functional, excitable cardiomyocytes (CMCs). However, recently it was shown that forced expression of only 4 transcrip- tion factors in adult fibroblasts reprogrammed these cells into induced pluripotent stem (iPS) cells, resembling many features of embryonic stem (ES) cells. However, to become a novel, clinically relevant cell type, the process of cardiomyogenic differenti- ation in these iPS cells should be as least as efficient as in ES or other stem cells. This is studied in Chapter IV, which describes a detailed comparison of genetic, electrop- hysiological, and structural aspects between mouse induced pluripotent stem (iPS) cells and mouse embryonic stem (ES) cells concerning their cardiomyogenic differen- tiation potential.
Transplantation of cells into damaged cardiac regions may regenerate or improve the electrophysiological properties of the infarcted myocardium. However, in order to maximize therapeutic efficiency of cell therapy, and minimize the risk of adverse
effects, these cells should functionally integrate with host cardiac tissue. Adult hu- man bone-marrow derived mesenchymal stem cells (MSCs) have been used in both experimental and clinical studies of cell transplantation, and they improved cardiac function in animals and patients with myocardial infarction. However, the process of electrical integration of these cells with recipient myocardial tissue is incompletely understood. Chapter V of this thesis evaluates the ongoing functional electrical inte- gration of transplanted adult human bone-marrow derived MSCs in a syncytium of cultured CMCs, and the role of gap junction coupling in this process.
Functional integration of transplanted cells with host myocardial tissue has been stu- died mainly at the level of electromechanical coupling. However, cardiac muscle has a typical anisotropic tissue structure, which influences both electrical and mecha- nical activation. Therefore, it seems that these transplanted cells should also align properly with native cardiac cells in order to restore tissue structure and contribute to anisotropic conduction. However, it is unknown how and to what extent alignment of transplanted cells affects the process of functional integration. These aspects are studied in Chapter VI, which explores the structural and functional effects of forced alignment of transplanted neonatal rat MSCs, undergoing cardiomyogenic differenti- ation, on functional integration with cultured cardiac tissue.
Epicardial cells are able to undergo epithelial-to-mesenchymal transformation (EMT), thereby contributing to cardiac development. Disturbances in this process of trans- formation are associated with seriously hampered cardiac function.120 In addition, transplantation of these epicardium-derived cells into ischemic myocardium was shown to preserve cardiac function and attenuate ventricular remodelling.121 Howe- ver, there is lacking knowledge regarding the electrophysiological properties of epi- cardial cells and whether EMT influences electrical conductivity of epicardial cells.
These aspects are studied in Chapter VII, by employing extracellular and intracel- lular electrophysiological techniques and co-cultures of nrCMCs with adult human epicardium-derived cells, before and after EMT.
Chapter VIII provides the summary and conclusions of this thesis, as well as future perspectives related to the integrative and functional electrophysiological aspects of cell modification and transplantation for the treatment of damaged myocardium.
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