Electrophysiological disturbances of coronary heart disease and gene,cell therapy

李中言,杨劲松

(吉林医药学院附属医院心内科，吉林 吉林 132013)

·综 述·

Electrophysiologicaldisturbancesofcoronaryheartdiseaseandgene,celltherapy

李中言,杨劲松

(吉林医药学院附属医院心内科，吉林 吉林 132013)

More recently,gene and cell therapy as potential therapeutic treatment modalities for patients with heart failure or ischemic heart disease were introduced.For gene and cell therapy to become successful it is not only necessary to select the most appropriate 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 therapy and to unravel the different processes playing a role in impulse conduction,it is important to consider normal electrophysiological characteristics of the heart first.

1 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 atrioventricular 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 coordinated,resulting in almost synchronous mechanical activation of the different ventricular segments.Of note,relatively small,physiological conduction delays can be measured between different ventricular segments,which together contribute to a process of sequential force generation[1].

This rapid,coordinated activation of the left ventricle is also referred to as synchronicity,and determines to a large extent the efficacy by which blood is extruded from the ventricles[2].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.

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.This process is referred to as electro-mechanical coupling and is the key mechanism by which the regulation contraction is managed in the heart.

Consequently,cardiomyocyte death will disrupt the three dimensional syncytium resulting in conduction abnormalities,the occurrence potentially lethal arrhythmias and ultimately in symptoms of heart failure.

2 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 pulmonary circulation by coordinated contractions.Underneath this basic principle of cardiac function lies the complex structure of the heart,which modifies both electrical and mechanical activation.Ventricular architecture has been described as a transmural spiral continuum between two helical fiber structures[2].In the long axis views of the ventricle,the fiber direction is mainly longitudinal in the endocardium (-60°) and gradually changes into a transverse (circumferential) direction in the midwall,after which it becomes longitudinal again in the epicardium (+60°).Moreover,short axis views of the ventricle shows diverging myofibers sheets separated by cleavage planes,associated with a change in orientation of less than 40°.Hence,tile activation wavefront propagates from the endocardium to the epicardium in a spiral-like fashion,guided by tile orientation of myofibers in the working myocardium.As a result of this fiber arrangement and associated electrical activation pattern,the left ventricular wall shortens,thickens,and twists along the long axis during cardiac activation,extruding a maximal volume of blood from the ventricles.In addition,this typical cardiac architecture also influences diastolic function[2].

3 Myocardial tissue structure and anisotropic propagation

During cardiac development,processes as cell differentiation,proliferation,migration,and integration,contribute to the formation of myocardial tissue[3-4].Among these newly formed cells are CMCs,which are initially round shaped,but become elongated through unidirectional growth and alignment in a specific direction,thereby 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 activation[5]and mechanical stretch.However,the role of the extracellular matrix in cell alignment is more evident,as shown by certain cardiac pathologies associated with extracellular matrix malformations,giving rise to increased structural heterogeneity.Later in cardiac development,intercalated disc components,such as gap junction proteins,become clustered at the longitudinal ends of CMCs.Consequently,these elongated rod-shaped cells are predominantly coupled in the longitudinal direction and organized in fiber bundles.Such CMCs are intertwined in an organized mesh of densely packed cells and therefore coupled to multiple neighbouring 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.

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 factors; cell geometry,cell size,and gap junction distribution patterns.

The degree of tissue anisotropy differs among the various regions in the heart.In particular,large variations can be found in both the anisotropy ratio and conduction velocity (CV) in different cardiac regions.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.These differences in CV seem to contribute to the specific roles of the various cardiac structures involved in electrical impulse propagation,together ensuring effective electrical and mechanical activation of the cardiac muscle.

4 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 behavior of myocardial tissue,and 3) the anisotropic tissue structure.At a more cellular level,propagation is influenced by factors as cell shape and volume,and accumulation of ion channels and gap junctions.

These gap junctions form intercellular channels that do not only allow Iow-resistance trafficking of electrical impulses but also the transfer of small molecules up to 1 KD between cytoplasmic compartments.These gap junctions are assembled by specific subtypes of connexons,following a site specific pattern,which allows distinct cardiac tissues to have different biophysical properties.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.Gap junction modulations are important to adapt effectively to physiological or pathophysiological changes,but cellular communication in the ventricles is controlled mainly by regulation of the number of functional gap junction channels.

Successful electrical impulse propagation depends not only on the presence of functional gap junctions between adjacent CMCs,but also on the excitability of these cells.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) repolarization,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.

5 Disturbances in impulse propagation

Electrical propagation in the heart is maintained by a harmonious interplay between ion cannels and gap junctions.However,electrical impulse propagation across cardiac tissue can be disturbed by different causes,which among others involve changes in excitability and gap junction coupling.Action potential generation is a sensitive process as it involves multiple ion channels which could all be affected by different circumstances,thereby decreasing the excitability of the myocardium.Secondly,diminished gap junction coupling will decrease intercellular conductance,and thereby 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.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 remodeling.In this thesis,especially these infarct related disturbances of electrical impulse initiation and propagation will be studied and discussed.

6 Myocardial ischemia and infarction

Once the myocardium becomes ischemic,especially in tile acute setting,the CMCs will rapidly uncouple by down regulating their connexin expression.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 results in conduction abnormalities that could eventually lead to decreased contractile function and increased arrhythmogenic risk.In addition,during acute ischemia,action potential characteristics will change due to electrical remodeling.However,after more than 30 minutes,CMCs will further depolarize,while necrosis is initiated,and conduction becomes blocked completely.At this point,an initially slow cascade of events starts transforming the endangered zone of excitable and well-coupled myocardium rate a non-excitable and poorly coupled mesh of myocardial scar fibroblasts,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.The process of infarct scar formation is a complex,multistage process,regulated by different mechanisms,serving mainly to restore structural integrity of the damaged heart[6].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.

Loss of excitation and reduced gap junction coupling,as a result of myocardial infarction,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 heterogeneity in orientation of these resident cells and thereby changing their degree of connectivity,which could affect their contribution to anisotropic conduction.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.These changes in ion channel properties do not only result in altered excitability; but also in altered refractoriness in these surviving CMCs.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 detrimental.However,over the last decade this view has changed and now infarcted myocardium is considered to be active and viable tissue,representing mainly scar fibroblasts and accumulating extracellular matrix,while still detrimental in nature.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.

7 Cell modification

It becomes 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[7].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 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 myocardium by genetic modification of resident cardiac cells with promising results.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[8].However,most studies 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 activation delay between different ventricular segments.Hence,a more synchronized activation pattern of the cardiac muscle might improve cardiac function.

Besides the treatment of tachyarrhythmias,genetic modification of myocardial tissue has also been proposed for the treatment of bradyarrhythmias.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 controlled induction of pacemaker activity in other cardiac cells may improve cardiac function by restoring normal heart rate.

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 detail,genetic studies have revealed that certain cardiac-specific transcription factors are essential for proper cardiac differentiation and development.Forced expression 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 target cells or even directly reprogram these cells into fully excitable and contractile CMCs.

Adult mouse and human somatic cells were reprogrammed into a pluripotent state by forced expression of only a small number of genetic factors[9].Such reprogrammed cells are now referred to as induced pluripotent stem (iPS) ceIls and appear to be very similar to embryonic stem (ES) cells in many aspects,including their potential to fully differentiate into functional excitable CMCs.This novel concept of reprogramming creates new perspectives with regard to patient specific diagnosis and treatment[10].In theory,autologous cardiac cells from diseased patients are easily available now for screening and transplantation 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.

In addition,iPS cell-derived CMCs should maintain long term phenotypic and genotypic 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 mechanistic insights in the differentiation processes of these cells[11-12].

8 Cell transplantation

Cell therapy for ischemic heart disease holds promise to regenerate infarcted myocardium,and thereby restore electrophysiological and contractile function[13].Adult CMCs are considered to be post-mitotic cells[14-15],and therefore stem or progenitor cells appear to be the ideal substrate to heal the infarcted myocardium.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 neovascularization,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 interesting aspects worth to be further investigated[16].

Concerning the electrophysiological aspects,these transplanted cells should couple to neighbouring cardiac cells,and,ideally,conduct the electrical 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 tissue 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.In addition,several studies have shown the beneficial effects of cell transplantation on conduction parameters in infarcted regions of the heart,associated with improved cardiac performance.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.These experiments highlighted the importance of gap junction coupling of transplanted cells with native cells to gain therapeutic benefit from these interventions.However,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.Interestingly,transplantation 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[17-18].This fall in gap junction coupling resulted most likely in decreased intercellular conductance,which was reflected by all 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.

[1] Remme E W,Lyseggen E,Helle-Valle T,et al.Mechanisms of preejection and postejection velocity spikes in left ventricular myocardium:interaction between wall deformation and valve events[J].Circulation，2008,118(4):373-380.

.[2] Bcckberg G,Hoffman J L,Mahajan A,et al.Cardiac mechanics revisited:the relationship of cardiac architecture to ventricular function[J].Circulation,2008,118(24):2571-2587.

[3] Zhou B,Ma Q,Rajagopal S,et al.Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart[J].Nature,2008,454(7200):109-113.

[4] Yang L,Soonpaa M H,Adler E D,et al.Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population[J].Nature,2008,453(7194):524-528.

[5] You J O,Rafat M,Ye G J,et al.Nanoengineering the heart：conductive scaffolds enhance connexin 43 expression[J].Nano Lett,2011,11(9):3643-3648.

[6] Nah D Y,Rhee M Y.The inflammatory response and cardiac repair after myocardial infarction[J].Korean Circ J,2009,39(10)：393-398.

[7] Davis J,Westfall M V,Townsend D,et al.Designing heart performance by gene transfer[J].Physiol Rev,2008,88(4):1567-1651.

[8] Sekar R B,Kizana E,Cho H C,et al.IK1 heterogeneity affects genesis and stability of spiral waves in cardiac myocyte monolayers[J].Circ Res,2009,104(3):355-364.

[9] Park I H,Zhao R,West J A,et al.Reprogramming of human somatic cells to pluripotency with defined factors[J].Nature,2008,451(7175):141-146.

[10] Park I H,Arora N,Huo H,et al.Disease-specific induced pluripotent stem cells[J].Cell,2008,134(5):877-886.

[11] Zhang J H,Wilson G F,Soerens A G,et al.Functional cardiomyocytes derived from human induced pluripotent stem cells[J].Circ Res,2009,104(4):e30-e41.

[12] Yoshida Y,Yamanaka S.iPS cells:a source of cardiac regeneration[J].J Mol Cell Cardiol,2011,50(2):327-332.

[13] Segers V F,Lee R T.Stem-cell therapy for cardiac disease[J].Nature,2008,451(7181):937-942.

[14] Chimenti I,Smith R R,Li T S,et al.Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice[J].Circ Res,2010,106(5):971-980.

[15] Gambini E,Pompilio G,Biondi A,et al.C-kit+ cardiac progenitors exhibit mesenchymal markers and preferential cardiovascular commitment[J].Cardiovasc Res,2011,89(2)：362-373.

[16] Dai B,Huang W,Xu M,et al.Reduced collagen deposition in infarcted myocardium facilitates induced pluripotent stem cell engraftment and angiomyogenesis for improvement of left ventricular function[J].J Am Coll Cardiol,2011,58(20):2118-2127.

[17] Coppen S R,Fukushima S,Shintani Y,et al.A factor underlying late-phase arrhythmogenicity after cell therapy to the heart:global downregulation of connexin43 in the host myocardium after skeletal myoblast transplantation[J].Circulation,2008,118(14 Suppl):S138-S144.

[18] Barnett P,van den Hoff M J.Cardiac regeneration:different cells same goal[J].Med Biol Eng Comput,2011,49(7):723-732.

1673-2995(2013)01-0033-06

冠心病电生理紊乱及基因、细胞疗法

Coronary artery disease is associated with electrophysiological disturbances.Numerous studies in the past few years have been demonstrated that the gene,cell therapy has to be considered as a safe therapeutic procedure in coronary heart disease.We reviewed the electrophysiological disturbances of coronary heart disease and gene,cell therapy.

coronary artery disease;electrophysiology;gene;cell;therapy

R541

A

李中言(1961-)，男(汉族)，主任医师，硕士.

2012-08-06)