Mitral valve prolapse (MVP) is a common and benign condition. However, some anatomic forms have been recently associated with life-threatening ventricular arrhythmias and sudden cardiac death. Imaging MVP holds the promise of individualized MVP risk assessment. Noninvasive imaging techniques available today are playing an increasingly important role in the diagnosis, prognosis and monitoring of MVP. In this article, we will review the current evidence on arrhythmogenic MVP, with special focus on the utility of echocardiography and CMR for identifying benign and “malignant” forms of MVP. The clinical relevance of this manuscript lies in the value of imaging technology to improve MVP risk prediction, including those arrhythmic-MVP cases with a higher risk of sudden cardiac death. It is characterized by fibromyxomatous changes in one or both mitral leaflets, which eventually causes a displacement into the left atrium3,4. Although MVP is a benign condition, it is the most common cause of isolated mitral regurgitation (MR) that requires surgical repair5. Other complications, such as congestive heart failure, endocarditis, stroke and tachyarrhythmias, have been associated with MVP. Furthermore, life-threatening ventricular arrhythmias and sudden cardiac death (SCD) have been recently linked with MVP patients. In fact, there is an important increased risk of atrial and ventricular tachyarrhythmias in MVP patients (50% to 60%) and SCD in these patients (0.4% to 2% per year)6–8, even in the absence of heart failure or significant MR.Mitral valve prolapse (MVP) is a common disorder affecting 2-3% of the general population9 were the first group to describe a subset of MVP patients at high risk of malignant arrhythmias with a specific phenotype, characterized by bileaflet involvement, female sex with mid-systolic click on auscultation, T-wave abnormalities on inferior leads and RBBB-type or polymorphic ventricular arrhythmias, resulting in a “new” MVP entity, called arrhythmogenic MVP.Sarano et al.3 . LV damage thus could play an important role in the development of cardiac symptoms in MVP patients, especially in arrhythmogenic MVP where the LV seems to have a pivotal role9.The normal mitral valve (MV) is a dynamic three-dimensional system, which consists of the mitral annulus, the anterior and posterior mitral leaflets, the chordae tendineae and the papillary muscles (PMs). Its proper function depends on the integrity and harmonious interaction of all these components. MVP entity, including arrhythmogenic MVP, affects all the components of the mitral valve system with histological, macroscopic-anatomical changes and finally, an altered function. Beyond the complex mitral apparatus, several studies have suggested a direct impact of the prolapsing valve on the left ventricular (LV) structure and adjacent myocardium to the MV, potentially causing a secondary cardiomyopathy10. Cardiac magnetic resonance (CMR) represents an exhaustive and unique imaging tool for the comprehensive assessment of MVP, with a special utility in arrhythmogenic MVP patients because it provides myocardial tissue characterization, high temporal and spatial resolution images and unlimited spatial planes without acoustic windows limitations.In this context, a detailed study of both the anatomy and function of the entire MV system and the LV myocardium is essential in the diagnosis and prognosis of patients with MVP. Echocardiography is usually the first-line imaging tool for the assessment of MVP patientsIn this article, we will review the current evidence on arrhythmogenic MVP, with special focus on the utility of echocardiography and CMR for identifying benign and “malignant” forms of MVP. The clinical relevance of this manuscript lies in the value of imaging technology to improve MVP risk prediction, including those arrhythmic-MVP cases with a higher risk of SCD. Noninvasive imaging will help to identify non-benign forms of MVP who are in need of intensive prevention or even, early therapeutic intervention.11 because it is an available, inexpensive and non-invasive technique with high temporal and spatial resolution. Echocardiography allows simultaneous morphological and functional assessment. Two-dimensional (2D TTE) and three-dimensional (3D TTE) echocardiography provides detailed assessment of MV function and morphology, LV size and function, and Doppler echocardiography allows hemodynamic quantification12,13.Transthoracic echocardiography (TTE) is the gold standard for diagnosis and follow-up of MVP14 and is increased in up to 40% of patients with severe MVP. In patients with MVP, leaflets are generally elongated and thickened, and the mitral annulus is enlarged15.MVP by echocardiography is defined by 2D TTE as a single or bileaflet displacement of at least 2 mm beyond the annular plane in parasternal long-axis view, independently of leaflet thickening. Echocardiography allows evaluating leaflet characteristics, especially length and thickness, mitral annulus diameter, and MR severity. Mid-leaflet thickness, which is normally 2–3 mm, is measured usually in parasternal long-axis view in diastole. This measurement depends on the acquisition setting (acoustic wavelength and harmonics)3. Regarding MVP geometry, Sanfilippo et al.3 described by 2D echocardiography a PM displacement or mechanical traction toward the mitral annulus due to a combination of leaflet and chordal extensibility, redundancy and elongation. Authors explained that a traction force over leaflet is due to the product of leaflet area and LV pressure and is increased in MVP due to larger leaflet area and annulus. Abnormal mechanical traction of the posterior papillary muscle could be associated with abnormal electrophysiological characteristics in MVP patients16.MVP is defined as classic when the leaflets thickness is >5 mm and non-classic if there is <5 mm-thickness3 demonstrated that it has a saddle-shaped with upward concave in the anterior-posterior axis and downward concave in the medium-lateral one. Anterior and posterior mitral leaflets are usually anatomically divided into three segments according to Carpentier’s nomenclature: three posterior scallops, the lateral (P1), middle (P2), and medial (P3) and three anterior scallops, the lateral (A1), middle (A2) and medial (A3)17,18.The use of 3D TTE imaging has shown the real geometry of mitral annulus. Classically, mitral annulus had been considered a planar structure, however, Levine et al.19. Scarce data exist on the 3D geometry of MAD and its functional implication. Recently described by Lee at al20 in a population of MVP by 3D TEE, MAD was present in 42% of patients with MVP, the disjunctive annulus is decoupled functionally from the ventricle, leading to paradoxical annular dynamics with systolic expansion and flattening.Mitral annular disjunction (MAD) is defined as a separation between the atrial wall–MV junction and LV attachment. This is a structural abnormality occurring in MVP, related recently to arrhythmic MVP with LV fibrosis in CMR studies18,21,22. Also, 3D TEE is superior in identifying the prolapsing segment and defining the MV anatomy over 2D TEE  is more effective in identifying MV anatomy. 3D TEE has the additional advantage of simulating the surgeon’s view and allows slicing the acquired 3D data in every dimension until the optimal and desired 2D view is obtainedr 2D TEE . Moreove23: 1) Barlow’s disease, characterized by excess tissue and significantly enlarged leaflets and mitral annulus, and 2) Fibroelastic deficiency, characterized by localized involvement of the leaflet with healthy adjacent segments17. This is important because the more extensive involvement of valvular and subvalvular apparatus, the higher the complication rates with poor surgical outcomes.For example, 3D TEE has been shown to be of value in distinguishing between two different MVP entities20,23. In Barlow’s disease (compared to Fibroelastic deficiency), there is an increased prolapse height and prolapse volume24, the mitral annulus is larger and present an abnormal enlargement, especially a intercommissural diameter in late-systole and saddle-shape accentuation in early-systole, with a global poor contraction. In severe myxomatous valve disease, the mitral annulus seems to increase flattening which may lead to severe MR and chordal rupture25.Thus, prolapsed volume and prolapsed height have recently been identified as possible discriminative factors for the diagnosis of Barlow’s disease and Fibroelastic deficiency13,18. The American Society of Echocardiography and European Association of Echocardiography recommendations for MR assessment highlight the importance of an integrated approach, including semiquantitative and quantitative methods18,26. The vena contracta width and proximal isovelocity surface area (PISA) are the preferred methods in patients with MVP. Phasic changes of MR occur in MVP and the effective regurgitant orifice (ERO) increases throughout systole, leading to inaccurate quantification of MR severity. However, properly timed measurements of PISA allow an accurate estimation of the overall ERO and regurgitant volume24,27.MR in MVP patients is a consequence of annular enlargement, leaflet redundancy and chordae elongation or rupture, leading to inadequate leaflet coaptation20. MVP patients revealed decreased longitudinal and circumferential peak longitudinal strain and absolute values of strain rate in septal segments28. Other studies in different MVP populations did not find decreased longitudinal function, probably related to early stages of LV remodelling, but they detected temporal patterns of myocardial deformation29. The authors suggested that the mechanical interaction between MVP and LV wall through chordae and PMs could potentially alter temporal segmental myocardial deformation patterns. Thus, the myocardial segments affected might present electrical vulnerability that could be linked with arrhythmias.Although echocardiography does not provide myocardial characterization as well as CMR, the novel myocardial deformation analysis by 2D speckle tracking echocardiography allows the identification of subclinical and specific myocardial deformation abnormalities in MVP. Subclinical LV longitudinal dysfunction has been described since the discovery of MR30,31, as a complement to the classic echocardiography. CMR also has the ability to acquire unlimited imaging planes and to overcome the limitation of poor acoustic windows, often presented in TTE. It is also less invasive than transesophageal echocardiography. Here, we explore the overall utility of CMR in MVP, with special interest on arrhythmogenic MVP.CMR is a non-invasive imaging technique that allows a comprehensive characterization of MVP, including assessment of MV morphology, LV structure, MR severity and myocardial tissue characteristics in a single examination. Because this technique provides a more profound look into the biology of the valve and its linked myocardium, it is becoming a valuable tool in complex MVP patients, especially in arrhythmic and genetic familial cases20  long-axis view, equivalent to a 3-chamber view on echocardiography20 . CMR hasFrom a technical point of view, all these measurements are obtained from a LVOT stack (6 to 8 non-gap 5 mm-slices) across the entire MV, perpendicular to the long axis of the valve using breath-hold, retrospectively electrocardiogram-gated steady state free-precession images. Further MV morphological characteristics have also been described in MVP, including : -The anterior and posterior displacement, defined as the maximum excursion of the leaflets during systole beyond the mitral annular diameter. -The distance between the PMs and the mitral leaflet coaptation point and the anterior and posterior leaflets, respectively. -The anterior and posterior leaflet thickening and length in diastole. -10. Mitral annulus is defined by a line connecting the inferolateral mitral annulus to the aortomitral junction. This measurement might be important when a MV prosthesis implantation or repair is planned.The mitral valve annulus dimensions, measured at the end-systole in the 2-, 3- and 4-chamber views33. These findings might be important because the prediction of the presence and severity of MR may be valuable for risk stratification, follow-up recommendations and surgical decision-making.Above all, anterior leaflet length, posterior leaflet thickness and displacement, and the presence of flail have been shown to be the best determinants of the MR severity in the context of MVP patients34,35.Interestingly, previous studies have suggested a direct impact of the prolapsing valve on ventricular structure, potentially causing a secondary cardiomyopathy. CMR imaging can provide quantitative determination of ventricular volumes, mass and function by high-resolution images, giving an overview of the LV remodelling. This is particularly useful for follow up of MVP patients with significant MR3)36,37. The extent and distribution of LV hypertrophy in patients with MVP is also important. Zia et al.38 showed that MVP is associated with focal basal LV hypertrophy, particularly in the inferolateral and anterolateral segments. Also, they reported that there is good correlation between the excursion of the mitral valve annulus and the degree of relative LV hypertrophy, suggesting that locally increased myocardial function could be responsible for this remodelling.LV volumes are calculated by manually traced of LV endocardial contours in end-diastolic and end-systolic phases on steady-state free precession cine images acquired across the entire LV (contiguous 10-15 short axis slices from base to apex). Using the Simpson’s method (LV end-diastolic volume-LV end-systolic volume/LV end-diastolic volume), the LV ejection fraction can be also obtained. LV mass is measured by tracing the LV epicardial contours and the total myocardial volume contained within the endo- and epicontours is multiplied by myocardial density  and the aortic stroke volume . The MR  0:none to trace (0% to 5%) 1:mild (5% to 16%) 2:moderate (16% to 25%) 3moderate to severe: (25% to 48%) 4:35.severe (>48%)37, presents the advantage to be independent of the effects of tricuspid and pulmonary regurgitation and allows correction for aortic regurgitation.It has been demonstrated that phase-contrast CMR imaging has low variability and excellent reproducibility20 demonstrated for the first time that CMR identifies myocardial fibrosis involving the PMs in MVP. Late gadolinium imaging to assess focal fibrosis is acquired in different LV planes 5 to 10 minutes after a bolus of contrast agent injection (Gadolinium 0.2 mmol/kg) using inversion recovery sequences.A unique feature of CMR is the ability to characterize myocardial or vascular tissues. Contrast agents allow the visualization of macroscopic scars or fibrotic tissue as bright areas into the normal myocardial detected as black using late gadolinium enhancement imaging . Focal f39. These increased extracelullar space may represent fibrosis or proteoglycan depositions, as demonstrated in previous studies using myocardial histological analysis. Recently, it has been described a relationship between chronic volume overload in asymptomatic chronic primary degenerative MR and myocardial fibrosis using T1 mapping techniques40. Moreover, the myocardial fibrosis was associated with reduced myocardial deformation and reduced exercise capacity. Thus, T1 mapping, by detecting increased extracelullar volume as a surrogate of diffuse fibrosis, could be considered as another valuable marker to decide the optimum surgery timing in asymptomatic patients with chronic severe primary degenerative MR.Another advantage of CMR is the ability of detecting increased extracelullar space by T1 mapping41,42. The most widely used is the MOLLI sequence (ECG-gated single shot modified Lock Locker inversion recovery)43. Using MOLLI sequences, T1 maps are acquired before and after a contrast infusion or bolus injection to compare myocardial relaxation times changes44. For the analysis, a region of interest is usually manually traced in the septum wall -β contributes to fibrosis and matrix remodelling in MVP50,51. Furthermore, expression of TGF-β was significantly increased in patients with SCD compared with controls, suggesting the role of TGF-β as a potential mediator of interstitial remodelling in sudden death52. Consequently, other mechanisms of arrhythmias may be involved besides focal scar, such as diffuse fibrosis, as arrhythmias may also been present in the absence of focal fibrosis. In conclusion, T1 mapping may become an additional marker of arrhythmic risk in MVP as a predictor of SCD.The authors suggested that diffuse fibrosis could be the consequence of profibrotic cytokine high levels. These hypotheses are based on previous animal and 53,54. More recently, several morphofunctional abnormalities of the MV that could explain a regional mechanical myocardial stretch have been described, including19  Mitr19 found that all these parameters were higher in arrhythmic MVP patients with focal scar compared to MVP cases without scar using CMR. In addition, histology of the mitral annulus showed longer MAD in sudden death patients with MVP than in patients without MVP. Indeed, the higher the magnitude of MAD, the higher the probability of having ventricular arrhythmias. Authors concluded that MAD is a constant feature of arrhythmic MVP with LV fibrosis. This concept of MAD was first introduced by Hutchins et al.55 in disease pathogenesis in patients with severe MR. The excessive mobility of the leaflets, caused by myxomatous degeneration, posterior systolic curling and MAD, accounts for a systolic mechanical stretch of the myocardial linked to the MV , eventually leading to myocardial hypertrophy and scarring. These mitral annulus abnormalities may identify MVP patients who would need arrhythmic risk stratification. Interestingly, a paradoxical systolic increase of the mitral annulus diameter was also observed in this study causing an alteration of the geometry and function of the mitral ring.Marra et al.56 They showed in a series of cases that the surgical correction of bileaflet MVP was associated with a reduction in malignant arrhythmias. MV repair could thus relieve the mechanical stretch of the myocardium, thus leading to a reduction of arrhythmias. All these findings by CMR and MVP studies provide a deep and better understanding of the pathophysiology of ventricular arrhythmias in MVP.The role of abnormal mechanical forces in the formation of ventricular arrhythmias was indirectly demonstrated by Vaidya et al.19. Different mechanisms of ventricular arrhythmias may be involved in MVP. Single ventricular premature beats (VPB) from the papillary muscle (RBB mostly with superior axis) may represent focal activity elicited by mechanical actions on this sensible area for arrhythmias57,58. In fact PMs sustained arrhythmias have been recognized as a different entity from fascicular tachycardia59,60.Beyond extracardiac causes as autonomic nervous dysfunction, the mechanism of arrhythmogenic MVP may be related to abnormalities in the MV system and the involved myocardium. As mentioned above, bileaflet involvement, larger MAD or severe MV geometry distortion may represent a subset of patients and risk markers to develop malignant ventricular arrhythmias45. Although a hidden cardiomyopathy in malignant MVP cannot be excluded, fibrosis might be a more advanced phase in more severe cases because of myocardial remodelling. Inducibility of sustained ventricular in MVP patients with VPB and symptoms of sustained ventricular arrhythmias and prior sustained ventricular tachyarrhythmias or ventricular fibrillation may support the difference of focal triggers versus substrate for reentry61.The infrequent malignant MVP, as compared to the MVP population with VPB, probably underlines the role of a substrate for sustained ventricular arrhythmias. Local fibrosis at the PMs, or more extensive fibrosis engaging inferoposterior wall, may well be the substrate. A patchier pattern of fibrosis, compared to that solid one of ischemic myocardial infarction, makes more feasible sustained rapid circuits, resulting more easily in ventricular fibrillation62. Antiarrhythmic effect of surgical reparation in early phases is still to be demonstrated since surgery in patients with sustained ventricular arrhythmia failed to avoid inducibility7. For other patients, like those with extensive scarring, frequent complex multifocal VPB or inducible sustained ventricular arrhythmias, symptoms of brain hypoperfusion, Implantable Cardioverter Defibrillator (ICD) implantation could be considered if the risk is evaluated to be high enough.Identification of these high-risk patients is important in order to consider different treatments. Ablation of the trigger could be feasible since it has been successfully reported in other patients with ventricular fibrillationAlthough MVP is a benign condition, there is a subset of patients at high risk of life-threatening ventricular arrhythmias and SCD with a specific phenotype, resulting in a “new” MVP entity, called arrhythmogenic MVP. Malignant arrhythmias in MVP may be generated due to the combination of abnormal LV myocardial substrate  and a continuous trigger . Noninvasive imaging technology, especially CMR, may help to identify non-benign forms of MVP who are in need of intensive prevention or even, early therapeutic intervention. The presence of focal fibrosis in the basal inferolateral LV wall, probably the area more related to the arrhythmias’ origin, diffuse LV fibrosis and morphological MV characteristics, such as increased MAD, diffuse leaflet thickening, elongation and redundancy of chordae, have been linked to arrhythmogenic MVP. Thus, a proper assessment of MVP for clinical decision-making and prognosis, should not be only limited to the MV, but rather include a comprehensive evaluation of the myocardial tissue composition and LV structure, as a potential cardiomyopathy.


To address this, we employed diffusion tensor magnetic resonance imaging to explore the architecture and tissue organization of the developing heart aged 95–143 DGA. We show that fractional anisotropy increases (from ~0.1 to ~0.5), diffusion coefficients decrease (from ~1 × 10−3mm2/sec to ~0.4 × 10−3mm2/sec), and fiber paths, extracted by tractography, increase linearly with gestation, indicative of the increasing organization of the ventricular myocytes. By 143 DGA, the developing heart has the classical helical organization observed in mature mammalian tissue. This was accompanied by an increase in connexin 43 and connexin 40 expression levels, suggesting their role in the development of the ventricular conduction system and that electrical propagation across the heart is facilitated in later gestation. Our findings highlight a key developmental window for the structural organization of the fetal heart.The developmental timeline of the human heart remains elusive. The heart takes on its characteristic four chambered appearance by ~56 days gestational age (DGA). However, owing to the complexities  of exploring development  Initially the heart develops from an early tube at ~20 days of gestational age (DGA)3 with the four-chambered structure being visible after ~56 DGA4.The known structure of the adult human heart  can only be identified during the later stages of gestation1. DT-MRI derived datasets can be used to describe the structure and organization of the human heart during gestation depending on its anisotropic and orthotropic properties. DT-MRI analysis has shown that the human fetal heart is highly isotropic in early gestational stages1. This means that diffusion moves equally in all directions within the tissue, as illustrated by the “chaotic” and non-organized cardiac structure we previously presented1. As the heart develops, we have previously demonstrated that its anisotropy increases with diffusion moving preferentially in one prominent direction, after ~140 DGA, creating a helical organization of the cardiac fibers with smooth angle changes1. These diffusion properties can be used to create computational models and algorithms able of computing the fiber tracks within a heart during development, as the ones presented in this paper. Fiber tracking, or tractography, is an MRI-based visualization technique for computing the location, orientation and anisotropy of tracts around any given tissue.The application of imaging technologies, such as diffusion tensor magnetic resonance imaging (DT-MRI), has previously been used to explore the detailed structure of the fetal human heart1. This helical structure and tissue organization are important for normal cardiac function. The orientation of cardiac myofibers within that helical organization, in the adult heart and the fetal human heart in later stages of gestation, is important for propagation of ventricular excitation5 and allows coordinated contraction of the ventricular tissue7. During diastole and systole, the ventricular wall thickness decreases as ventricular volume increases8. This is facilitated by the structure of the ventricular myocardium, which consists of plate like structures running within the helical organization9. Any changes to this structure can lead to fibrosis and remodeling of the helical organization, affecting normal cardiac function10. Hence, understanding the rate of development of this helical structure and the mechanisms involved, in the human, may elucidate important developmental processes crucial for normal cardiac function, or even pathology.Previously, we demonstrated that the smooth helical organization  can only be seen in the fetal heart from ~140 DGA11, alone does not provide a complete understanding of human heart development. The exploration of the molecular mechanisms necessary for propagation of excitation through the ventricular tissue and their developmental regulation will help elucidate the process of how ventricular fiber organization occurs. Thus, studying the expression of gap junctions found in the ventricular myocardium during fetal development is necessary, as it is known that gap junctions between myocytes facilitate the propagation of excitation within the myocardium13. Gap junctions are channels called connexons which are hexamers of proteins called connexins. The most abundant connexin found in the ventricular myocardium is connexin 43 (Cx43). Its expression and distribution throughout the ventricular tissue plays a crucial role in the development of the ventricular structure and its remodeling14. Thus, Cx43 expression during early fetal life could facilitate ventricular structural organization by enabling myocyte-to-myocyte electrical communication, therefore promoting propagation properties within the developing heart.Knowledge of the structure of the fetal human heart, derived from DT-MRI15. Cx40 is the main gap junction protein found in the ventricular conduction system (the His bundle and the upper parts of the bundle branches), and it is thought to play a developmental role in the appearance of Purkinje fibers during mammalian gestation16.Cardiomyocytes co-express connexin 43 (Cx43) with connexin 40 (Cx40), another gap junction expressed in the mammalian heart. Their pattern of co-expression is thought to be important for the maintenance of normal heart rateex vivo fetal hearts by DT-MRI. DT-MRI has been validated as providing a measure of cardiomyocyte orientation19 and an index of the helical structure of the ventricular wall21, and has provided measurements of myocardial fiber and lamellar orientations in mammalian ventricular myocardium22. However, we currently lack an understanding of how the detailed microstructure of the ventricular myocardium develops over gestation. Previous studies have used a very limited sample size11, from which statistically robust conclusions cannot be made. Herein, we use DT-MRI protocols (with a cubic voxel size of 100 µm) to quantify the development of the ventricular wall, by means of diffusion properties, in 23 fetal human hearts aged from 95 to 143 DGA, thereby being the first study to provide a complete developmental timeline of the human ventricular development across the first and second trimester. We have also quantified the expression of Cx43 and Cx40 in developing human hearts from 67 to 136 DGA. We correlate the increased expression of these two connexins (Cx43 and Cx40) with the increased structural organization of computed cardiac fiber paths observed with gestational age.In this study, we quantify the developmental changes of myocardial architecture of 1 and largely resembles mature tissue. A related measure, ADC, has yet to be applied to human fetal cardiac tissue. ADC quantifies the diffusion rate within a tissue, and is thought to be a more biologically-relevant quantification of DT-MRI datasets. In order to provide a full developmental timeline of ventricular wall maturation, including left/right comparisons, 23 hearts were scanned and FA and ADC quantified.Diffusion properties can be used to calculate the development of the ventricular wall. We have previously shown that by ~140 DGA, the ventricular wall (as measured by FA) has become more anisotropic = 20.22, p = 0.0002, R2 = 0.49 for the right ventricle; F = 15.92, p = 0.0007, R2 = 0.43 for the left ventricle). There was no significant difference between the regression slopes for both ventricles <1), suggesting a similar rate of right/left ventricular development.FA was estimated from DT-MRI datasets for the segmented ventricles (right and left) and increased with gestation from <0.1 to 0.6 as gestation approaches the third trimester  = 8.58, p = 0.008, R2 = 0.29 for the right ventricle and F = 8.38, p = 0.009, R2 = 0.29 for the left ventricle). The ADC data suggest that there is more variance at younger developmental stages, linked to the diffusion within the radial direction (measured by changes in λ2 and λ3). As the complexity of the tissue increases, diffusion becomes more restricted and as a result ADC reduces.Ventricular ADC was also measured and decreased with age from approximately 1 × 10Tractography analysis of DT-MRI derived datasets is more commonly associated with exploring fiber tracts within the brain, although recently their application has extended to the heart. Tractography works by computing the preferred orientation of subsequent voxels; those that follow a connected ‘path’ are likely anatomically linked. We hypothesized that tractography would be particularly useful for the study of development; fibers that were not fully developed would show reduced fiber complexity, length and density. = 138.7, p < 0.0001, R2 = 0.87) and volume  = 308, p < 0.0001, R2 = 0.94). Together, this confirms that within the first trimester, the ventricular wall has yet to take on any complex and regular features, possibly as the myocardium is still dis-organized. This is particularly evident between 124 and 128 DGA, where interventricular septum and ventricular walls rapidly become ordered and have distinct fiber tracts  = 14.81, p = 0.0009, R2 = 0.41) and ADC  = 6.37, p = 0.0196, R2 = 0.23). Given ADC measures diffusion in three directions , we sought to test whether the decrease in ADC was being driven predominately by diffusion in the axial (λ1) or the radial orientation (λ2 and λ3) by characterization of axial diffusion  and radial diffusion  (as in equation [2]) components  = 2.99, p = 0.099) whereas RD was significantly negatively correlated  = 7.85, p = 0.011, R2 = 0.27). This potentially reflects increasing organization of the ventricular tissue with development, given there is less ‘branching’ of myocytes owing to their more regular orientation.Quantitative analysis of the diffusion properties of the cardiac fiber paths revealed a consistent increase in the FA of ventricular fibers and a decrease in ADC with gestational age  = 12.45, p = 0.0002). Cx43 expression was significantly greater between 67–73 DGA and 107–136 DGA (p = 0.001), and between 96 DGA and 107–136 DGA (p = 0.0004), highlighting the potential role of Cx43 in fetal heart maturation via gap junction-mediated cellular signaling pathways.The conduction of electrical signaling within the ventricles is dependent upon gap junction signaling between myocytes, of which the most abundant is Cx43. Deletion of Cx43 is known to severely alter the geometric anatomy of, particularly, the right ventricle26 has a role in the formation of the mammalian ventricular conduction system during development and its loss results in cardiac abnormalities27. Although the absence of Cx40 does not affect the gross structure of the heart, it results in conduction deficits with a reduced cardiac conduction velocity28. We therefore quantified Cx40 expression in developing hearts aged 67–136 DGA  = 9.43, p = 0.001; 67–73 vs. 107–136 DGA p = 0.0006).It has also been shown that expression of Cx40DGA Fig. . Cx40 inWithin the current study, we have employed high-resolution diffusion tensor magnetic resonance imaging (DT-MRI) to image the human heart during development ) and visualize computed cardiac fiber path lines Fig.  of ventr30. DT-MRI derived datasets can provide directional information of tissue fibers and have been used to predominately map connections within the brain31 and heart32. Such studies have combined DT-MRI with tractography to map normal brain and heart physiology (creating population-averaged atlases) and to examine disease states. Availability of detailed atlases33 of the structure of complex organs with 3D visualizations of their fibers is important and necessary for guiding the interpretation of clinical data and aiding medical diagnosis and treatment. As yet these approaches have not been applied to the developing human heart, during gestation.DT-MRI has developed into a useful tool for the investigation of changes within tissue microstructure1 and ref.11) to allow statistical analysis of observed developmental changes. Our study uses indices, such as ADC, AD and RD, to quantify the developing myocardial tissue architecture and relate these changes to an increase of Connexin 43 (Cx43) expression, the main connexin protein found in the ventricular myocardium, and Connexin 40 (Cx40), the main connexin protein expressed in the ventricular conduction system.This is a first preliminary application of DT-MRI derived tractography using a sufficient number of human fetal hearts  also has an effect on the ADC, which was found to decrease with gestational age. This would account for the changes of the radial diffusion (RD) we observed, which occurs perpendicularly to the principal diffusion and it is dependent on the secondary and tertiary eigenvectors.34. Our tractographic analysis has revealed an increase in the myocardial organization, evidenced by an increase in the density of fiber paths and their length across gestation . These structural changes alter the pattern of propagation, which may lead to arrhythmias41. Once the physiologically normal cardiac organization , we believe that at full term multiple abnormalities would have been present. Although this finding was serendipitous, it does represent 9% of our total sample, which may highlight new and important research avenues for a role of connexins in normal heart development.During the process of estimating the expression changes of Cx43 during gestation, we identified one heart aged 97 DGA which did not express the protein Fig. . This he43. Previous studies have demonstrated that conduction pathologies (such as right bundle branch block or slower conduction velocity) are present in Cx40 deficient mice45. Our study revealed a gradual increase in the expression levels of Cx40 during the development of the human fetal heart. Our preliminary study may demonstrate the requirement for Cx40 at early human ventricular development but the role of other proteins and their expression profiles should be further investigated.Effective cardiac conduction is aided by tissue geometry  and the distribution of gap junctions. Connexin 40 is expressed in the ventricular conduction system and the fast-conducting atrial myocardium. Later in development of the mammalian heart, Cx40 can be found expressed in the trabecular network and the Purkinje fibers networkIn summary, this is the first study that structural data (acquired from DT-MRI) and protein expression data were combined with computational modelling in an effort to map human heart development. We have used DT-MRI and visualization tools to characterize and quantify the development of the structure and organization of the human fetal ventricular myocardium. We have identified the development of the organization of the ventricular streamlines (cardiac fibers) and we have shown an increase in their length and density relative to gestational age. We have also quantified the expression of Cx43 and Cx40 and its increase during gestation in the human fetal heart. Although preliminary, the current study presents an advancement in our understanding of fetal cardiac development, and future studies will expand upon our themes. Further research will also interpret how this developmental timeline relates to clinical cases such as congenital heart block, to aid in the early identification of developmental abnormalities.23 human fetal hearts with an age range from 95 to 143 days gestational age (DGA) were obtained for DT-MRI. The hearts were fixed in 4% formaldehyde before used for MRI experiments. All samples were clinically assessed and found free of any visually identifiable abnormalities and dysmorphism.Temporary storage of tissue was in premises licensed by the 2004 Human Tissues Act (UK) and all protocols had ethical committee approval. The samples were acquired following elective termination of pregnancy, with informed written consent obtained from all subjects. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by NHS Lothian Research and Development, the University of Edinburgh Research Governance Hope and the University of Leeds ethical committee (Study Code LREC 08/S1101/1).The samples were obtained legally under the U.K. Abortion Act of 1967. In England, Wales and Scotland the Abortion Act 1967 allows a pregnancy to be terminated by a registered medical practitioner if the pregnancy has not exceeded its twenty-fourth week of gestation. All samples were collected after informed consent was secured by both parents (when possible) or just the mother (in the case of a single parent). All samples used in this study originated from abortions due to social reasons. Any samples from abortions performed due to medical reasons were not made available to the study. Due to this reason, it has taken us more than 3 years to collect as many samples as we could. All mothers and fetuses were being medically examined throughout their pregnancy until the time of the elective termination. No defects were seen using ultrasound in utero during gestation. All fetuses appeared morphologically normal during regular checks with the midwife and upon medical termination of pregnancy as certified by the physician. Furthermore, when MRI was performed all hearts were found structurally normal. Gestational age was measured by ultrasound scan before the procedure and confirmed by measuring foot length afterwards.1. For further details go to Supplementary Material The hearts were imaged in Fomblin . The MRI acquisition protocol used here has been described in detail before1.We have previously described mathematical approaches to extract local fiber orientation and organization, and values of fractional anisotropy  for the visualization of the ventricles and their boundaries using DSI Studio  by extracting the intensity values for every image voxel and their b factors using the Stejskal-Tanner modelUsing DSI Studio, we characterized and extracted (in a semi-automated manner) the primary  and secondary  diffusion direction of the segmented ventricles (right and left) throughout gestation.λ1 and radial diffusion (RD) is represented byAxial diffusion (AD) is represented by the primary eigenvector 31 and humans47. In this study tractography was used to join voxels with close local average myofiber orientation into a path, starting from random seed point. These fiber paths, which reconstruct the global helical organization of the ventricular myocardium, are quantified by their number and lengths and represented as color-coded lines.The computed cardiac fiber paths follow the local fiber orientations as calculated from the diffusion data. This method is known as tractography and it is a computational visualization method for the 3D representation of vector fields. Tractography is used to study the structure and architecture of axonal tracts in the brain of animals47 using a step size equal to half the MRI resolution . The seed orientation was set to track the primary eigenvector field and its position was set to the subvoxel level for the detection of micro-structural features.Tractography uses Euler’s integration algorithm48. For further details on the western blotting protocol used in this study go to Supplementary Material The Western blotting protocol used here has been described in detail elsewhere2. Protein expression was analyzed by one-way ANOVA, followed by Tukey’s post hoc tests. In all cases, α was set at <0.05. Graphs and statistics were prepared using GraphPad Prism version 6.To assess differences between myocardial development across age, linear regression analysis was performed. The sign, significance and magnitudes of correlations between age and FA, ADC and diffusivity were tested and the goodness of fit is expressed as RSupplementary Material


Previous serological studies have shown controversial results whether defibrillation or cardioversion can cause myocardial injury. Cardiovascular Magnetic Resonance (CMR) can be used to detect myocardial edema, hyperemia and capillary leak as features of acute myocardial injury. The aim of this study was to assess for myocardial and skeletal muscle injury in swine following transthoracic shocks.Seventeen anaesthetized swine were examined, with 11 undergoing five synchronized transthoracic shocks (200J). Myocardial and skeletal muscle injury were assessed at baseline and up to 5h post-shock employing T1 mapping, T2 mapping, early and late gadolinium enhancement. Serologic markers  and myocardial tissue were assessed by standard histology methods.In myocardial regions within the shock path, T1 and T2 were significantly increased compared to remote myocardium in the same animals. The early gadolinium enhancement ratio between the left-ventricular myocardium and the right pectoral muscle was also increased compared to control animals. After the shocks cFABP and CK were significantly elevated. After shock application, the regions identified as abnormal by CMR showed significantly increased interstitial and myocardial cell areas in histological analysis. This increased cell area suggests significant cellular and interstitial edema.Our pilot study data indicate that serial defibrillator shocks lead to acute skeletal muscle and myocardial injury. CMR is a useful tool to detect and localize myocardial and skeletal muscle injury early after transthoracic shocks in vivo. In the future the technique could potentially be used as an additional tool for quality control such as verifying insufficient local shock application in non-responders after cardioversion or to develop safer shock forms. Electric defibrillation is an important and often life-saving intervention in patients with fatal pulseless and shockable arrhythmia. Electric cardioversion is typically applied for converting atrial fibrillation, flutter and other (supra-)ventricular tachycardia to sinus rhythm.While its clinical role is well defined, there are no conclusive data on potential injury to the heart or the path of the electrical stimulus through the thorax.Myocardial injury has been investigated mainly with serologic markers such as troponin (Tn) , creatinCardiovascular magnetic resonance imaging (CMR) allows for the in vivo visualization of acute myocardial injury. Acute myocardial injury often features edema, hyperaemia and capillary leak, all which can be detected by CMR using specific sequences . AlthougThe aim of the present study was to assess for the presence and severity of myocardial injury caused by repeated biphasic synchronized transthoracic shocks.This study was conducted in accordance with the ‘‘Guide to the Care and Use of Experimental Animals” by the Canadian Council on Animal Care. It was approved by the ‘‘Animal Care and Use Board” of the Montreal Heart Institute [#20125003]. Animals were bred at Ferme Lavallée, Quebec, Canada for research and food industry purposes. Animals were brought to the research institute at least 48h before the experiment in groups of 2 or more, and were fed commercial feed (Harlan #7037) twice daily with free access to water up to the start of the experiments.Seventeen female common landrace swine (32.2±1.3kg body weight) were studied. After premedication  and induction with 2-4mg/kg propofol IV, the animals were intubated. Anaesthesia was maintained with a continuous propofol infusion (12 to 37mg/kg/hr) as required. The femoral artery and vein were cannulated for fluid infusion, blood sampling, as well as invasive blood pressure monitoring. Bolus injections of phenylephrine were administered when required.The animals were placed in a recumbent position in a clinical 3 Tesla MRI . Using a clinical device , five serial biphasic synchronized transthoracic shocks (200J per shock) were applied to eleven animals within five minutes. Pads were placed on the right pectoral muscle in proximity to the axilla, as well as on the left postero-lateral chest wall anterior of the scapula. Six animals served as a control group. Serology samples and CMR images were obtained at baseline as well as 1h, 3h, and 5h after shock application. At all-time points, cardiac function was measured using standard cine CMR imaging covering the entire left ventricle, and the myocardium was assessed with edema-sensitive native T1 and T2 mapping (Siemens Cardiac Mapping Works in Progress package “Quantitative Cardiac Parameter Mapping T1/T2/T2*”). At the end of the study, 5 hours after shock application, early gadolinium enhancement (EGE) images were obtained prior to and within 3 minutes after i.v. administration of 0.1mmol/kg gadolinium  to assess hyperaemia and capillary leak. Late gadolinium enhancement (LGE) images were obtained 5 and 10 minutes post-contrast respectively to detect scar. Sequence details are in Following imaging, animals were euthanized with 200mg propofol and 40mmol KCl IV. A sample of the right pectoral muscle and the entire heart were preserved in 10% formalin. After identifying regions of interest with CMR, myocardial tissue samples were obtained from these segments. All samples were stained according to a standard hematoxylin-eosine protocol.Blood was collected and plasma isolated by centrifugation . Porcine Troponin I (TnI) ELISA-kit  as well as porcine cardiac fatty acid binding protein  tests were performed for each sample at baseline and 1, 3 and 5 hours post-shock, according to the operation manuals. Creatine Kinase  and the porcine cardiac isoform CK-MB  were measured at baseline and 5h post shock.42, Release 4.1.5, Circle CVI Inc., Calgary, Canada). For cardiac function parameters, epi- and endocardial contours were traced in systole and diastole. For T1 and T2 maps, regions of interest (ROI) were manually defined by tracing visually affected right and left pectoral muscle; epi- and endocardial contours were drawn to include the entire left ventricular myocardium. Additionally, a ROI with a visibly increased T1 or T2 as compared with remote myocardium was selected by the consensus of three readers. These ROI were then compared to remote myocardium in the same animal and to the entire myocardium of the control animals, averaging the T1 or T2 of the ROI or remote tissue respectively of both slices. EGE was calculated by normalizing myocardial signal intensity (SI) to skeletal muscle [All images were analyzed using certified evaluation software . For assessing changes of the interstitial space fraction of the samples, we performed a semi-quantitative planimetry of three representative areas of each sample slice .Results are displayed as mean values ± SEM. Data was examined for normality with a d’Agostini Pearson omnibus test. For normal distributed data, repeated measures were assessed with a paired t-tests or repeated measures ANOVA with Bonferroni correction if appropriate. Comparisons between the two groups were conducted at each time point using independent t-tests. Otherwise, if the normality test failed, Mann-Whitney U, Friedman’s with Dunn’s post-testing, or Wilcoxon tests were used. Statistical analysis was performed with Graphpad Prism . P<0.05 was deemed significant.One animal died due to ventricular fibrillation after an un-synchronized shock, resulting in 10 animals with 5 serial shocks and 6 control animals.There was no significant change in heart rate within or between the groups. Six animals in the shock group required phenylephrine support resulting in a mean total dose of 125± 68μg for the group versus control animals in the first 30 minutes after the shocks (p = 0.03) in order to maintain a mean blood pressure >50mmHg. None of the control animals required any vasopressor support.Propofol requirements of the shocked animals (5430±1148mg) were not different from the control group . Also, there was no difference in fluid regimen with lactated Ringer’s solution between groups (shock 2313±205ml vs. control 2158±216ml p = 0.63).There was a small change in end-diastolic volume , with boDefibrillation pad locations were visible by increased T1  and T2 iIn all shocked pigs, a marked regional increase in native T1 and T2 values was detected, located in the anterior and inferior wall (n = 8), in the septum and inferior wall , n = 1 aInterestingly, one animal showed an abnormal path of tissue changes compared to the other animals. Instead, in this animal T1 and T2 visually increased in the minor pectoral muscle and the intercostal muscles over the thorax, indicating that most of the current may have passed over the chest wall. In the anterior wall increased T2 was observed .P<0.01).In the quantitative analysis there was no significant difference between native T1 of the ROI of the shocked animals and the controls (p>0.17), but we observed variation in T1 in the control animals . HoweverP<0.05, T2 of the ROI was significantly longer post shock than in remote myocardium of the same animals P<0.05, . NeitherThe EGE ratio  was significantly higher in the global LV myocardium and the right pectoral muscle of the shocked pigs than in the controls p<0.05, . The incNo animal showed signs for pre-existing scar or fibrosis in LGE images.<0.01; The mean interstitial space in LV myocardial tissue obtained from regions with abnormal T1 or T2 was larger in pigs of the shock group than in control animals , as well as compared to the control animals p<0.01, . Total CTo our knowledge we have detected and reliably localized acute myocardial and skeletal muscle injury after serial transthoracic shocks in-vivo the first time using a comprehensive CMR study with state-of the-art clinical sequences. The presence and extent of acute injury detected by CMR was verified by histology and serology.Furthermore, our study indicates a utility of CMR to detect skeletal muscle and myocardial injury after serial transthoracic shocks in vivo, potentially to detect insufficient shock application (non-responders after cardioversion) or to further improve the devices (different shock forms) and protocols .All these sequences are currently used for human assessment, and thus our technique has a high potential to be translated into clinical use.As recent serologic studies failed to indisputably show myocardial injury, we used several CMR techniques to not only detect but also localize the injury caused by serial transthoracic shocks. First, we observed an increased early gadolinium enhancement ratio in the LV myocardium of animals with shock application. This is consistent with previously published data from human studies, where an EGE ratio of 4 or more indicates hyperaemia and capillary leak in inflammatory myocardial disease . FurtherOf note, the increase of T1 and T2 in the affected regions did not reach statistical significance when compared to control animals, which may be due to the small sample size. The visible decline in T1 and T2 in the control animals, as well as in the remote myocardium may be a result of too restrictive fluid administration during the experiments in both groups.The significant injury of the right pectoral muscle from both the T1 and T2 analyses after the shocks show electroporetic skeletal muscle injury ,17 that We found a decrease in EDV and a trend towards a lower cardiac output after shock administration. We interpret these findings as reflective of myocardial edema with increased stiffness and resulting impaired left-ventricular filling. The absence of late gadolinium enhancement excludes pre-existing myocardial injury.Cardiac troponins are sensitive serologic markers for myocardial damage. Current literature does not conclusively support that the application of transthoracic electric shocks leads to myocardial injury. While there is data indicating an elevation in serum troponin after transthoracic shocks, most of these studies show only small and clinically insignificant increases ,18. In aIn accordance with existing literature, we did not find changes in porcine TnI levels. In the context of our findings and current literature we doubt that cardiac troponin assays are suitable markers to assess acute myocardial electroporation injury. However, more data is needed to support this assumption. The increase in CK activity in our study may be partially related to myocardial injury but are expected to be mainly due to skeletal muscle injury.Cardiac Fatty Acid Binding Protein (cFABP) is a marker that is elevated early after myocardial injury and after transthoracic shocks ,21, and Compared to serologic markers, histology is a more reliable test for the detection and localization of tissue injury, if the sample was taken from truly affected tissue. We show that histologic samples which were regionally matched with abnormal tissue characteristics in CMR images, had an increased proportion of interstitial space in comparison to samples of control animals. This increase may represent capillary leakage, resulting in edema and longer native T1 and T2 times. We also found evidence for intracellular edema shown by an increased cell area in the shocked pigs, likely caused by electroporation injury of the cellular membranes and the subsequent water influx.Skeletal muscle injury was much more pronounced than the myocardial injury, as seen by higher T1 and T2, suggesting that the skeletal muscle under the defibrillation pad significantly scavenges and attenuates the energy that reaches the myocardium. Similarly, in the histology samples, disruption of cell integrity was observed in the right pectoral muscles of the shocked animals whereas the cardiomyocytes only exhibited signs of cellular edema.The aim of this pilot study was to test the feasibility of CMR to detect shock related injuries. Considering the weight of the animals (32.2±1.3kg), relatively high cumulative energies were used (>4J/kg per shock). However, guidelines stipulate that when using an automated external defibrillator, adult pads are to be used in children, when paediatric pads are not available ,24. The Our data indicate that serial applications of defibrillator shocks can lead to acute skeletal muscle and myocardial injury detectable by CMR scans in-vivo. In the future the technique could potentially be used as an additional tool for quality control such as verifying insufficient local shock application in non-responders after cardioversion or to develop safer shock forms. There is suspicion by clinicians that a fraction of all applied shocks does not correctly through the desired route through the chest, therefore such a diagnostic tool may indeed be helpful.S1 File(XLSX)Click here for additional data file.


E/A), tissue Doppler , and left atrial volume index in 176 RA patients. Relationships of traditional cardiovascular risk factors and RA characteristics with LV diastolic function and dysfunction according to previous and current criteria were determined in multivariate regression models. Waist-hip ratio was associated with E/A (standardised β (SE) = −0.28 ± 0.09, p = 0.0002) and lateral e′ (standardised β (SE) = 0.26 ± 0.09, p = 0.01); low diastolic blood pressure was related to E/e′ (standardised β (SE) = −0.16 ± 0.08, p = 0.04). Diastolic dysfunction prevalence differed upon applying previous (59%) compared to current (22%) criteria (p < 0.0001). One SD increase in waist-hip ratio was associated with diastolic dysfunction when applying current criteria (OR = 2.61 (95% CI = 1.51–4.52), p = 0.0006), whereas one SD increase in diastolic blood pressure was inversely related to diastolic dysfunction upon using previous criteria (OR = 0.57 (95% CI = 0.40–0.81), p = 0.002). In conclusion, application of current and previous diastolic dysfunction criteria markedly alters the prevalence and risk factors associated with diastolic dysfunction in RA.This study compared the estimated prevalence and potential determinants of left ventricular (LV) diastolic dysfunction upon applying different classification criteria in rheumatoid arthritis (RA). LV diastolic function was assessed echocardiographically by pulsed Doppler ( Rheumatoid arthritis (RA) increases the risk of heart failure 2-fold . Heart fα [In RA, traditional cardiovascular risk factors and coronary artery disease do not fully explain all changes in diastolic dysfunction . In thisα  have beeα , which iα , 19, 23 α , 18. TheE) to atrial contraction velocity (A) (E/A) ratio as a parameter of diastolic function [E/early peak velocity during diastole at the mitral annulus (e′) (E/e′) ratio were proven to more reliably represent diastolic function as assessed by invasive pressure-volume analysis [E/A ratio was no longer considered in patients with an ejection fraction of ≥50% and a relatively high cut-off value for raised E/e′ of 14 cm/sec was proposed [Diastolic dysfunction is most frequently assessed by echocardiography. Importantly, in the present context, several RA investigations focused on the pulsed Doppler determined early filling velocity  independent relationships of traditional cardiovascular risk factors and disease characteristics with pulsed and tissue Doppler measures of diastolic function and (2) whether application of different diastolic dysfunction classification criteria sets impacts on the estimated prevalence and associated risk factors of diastolic dysfunction in patients with RA.We recruited 186 patients that met the 2010 American College of Rheumatology/European League against Rheumatism criteria for RA  at the MBaseline characteristics were assessed using standard approaches as previously reported . DemograRA disease duration, rheumatoid factor, and anti-citrullinated peptide antibody (ACPA) status were assessed. Disease activity was estimated by the disease activity score in 28 joints (DAS28) and the clinical disease activity index (CDAI) . Current2 for women and >115 g/m2 for men [Echocardiography was performed using SonoSite's M-Turbo ultrasound  with the patient in the partial left decubitus position according to the American Society of Echocardiography convention . LV dime for men .E) and late  diastolic wave were measured and the ratio of early and late diastolic filling (E/A) was calculated. To perform tissue Doppler imaging, the velocity of myocardial tissue lengthening was measured by placing the curser at the septal and lateral corners of the mitral annulus in the apical four-chamber view. To determine diastolic function using tissue Doppler imaging, the peak velocities during early (e′) and late (a′) diastole were recorded. Because mitral E is dependent on ventricular relaxation as well as left atrial driving forces (pressures), while e′ is dependent on relaxation alone, E/e′ is considered to be an index of left ventricular filling pressures. The E/e′ ratio was calculated as mitral E/the average of septal and lateral e′. Left atrial area was determined using planimetry at end-systole in the apical four-chamber or two-chamber view from which left atrial volume was calculated by the area-length method and was indexed to body surface area (LAVI). The presence of diastolic dysfunction was determined using previously [E/A was <0.75 and E/e′ < 10, moderate diastolic dysfunction was diagnosed when 0.75 ≤ E/A < 1.5 and E/e′ ≥ 10, and severe diastolic dysfunction was diagnosed when E/A was ≥1.5 and E/e′ ≥ 10 [E/e′ > 14, lateral e′ < 10 cm/sec or septal e′ < 7 cm/sec, and left atrial volume index > 34 ml/m2; in those with an ejection fraction < 50%, mild diastolic dysfunction was diagnosed when E/A was <0.8, severe diastolic dysfunction was diagnosed when E/A was >2, and moderate diastolic dysfunction was diagnosed when E/A was >0.8 and <2, E/e′ > 14, and left atrial volume index > 34 ml/m2 [Left ventricular diastolic function was assessed using pulsed Doppler, tissue Doppler imaging, and left atrial volume . Transmieviously  and curreviously . Briefly/e′ ≥ 10 . Accordi34 ml/m2 .E, A, and e′ were 0.71, 0.84, 0.81, 0.95, 0.92, and 0.92, respectively  and the coefficients of variation for LV end-diastolic diameter, septal wall thickness, posterior wall thickness, E, A, and e′ were 2.7%, 2.9%, 2.6%, 1.9%, 3.9%, and 4.2%, respectively. There were no significant differences between the measures on unpaired t-tests . For the intraobserver variability, Pearson's correlations for repeat measures ranged between 0.74 and 0.98 for one observer and between 0.82 and 0.94 for the other observer for all the aforementioned measures . The respective coefficients of variation ranged between 1.4% and 4.7% for one observer and between 1.2% and 5.4% for the other observer for all the aforementioned measures. There were no differences in the repeat measures on paired t-tests for either observer .Echocardiography was performed by two experienced operators that were blinded to the clinical data and cardiovascular disease risk factor profiles of the patients. All echocardiographic images were reviewed by a single experienced operator. Intra- and interobserver studies were conducted on 20 individuals. For the interobserver variability, Pearson's correlation coefficients for LV end-diastolic diameter, septal wall thickness, posterior wall thickness, E/A ratio , lateral e′ , septal e′ , and LAVI ; relative wall thickness was not associated with any LV diastolic function parameters. In age-, sex-, and race-adjusted analysis, neither LV mass index nor relative wall thickness was associated with diastolic dysfunction parameters (data not shown). Nevertheless, as LV mass index was more strongly and consistently associated with diastolic function parameters than relative wall thickness in univariate analysis, LV mass index was forced in additional regression models with diastolic dysfunction as dependent variable. Dyslipidemia, smoking, and diabetes were added as independent variables into final models on diastolic dysfunction among all RA patients. As the impact of risk factors on cardiovascular disease can differ in women compared to men, the independent associations of baseline characteristics with diastolic dysfunction were also assessed in a sensitivity analysis among women. Lastly, sensitivity analysis was performed in patients without established cardiovascular disease (n = 9) and in those with an ejection fraction of ≥50% (isolated diastolic dysfunction). Significance was set at p ≤ 0.05.Database management and statistical analyses were performed using SAS software, version 9.4 . Descriptive data are presented as mean (SD), median (interquartile range (IQR)), or proportions for normally distributed, nonnormally distributed, and categorical variables, respectively. Nonnormally distributed variables were logarithmically transformed in order to improve skewness and kurtosis for statistical analysis. Potential relationships of baseline characteristics including traditional cardiovascular risk factors and RA features with markers of diastolic function (as well as LV mass parameters) were first assessed in age-, sex-, and race-adjusted linear regression models. The independent associations of baseline characteristics with diastolic function parameters and diastolic dysfunction according to previous and current diastolic dysfunction classification criteria sets were subsequently determined in linear and backward logistic regression models, respectively. As LV mass and concentric hypertrophy are strongly associated with diastolic function, the associations of LV mass index and relative wall thickness with diastolic function parameters were determined. LV mass index was associated with E/e′ ratio was ≥10 and >14 in 58% and 20% of participants, respectively. Upon applying previously reported criteria [The mean (SD) ejection fraction was 58.7 (13.2) %; 21% of participants had an ejection fraction of <50% and 19.3% left ventricular hypertrophy. The criteria , 58.6% ocriteria  were appp < 0.0001).The estimated proportion of RA persons with diastolic dysfunction differed significantly when applying previous  comparedhttps://doi.org/10.1155/2017/2323410. Age was associated with all markers of diastolic function  and LV mass index  but not relative wall thickness (p = 0.4). Female sex  and heart rate  were associated with E/A. Estimated glomerular filtration rate was directly related to E/A , lateral e′ , and septal e′  and inversely associated with left atrial volume index  and LV mass index . Waist-hip ratio (WHR) comprised the adiposity marker that was most consistently associated diastolic function marker and was therefore included in subsequent analysis. Waist-hip ratio was associated with low E/A  and decreased lateral e′  and with increased LAVI  and LV mass index . Diastolic blood pressure was related to E/e′  and lateral e′  and hypertension was related to LAVI . Triglycerides were inversely related to lateral e′  and directly related to LAVI . HOMA-IR was inversely associated with E/A  and lateral e′  and directly with LVAI .The significant age-, sex-, and race-adjusted associations of baseline characteristics with markers of diastolic function and LV geometry are shown in Supplementary Table  1 in Supplementary Material available online at E/A , lateral e′ , left atrial volume index , and LV mass index . DAS 28 , erythrocyte sedimentation rate , and C-reactive protein  were related to E/A. ACPA status  and white cell count  related to relative wall thickness. The presence of extra-articular manifestations was associated with LAVI .Among RA characteristics, disease duration was most consistently associated with diastolic function markers including Baseline characteristics that were not significantly related to diastolic function parameters are given in Supplementary Table  2.β (SE) to allow for comparison of the contributions of the different baseline recorded characteristics to the variation in diastolic function. Age at disease onset (β (SE) = −0.31 (0.11), p = 0.0006), race (β (SE) = 0.19 (0.09), p = 0.02), heart rate (β (SE) = −0.21 (0.08), p = 0.01), and waist-hip ratio (β (SE) = −0.28 (0.09), p = 0.002) remained significantly associated with E/A. Age (β (SE) = 0.19 (0.08), p = 0.01) and diastolic blood pressure (β (SE) = −0.16 (0.08), p = 0.04) were associated with E/e′. Age at disease onset (β (SE) = −0.34 (0.11), p = 0.002) and waist-hip ratio (β (SE) = −0.26 (0.09), p = 0.01) were associated with lateral e′. Age at disease onset (β (SE) = −0.46 (0.10), p < 0.0001), disease duration (β (SE) = −0.35 (0.09), p = 0.0003), and azathioprine use (β (SE) = −0.24 (0.08), p = 0.005) were associated with septal e′. Race (β (SE) = −0.22 (0.09), p = 0.01), waist-hip ratio (β (SE) = 0.27 (0.11), p = 0.01), and triglyceride concentrations (β (SE) = 0.20 (0.09), p = 0.03) were associated with left atrial volume index.Clinical characteristics that were associated with diastolic function markers in the previous analysis were then entered into separate linear regression models with diastolic function markers as dependent variables. p = 0.01) and the waist-hip ratio (OR = 2.61 (95% CI = 1.51–4.52), p = 0.0006) were associated with diastolic dysfunction upon using the current criteria; these relationships were materially unaltered after adding LV mass index  as an additional potential confounder to the model. Upon applying the previous criteria, only a low diastolic blood pressure (OR = 0.57 (95% CI = 0.40–0.81), p = 0.002) was associated with diastolic dysfunction; low diastolic blood pressure remained independently associated with diastolic dysfunction (according to previous criteria) when LV mass index (p = 0.003) was added to the model. When adding smoking, dyslipidemia, and diabetes to the models, age at disease onset (OR = 2.27 (95% CI = 1.18–4.37), p = 0.01) and waist-hip ratio (OR = 2.44 (95% CI = 1.39–4.27), p = 0.002) remained associated with diastolic dysfunction according to previous criteria and low diastolic blood pressure (OR = 0.56 (95% CI = 0.39–0.81), p = 0.002) remained related to diastolic dysfunction according to recent criteria. After exclusion of patients with established cardiovascular disease (n = 9), the results were materially unaltered (data not shown).Baseline characteristics that remained associated with diastolic function markers in p = 0.0002) was associated with diastolic dysfunction according to current criteria and a low diastolic blood pressure (OR = 0.62 (95% CI = 0.42–0.92), p = 0.02) was related to diastolic dysfunction according to previous criteria. When adding LV mass index to the models, these associations remained significant. Sensitivity analysis was not performed in men due to the small numbers involved (n = 32).As given in p = 0.01). When LV mass index was added to the model, age at disease onset remained associated (p = 0.01) with diastolic dysfunction according to the current criteria. Additionally, waist-hip ratio (OR = 1.99 (95% CI = 1.04–3.80), p = 0.03) and disease duration (OR = 1.99 (95% CI = 1.01–3.93), p = 0.05) were related to diastolic dysfunction according to the current criteria. A low diastolic blood pressure was significantly associated with diastolic dysfunction according to previous criteria before (OR = 0.59 (95% CI = 0.40–0.89), p = 0.01) and after (OR = 0.60 (95% CI = 0.40–0.91), p = 0.01) LV mass index was added to the model.In When azathioprine was omitted as independent variable from the models in Tables E/A ratio, lateral e′, and left atrial volume index, whereas low diastolic blood pressure related to the E/e′ ratio. We also report for the first time that the estimated prevalence of diastolic dysfunction is markedly lower upon application of the recently reported classification criteria by Nagueh and colleagues [This study examined the potential contribution of comprehensively assessed traditional cardiovascular risk factors and disease characteristics to LV diastolic function in patients with RA. Our analysis revealed that waist-hip ratio was independently associated with the lleagues  comparedlleagues  (22.0% vE/A ratio and e′ to a similar extent as age at disease onset, and diastolic blood pressure was as strongly related to E/e′ as age. Adequate LV diastolic function depends on active and passive processes. The active process comprises high energy requiring relaxation [E/A ratio and e′, and diastolic blood pressure was inversely associated with E/e′. In this regard, a low E/A ratio and e′ represent impaired relaxation, whereas a large E/e′ ratio is an index of filling pressure and is more strongly associated with fibrosis as part of myocardial stiffness [We found that waist-hip ratio contributed to the variability of laxation . It involaxation . The paslaxation . In the tiffness , 41. TakE/A ratio is not included as a criterion among patients with an ejection fraction ≥ 50% in the recent recommendations. Additionally, the E/e′ ratio is considered as a criterion when its value is ≥10 cm/sec in the previous compared to >14 cm/sec in the recent recommendations. In our study, waist-hip ratio was related to the E/A ratio and diastolic dysfunction according to the recent but not previous criteria. On the other hand, diastolic blood pressure was associated with the E/e′ ratio and diastolic dysfunction according to the previous but not current criteria. Moreover, 20% of our patients had E/e′ ratio of >14 and 58% experienced E/e′ ratio of ≥10. These findings suggest that the reduced sensitivity in classifying patients as having diastolic dysfunction by the recent criteria set is at least partly due to the high threshold that is used upon considering an increase in E/e′ ratio. Our results indicate the need for further studies aimed at determining adequate heart failure risk stratification in RA. Interestingly, a recent population study similarly reported marked differences in the prevalence and predictors of diastolic dysfunction according to different classification criteria [The question arises as to why there is a 2- to 3-fold difference in estimated diastolic dysfunction prevalence as well as a consistent disparity in associated risk factors by applied classification criteria in RA. Both criteria sets include pulsed and tissue Doppler measurements. However, in contrast to previously reported criteria, the criteria .In the present study, 1 SD increase in diastolic blood pressure reduced the odds ratio for diastolic dysfunction according to previous criteria to 0.57, whereas 1 SD increase in waist-hip ratio increased the odds ratio for diastolic dysfunction according to current criteria 2.61-fold in patients with RA. These relationships were independent of LV mass index and reproduced in sensitivity analyses among women and patients without established cardiovascular disease as well as those with isolated diastolic dysfunction. In aged subjects, low diastolic blood pressure forms part of an increased pulse pressure, which is mediated by increased arterial stiffness and wave reflection . Neithere′ only. RA duration was associated with diastolic dysfunction according to the new criteria only in those with an ejection fraction ≥ 50% and after adjustment for LV mass index. DAS28, erythrocyte sedimentation rate, and C-reactive protein concentrations were associated with the E/A ratio in age-, sex-, and race-adjusted analysis but not in fully adjusted models.An association between RA duration and diastolic function parameters or/and dysfunction according to reported criteria was previously reported in some –16 but nE/A ratio may be affected by pseudonormalization [E/A ratio is not included among the new diastolic dysfunction classification criteria upon their application in persons without systolic dysfunction [Our study design was cross-sectional, thereby precluding drawing conclusions on cause and effect relationships. Our findings require further evaluation in future longitudinal studies. We did not record echocardiographically determined tricuspid regurgitation velocity. The left atrial volume index was remarkably low in this RA study. Upon application of diastolic classification criteria, we used left atrial volume index threshold values as given in recent recommendations that are largely based on data obtained in American populations . In thislization , which cfunction , our resIn conclusion, central adiposity and low diastolic blood pressure are independently associated with diastolic function in patients with RA. The application of different reported criteria markedly influences the estimated prevalence and associated risk factors of diastolic dysfunction in RA. Future studies are urgently needed in order to further improve our understanding of heart failure risk and its stratification among patients with RA.Supplementary Table 1: Traditional risk factors and RA characteristics associated with diastolic function and left ventricular geometry. Supplemental Table 2: Non-significant associations of traditional risk factors and RA characteristics with markers of diastolic function and left ventricular geometry.


Background: Myocardial fibrosis (MF) is a risk factor for poor prognosis in dilated cardiomyopathy (DCM). Late gadolinium enhancement (LGE) of the myocardium on cardiac magnetic resonance (CMR) represents MF. We examined whether the LGE amount increases the incidence of adverse cardiovascular events in patients with stage C or D heart failure (HF).Methods: Eighty-four consecutive patients with stage C or D HF, either ischemic or non-ischemic, were enrolled. Comprehensive clinical and CMR evaluations were performed. All patients were followed up for a composite endpoint of cardiovascular death, heart transplantation, and cardiac resynchronization therapy with defibrillator (CRT-D).Results: LGE was present in 79.7% of the end-stage HF patients. LGE distribution patterns were mid-wall, epi-myocardial, endo-myocardial, and the morphological patterns were patchy, transmural, and diffuse. During the average follow-up of 544 days, 13 (15.5%) patients had endpoint events: 7 patients cardiac death, 2 patients heart transplantation, and 4 patients underwent CRT-D implantation. On univariate analysis, LGE quantification on cardiac magnetic resonance, blood urine nitrogen, QRS duration on electrocardiogram, left ventricular end-diastolic diameter (LVEDD), and left ventricular end-diastolic volume (LVEDV) on CMR had the strongest associations with the composite endpoint events. However, on multivariate analysis for both Model I  and Model II , LGE amount was a significant risk factor for composite endpoint events .Conclusion: LGE amount from high-scale threshold on CMR increased the incidence of adverse cardiovascular events for patients in either stage C or D HF. End-stage heart failure is associated with significant morbidity and mortality  and non-ischemia-dilated cardiomyopathy  performed a prospective observational cohort study. The study enrolled 84 consecutive heart failure patients between January 1st, 2014 and December 31st, 2014, with ischemic or non-ischemic heart disease as defined by the World Health Organization/International Society and the Federation of Cardiology criteria  examinations were performed using a 3.0-T Magnetic Resonance Imaging (MRI) system  with a 32-channel phased-array coil. True fast imaging with steady state free precession breath-hold cine images were acquired in the short-axis, and 2-, 3-, and 4-chamber views encompassing the entire left ventricle (LV) volume from the apex to the base. Diastolic phase myocardial delayed enhancement images in the same orientations as in the cine images were acquired 10 min after intravenous infusion with gadolinium chelate contrast agent  with a prospectively ECG-gated gradient echo sequence with an inversion prepulse. Inversion times were optimized to null normal myocardium. Imaging parameters were as follows: repetition time/echo time, 4.1/1.6 ms; flip angle, 20°; image matrix, 256 × 130; section thickness, 8 mm (contiguous short axis) or 5 mm (long axis images), with no intersection gap.2 was defined within the normal remote myocardium, in an area with uniform myocardial suppression, free of artifacts. The commercial software automatically calculated the %Myo .The QMASS commercial software package  was used to analyze the CMR DICOM images. The cardiac functional indexes, specifically the left ventricular ejection fraction (LVEF), volumes, and mass were quantified from cine images by using standard methods. The volume and mass of the LV was normalized to the body surface area. Two experienced observers, blinded to the clinical outcome independently determined the dichotomous presence or absence of LGE by reviewing all short and long axis contrast-enhanced images; regions of elevated signal intensity had to be confirmed in two spatial orientations. Once the LGE was present, the distribution of the LGE was recorded in the 17 segments according to the standards of the American Heart Association (AHA). Furthermore, the quantity of hyper-enhancement was defined as regions with an abnormally increased signal intensity >2SD and >6SD of the peak remote. According to other studies , including cardiovascular death, cardiac transplantation, and cardiac resynchronization therapy with defibrillator (CRT-D). CRT-D implantation was based on information obtained from medical records and telephone calls. CRT-Ds were only implanted in patients who fit the guideline recommendation criteria for CRT-Ds and who had recurrent admissions for heart failure, despite undergoing at least 6 months of optimal medical therapy , while categorical variables are expressed as a frequency (%). Continuous variables were compared using an unpaired Student t-test or a Mann-Whitney nonparametric test, as appropriate. Categorical variables were performed using the Pearson Chi-Square test and the Fisher exact test. A P < 0.05 was deemed statistically significant and all statistical tests were 2-sided. Receiver operating characteristic (ROC) curves constructed using Bootstrap resampling (times = 500) were used to find the optimal cut-off values for LGE quantification (with maximizing the sum of sensitivity and specificity) as measured by 6SD methods to predict MACE. Survival estimates and cumulative event rates were compared by the Kaplan–Meier method by using the time-to-first event for each endpoint. The log-rank test was used to compare the Kaplan–Meier survival curves. Cox regression analyses were used to estimate the hazard ratio (HR) for adverse events and their corresponding 95% confidence intervals (CI). Univariate Cox regression analyses were used to examine all significant variables. We also adjusted for variables that, when added to this model, changed the Matched Hazard Ratio by at least 10%. All analyses were performed using Empower (R)  and R (http://www.R-project.org).Continuous variables are expressed as mean ± standard deviations  at the time of CMR imaging, in addition, 65 of the these patients (77.4%) were male. The mean LVEF on CMR was 16.4 ± 8.8%, and all patients at presentation had either stage C or stage D heart failure. After extensive cardiac examination of all patients at baseline, 57 patients (67.9%) were diagnosed with non-ischemic dilated cardiomyopathy, and 27 patients (32.1%) were diagnosed with ischemic dilated cardiomyopathy, and atrial fibrillation was observed on the electrocardiogram among 20.5% of patients with heart failure, Table .5 ± 12.3p < 0.001). However, although the LVEF on CMR was lower in patients with events, these differences were not statistically significant . LGE distribution pattern was mid-wall and epi-myocardial in 24  patients, endo-myocardium in 12  patients, patchy in 6  patients, transmural in 20  patients, and diffuse in 5  patients , 13 (15.5%) patients developed major adverse cardiac events; in addition, 7 patients died, 2 patients underwent cardiac transplantation, and 4 patients underwent CRT-D implantation.P = 0.305). In the multivariable analysis shown in Table adj 1.44/10% increase in 6SD %Myo, 95%CI 1.05–1.98, P = 0.022) after adjusting for the patients' age, sex, and body mass index (BMI), and in Model II  after adjusting for patients' age, BMI, sex, blood urea nitrogen (BUN), QRS duration, and Atrial fibrillation (%) on EKG, etiology of heart failure, LVEF, CMR- LVEDD, and LVEDV.The results of the univariate analyses of major adverse events during the follow-up period are summarized in Table P < 0.001).ROC curves of LGE quantification were used to determine the optimal cut-off values for prediction of events during follow-up by using a bootstrap resampling times = 500) showed an area under curve of 0.74  and the full width at half maximum (FWHM) method  and reactive  fibrosis, leading to expanded extracellular matrix , in Model I, and 1.045 (P = 0.022) in Model II for cardiovascular death and cardiac transplantation or implanted CRT-D. Our results also indicated that LGE cutoff values of 10.91% (6SD method) for patients with stage C or stage D heart failure are associated with poor outcomes.The major finding of our study was that the amount of LGE from high-scale threshold on CMR increased the likelihood for adverse cardiovascular outcomes in patients with stage C or stage D heart failure. This association was independent of the LVEF and the etiology of heart failure . Although the study populations were very different, the results of patients with hypertrophic cardiomyopathy (HCM) in their study and those with stage C or D heart failure in our study were consistent with the results reported by Chan et al. (P = 0.022), even after the adjustment for other relevant variables, including LVEF. The Bootstrap resampling (times = 500) was used to find the optimal cut-off value and the 6SD LGE >10.91% of LV mass conferred the high risk of major adverse events in patients with stage C or D heart failure.Perazzolo Marra et al.  evaluaten et al. . Our datThe current assessment of the prognosis of patients with stage C or D heart failure, either ischemic or non-ischemic in etiology, is purely based on a decreased LVEF  with end-stage heart failure. Further research efforts should include a prospectively enrolled cohort to determine whether CMR in combination with other novel markers could help identify patients with stage C or D heart failure who may benefit from additional device treatment.The major limitation of our study is the relatively small cohort size and short follow-up period. Furthermore, there were only two cases of heart transplantations in our study. The pathological report for the recipient's heart further confirmed our diagnosis of IDCM and NIDCM. We will continue to follow the study population to collect additional pathological results in support of our initial diagnosis. Finally, we quantified LGE using a signal intensity threshold of 6SD above a remote reference region. The traditional LGE measurement method cannot detect small patches as well as diffuse microscopic fibrosis. The T1 mapping technique may enable us to improve the clinical assessment of diffuse myocardial fibrosis.TL and XM conceived and designed the experiments, and wrote the manuscript together. They have contributed equally to the work. TL and JD are responsible for the data and result. WL, DS, TYL, JK, and XL performed the experiment. SL and JL analyzed the data. LZ and ZF provide reagents and analysis tools. ZS and LX helped to revise the manuscript.This study was supported by the China Natural Science Funding (81101173 and 81470429) and the High Levels of Health Technical Personnel in Beijing City Health System (2013-3-005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.