It might reasonably be thought that those who diagnose and treat patients with congenitally malformed hearts would, by now, have reached consensus concerning the most appropriate way of describing the malformations with which they are confronted. It is certainly the case that nomenclature is far less contentious now than was the case a decade ago. It would be a brave person, nonetheless, who stated that the field of description and categorization was now fully resolved. There are still major differences of opinion as how best to cope with certain topics, such as those patients who have so- called visceral heterotaxy. In this review, we outline a system for description that accounts well for such topics. Indeed, it provides a means of cataloguing and describing all congenital cardiac malformations, even if the combination of lesions has never previously been encountered. In reality, there is no right or wrong way of describing the hearts, simply different ways.2 Even thIn terms of its basic make-up, the heart has three building blocks, namely the atriums, the ventricular mass, and the arterial trunks. The first systems of categorization based on recognition of the limited potential for variation in each of these cardiac segments were developed independently in the 1960s by two groups: one based in the United States of America, and led by Richard Van Praagh, Both of All of this changed with the advent of cross-sectional echocardiography. Since the mid 1970s, it has been possible with precision to determine how atriums are, or are not, joined to ventricles, and similarly to establish the precise morphology found at the ventriculo-arterial junctions. Thus, the system with which we have been involved was produced concomitantly with the development of echocardiography, with attention concentrated on the potential variations to be found across the atrioventricular and ventriculo-arterial junctions. The system was called, and is still called, sequential segmental analysis.5–7 It shDuring its evolution, the system has followed some basic and simple rules. From the outset, categories have been based on recognizable anatomical features, eschewing speculative embryological assumptions. Emphasis is placed on the morphology of the cardiac components, the way they are joined or not joined together, and the relations between them, as three different facets of the cardiac make-up. Any system that separates these features one from the other, does not use one to determine another, and describes them with mutually exclusive terms, must perforce be unambiguous. The clarity of the system then depends upon its design. Some systems opt for brevity, with formidable codifications constructed to achieve this aim. But clarThe system depends first upon the establishment of the arrangement of the atrial chambers. Attention is then concentrated on the anatomical nature of the junctions between the atrial myocardium and the ventricular myocardial mass. This feature, described as a type of connection, is separate from the additional feature of the morphology of the valve or valves that guard the junctions . The norImplicit in the system is the ability to distinguish the morphology of the individual atriums and ventricles, along with the pattern of branching of the arterial trunks taking origin from the ventricles. This is not as straightforward as it may seem, since often, in congenitally malformed hearts, the chambers or arterial trunks may lack some of the morphological features that most obviously characterize them in the normal heart. For example, the most obvious feature of the morphologically left atrium in the normal heart is the connection to it of the pulmonary veins. Hearts with totally anomalous pulmonary venous connection, for example, lack such a feature. In spite of this lack of pulmonary venous connection, it is almost always still possible to identify the left atrium. Considerations of this type prompted the concept now used for recognition of the cardiac chambers and great arteries. Called the morphological method, the prinThe morphologically left appendage, in contrast, is much narrower and tubular. It has a narrow junction with the remainder of the atrium. The pectinate muscles are confined within the appendage, with the posterior aspect of the morphologically left vestibule, also containing the coronary sinus, being smooth walled as it merges with the body of the atrium [The morphological method also shows its value when applied to the ventricular mass, which extends from the atrioventricular to the ventriculo-arterial junctions. Within the ventricular mass as thus defined, there are almost always two ventricles. Description of ventricles, no matter how malformed they may be, is facilitated if they are analysed as possessing three components. These are, first, the inlet, extending from the atrioventricular junction to the distal attachment of the atrioventricular valvar tension apparatus. The second part is the apical trabecular component. The third is the outlet component, supporting the leaflets of the arterial valve. Of these three components, it is the apical trabecular component that is most universally present in normal as well as in malformed and incomplete ventricles, and which most readily differentiates morphologically right from left ventricles . This isIn order fully to describe any ventricle, account must also be taken of its size. It is then necessary further to describe the way that the two ventricles themselves are related within the ventricular mass. This feature is described in terms of ventricular topology, since two basic patterns are found that cannot be changed without physically taking apart the ventricular components and reassembling them. The two patterns are mirror images of each other. They can be conceptualized in terms of the way that, figuratively speaking, the palmar surface of the hands can be placed upon the septal surface of the morphologically right ventricle. In the morphologically right ventricle of the normal heart, irrespective of its position in space, only the palmar surface of the right hand can be placed on the septal surface such that the thumb occupies the inlet and the fingers fit into the outlet. The palmar surface of the left hand then fits in comparable fashion within the morphologically left ventricle, but it is the right hand that is taken as the arbiter for the purposes of categorization. The usual pattern, therefore, can be described as right hand ventricular topology. The other pattern, the mirror image of the right hand prototype, is then described as left hand ventricular topology. In this left hand pattern, seen typically in the mirror-imaged normal heart, or in the variant of congenitally corrected transposition found with usual atrial arrangement, it is the palmar surface of the left hand that fits on the septal surface of the morphologically right ventricle with the thumb in the inlet and the fingers in the outlet. These two topological patterns can always be distinguished irrespective of the location occupied in space by the ventricular mass itself. Component make-up, trabecular pattern, topology, and size are independent features of the ventricles. On occasion, all may need separate description in order to remove any potential for confusion.Only rarely will hearts be found with a solitary ventricle. Sometimes this may be because a right or left ventricle is so small that it cannot be recognized with usual clinical investigatory techniques. There is, nonetheless, a third pattern of apical ventricular morphology that is found in hearts possessing a truly single ventricle. This is when the apical component is of neither right or left type, but is very coarsely trabeculated, and crossed by multiple large muscle bundles. Such a solitary ventricle has an indeterminate apical morphology . Analysi10When determining the morphology of the arterial trunks, there are no intrinsic features that enable an aorta to be distinguished from a pulmonary trunk, or from a common or solitary arterial trunk. The branching pattern of the trunks themselves, nonetheless, is always sufficiently characteristic to permit these distinctions. The aorta gives rise to at least one coronary artery and the bulk of the systemic arteries. The pulmonary trunk gives rise directly to both, or one or other, of the pulmonary arteries. A common trunk supplies directly the coronary, systemic and pulmonary arteries. A solitary arterial trunk exists in the absence of the proximal portion of the pulmonary trunk. In such circumstances, it is impossible to state with certainty whether the persisting trunk is common or aortic. Even in the rare cases that have transgressed one of these rules, examination of the overall branching pattern has always permitted us to distinguish the nature of the arterial trunk.The cornerstone of any system of sequential analysis must be accurate establishment of the arrangement of the atrial chambers, since this is the starting point for subsequent analysis. When this arrangement is assessed on the basis of the morphology of the junction of the appendages with the rest of the atriums, then since all hearts have two atrial appendages, each of which can only be of morphologically right or left type, there are only four possible patterns . The mosIn the ideal world, the arrangement of the appendages will be recognized by direct examination of the extent of the pectinate muscles round the vestibules . In skilGenerally speaking, isomerism of the right atrial appendages is associated with absence of the spleen, while isomerism of the left atrial appendages is associated with multiple spleens. Patients with isomerism of the atrial appendages, therefore, are frequently grouped together, from the cardiac standpoint, under the banner of the splenic syndromes. This approach is much less accurate than describing the syndromes directly in terms of isomerism of the atrial appendages, since the correlation between isomerism of the right atrial appendages and absence of the spleen, and between isomerism of the left atrial appendages and multiple spleens, is far from perfect. IsomerisIn the normal heart, the atrial myocardium is contiguous with the ventricular mass around the orifices of the mitral and tricuspid valves. Other than at the site of the penetration of the bundle of His, electrical insulation is provided at these junctions by the fibrofatty atrioventricular grooves. In order to analyse accurately the morphology of the atrioventricular junctions in abnormal hearts, it is first necessary to know the atrial arrangement. Equally, it is necessary to know the morphology of the ventricular mass so as to establish which atrium is connected to which ventricle. With this information to hand, it is then possible to define the specific patterns of union or non-union across the junctions, and to determine the morphology of the valves guarding the atrioventricular junctions. In hearts with complex malformations, it is also necessary, on occasion, to describe the precise topology of the ventricular mass, and to specify the relationships of the ventricles themselves.These patterns depend on the way that the myocardium of the atrial chambers is joined to the ventricular myocardium around the entirety of the atrioventricular junctions. The cavities of the atrial chambers are potentially connected to the underlying ventricular cavities via the atrioventricular orifices. In every heart, since there are two atrial chambers, there is the possibility for two atrioventricular connections, which will be right-sided and left-sided. This is the case irrespective of whether the junctions themselves are guarded by two valves or a common valve. One of the junctions may be blocked by an imperforate valvar membrane, but this does not alter the fact that, in such a setting, there are still two potential atrioventricular connections. In some hearts, in contrast, this possibility is not fulfilled. This is because one of the connections is absent. The atrial myocardium on that side then has no direct connection with the underlying ventricular myocardium, being separated from the ventricular mass by much more extensive formation than normal of the fibrofatty tissue of the atrioventricular groove. This arrangement is the most common pattern producing atrioventricular valvar atresia.When atrioventricular connections are defined in this fashion, all hearts fit into one of three groups. In the first group, by far the most common, the cavity of each atrial chamber is in actual or potential, and separate, connection with the cavity of an underlying ventricle. The feature of the second group is that only one of the ventricles, if indeed two are present, is in communication with the atrial cavities. There is then an even rarer third group. This is seen when one atrioventricular connection is absent, and the solitary atrioventricular junction, via a straddling valve, is connected to two ventricles. This arrangement is uniatrial but biventricular.There are 3 possible arrangements in hearts with each atrium joined to its own ventricle. These depend on the morphology of the chambers connected together. The first pattern is seen when the atriums are joined to morphologically appropriate ventricles, irrespective of the topology or relationship of the ventricles, or of the morphology of the valves guarding the junctions. This arrangement produces concordant atrioventricular connections, and exists with either usually arranged atrial appendages, or in the mirror-imaged arrangement . The secThese first two arrangements are found when the atrial appendages are lateralized. The other biventricular atrioventricular arrangement is found in hearts with isomeric appendages, be they of right or left morphology. This third arrangement cannot accurately be described in terms of concordant or discordant connections. Rather, it is a discrete biventricular pattern in its own right, which is mixed. It, too, is independent of ventricular relationships and atrioventricular valvar morphologies, and requires specification of ventricular topology to make the description complete.There are also 3 possible junctional arrangements that produce univentricular atrioventricular connections . The firDescribing the fashion in which the atriums are joined to the ventricles across the atrioventricular junctions accounts only for the way in which the atrial musculature inserts into the base of the ventricular mass. The morphology of the valves guarding the overall atrioventricular junctional area is independent of this feature, within the constraints imposed by the pattern of the junctions itself. When the cavities of both atriums are joined directly to the ventricular mass, the right- and left-sided atrioventricular junctions may be guarded by two patent valves, by one patent valve and one imperforate valve, by a common valve, or by straddling and overriding valves. These arrangements of the valves can be found with concordant, discordant, biventricular and mixed, or double inlet types of connection. Either the right- or left-sided valve may be imperforate, producing atresia but in the setting of a potential as opposed to an absent atrioventricular connection. A common valve guards both right- and left-sided atrioventricular junctions, irrespective of its morphology. A valve straddles when its tension apparatus is attached to both sides of a septum within the ventricular mass. It overrides when the atrioventricular junction is connected to ventricles on both sides of a septal structure. A right-sided valve, a left-sided valve, or a common valve can straddle, can override, or can straddle and override. Very rarely, both right- and left-sided valves may straddle and/or override in the same heart. When one atrioventricular connection is absent, then the possible modes of connection are greatly reduced. This is because the valve, of necessity, is solitary. The single valve is usually committed in its entirety to one ventricle. More rarely, it may straddle, override, or straddle and override. These latter patterns produce the extremely rare group of uniatrial but biventricular connections.A valve that overrides has an additional influence on description, since the degree of commitment of the overriding atrioventricular junction to the ventricles determines the precise fashion in which the atriums and ventricles are joined together. Hearts with two valves, in which one valve is overriding, are anatomically intermediate between those with, on the one hand, biventricular and, on the other hand, univentricular atrioventricular connections. When most of an overriding junction is connected to a ventricle that is also joined to the other atrium, the pattern is effectively double inlet. If the overriding junction is connected mostly to a ventricle not itself joined to the other atrium, then the pattern is more akin to concordant, discordant, or mixed connections, and is categorized in one of these fashions.When describing atrioventricular valves, the adjectives mitral and tricuspid are strictly accurate only in hearts with biventricular atrioventricular connections having separate junctions, each guarded by its own valve. In this context, the tricuspid valve is always found in the morphologically right ventricle, and the mitral valve in the morphologically left ventricle. In hearts with biventricular atrioventricular connections but with a common junction, in contrast, it is incorrect to consider the common valve as having mitral and tricuspid components, even when it is divided into right and left components. These right- and left-sided components, particularly on the left side, bear scant resemblance to the normal atrioventricular valves. In hearts with double inlet, the two valves are again better considered as right- and left-sided valves rather than as mitral or tricuspid, as is the case when one connection is absent. Valves can always accurately be described as being right or left sided.Even in the normal heart, the ventricular spatial relationships are complex. The inlet portions are more or less to the right and left, with the inferior part of the muscular ventricular septum lying in an approximately sagittal plane. The outlet portions are more or less anteroposteriorly related, with the septum between them in an approximately frontal plane. The trabecular portions extend between these two components, with the trabecular muscular septum spiralling between the inlet and outlet components. It is understandable that there is a desire to have a shorthand term to describe such complex spatial arrangements. We use the concept of ventricular topology for this purpose. When applying this concept, the morphologically right ventricle is considered in the way the palmar surfaces of the hands can be applied to its septal surface such that the thumb is in the inlet, and the fingers in the outlet, with the wrist occupying the apical trabecular component. This produces right hand and left hand patterns. In persons with usually arranged atriums and discordant atrioventricular connections, the ventricular mass almost always shows left-handed topological pattern, whereas right-handed ventricular topology is usually found with the combination of mirror-imaged atriums and discordant atrioventricular connections. Although these combinations are almost always present, exceptions can occur. When noting such unexpected ventricular relationships as a feature independent of the topology, we account for right-left, anterior-posterior and superior-inferior coordinates. And, should it be necessary, we describe the position of the three ventricular components separately, and relative to each other.In hearts with disharmonious arrangements in the setting of usual atrial arrangement and discordant atrioventricular connections, the distal parts of the ventricles are usually rotated so that the morphologically right ventricular trabecular and outlet components are to the right of their morphologically left ventricular counterparts, giving the impression of normal relationships. In such criss-cross hearts seen with usual atrial arrangement and concordant atrioventricular connections, the ventricular rotation gives a spurious impression of left-handed topology. In cases with extreme rotation, the inlet of the morphologically right ventricle may also be right sided in association with discordant atrioventricular connections. Provided relationships are described accurately, and separately, from the connections and the ventricular topology, then none of these unusual and apparently complex hearts will be difficult either to diagnose or to categorize. In addition to these problematic criss-cross hearts, we have already discussed how description of ventricular topology is essential when accounting for the combination of isomeric appendages with biventricular mixed atrioventricular connections. This is because, in this situation, the same terms would appropriately be used to describe the heart in which the left-sided atrium was connected to a morphologically right ventricle as well as the heart in which the left-sided atrium was connected to a morphologically left ventricle. The arrangements are differentiated simply by describing also the ventricular topology.Both the position and the relationships of incomplete ventricles need to be described in hearts with univentricular atrioventricular connections. Here, the relationships are independent of both the connections and the ventricular morphology. While, usually, the incomplete right ventricle is anterior and right sided in classical tricuspid atresia, it can be anterior and left sided without in any way altering the clinical presentation and haemodynamic findings. Similarly, in hearts with double inlet ventricle, the position of the incomplete ventricle plays only a minor role in determining the clinical presentation. When we describe the position of incomplete ventricles, therefore, we simply account for their location relative to the dominant ventricle, taking note again when necessary of right-left, anterior-posterior and superior-inferior coordinates. On occasion, it may also be advantageous to describe separately the position of trabecular and outlet components of an incomplete ventricle.Most polemics concerning the ventriculo-arterial junctions devolved upon the failure to distinguish between the way the arterial trunks arose from the ventricular mass as opposed to their relations to each other, along with undue emphasis on the nature of the infundibulums supporting their arterial valves. When these features are described independently, following the precepts of the morphological method, then all potential for disagreement is removed.As with analysis of the atrioventricular junctions, it is necessary to account separately for the way the arteries take origin, and the nature of the valves guarding the ventriculo-arterial junctions. There are four possible types of origin. Concordant ventriculo-arterial connections exist when the aorta arises from a morphologically left ventricle, and the pulmonary trunk from a morphologically right ventricle, be the ventricles complete or incomplete. The arrangement where the aorta arises from a morphologically right ventricle or its rudiment, and the pulmonary trunk from a morphologically left ventricle or its rudiment, produces discordant ventriculo-arterial connections. Double outlet connection is found when both arteries are connected to the same ventricle, which may be of right ventricular, left ventricular or indeterminate ventricular pattern. As with atrioventricular valves, overriding arterial valves are assigned to the ventricle supporting the greater parts of their circumference. The fourth ventriculo-arterial connection is single outlet from the heart. This may take one of four forms. A common trunk exists when both ventricles are connected via a common arterial valve to one trunk that gives rise directly to the coronary arteries, at least one pulmonary artery, and the majority of the systemic circulation. A solitary arterial trunk exists when it is not possible to identify any remnant of an atretic pulmonary trunk within the pericardial cavity. The other forms of single outlet are single pulmonary trunk with aortic atresia, or single aortic trunk with pulmonary atresia. These latter two categories describe only those arrangements in which, using clinical tecniques, it is not possible to establish the precise connection of the atretic arterial trunk to a ventricular cavity. If its ventricular origin can be established, but is found to be imperforate, then the connection is described, along with the presence of an imperforate valve. It is also necessary in hearts with single outlet to describe the ventricular origin of the arterial trunk. This may be exclusively from a right or a left ventricle, but more usually the trunk overrides the septum, taking its origin from both ventricles.There are fewer morphologies for the valves at the ventriculo-arterial than at the atrioventricular junctions. A common arterial valve can only exist with a specific type of single outlet, namely common arterial trunk. Straddling of an arterial valve is impossible because it has no tension apparatus. Thus, the possible patterns are two perforate valves, one or both of which may override, or one perforate and one imperforate valve. As with overriding atrioventricular valves, the degree of override of an arterial valve determines the precise origin of the arterial trunk from the ventricular mass, the overriding valve, or valves, being assigned to the ventricle supporting the greater part of its circumference. When making this decision, as with atrioventricular connections, we err on the side of the more usually encountered pattern.Relationships are usually described at valvar level, and many systems for nomenclature have been constructed on this basis. It is our practise to describe arterial valvar relationships in terms of both right-left and anterior- posterior coordinates. In this way, aortic valvar position is described relative to the pulmonary trunk in terms of eight positions of a compass, using the simple terms left, right, anterior, posterior and side-by-side in their various combinations. As long as we then remember that these describe only arterial valvar relations, and convey no information about either the origin of the arterial trunks from the ventricular mass, or the morphology of the ventricular outflow tracts, we have no fear of producing confusion.From the stance of positions of the arterial trunks, the possibilities are either for the pulmonary trunk to spiral round the aorta as it ascends from the base of the ventricles, or for the two trunks to ascend in parallel fashion. It is rarely necessary to describe these relationships. Spiralling trunks are associated most frequently with concordant ventriculo-arterial connections, and parallel trunks with discordant or double outlet connections, but again there is no predictive value in these relationships. In almost all hearts, the aortic arch crosses superiorly to the bifurcation of the pulmonary arteries. The side of the aortic arch depends on whether it passes to the right or left of the trachea. The position of the descending aorta is defined relative to the vertebral column.The infundibular regions are no more and no less than the outlet components of the ventricular mass. If the infundibular structures are recognized for what they are, and their morphology described as such, then they, too, provide no problems in recognition and description. The morphology of the ventricular outlet portions is variable for any heart. Potentially, each ventricle can possess a complete muscular funnel as its outlet portion, and then each arterial valve can be said to have a complete infundibulum. When considered as a whole, the outlet portions of the ventricular mass in the setting of bilateral infundibulums have three discrete parts. Two of the parts form the anterior and posterior halves of the funnels of muscle supporting the arterial valves. The anterior, parietal, part is the free anterior ventricular wall. The posterior part is the inner heart curvature, a structure that separates the leaflets of the arterial from those of the atrioventricular valves. We term this component the ventriculo-infundibular fold. The third part is the septum that separates the two subarterial outlets, which we designate the outlet, or infundibular, septum. It is possible, albeit rarely, for both arterial valves to be separated from both atrioventricular valves by the ventriculo-infundibular fold, but for the arterial valves to be in fibrous continuity with one another because of the absence of the outlet septum. In most hearts, however, some part of the infundibular musculature is effaced so that fibrous continuity occurs between the leaflets of one of the arterial and the atrioventricular valves. Most frequently, it is the morphologically left ventricular part of the ventriculo-infundibular fold that is attenuated. As a result, there is fibrous continuity between the leaflets of the mitral valve and the arterial valve supported by the left ventricle. Whether the arterial valve is aortic or pulmonary will depend on the ventriculo-arterial connections present. In the usual arrangement, the morphologically right ventricular part of the ventricular-infundibular fold persists, so that there is tricuspid-arterial valvar discontinuity. Depending on the integrity of the outlet septum, there is usually a completely muscular outflow tract, or infundibulum, in the morphologically right ventricle. When both outlet portions are connected to the morphologically right ventricle, then the ventriculo-infundibular fold can persist in its entirety, producing discontinuity bilaterally between the leaflets of the atrioventricular and arterial valves. But many hearts in which both arterial valves are connected unequivocally to the right ventricle have fibrous continuity between at least one arterial valve and an atrioventricular valve. It makes little sense to deny the presence of origin of both arterial trunks from the right ventricle in this setting. This situation is yet another example of the controversy generated when one feature of cardiac morphology is determined on the basis of a second, unrelated, feature. When both arterial trunks take their origin from the morphologically left ventricle, the tendency is for there to be continuity between the leaflets of both arterial valves and both atrioventricular valves. Even then, in some instances, the ventriculo-infundibular fold may persist in part or in its whole.It is usually the state of the ventriculo-infundibular fold, therefore, that is the determining feature of infundibular morphology. Ignoring the rare situation of complete absence of the outlet septum, and considering morphology from the standpoint of the arterial valves, there are four possible arrangements. First, there may be a complete subpulmonary infundibulum, with continuity between the leaflets of the aortic and atrioventricular valves. Second, there may be a complete subaortic infundibulum, with continuity between the pulmonary and the atrioventricular valves. Third, there may be bilateral infundibulums, with absence of continuity between the leaflets of the arterial and atrioventricular valves. Fourth, there may be bilaterally deficient infundibulums, with continuity bilaterally between the arterial and the atrioventricular valves. In themselves, these terms are not specific. For specificity, it is also necessary to know which arterial valve takes origin from which ventricle. This emphasizes the fact that infundibular morphology is independent of the ventriculo-arterial connections.The majority of patients seen with congenitally malformed hearts will have their cardiac segments joined together in usual fashion, together with normal morphology and relations. In such a setting, the associated malformation will be the anomaly. It is also necessary, nonetheless, to pay attention to the position of the heart within the chest, and the orientation of the cardiac apex, recognising that the heart may be positioned ectopically outside the thoracic cavity. An abnormal position of the heart within the chest is another associated malformation, and should not be elevated to a prime diagnosis. This is not to decry the importance of an abnormal cardiac position, if only to aid in interpretation of the electrocardiogram. Knowing that the heart is malpositioned, however, gives no information concerning its internal architecture. Full sequential segmental analysis is needed to establish the cardiac structure, and not the other way round. The heart can be located mostly in the left hemithorax, mostly in the right hemithorax, or centrally positioned in the mediastinum. The cardiac apex can then point to the left, to the right, or to the middle. The orientation of the apex is independent of cardiac position. Both of these features are independent of the arrangement of the atrial appendages, and of the thoracic and abdominal organs. Describing a right-sided heart, with leftward apex, should be understandable by all, even including the patient, or his or her parents.The system we have described is simple, and accounts for all malformations, even if a combination of lesions has never previously be en encountered or described. It depends only on recognition of the anatomy as it is observed. With the newly available techniques of three-dimensional imaging, all the features described should be seen as readily by the clinician as by the morphologist. Thus, there is hope that polemics concerning description of congenitally malformed hearts will now be viewed as part of the history of evolution of our specialty.


Mechanisms underlying inotropic failure in septic shock are incompletely understood. We previously identified the presence of exosomes in the plasma of septic shock patients. These exosomes are released mainly by platelets, produce superoxide, and induce apoptosis in vascular cells by a redox-dependent pathway. We hypothesized that circulating platelet-derived exosomes could contribute to inotropic dysfunction of sepsis.We collected blood samples from 55 patients with septic shock and 12 healthy volunteers for exosome separation. Exosomes from septic patients and healthy individuals were investigated concerning their myocardial depressant effect in isolated heart and papillary muscle preparations.Exosomes from the plasma of septic patients significantly decreased positive and negative derivatives of left ventricular pressure in isolated rabbit hearts or developed tension and its first positive derivative in papillary muscles. Exosomes from healthy individuals decreased these variables non-significantly. In hearts from rabbits previously exposed to endotoxin, septic exosomes decreased positive and negative derivatives of ventricular pressure. This negative inotropic effect was fully reversible upon withdrawal of exosomes. Nitric oxide (NO) production from exosomes derived from septic shock patients was demonstrated by fluorescence. Also, there was an increase in myocardial nitrate content after exposure to septic exosomes.in vivo exposure to lipopolysaccharide. The generation of NO by septic exosomes and the increased myocardial nitrate content after incubation with exosomes from septic patients suggest an NO-dependent mechanism that may contribute to myocardial dysfunction of sepsis.Circulating platelet-derived exosomes from septic patients induced myocardial dysfunction in isolated heart and papillary muscle preparations, a phenomenon enhanced by previous  Septic shock remains one of the most challenging medical conditions, with increasing incidence over the last years. The tendency of such an increase is probably due to progressive aging of the population, improvements in critical care support, and progress in chemotherapy and immunosuppressive therapies, with increased life expectancy of immunosupressed patients and patients with malignancies. Although much has been learned about the pathophysiology of sepsis in the last decade, the mortality of this condition is still elevated.One of the most important features of sepsis is myocardial dysfunction -3. StudiSeveral hypotheses have been formulated to explain the decrease in myocardial function in sepsis. Cytokines such as interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-α)  and, morA previous report from our laboratory showed the presence in the plasma of septic patients of microparticles characterized as exosomes . These pAll chemicals and reagents were purchased from Sigma-Aldrich  unless otherwise specified.We collected blood samples (50 mL) of 55 patients from the Emergency Medicine Intensive Care Unit of Hospital das Clinicas, University of São Paulo, Brazil, with early (less than 48 hours) diagnosis of septic shock defined according to the criteria of the American College of Chest Physicians and the Society of Critical Care Medicine . Twelve g for 10 minutes. Three (3.0) mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL aprotinin, and pepstatin were added to the supernatant, which then was sequentially filtered through 1-, 0.45-, and 0.22-μm nylon filters to remove platelets, cellular fragments, and apoptotic bodies. The remaining cell-free plasma was ultracentrifuged at 140,000 g for 150 minutes. The pellet containing exosomes first was washed with Tris (50 mM)-buffered saline (NaCl 150 mM) with ethylenediaminetetraacetic acid (EDTA) (0.1 mM) to avoid contamination with plasma proteins and then was resuspended with the same buffer. Protein concentration was measured by the Bradford method . After separation procedures, healthy individuals generated samples with 5.8 ± 0.96 mg of exosome protein per milliliter of solution, whereas septic patients generated samples with 16.5 ± 0.99 mg of exosome protein per milliliter of solution .The technique for isolating exosomes was performed as described previously . Brieflyg for 2 minutes to obtain platelet-poor plasma. A buffer containing Tris-NaCl-EDTA and PMSF 3 mmol/L was added to plasma, and another centrifugation  was performed. The pellet was then resuspended in 100 μL of the same buffer. Under FORMVAR® grids , 10 μL of exosome samples was deposited for 30 seconds for film absorption. Thereafter, 1% phosphotungstic acid (10 μL) was added to the grids, and these samples were submitted to transmission electron microscopy in a Philips EM-420 microscope .In experiments involving electron microscopy, exosome separation was performed without the steps of filtration and ultracentrifugation, in order to avoid aggregation of exosomes. Briefly, the plasma of septic patients was centrifuged at 11,000 2 1.25 mM, NaHCO3 25 mM, MgSO4 1.2 mM, NaH2PO4 1.15 mM, and glucose 11 nM) at pH 7.40 equilibrated with 95% O2/5% CO2 and at a constant temperature of 37°C, in order to maintain the heart with a retrograde coronary perfusion, according to the Langendorff technique. The heart was excised, an incision was made in the left atrium, and a ventricular drain as well as a fluid-filled latex balloon were inserted through the mitral orifice into the left ventricle. The balloon was filled to achieve a constant diastolic pressure of 5 mm Hg. We recorded left ventricular pressure, and the maximal positive (+dP/dtmax) and negative (-dP/dtmax) left ventricular pressure derivatives were electronically derived from the left ventricular signal by means of a data acquisition system . Hearts were maintained at 37°C throughout the experiment by enclosing the heart in a temperature-regulated double-glass chamber.Fifty male New Zealand rabbits weighing 2.5 to 3.5 kg were anesthetized with chloralose (60 mg/kg) and urethane (600 mg/kg) and were heparinized (250 IU). We performed a tracheotomy and submitted the rabbits to mechanical ventilation throughout the entire procedure. A torachotomy was performed, and the aorta was cannulated and perfused at a constant flow of 15 mL/minute with Krebs-Henseleit solution  to achieve a concentration equal to one half of plasma concentration. This concentration was chosen after preliminary experiments depicted similar effects of exosome infusion in concentrations varying from 0.5 to 1× plasma concentrations. The system was maintained under perfusion for 20 minutes. After this period, the system was again perfused for 15 minutes with Krebs-Henseleit solution free of exosomes. In another set of experiments, the system was perfused 20 minutes before and during exosomes infusion with specific reactive oxygen species (ROS) inhibitors (Table Escherichia coli serotype 026:B6) in a concentration of 1 mg/kg 6 hours before sacrifice. Three hours after exposure, rabbits were resuscitated with 8 mL/kg saline to reduce hypovolemia, which is a hallmark of this model. After 6 hours of LPS exposure, the hearts were removed and the experiments were performed as already described.To investigate the effects of exosome infusion in an experimental model more closely related to human septic shock, rabbits were challenged with endotoxin (lipopolysaccharide [LPS] 2 and 5% CO2) Krebs-Henseleit solution  in a bath at 29°C. The left ventricular chamber was opened, and anterior and posterior papillary muscles were dissected. Two stainless steel rings held the extremities of the muscles, and the muscles were vertically mounted in an organ bath filled with 35 mL of the same Krebs-Henseleit solution. The upper extremity of the muscle was connected by the ring to a force transducer , and the inferior ring was connected to a micromanipulator . The muscles were stimulated electrically at 0.2 Hz and at a voltage approximately 10% above threshold by rectangular pulses of 5-millisecond duration through two longitudinally placed platinum electrodes. The preparation was stabilized for 30 minutes at the muscle length at which the maximal active tension was developed. After stabilization, exosomes from septic patients and healthy volunteers in a concentration equal to one half of plasma concentration were incubated in bathing solution and maintained for 45 minutes. The parameters recorded were developed tension (DT), resting tension (RT), positive temporal derivative of developed tension (+dT/dt), and negative temporal derivative of developed tension (-dT/dt). These two derivatives report temporal variations in contraction and relaxation myocardial capabilities, respectively.Ten Wistar male rats weighing 350 to 450 g were anesthetized with ketamine and xylazine (0.1 mL per 100 mg of body weight each). A torachotomy was performed, and the heart was excised and immediately immersed in oxygenated  were performed. In other experiments, vascular rings were incubated with exosomes overnight and analyzed the next day as described above.Rat thoracic aortas from 10 Wistar rats were carefully dissected and divided in four 5-mm-long rings that were suspended in two intraluminal parallel wires, introduced in an organ bath containing Krebs-Hepes at a constant temperature of 37°C, and equilibrated with 95% O2 under neutral pH, yielding the highly fluorescent DAF-2T. DAF-2 (5 μM) was added to a suspension containing 10 μg of exosome protein in 100 μL of Krebs buffer (pH 7.4). After a stabilization period of 5 minutes, fluorescence measurements were acquired at 37°C in an Amersham FARCyte/Tecan Ultra fluorescence plate reader . Excitation wavelength was set to 495 nm, emission was set to 520 nm, and measurements corresponding to 40-microsecond integration of signals were obtained by 10 flashes. In some experiments when indicated, N(G)-nitro-L-arginine methyl ester (L-NAME) (100 μM) or superoxide dismutase (SOD) (250 IU/mL) was added to the exosomes 20 minutes before DAF-2. Autofluorescence was corrected for by the inclusion of parallel blanks and did not exceed 10% of the total fluorescence. Data are expressed as arbitrary fluorescence units per milligram of exosome protein.Recently, observations from the laboratory of one of the authors (MJ) demonstrated the presence of inducible NO synthase (NOS) in exosomes from septic shock patients . Thus, w3 and HCl (at 95°C) as reductants. Results were normalized for protein concentration.Samples of exosomes from septic patients were separated from their protein content by diluting the samples in an equal volume of trichloroacetic acid (10%) followed by centrifugation at 10,000 rpm for 10 minutes. Their intrinsic nitrate content was measured by chemiluminescence in a Sievers analyzer  with VClg for 5 minutes at 4°C. The supernatant was collected, and protein was quantified by the Bradford method. Samples (20 μL) had their nitrate content measured as described before, and the results were normalized for myocardial protein concentration.In other experiments, isolated rabbit heart preparations were exposed for 45 minutes to exosomes of septic patients and healthy volunteers. After exposure, the system was perfused for 5 minutes with Krebs-Henseleit solution at 4°C free of exosomes. The heart was removed, and myocardial tissue was carefully dissected and immersed in liquid nitrogen. The hearts were minced under liquid nitrogen, and the homogenate was resuspended in 1 mL of Tris HCl buffer  containing mercaptoethanol (0.1%) and PMSF (1 mM) and centrifuged at 5,000 t test and paired t test as indicated, and a p value of less than 0.05 was considered significant. Means within group and between groups as well as the factor × time interaction were analyzed using two-way analysis of variance  with Bonferroni's correction for multiple comparisons. Post hoc analysis was performed with the Tukey test. The software used was Sigma Stat 2.0 software .Data were considered normal using the Kolmogorov-Smirnov goodness-of-fit model and are presented as mean ± standard error of the mean. Single means were compared with the Student Figure Table max in isolated heart preparations. Incubation of the recirculating system with exosomes derived from septic patients at 0.5× plasma concentration induced a statistically significant decrease in myocardial contractility assessed through both positive and negative derivatives of left ventricular pressure when compared with baseline. This effect, however, was not statistically significant when compared with control exosomes. After 20 minutes, the exosome preparation was removed from the system and there was a spontaneous return of myocardial function to baseline levels after 15 minutes, indicating that the effects are reversible. Incubation with exosomes obtained from healthy individuals induced a non-significant decrease in myocardial contractility .Figure max, an effect not dependent on a direct action of the inhibitor on myocardial performance, since preincubation of the heart with apocynin before exosome exposure did not induce myocardial dysfunction (data not shown). We postulated that this effect could be mediated by increased availability of NO, due to apocynin-mediated decrease in superoxide production. Nevertheless, an apparent obstacle to this proposal was that NOS inhibition did not correct the loss in myocardial contractility. This result, however, should be taken with caution since NOS inhibition can promote significant vasoconstriction and lead to myocardial dysfunction [To identify the mechanisms inducing exosome-dependent myocardial dysfunction, we performed experiments in which the perfusate was preincubated for 20 minutes with ROS inhibitors or redox-active compounds Table  before efunction .max, 1,743.4 ± 266 mm Hg/s; -dP/dtmax, 1,225.1 ± 231 mm Hg/s). Despite the baseline dysfunction, infusion of exosomes obtained from septic patients in endotoxemic hearts induced a significant decrease in contractility when compared with baseline and, in addition, a significant decrease in +dP/dtmax as compared with infusion of septic and healthy exosomes in normal hearts not previously exposed to LPS. These results indicate that, in myocardial tissue primed by prior LPS exposure, exosomes may induce a more important negative inotropic effect.Hearts from endotoxemic rabbits were exposed to exosomes from septic individuals Figure . The meaAlthough isolated heart preparations are well-established systems for measuring myocardial performance, they may be subject to several pitfalls. To expand our results with isolated hearts, we incubated isolated rat papillary muscle preparations for 45 minutes with exosomes from septic patients and healthy volunteers at 0.5× plasma concentration. Exosomes from septic patients induced a statistically significant decrease in maximal DT and +dT/dt Figure , whereasStandard curves of norepinephrine-mediated contraction and acetylcholine-mediated relaxation did not depict a significant direct effect of exosomes in vascular tone (data not shown). These data were assessed with concentration of exosomes equal to plasma concentrations and different periods of incubation (2 hours and overnight incubation).Figure Exosomes from septic shock patients had an intrinsic nitrate content of 0.026 ± 8.5 μM per microgram of exosome protein. The nitrate myocardium content of hearts incubated for 45 minutes with normal and septic exosomes is demonstrated in Figure In this study, we showed for the first time that (patho)physiologically relevant concentrations of platelet-derived exosomes from septic patients induce a decrease in myocardial contractility in isolated heart and papillary muscle preparations. The effect of exosomes is enhanced by previous exposure to LPS. Therefore, exosomes may be implicated in myocardial dysfunction of sepsis. Exosomes also contain NOS, generate NO, and induce myocardial NO production. These data provide evidence that NO may be a pathway for septic exosome-derived myocardial dysfunction.In our Langendorff model, exosomes induced a significant decrease in myocardial contractility assessed by positive derivative of ventricular pressure. However, in these preparations, myocardial contractility is dependent on several variables, such as coronary perfusion pressure and heart rate, and compounds affecting these indices may not have an intrinsic myocardial depressant effect. Thus, we decided to perform some experiments in a system in which the effect of exosomes in myocardial contractility could be measured. The experiments with isolated papillary muscles demonstrated unequivocally an inhibitory effect of exosomes in myocardial function. However, in isolated papillary muscles, exosomes decreased myocardial performance only in the DT and its positive temporal derivative. This finding may be due to the possible dependence of myocardial relaxation on some peculiarities of the isolated heart, such as changes in transmural perfusion. The lack of a direct effect of exosomes in vascular tone evidenced in this study argues against a direct effect of exosomes in coronary tone. This result also contrasts with the decreased relaxation verified after exposure of vessels to other types of microparticles, such as vesicular particles exposing phosphatydilserine .In LPS-treated rabbits, exosomes induced more significant decreases in myocardial function. It is noteworthy that these preparations had baseline levels of dP/dt, on average, 65% lower than normal hearts. The enhanced effect of exosomes in myocardial contractility of septic hearts may be partially explained by the results obtained by Clayton and colleagues  which suAlthough the experiments with inhibitors could not clarify the precise mechanism of myocardial dysfunction, they were important for ruling out several possible pathways. In particular, experiments with apocynin, a specific NADPH oxidase inhibitor, called our attention to the possibility of NO-mediated dysfunction. Our results with L-monomethyl-arginine do not rule out NO as the mediator of myocardial dysfunction, probably due to coronary vasoconstriction induced by NO antagonism .NO has been implicated for a long time in sepsis-induced myocardial dysfunction. Classic studies have linked cytokine production to cardiac dysfunction mediated by myocardial NOS ,20. One Inducible NOS (NOS2) has also been linked to myocardial dysfunction of sepsis -23. MiceAnother possible source of myocardial depression is peroxynitrite and/or related toxic by-products of reaction between NO and superoxide. Ferdinandy and colleagues  demonstrAlthough our results are encouraging, some study limitations should be considered. First, the isolated heart or papillary muscle preparations provide interaction of exosomes with different cell types. Thus, our results should not be taken to represent exclusively the effects of exosomes with cardiomyocytes. Particularly, in isolated hearts, effects of exosomes on coronary vascular tone could account for the observed dysfunction, a limitation overcome in the papillary muscle preparation and also discouraged by the absence of an effect of exosomes on isolated vessels. On the other hand, taken together, the strength and reproducibility of the effects in both preparations suggest at least some direct effect of exosomes on myocardial contractility. A puzzling question was the observed effect of healthy exosomes in the myocardium. Although this effect was small and not statistically significant versus baseline, it was sufficiently evident to jeopardize a statistical difference with septic exosomes. Indeed, there is evidence in the literature of a cellular effect of 'sham' exosomes, mainly related to ROS production ,24. Thusin vivo is not yet established, the data depicted here indicate that exosomes present in the circulation may represent a novel redox-signaling microenvironment and contribute to myocardial dysfunction of sepsis.This study showed that (patho)physiologically relevant concentrations of exosomes derived from platelets of patients with septic shock may induce myocardial dysfunction in isolated rabbit hearts and isolated papillary muscle preparations. The increase in myocardial dysfunction after LPS exposure and the increase in myocardial content of NO derivatives as well as the production of NO by exosomes from septic patients suggest that NO may be a significant contributor to this phenomenon. Although the significance of this dysfunction • Previous data have demonstrated the presence of exosomes in the plasma of septic shock patients.• Exosomes derived from the platelets of septic shock patients induce myocardial dysfunction in isolated heart and isolated papillary muscle preparations.• Previous exposure of hearts to lipopolysaccharide increases exosome-derived myocardial dysfunction.• Data supporting nitric oxide (NO) production by exosomes and the increase in myocardial NO content after exosome exposure suggest that NO may be associated with the induction of myocardial derangement in this setting.• Exosomes may represent a novel vascular redox-active pathway in sepsis.max = maximal positive derivative of left ventricular pressure; -dP/dtmax = maximal negative derivative of left ventricular pressure; DT = developed tension; +dT/dt = positive temporal derivative of developed tension; EDTA = ethylenediaminetetraacetic acid; IL = interleukin; L-NAME = N(G)-nitro-L-arginine methyl ester; LPS = lipopolysaccharide; NO = nitric oxide; NOS = nitric oxide synthase; NOS2 = inducible nitric oxide synthase; PMSF = phenylmethylsulfonyl fluoride; ROS = reactive oxygen species; RT = resting tension; SOD = superoxide dismutase; TNF-α = tumor necrosis factor-alpha.DAF = 4,5-diaminofluorescein; +dP/dtThe authors declare that they have no competing interests.LCPA conceived the study, carried out the experiments, analyzed and interpreted the data, and wrote the manuscript. MJ helped to conceive the study, carried out the experiments in ROS production, analyzed and interpreted the data, and corrected the final version of the manuscript. VP carried out the experiments with isolated heart preparations, analyzed the data, and helped to write the draft of the manuscript. MAP carried out the experiments, helped to write the draft of the manuscript, and corrected the final version. EB carried out organ chamber experiments and collected, analyzed, and interpreted the data. PJFT participated in the design of the study, carried out papillary muscle experiments, and analyzed and interpreted the data. FRML conceived and supervised the study, analyzed and interpreted the data, and corrected the final version of the manuscript. All authors read and approved the final manuscript.


A number of “interesting” risk markers have been proposed as providing prognostic informations in acute coronary syndromes (ACS). Elevation in plasma inflammatory and necrosis biomarkers have been related to future cardiovascular events in individuals with or without prior myocardial infarction. Recently BNP and pro-BNP are entered in clinical practice to recognize patients at major risk, providing incremental information respect to the traditional markers. Together with these laboratory indexes, a few of promising laboratory markers once easily available, could become usefull in identification of patients at high risk.Several studies evaluated many markers of platelet aggregation, endothelial dysfunction and vascular thrombosis, but it is not yet clear whether each of the proposed markers may provide incremental predictive information.We describe, following the most studies reported in literature, the laboratory markers with potential clinical and prognostic power that could early help physicians in the identification of patients with impaired coronary disease and more narrowed coronary arteries. Acute coronary syndromes without ST elevation (ACS) are heterogeneous disorders ranging from unstable angina pectoris (UA) without evidence of myocardial necrosis to myocardial infarction (MI) associated with a significant increase of troponin and isoenzime MB creatinkinase.1Because of heterogeneous clinical presentation, ACS encompasses a wide range of events and different prognosis in relation to plaque type lesions and coronaric atherosclerosis diffusion. Risk assessment is based on clinical history and examination, electrocardiographic (ECG) characteristics and markers of myocardial damage, but it remains relatively inaccurate.Recently, several biomarkers have been purposed to identify patients at high risk needing for more aggressive therapy or early angiography, to detect their risk profile and optimise the treatment.In this report we review, on the basis of more recent literature, the biomarkers with potential clinical and prognostic significance able to recognize patients with more advanced coronary disease; therefore, we describe the eventual possibility to identify, by laboratory research at hospital admission, patients candidates for angiographic study that would more benefit from aggressive behaviour. Advances in understanding of coronary disease have focused attention on the plaque composition rather than degree of stenosis; non stenotic lesions are more frequently implied in ACS, nonocclusive plaques may become complicated by thrombus and rapidly progress to total occlusion. Five principal causes of ACS habe been described, they include: 1) plaque rupture with acute thrombosis by formation of blood clot that gradually occludes the lumen, the pivotal event in the atherothrombotic process; 2) mechanical obstruction due to progressive increase of plaque volume; 3) inflammation, by local and sistemic production of mediators as cytokines growth factors and enzymes that activate the plaque and disrupt the fibrous cap; 4) dynamic obstruction influenced by endothelial dysfunction leading to coronary vasocostriction; 5) plaque embolization at distant level from atherothrombotic coronary occlusion.5Traditional cardiac risk factors that predispose to atherosclerosis are only a part of the equation and must be considered within the setting of other systemic and local contributors that may adversely affect coronary plaque progression and thrombotic complication. The efforts must be addressed in early detection of culprit plaque, by more sensitive serum markers assessment to find and treat the vulnerable patients at elevated risk.Recently AHA/ACC have purposed a TIMI risk score for identification of patients with major risk, however this classification is not completely able to identify all high risk subjects. Hence, the introduction of other markers easily available could bring to worth a better risk profile evaluation.6Classical cardiac markers usually considered as strong predictors among patients with coronary disease, are Troponin T (TnT) and C-reactive Protein (CRP), both related to increased risk of recurrent ischemic events and cardiovascular death.Elevated TnT levels appear related to more ‘active’ lesions, which in turn are associated with increased long-term coronary instability. Angiographic studies have shown an association between increased troponin levels and complex lesions, more severe coronary artery disease, decreased coronary blood flow and greater activation of the coagulation system compared to patients with normal troponin levels.7Recently, a substudy from GUSTO-IV clearly demonstrated that TnT is associated with early and late risk of mortality and myocardial infarction, probably due to greater microembolization risk or to major extension of coronary atherosclerosis.The correlation between elevated CRP levels and prognosis in coronary disease may be mediated by the vascular effects of proinflammatory cytokines, including tumor necrosis factor TNF-α, interleukin IL-1 and IL-6, which induce the hepatic production of CRP.10Some works have indicated that the protein doesn’t reflect only inflammation stage that concur to atherosclerotic process, but it could have a direct role in the plaque rupture: unstable atherosclerotic plaques have an increased number of macrophages that seem to be most abundant in the vulnerable shoulder region of the fibrous cap, overlying the core of atheroma within the vessel wall.12CRP increase appears related to incidence of revascularization in patients with ACS and increased risk of recurrent ischemia. However, studies are not in accordance: Toss et al. recruited no correlation; on the other hand, Liuzzo et al. showed worse prognosis in patients with UA.15The highest values in CRP have been found in patients with the largest increase in troponin concentrations. High CRP levels are independent predictors of death in these patients, but are not associated with a greater risk of MI. These apparently contradictory findings may be explained by hypothesizing a strong influence of troponin values on CRP increase in ACS.16Many studies have been shown that elevation of BNP as well as NT-proBNP levels obtained after the acute phase  in patients with a broad range of ACS independently predicts mortality.21Data from both experimental and clinical studies suggest that the plasma levels of BNP may reflect the size or severity of ischemic insult, even in absence of myocardial necrosis. Specifically, in human models of ischemia, BNP levels have been shown to increase transiently both after exercise in patients with stable coronary disease and after uncomplicated coronary angioplasty, despite stable intracardiac filling pressures.24All these data suggest that BNP could represent not only a marker of LV enlargement but also a marker of coronary disease extension: BNP could increase during ischemic damage in patients with CAD, suggesting that elevated levels are associated with larger extent as well as greater severity of ischemia, independently on LV systolic dysfunction. Biomarkers of platelet activation may provide clues regarding the extent of platelet activation in patients with ACS and predict the severity of the inflammatory response. Rupture or erosion of an atheromatous plaque exposes subendothelial proteins such as collagen and von Willebrand factor, leading to adhesion via the platelets surface receptors. This adhesion causes platelet activation which triggers a conformational change in the platelet glycoprotein IIb/IIIa receptor and generates the enzyme thrombin from circulating prothrombin which converts fibrinogen to fibrin. Fibrin and von Willebrand factor bind to the GP IIb/IIIa receptor and trap multiple activated platelets leading to their aggregation.Soluble CD40 ligand is a transmembrane glycoprotein of the tumor necrosis factor family. CD40L has been shown to promote atherosclerosis and plaque instability,27In patients with ACS, sCD40L identifies a subgroup at increased 6-month risk of death or non-fatal MI.P-selectin is a transmembrane cell adhesion molecule stored in endothelial cells and in platelet granules. Endothelial cell P-selectin mediates tethering and rolling of leukocytes along the activated endothelium, whereas platelet P-selectin mediates formation of platelet aggregates in pulsatile high-shear stress conditions.Attempts to use plasma P-selectin for risk stratification in ACS have yielded conflicting results. Some studies have suggested that P-selectin may be useful in risk stratification for patients presenting with chest pain;32Tissue plasminogen activator (t-PA) is a glycoprotein produced mainly by vascular endothelial cells.35A combined analysis of studies evaluating t-PA in coronary diseases, adjusted for some risk factors, suggests that CHD risk is about 50% greater in those with increased levels: a comparison of those in the top third with those in the bottom third of baseline t-PA antigen values yielded an odds ratio for CHD of 2.20  after adjustment for age and town only, and of 1.48  after further adjustment for classical CHD risk factors.37Epidemiological studies of t-PA antigen consistently show strong correlations with PAI-1 activity or antigen.39What is the potential biological significance of the positive association between circulating t-PA antigen and risk is still unknown: t-PA is released from vascular endothelium, hence increased circulating levels may be a marker of endothelial disturbance. In one study, circulating t-PA antigen correlated strongly with circulating levels of von Willebrand factor (vWF), another endothelial release product, as well as with risk markers associated with endothelial dysfunction. Tissue plasminogen activator and PAI-1 may partly reflect their mutual association with the inflammatory response. While increased circulating free t-PA might increase fibrin lysis, no significant association of t-PA antigen was observed with fibrin D-dimer levels.Metalloproteinases (MMPs) are zinc-dependent endoproteases with collagenase and/or gelatinase activity. Degradation of collagen fibrils compromises plaque stability and the integrity of the endothelial basament membrane, predisposing advanced atheromas to rupture.41There are many types of MMPs, the most known are MMP-2, a gelatinase capably to degrade type IV collagen, represented in the subendothelial basement membrane. MMP-8 is a collagenase produced by neutrophils, but also by endothelial cells, smooth muscle cells and macrophages in plaques. MMP-9 is a gelatinase widely implicated in ventricular remodeling and the development of heart failure.Some groups have found that MMP-1, -2 and -9 are not elevated at the presentation, but that they increase during the subsequent 7 to 14 days.44Another study reports an association between MMP levels during ACS and cardiovascular outcomes. In a study of 24 patients with ACS, elevation in MMP-1 at 7 and 14 days after ACS were negatively correlated with left ventricular ejection fraction.It has recently recognized that polymorphonuclear neutrophils (PMNs) are critical mediators in ACS, because of their recruitment and activation before platelet aggregation.49Elevated MPO plasma levels in patients with unstable angina and acute myocardial infarction have been shown. Biasucci et al. demonstrated that neutrophils are activated in acute coronary syndromes, may be not only secondary to ischemia-reperfusion injury.50Some authors found that MPO serum levels were equally distributed among patients with low and high Troponin T (TnT) serum levels, which indicates that elevated MPO levels are not temporary related to myocardial injury.49Pregnancy-associated plasma protein A (PAPP-A) is a peptide specifically elevated in pregnancy, specially in the first trimester and it is used for the screening of chromosomal abnormalities. PAPP-A is a zinc-binding metalloprotease that indirectly activates insuline-like growth factor (IGF), a potent mitogen and chemotactic agent for vascular smooth muscle cells. Because inhibition of IGF signaling has been shown to delay atherosclerosis, it is possible that PAPP-A indirectly promotes atherosclerosis or restenosis by increasing the activity of IGF. Infact, PAPP-A levels after angioplasty have been implicated as a possible mechanism for restenosis.51In the same study has been found that PAPP-A was not associated with TnT or creatine-kinase-MB elevation, suggesting that PAPP-A is not useful for detectable myocardial infarction, though it may have diagnostic value for identifying patients with ACS. Other studies would confirm the hypothesis that PAPP-A is an independent predictor of cardiovascular events, regardless of troponin status.53These studies suggest that PAPP-A may define subsequent cardiovascular risk in patients with unstable angina and no troponin elevation. Moreover, increased serum levels of PAPP-A may reflect instability of atherosclerotic plaques.54In the last few years, several studies demonstrated that raised levels of interleukin-6 are common in unstable angina, correlate with C-reactive protein, and are associated with prognosis, thus confirming the importance of the cytokine pathway for the production by the liver of acute-phase proteins and strengthening the importance of inflammation in this syndrome. Therefore, interleukin (IL)-18, originally identified as an interferon (IFN)-γ-inducing factor in Kupffer cells and macrophages, has been proved to play a central role in the inflammatory cascade. Furthermore, IL-18 acts in synergy with IL-12 to promote the development of T helper 1 responses.56The ST2 gene is a member of the interleukin-1 receptor host defense-inflammation family. The protein product of ST2 encodes a transmembrane receptor form (ST2L) and a truncated soluble receptor form (ST2) that can be detected in human serum. It is known that ST2 directs Th2 lymphocyte function; this finding suggests that ST2 is induced in conditions of myocardial overload such as myocardial infarction, when the remaining viable myocardium must bear more stress. Indeed, soluble ST2 levels are increased in the serum of patients one day after myocardial infarction. Furthermore, ST2 serum levels predict outcome in patients with heart failure, and a change in ST2 over time is also associated with prognosis.58Several emerging laboratory markers are now disposal for diagnosis and prognosis of ACS. Some common parameters are actually recommended for risk stratification , some others could become important to identify patients at higher risk: the more promising seem to be parameters of platelet activation (P-selectin and t-PA) and inflammatory markers mediators (MPO) .Every cited markers could represent a specific index of atherosclerotic process and plaque activation, physicians effort could address in the discover and identification of laboratory parameters with both easy sample and clinical significance application. The combination approach could permit in the future to encompass from the laboratory to the clinical aspects and to recognize patients with more dangerous coronary lesions and coronary disease severity.


Drug refractory ventricular tachycardia (VT) occurring as a storm after acute myocardial infarction has grave prognosis. We report a case of a middle-aged lady who presented with drug refractory VT that lead to persistent electrical storm two weeks after an anterior wall myocardial infarction. She underwent a successful catheter ablation of VT followed a few days later by implantation of an AICD. Catheter ablation of the VT could control the persistent electrical storm and the patient was free from a recurrence of VT at three month follow up. The incidence of incessant drug refractory ventricular tachycardia (VT) after a remote myocardial infarction (MI) is quoted to be 10% . The sucA 62 years old lady was admitted to an outside facility with evolved anterior wall myocardial infarction and had undergone rescue angioplasty and stenting of single vessel disease in left anterior descending coronary artery (LAD) under cover of Eptifibatide. Ten days after the stenting she developed recurrent episodes of monomorphic VT , suggesting a driver role in the sustenance of the tachycardia  and 7.5F Navistar F curve irrigated tip catheter. A bipolar LV voltage map was created to define the scar and border zone of MI. The infarct area was defined during sinus rhythm by electrograms with an amplitude ≤ 1.5 mV; dense scars were defined by electrograms with an amplitude ≤ 0.5 mV. Once the map was completed, amplitude scale was adjusted (0.2 to 0.5 mV), setting the value for scar at ≤ 0.2 mV to identify conducting channels within the scar area. They were marked out as corridors of continuous electrograms differentiated from the surrounding scar tissue by a higher amplitude, bounded by two scar areas, or by one scar area and the mitral annulus, and connected to normal myocardium by at least two sites . A largehycardia . RF lesihycardia . After tElectrical storm, defined originally as = 3 distinct episodes of VT/VF within 24-hour period that occurs in ICD recipients, can rarely occur in the few weeks following AMI . The acuRF ablation of VT soon after myocardial infarction is more difficult and often challenging, but a need for an attempt at ablation always exists since the mortality rate remains high even with combination antiarrhythmic drug therapy and left stellate ganglionic blockade. Both activation mapping and approaches during sinus rhythm have been shown to be effective methods for ablation of VT. Previous studies conducted on arrhythmias soon after experimental acute myocardial infarction have suggested different kinds of mechanisms responsible for its occurrence. They include enhanced automaticity of subendocardial Purkinje fibers that survived myocardial infarction , triggerIncessant ventricular tachyarrhythmia soon after MI leading to drug-resistant electrical storm is a rare but technically challenging entity. A search for various triggers that initiate refractory VT should be meticulously made to enhance the success rates of catheter ablation of the VT and to use it as a bailout therapy in these patients.


Light chain amyloidosis (AL) is a rare but often fatal disease due to intractable heart failure. Amyloid deposition leads to diastolic dysfunction and often preserved ejection fraction. We hypothesize that AL is associated with regional systolic dyssynchrony. The aim is to compare left ventricular (LV) regional synchrony in AL subjects versus healthy controls using 16-segment dyssynchrony index measured from 3-dimension-al (3D) echocardiography.Cardiac 3D echocardiography full volumes were acquired in 10 biopsy-proven AL subjects  and 10 healthy controls . The LV was subdivided into 16 segments and the time from end-diastole to the minimal systolic volume for each of the 16 segments was expressed as a percent of the cycle length. The standard deviations of these times provided a 16-segment dyssynchrony index (16-SD%). 16-SD% was compared between healthy and AL subjects.Left ventricular ejection fraction was comparable . 16-SD% was significantly higher in AL versus healthy subjects . 16-SD% correlated with left ventricular mass index  but not to left ventricular ejection fraction.Light chain amyloidosis is associated with left ventricular regional systolic dyssynchrony. Regional dyssynchrony may be an unrecognized mechanism of heart failure in AL subjects. Light chain amyloidosis (AL) is a rare multiorgan disease with extracellular deposition of fibrillar amyloid proteins derived from immunoglobulin light chains ,2. AmyloTen consecutive biopsy-proven AL subjects undergoing workup at 1 institution and 10 healthy controls underwent 3D echocardiography . The diagnosis was initially confirmed by biopsy for light chain amyloid in kidneys (n = 4), cardiac (n = 3), bone marrow (n = 2), gastrointestinal tract (1), fat pad, axillary mass (n = 1 each). Among AL subjects, 7 had cardiac involvement as defined by cardiac biopsy or subendocardial late gadolinium enhancement on routine magnetic resonance imaging ,17. ThreThe study was approved by the local Institutional Review Boards (IRB) and is in compliance with the Helsinki Declaration. All healthy controls gave informed consent. 9 AL subjects signed informed consent as part of a prospective observational study of biopsy-proven AL subjects. 1 AL subject who had tissue biopsy confirmation of the disease at post-mortem did not provide informed consent and waiver of consent authorization was obtained from the IRB.In all subjects, cardiac 3D full-volume datasets were acquired from the apical window using either an IE33 or Sonos 7500 echocardiograph and X3-1 and X4-2 full matrix-array transducer . The full volume data sets consisted of 4 real-time subvolumes acquired during 4 cardiac cycles that are subsequently combined to create a full 3D pyramidal data set. The data sets all had evaluable endocardial borders.The 3D volume dataset were analyzed by software  similar to previously published procedure . In brieThe LV was divided into 16 segments (excluding the apex) as per American Society of Echocardiography recommendations . The volTwo investigators (RQM and LH) trained in 3D volume analyses independently measured the 3D dataset blinded to disease condition and measured the subjects in random order. The first investigator repeated the measurement greater than a week after the first measurement in random order and still blinded to subject condition.Left ventricular mass index (LVMI) was calculated by Devereux's formula using the diastolic left ventricular internal diameter, anteroseptal thickness and inferolateral thickness . Left atData are expressed as mean ± standard deviation. Continuous variables were compared by unpaired Student's t-test for normally distributed data -or Mann-Whitney rank-sum test for non-normally distributed data. Correlation analysis was performed using Pearson's correlation. Intraobserver and interobserver agreement was assessed using intraclass correlation coefficient (ICC) analyses and method of differences by Bland-Altman. For the ICC, a two-way mixed model absolute agreement type was used . In thisEight AL patients had New York Heart Association functional classification I, with 1 each presenting in Class III and IV heart failure. Left ventricular ejection fraction was 62.4 ± 0.6% in control subjects and 58.6 ± 2.8% for AL (p = NS) (Table There was higher 16-SD% in AL subjects compared to controls Table . There wIntraclass correlation coefficient was 0.625 (p = 0.001) for intraobserver and 0.606 (p = 0.002) for interobserver differences in 16-SD%. The ICC together with the Bland-Altman analysis Figure  show gooLight chain amyloidosis is an often fatal disease with about 2500 new cases per year in the United States ,32-35. PHeart failure is an important cause of morbidity and mortality in AL amyloidosis. There is increasing recognition that timing of regional ventricular contraction or ventricular synchrony is important in the development of cardiac dysfunction and heart failure. Both interventricular and intraventricular dyssynchrony reduce ventricular efficiency and performance . TreatmeThe current finding needs to be related to the recent study of Bellavia, et al.  that utiThe 3D echocardiographic method of assessing ventricular dyssynchrony appears to have good, although not excellent, agreement when measured by the same or different observers based on intraclass correlation coefficient and Bland-Altman analysis. The reliability of this technique for use in AL amyloidosis subjects needs to be validated in a larger number of subjects. In future studies, comparing the presence and degree of dyssynchrony between amyloid subjects and other benign conditions presenting with ventricular thickening such as hypertension may be useful in determining whether this novel measure is clinically useful in the early diagnosis of AL cardiac amyloidosis.Intraventricular segmental dyssynchrony is demonstrated for the first time in light chain amyloidosis subjects compared to healthy controls with higher temporal pattern of dispersion of regional volume systolic change (16-SD%) on 3D echocardiography. Since left ventricular ejection fraction is relatively preserved in this cohort of AL subjects, regional intraventricular dyssynchrony by 3D echocardiography is a novel technique that may allow earlier detection of systolic dysfunction in this disease condition.The authors declare that they have no competing interests.RQM and PH contributed to conception and design of the study, data acquisition and analyses and drafting of the manuscript. LH, TW, MB and ST contributed to acquisition of data and manuscript review. All authors gave final approval of the manuscript submitted.Normal control subject. Top panel shows motion of the left ventricle with regions color-coded by standard ASE segmentation. The mesh represents the end-diastolic position of the segments. Lower panel shows the volume of each segment as a percent of end-diastolic volume by time normalized to the cardiac cycle length. Note that the timing of peak systolic volume change occurs almost simultaneously in this normal control subject.Click here for fileAL patient. A similar 3D echocardiographic acquisition as in Movie 1 but in an AL patient. Note that there is less synchronous regional motion. Bottom panel shows the dispersion of timing of peak systolic volume change in the different segments.Click here for file


Aim of this study was to elucidate the relation between localised inflammatory heart disease and endothelial dysfunction in the peripheral circulation, considering circulating cytokines as a potential link.In 38 patients with non-ischemic heart disease, myocardial biopsies were examined for myocardial inflammation (immunohistology) and virus persistence (PCR). Cytokines  were measured by ELISA in venous serum. Endothelial function of the radial artery was examined by ultrasound, measuring diameter changes in response to reactive hyperemia (FMD), compared to glyceroltrinitrate (GTN-MD). Patients with EF < 35% were excluded.Age 44 ± 14 years, 19 male, 19 female, EF 63.5[16]%. FMD 4.38 [4.82]%. 30 patients had myocardial inflammation (8 not), 23 virus persistence (15 not). FMD correlated significantly with sIL-12p7 , but not with other cytokines. sIL-12p7 levels were significantly higher in patients with severely impaired FMD (n = 17), compared with normal FMD (n = 21): 10.70 [10.72] vs. 4.33 [7.81] pg/ml (p = 0.002). Endothelium independent vasodilation (GTN-MD 23.67 [8.21]%) was not impaired.Endothelial dysfunction of peripheral arteries in patients with non-ischemic cardiomyopathy is associated with elevated serum concentrations of sIL-12p7, but not of other cytokines. Circulating sIL-12p7 may partly explain, that endothelial dysfunction is not restricted to the coronary circulation, but involves systemic arteries. Endothelial dysfunction in patients with non-ischemic heart disease, which has been observed in the coronary circulation  as well We included 38 patients with non-ischemic heart disease, considering history, physical examination and non-invasive tests, that were referred to our hospital for acquisition and examination of myocardial biopsies. Inclusion criteria were 1. suspected cardiomyopathy by history and symptoms  or by history and ECG-changes  and 2. echocardiographic regional wall motion abnormalities or global left ventricular dysfunction. Duration of symptoms was at least 3 months. By left ventricular catheterization and angiography, coronary artery disease was excluded, left ventricular ejection fraction and pressures were measured. Right-ventricular catheterization was performed to obtain endomyocardial biopsies and perform hemodynamic measurements. To minimize other confounding factors on endothelial dysfunction, patients with coronary artery disease -11, diabInformed consent was obtained from all patients.Endothelial function of the radial artery was measured, as published previously ,3. By hiFMD represents the percentage of diameter increase caused by shear stress, compared to baseline. GTN-MD represents the percentage of diameter increase induced by GTN, compared to baseline.Venous blood samples were obtained from the cubital vein from each patient by standard venous puncture. Samples were immediately centrifuged and the serum stored at -80°C. Cytokine measurements  were performed with standard sandwich enzyme immunoassay (ELISA) kits according to the manufacturer's instructions: Hölzel Diagnostica Handels GmbH Pelikine Compact TM human IFN ELISA kits (sensitivity 1pg/ml), Hölzel Diagnostica Handels GmbH Pelikine Compact TM human IL-4 ELISA kit (sensitivity 0.2 pg/ml), Genzyme Diagnostics PredictaHuman Total Interleukin 12 ELISA kit (sensitivity 10 pg/ml).Endomyocardial biopsies from the right ventricular septum were obtained by standard percutaneous transvenous femoral approach with a standard bioptome.2). Endothelial expression of HLA-1, HLA-DR and ICAM-1 was scaled , according to intensity of immunoperoxidase staining. Myocardial inflammation was confirmed in myocardial biopsies, if more than 7 CD3+ lymphocytes per 1 mm2 tissue were identified and/or if endothelial expression of cell adhesion molecules was enhanced. Myocardial biopsies were examined and analyzed by two independent blinded observers.For immunohistological evaluation, the samples were prepared and evaluated as published previously -3,29,30.p rank-order analysis, calculating the coefficient of correlation r. Statistical significance was inferred at p < 0.05.Statistical analysis was performed with the SPSS Inc.  software, version 12.0 for IBM-PC. Descriptive data are expressed as median and interquartile range. Quantitative data were compared to qualitative data by use of the Kruskal-Wallis test on rank sums for independent samples, followed by a post hoc multiple comparison procedure, if appropriate. To compare quantitative data of two groups, the Mann Whitney U test was applied. Quantitative data were correlated by use of the Spearman Mean age of the 38 patients was 44.5518 years, 19 were male, 19 female. At the time of investigation, all patients were normotensive (9 treated for hypertension) and had normal lipid-levels (8 treated with statins), 4 were smokers. Of the patients on standard cardiovascular medication, 11 were treated with ACE-inhibitors, 1 with AT1-antagonists, 9 with betablockers, 2 with calciumantagonists and 5 with digitalis glucosides.29 patients presented with chest pain (angina), 12 with palpitations, 34 with fatigue or exercise intolerance. At the time of inclusion in the study, all patients were in NYHA-stage I–II.ST-segment or T-wave changes were documented in the ECGs of 11 patients, rhythm disturbances in 9 patients. Echocardiography revealed regional wall motion abnormalities or an impaired global systolic left ventricular function, according to the inclusion criteria. Pericardial effusions were not observed.2).Left ventricular ejection fraction (EF) was 63.5[16]%, left ventricular enddiastolic pressure (LVEDP) 74 mmHg, pulmonary capillary wedge pressure (PCWP) 66 mmHg; cardiac output (CO) 5.652 l/min and cardiac index (CI) 31  was 64, leukocyte counts were 0.8 [1.2] CD2+, 0.8 [1.1] CD3+, 0.3 [0.5] CD4+, 0.3 [0.4] CD8+, 0.8 [1.3] CD45RO and 0.95 [1.2] macrophages. Myocyte necrosis were not observed in this study population.Serum concentration for sIL-4 was 37.65 [39.57], for IFN-g 4.75 [4.5], for IFN-b 34.90 [25.4], for IFN-a 1.55 [1.57], for sIL-12p7 8.31 [9.64] and for TNF-a 7.09 [11.67] pg/ml.Serum concentrations of TNFa correlated significantly with myocardial CD3+ lymphocytes , no correlations were observed between TNFa and other myocardial leukocytes or other cytokines and myocardial leukocytes. No correlation was observed between cytokine concentrations and endothelial activation. Concentrations of IFNg correlated significantly with PCWP , no correlations were observed between the other cytokines and hemodynamic parameters. Concentrations of IFNb and IFNa were inversely correlated , no correlations were observed between the other cytokines.CRP-levels were below 6 mg/l and white cell counts were normal in the study population.Heart rate and blood-pressure (systolic and diastolic) did not change significantly during measurements with reactive hyperemia and after application of glyceroltrinitrate. Adequate reactive hyperemia was achieved in all subjects. Mean resting diameter of the radial artery was 2.34[1] mm at baseline, after reactive hyperemia the mean diameter increased to 2.52[1] mm and after glyceroltrinitrate (GTN) to 2.93[1] mm.Endothelial function, as determined by FMD of the radial artery, was impaired in the study population (FMD 4.38 4.82]%). sIL-12p7 levels were significantly higher in patients with severely impaired FMD , compared with normal or moderately impaired FMD : 10.70 [10.72] versus 4.33 [7.81] pg/ml (p = 0.002) . sIL-1In the patient subgroup with myocardial inflammation, similar correlations were observed as for the total study population, additionally however, we found a significant correlation between FMD and TNFa .Endothelium independent vasodilation in response to GTN was not significantly impaired in the study population (GTN-MD 23.67 [8.21]%). No significant correlations were observed between GTN-MD and sIL-4, IFN-g, IFN-b, IFN-a, sIL-12p7 and TNF-a. GTN-MD correlated significantly with myocardial CD3+  and CD45RO leukocytes .Age, ejection fraction and other hemodynamic measurements did not vary extensively among the study population, therefor, therse parameters had no significant impact on endothelial function or cytokine concentration or endothelial activation, with the exception of a correlation between PCWP and IFNg .Endothelial dysfunction of peripheral arteries has been associated with myocardial inflammation and myocardial virus persistence, even in the absence of severely impaired left ventricular function -3. The lWe found, that endothelial dysfunction of peripheral arteries in patients with non-ischemic heart disease is associated with elevated sIL-12p7 concentrations in venous serum. sIL-12p7 activates NK-cells and displays proinflammatory actions which may potentially result in endothelial dysfunction. For sIL-4, IFN-g, IFN-b and IFN-a no association with endothelial dysfunction was observed. For s-IL-4 this was expected, as it inhibits cytokine and protease production and therefor may protect endothelium. IFN-g can display proinflammatory as well as antiinflammatory properties, which may explain, that a correlation with endothelial function was observed for sIL-12p7 but not for IFN-g.In patients with myocardial inflammation, there was also a significant correlation between TNFa and endothelial function, impaired FMD is associated with lower TNFa concentrations. TNFa activates macrophages, induces expression of cellular adhesion molecules, and enhances prostaglandine and metalloproteinase production. It is elevated in patients with heart failure ,21, but The association between endothelial function and circulating cytokines in patients with non-ischemic cardiomyopathy helps to understand pathomechanisms in myocardial disease and may explain symptoms. Whether cytokine production is localized to areas of inflammation or virus persistence in the heart or if it is due to generalized vascular inflammation cannot be differentiated by this study. Endothelial dysfunction may be induced by circulating cytokines through decreased production or decreased bioavailability of endothelium derived nitric oxide.Elevated cytokine sIL-12p7 concentrations in the peripheral circulation in patients with non-ischemic heart disease are associated with systemic endothelial dysfunction. These circulating cytokines may explain, that in patients with non-ischemic heart disease endothelial dysfunction is not restricted to the coronary circulation, but also involves systemic arteries. This finding is clinically important, as endothelial function represents an important predictor of prognosis -12 and mThe author(s) declare that they have no competing interests.