Aortic Stenosis (AS) is the most frequent valvulopathy in the western world. Traditionally aortic valve replacement (AVR) has been recommended immediately after the onset of heart failure (HF) symptoms. However, recent evidence suggests that AVR outcome can be improved if performed earlier. After AVR, the process of left ventricle (LV) reverse remodelling (RR) is variable and frequently incomplete. In this study, we aimed at detecting mechanism underlying the process of LV RR regarding myocardial structural, functional and molecular changes before the onset of HF symptoms. Wistar-Han rats were subjected to 7-weeks of ascending aortic-banding followed by a 2-week period of debanding to resemble AS-induced LV remodelling and the early events of AVR-induced RR, respectively. This resulted in 3 groups: Sham (n = 10), Banding  and Debanding . Concentric hypertrophy and diastolic dysfunction (DD) were patent in the Ba group. Aortic-debanding induced RR, which promoted LV functional recovery, while cardiac structure did not normalise. Cardiac parameters of RV dysfunction, assessed by echocardiography and at the cardiomyocyte level prevailed altered after debanding. After debanding, these alterations were accompanied by persistent changes in pathways associated to myocardial hypertrophy, fibrosis and LV inflammation. Aortic banding induced pulmonary arterial wall thickness to increase and correlates negatively with effort intolerance and positively with E/e′ and left atrial area. We described dysregulated pathways in LV and RV remodelling and RR after AVR. Importantly we showed important RV-side effects of aortic constriction, highlighting the impact that LV-reverse remodelling has on both ventricles. One of these changes is ventricular hypertrophy, which is perceived as an initial compensatory mechanism to normalise increased wall stress. However, over time LV hypertrophy becomes decompensated3 and results in diastolic dysfunction (DD), with an increase in LV stiffness, an abnormal filling and relaxation pattern, followed by an enlargement of the left atrium and pulmonary congestion4. Myocardial maladaptive remodelling is one of the principal pathological markers of cardiovascular disease progression/severity, and its prevention or reversal is a desirable strategy. Currently, the most effective treatment to AS is the surgical alleviation of pressure overload subsequently to aortic valve replacement (AVR). AVR allows the myocardium to undergo a process named reverse remodelling (RR), which usually results in an improvement of cardiac structure and function. However, the process of RR is frequently incomplete, and the underlying mechanisms remain to be clarified as patients show an extremely variable myocardial response during RR, ranging from partial to total recovery of cardiac function and structure.Ventricular remodelling includes structural and functional changes taking place in the ventricle in response to chronic pressure overload. Aortic stenosis (AS) is the most common valvulopathy, whereby a stenotic valve increases afterload and imposes additional hemodynamic stress on the left ventricle (LV). LV overload activates several molecular and cellular pathways that trigger remodelling through morphological and functional alterations5. Diastolic dysfunction, associated with impaired active relaxation, is a feature of most AS patients. Several studies showed improvement of diastolic function7 including active relaxation early after AVR8, however, the percentage of patients with moderate to severe DD increases 10-years after AVR9.Incomplete functional recovery is an indicator of poor prognosis, associated with persisting symptoms and increased mortality10. Indeed, LV mass regression, which usually does not exceed 31% at 6 months post-AVR, is assumed to be a favourable marker of LV RR12.In incomplete structural recovery after AVR, AS patients with greater LV mass regression associate positively with lower rates of rehospitalisation13, and in AS deterioration of RV function after AVR has been shown14. Nevertheless, knowledge about RV function and structure in this context remains scarce, especially in a modulable experimental context.RV failure is a frequent complication following LV assist device (LVAD) implantation16.Currently, most of the cellular and molecular information regarding RR derives from samples of patients undergoing RR after LVAD implantation in which myocardial sample are available before and after unloading the heart. However, these “end-line” patients suffer from advanced heart failure (HF) characterised by chamber dilatation and reduced ejection fraction (EF), and therefore they do not represent the typical phenotype of AS-induced myocardial remodelling. Thus, we aimed to describe the molecular pathways underlying myocardial remodelling in the presence of DD and preserved EF as well as to highlight the early biventricular changes invoked by myocardial RR focusing on cardiomyocytes’ myofilaments, calcium-handling, signalling pathways and extracellular matrix. To achieve these goals, we selected a rat model of aortic banding, which is widely used to impose chronic pressure overload, therefore mimicking AS-induced remodelling during increased afterload, followed by aortic debandingto trigger RR upon overload reliefEchocardiography of banded animals revealed ventricular concentric hypertrophy, based on thicker LV wall and decreased LV dimensions Table . In respThe debanding group showed an overall imrpovement in haemodynamic parameters  and partial regression of hypertrophy as confirmed by thinner LV wall and decreased LV mass compared to banding rats. Some of these variables did not normalise completely, persisting significantly different from the sham group Table . In the Cardiomyocyte area increased in the Ba relative to the sham group and was attenuated in the Deb group Fig. . AccordiWhen compared to sham, Ba rats displayed increased LV cardiomyocyte stiffness, as shown by a steeper passive tension versus sarcomere length (SL) relation Fig. . This stprocollagen type I and type III and Lox expression . Interestingly, we found that, animals that presented a worse pattern of LV RR (Deb 1) displayed increased levels of STAT3 and GSK3β activation as well as overexpression of IL-6, TGF-β, Galectin and TIMP2 Fig. .S100a1 after pressure-overload relief , myofilament cooperativity (nHill) and a reduction of the rate constant of force redevelopment  and mTOR, P38 activation and AKT were not significantly altered Fig. , but theBanding group showed increased RV cardiomyocyte area and fibrosis Fig. . These vWhen compared to sham, Ba rats displayed increased RV cardiomyocyte stiffness (as shown by a steeper passive tension versus SL relation) and decreased Ktr Fig. .Figure 4Aortic constriction relief slightly increased Ktr towards normalisation and induced a trend to cardiomyocytes’ active tension to decrease when compared to sham group Fig. .Regarding the expression of proteins associated with calcium homeostasis, Ba animals presented reduced content of NCX and Junctophilin Fig. . After dThe thickness of pulmonary arteries induced by aortic constriction normalized completely after debanding. Interestingly, the thickness of the pulmonary arteries correlates negatively with exercise capacity and positively with E/e′ and indexed LAA Fig. .Figure 5The underlying mechanisms associated with incomplete myocardial RR need to be clarified to successfully potentiate myocardial recovery after AVR with a special emphasis on RV involvement. Banding rats resemble AS patients by presenting LV concentric hypertrophy, DD , pulmonary congestion and effort intolerance. Besides LV alteration, aortic constriction-induced several RV abnormalities.S100a1 expression that potentially restrains calcium leak by the ryanodine receptor (RyR) and improves calcium reuptake by the sarcoplasmic reticulum (SR). Importantly, this model presents significant RV abnormalities. Some of these abnormalities do not revert during RR, such as hypertrophy, fibrosis, right-atria enlargement and systolic and diastolic markers of cardiac dysfunction. At the cell level, and similarly to the LV, RV cardiomyocytes from banding rats were stiffer and showed a reduced rate of force redevelopment (Ktr). This manuscript provides novel insights on LV and RV changes secondary to chronic pressure overload and highlights the involvement of both ventricles in chronic pressure overload conditions imposed on the LV, such as in the case of aortic stenosis.Two weeks after overload relief, reduction of aortic velocity trigger reverse remodelling and cardiac function amelioration (more evident on left heart). On the LV, myocardial hypertrophy, fibrosis and atrium area enlargement remained altered after pressure-overload relief. In addition to sustained LV fibrosis, the incomplete reversion of titin PEVK phosphorylation contributed to the persistent myocardial stiffness in debanding rats. The improvement of relaxation during RR is probably related to the reduced CAMKii activity, and increased Serca2a and 17. Therefore, we aimed to detect early changes that might represent important time points for therapeutic interventions aiming to reverse the progression of the disease. Traditionally AVR has been recommended only after the onset of HF symptoms and a mean gradient higher than 40 mmHg18. However, recent evidence suggests that AVR outcome can be improved if performed earlier, as soon as diastolic function starts to decline20. Thus, in our aortic-banding model , and the hyperphosphorylation of Titin S26-PEVK segment contributed to increased LV myocardial stiffness by augmenting myocardial fibrosis, collagen cross-linking and cardiomyocyte passive tension, respectively. The LV delayed relaxation in Ba rats can be attributed to the reduction of SERCA2a/PLB, hindering Ca2+ reuptake to the SR.Diastolic dysfunction is frequently found in AS patients with normal systolic performance. In severe AS with preserved EF, symptomatic patients show impaired diastolic function and atrial dilatationdel Fig. , we monidel Fig. . The mec25. Furthermore, it has been postulated that alterations in myofilamentary proteins are important for the development of familial hypertrophic cardiomyopathy26.In the hypertrophic and hypercontractile myocardium of Ba rats, active tension, calcium sensitivity and cooperativity increased while Ktr decreased. Myocardial hypercontractility occurs in parallel with changes in myofilamentary proteins that increase force generation at a given calcium concentration27, when the RV is overloaded, it develops several abnormalities, such as hypertrophy and deterioration of systolic and diastolic function. Similar results were recently obtained in patients who underwent AVR, and no significant change in RV function was observed28. Calcium overload and impaired ventricular relaxation can promote pulmonary congestion29 as observed in our banding animals and strengthening our positive correlation data between arterial wall thickness and some important parameters of diastolic dysfunction, namely E/e′ and LAA. Therefore, pulmonary vascular remodelling can be a consequence of the sustained increase in pulmonary venous pressure30. From the epidemiologic-standpoint, pulmonary hypertension is frequently present in AS patients and the underlying reason may be attributed to DD31. Interestingly in our banding rats, pulmonary arterial wall thickness correlates negatively with VO2 max and positively with DD, showing an interplay between these parameters. In the AS clinical scenario, pulmonary hypertension and DD can affect the quality of life of these patients by limiting their functional capacity, mainly during exercise33.Importantly, in agreement with a previous studyAortic constriction triggered RV cardiomyocyte hypertrophy and downregulation or inactivity of anti-hypertrophic proteins such as muRF1, myostatin and GSK3β and activation of the pro-hypertrophic ERK. Despite the unquestionable involvement of these proteins on hypertrophic response to afterload, the activation of the well-recognised pro-hypertrophic agent, mTOR, decreased, AKT was unaltered, and the transcription factor 1 (FOXO1) decreased in the RV of Ba rats. Thus, in RV cardiomyocytes’ hypertrophy is probably mediated by activation of GSK3β, ERK, muRF1, myostatin and FOXO1.In addition to RV myofilamentary passive tension in the banding group, myocardial fibrosis further contributed to RV stiffness. Similar to LV, RV cardiomyocytes showed a reduction in the rate of tension development with no significant changes in active tension.36 and most of the available information on RR derives from LVAD studies. However, RR-induced by LVAD is imposed on a totally different phenotype characterised by dilated hypertrophy and systolic dysfunction46. This is markedly contrasting to the concentric hypertrophy and DD patent in our animal model that was designed to resemble AVR-induced RR and to assess the early events of myocardial RR.Notwithstanding the myocardial remodelling changes imposed by chronic pressure overload, our main goal was to describe the changes during RR. This is quite pertinent since only a few studies describe the changes induced by aortic debanding at an early stage of the disease47. After debanding, the pressure relief allowed the myocardium to remodel as denoted by the partial recovery of LV mass, normalisation of LV volumes and amelioration of LV diastolic dysfunction. Although, RV abnormalities induced by LV pressure overload prevail during myocardium RR, independently of pulmonary edema reduction. This data shows that after aortic pressure relief, the RV takes longer than the LV to recover. Recent studies has shown RV function deterioration after aortic valve replacement14. The mechanism that underlies RV dysfunction post-surgery were not addressed nor discussed in the existing literature. Herein, we were able to describe major changes at cardiomyocyte, myofilaments and calcium levels, adding relevant information to the state of the art.Clinically, the classical parameters to evaluate the success of RR are EF, LV end-diastolic and end-systolic volumes, LV mass and, more recently, diastolic function49, we speculated that these kinases (not assessed in our study) might have contributed for the persistent GSK3β activation after overload relief.We expected that, due to the early nature of our intervention, Deb animals would normalise hypertrophy by reducing AKT pro-hypertrophic signalling pathway, namely phosphorylation of GSK3β and mTOR. However, despite the reduction of AKT and mTOR activation, LV mass and GSK3β phosphorylation remained increased in Deb rats compared to Sham, rats. Since the anti-hypertrophic effects of GSK3β can be blocked by kinases other than AKT or CAMKiiDuring RR, RV cardiomyocytes’ hypertrophy and fibrosis remained high. The downregulation or decreased activity of anti-hypertrophic proteins such as myostatin, GSK3β or FOXO1 could underlie the persistent hypertrophy. Nevertheless, mTOR activation remained decrease and AKT and ERK increased compared to sham, highlighting the complex interaction between pro- and anti-hypertrophic pathways.50. Of note, after AVR, the survival rate is lower in patients with severe fibrosis47. Myocardial fibrosis results from an excessive collagen deposition due to an imbalance in its turnover, and to increased collagen cross-linking52. Although in our study collagen deposition was not modified after overload relief in none of the ventricles, we recognise that a longer period could be necessary to report a significant decrease of myocardial fibrosis. On the other hand, the interplay between fibrosis and inflammation can regulate extracellular matrix remodelling. In fact, IL-6-induced STAT3 activation can promote fibrosis, at least indirectly, by enhancing TGF-β signalling27 or TIMP-1 expression53. Thus, the activation of the IL6-STAT3 proinflammatory and profibrotic pathways could contribute to the maintenance of myocardial fibrosis in Deb rats. Notwithstanding, rats that showed a worse pattern of reverse remodelling present increased activation of proinflammatory and profibrotic pathways .In addition to LV hypertrophy, myocardial stiffness represents an important postoperative clinical marker to determine the RR outcomes after AVR as it strongly associates with DD54. In LV from debanding group, we observed a non-significant attenuation of passive tension that was, at least in part, consequence of S26-PEVK segment phosphorylation, decreasing PEVK persistence length, and increasing titin-based passive force55. Intriguingly, when compared to Sh rats, PKCα normalised and CAMKii is still upregulated. PKCα and CAMKii have opposite effects on titin passive tension, while the former increases, the latter decreases stiffness58. Thus, we propose that the balance of both kinases could contribute to the titin based-passive tension in Deb group. Indeed, titin-derived stiffness results from a fine balance between kinases activity in over 2000 residues that compose the PEVK and others titin-segments, whose numerous potential phosphorylation sites still await validation59.Apart from the extracellular matrix contribution, titin and other myofilamentary proteins also impact myocardial stiffness. Indeed, relatively few information is available about titin changes in the context of AVR-induced LV RR12. Indeed, Bjørnstad et al. demonstrated the upregulation of β-myosin heavy chain to persist after mice aortic debanding16. In our study, if the decrease of Ktr is detrimental or beneficial in LV remodelling or LV RR is still unknown. However, the translation/clinical importance of this finding is relative since humans express predominantly the β-myosin heavy chain isoform in ventricular myocytes as opposed to the α-myosin heavy chain isoform that predominates in rodents60.Regarding myofilamentary changes during RR, we have shown that calcium sensitivity decreased, the myocardium contractility was reduced, and myofilaments active tension and cooperativity was attenuated. In parallel, Ktr sustained decrease meaning that the rate of crossbridges recycling was still reduced, maybe due to a shift towards β-myosin heavy chain, which impairs cross-bridge cycling29 and thus contribute to the improved relaxation observed during RR.Finally, another important contributor for DD in AS is myocardial active relaxation associated with calcium-handling. Two-weeks of RR triggered myocardial relaxation to recover to sham values possibly a consequence of the decrease in CaMKii phosphorylation or the up-regulation of calcium-binding protein S100A1. These changes can prevent diastolic SR calcium release and calcium leakin vitro modulation of myofilamentary proteins function or targeting right ventricular changes.This study aimed to provide an overview of the major biventricular changes taking place during myocardial reverse remodelling. Thus, this study represents an initial approach to describe important signalling pathways related to cardiomyocytes’ myofilaments, calcium-handling, hypertrophic and inflammatory signalling pathways as well as to the extracellular matrix in an early stage of AS progression. Thus, its nature is essentially descriptive, opening novel perspectives for the scientific and medical community to carry out future studies, for instance, focusing on the This work describes several mechanisms underlying cardiac remodeling and RR and highlights that this model nicely recreates important phenotypic features  of chronic pressure overload and its relief, respectively. These rats demonstrate LV concentric hypertrophy and DD, RV dysfunction, remodeling of pulmonary arteries and, importantly, exercise intolerance. After debanding, the normalisation of cardiac function was not paralleled by the reversion of alterations in cardiac structure, myocardial fibrosis and inflammation. Importantly, banding-induced changes in RV function and structure persisted after debanding, despite the complete reversal of pulmonary vascular remodelling, demonstrating a possible masked effect of RV dysfunction.nd and 3rd intercostal space aortic banding was surgically made with a 22-gauge blunt needle placed parallel to the aorta. A ligature  was firmly tied around both, and the needle was subsequently removed . In the sham group, the suture was kept loose . The thorax was closed and the animal allowed to recovered with proper analgesia . The cardiac function and structure were followed by echocardiography. When LV mass was ≥25%, and DD was observed (8–9 weeks)  were anasthetized by inhalation of 8% sevoflurane, orotracheally intubated and mechanically ventilated . Anaesthesia was maintained with sevoflurane (2.5–3%). The animals were placed in right-lateral decubitus on a heating pad. After a small incision between the 2ks) Fig. , a secon®). The treadmill was tilted to 10°. The adaptation was carried out at a speed of 15 cm.s−1 for 3 minutes. The maximum stress test started at a speed of 30 cm.s−1, with increments of 5 cm.s−1 every minute until the animals reached maximal aerobic capacity (VO2max).Aerobic capacity was evaluated on a treadmill chamber coupled to a gas analyser  was used for the echocardiographic studies. M-mode was used to determine systolic and diastolic wall thickness and cavity dimensions. Ejection fraction, fractional shortening and LV mass were calculated as previously described61.For assessing Mitral flow velocity tracings were obtained with pulsed-wave Doppler, peak systolic tissue velocity and E′ were measured with tissue Doppler. Atrial dimensions and TAPSE were measured in the four-chamber view. Acquisitions were made using an echocardiograph (Siemens Acuson Sequoia C512). Recordings were averaged from three consecutive heartbeats.847. 1.4 F, Millar instruments. Baseline and inferior vena cava occlusion recordings were obtained with ventilation suspended at end-expiration. Data was continuously acquired  at 1000 Hz  and analysed off-line by PVAN software (Millar Instruments). Parallel conductance was computed after hypertonic saline bolus. In the end, while anesthetised, the animals were sacrificed by exsanguination, the tissues were collected, weighed and properly stored for molecular and functional studies.For the haemodynamic evaluation, femoral vein was catheterized for fluid administration, and a left lateral thoracotomy exposed the heart allowing for the insertion of a catheter into the LV (SPR-The right ventricle (RV), LV and lungs were fixed in formalin, dehydrated in ethanol, cleared in xylol and impregnated in paraffin. Five-micrometer slides were dewaxed, rehydrated, and stained withHaematoxylin-Eosin (HE), to assess cardiomyocyte area, or Picrosirius Red, to assess myocardial fibrosis, and finally mounted with Entellan®. An optic microscope , equipped with a photographic camera  was used to visualise and photograph the histological preparations. The area of 60 cardiomyocytes per animal was measured using Cell^B software (Olympus). To calculate the area of fibrosis eight fields per animal were photographed and analysed with Image-Pro Pus 6 software . To assess pulmonary arteries remodelling, HE staining was used, and artery medial wall thickness (WT) was expressed as follows: %WT = [/Arterial external diameter] × 100. Analysis was performed in a blind mode. We attributed a code to each sample and the correspondence to the experimental group was done after analysis of the results.24 using a force transducer from Aurora Scientific Inc. (Model 403A) and a length controller (Model 315C-I). Briefly, a passive tension-length relation was done stretching the cell until a SL of 2.2 µm. Maximal activation at pCa 4.5 was used to calculate maximal calcium-activated isometric force  and the slack test  allowed to measure the rate of force-redevelopment (Ktr). A relaxing solution pCa 9.0 was used to determine passive tension (Tp). In the end, the cell was activated (pCa = 4.5) to access the cardiomyocyte functional stability and integrity. Lastly, the dimensions of the cell were measured, and the force values were normalized to the cardiomyocyte cross-sectional area. Data acquisition was made by ASI 600 A program with a sampling frequency of 2 KHz.After defrosted in 2.5 mL of calcium-free relaxing solution LV samples were mechanically disrupted, and the cardiomyocyte suspension incubated with 0.5% Triton X-100 for 5 minutes at room temperature to permeabilize the membranes. To remove the detergent, the cells were washed with relax solution and centrifuged  4 to 5 times. Force measurements were performed as described previously® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0; Sigma-Aldrich, R0278,) and protein concentration was determined based on the method of Bradford . Twenty micrograms (20 μg) of protein of each sample was prepared with Laemmli buffer  randomly loaded and separated by SDS-PAGE . After transferring to a nitrocellulose membrane (Bio-Rad 1620115 and 1620112) and blocking with 5% BSA  in TBS-0.5%Tween  the membranes were cut and incubated with primary antibodies overnight at 4 °C. NCX , AKT , p-AKT , Junctophilin-2 , TIMP-1 (abcam ab38978), TIMP-2 (abcam ab1828), Galectin-3 , MMP-2 (abcam 37150), MMP-9 (abcam ab38898), mTOR , p-mTOR , GSK3β , p-GSK3β , PKC  and p-PKC (abcam ab23513); IL-6  Stat3  p-Stat3  SERCA2a ; PLB , p-PLB  Camkii  and p-Camkii . Finally, membranes were incubated for an hour at room temperature with secondary antibodies . The signal was detected by an image acquisition system (Odyssey Infrared Imaging System LI-COR Biosciences at 700 or 800 nm) and the image acquired prior to signal saturation. The signal was normalised to total protein density (ponceauS (Sigma-Aldrich P7170) as previously described62.Tissue was homogenized in RIPA buffer  . To determined titin isoforms the gels were stained with Coomassie brilliant blue. For titin Western blotting (WB) LV samples were run on 1.8% agarose gels and transferred to PVDF membrane . The blots were stained with Ponceau S solution (Sigma-Aldrich P7170) to visualise total transferred protein. The blots were then probed with rabbit polyclonal antibodies against titin’s pS26  and pS170 . Bands were visualised using the LAS-4000 Image Reader . Densitometry was performed using Multi Gauge version 3.2 software (Fuji Science Imaging Systems).For titin analyses, cardiac muscle tissue was homogenized in modified 18S. Specific PCR primer pairs for the studied genes were 18S, Procollagen-type-I alpha1 and Type-III aplha1, lysyl oxidase (Lox), transforming growth factor beta (Tgf-β) and calcium-binding protein A1 (S100a1).For gene expression analyses, RNA was extracted with TriPure (Roche). RT-PCR was performed with total RNA, followed by real-time PCR analyses using the SYBR Green method in a StepOne Plus, Applied Biosystems. For the animal studies, results are relative to the mean obtained for the SHAM group (set as arbitrary units) and normalised for Results are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism software. Shapiro-Wilk was used to assess a parametric distribution. Comparisons were performed by one or two-Way ANOVA and appropriate post-hoc tests were used. The probability values < 0.05 were considered significant.Supplements SREP-18-36653A


The leading cause of mortality of thalassemia major patients is iron overload cardiomyopathy. Early diagnosis with searching for left ventricular diastolic dysfunction before the systolic dysfunction ensued might yield better prognosis. This study aimed to define the prevalence of the left ventricular diastolic dysfunction (LVDD) in thalassemia major patients with normal left ventricular systolic function and the associated factors.Adult thalassemia major patients with normal left ventricular systolic function who were referred for cardiac T2* at Siriraj Hospital – Thailand’s largest national tertiary referral center – during the October 2014 to January 2017 study period. Left ventricular diastolic function was defined by mitral valve filling parameters and left atrial volume index using CMR. Patients with moderate to severe valvular heart disease, pericardial disease, or incomplete data were excluded. Baseline characteristics, comorbid diseases, current medication, and laboratory results were recorded and analyzed.One hundred and sixteen patients were included, with a mean age of 27.5 ± 13.5 years, 57.8% were female, and 87.9% were transfusion dependent. Proportions of homozygous beta-thalassemia and beta-thalassemia hemoglobin E were 12.1 and 87.9%, respectively. The baseline hematocrit was 26.3 ± 3.3%. The prevalence of LVDD was 20.7% (95% CI: 13.7–29.2%). Cardiac T2* was abnormal in 7.8% (95% CI: 3.6–14.2%). Multivariate analysis revealed age, body surface area, homozygous beta-thalassemia, splenectomy, heart rate, and diastolic blood pressure to be significantly associated with LVDD.LVDD already exists from the early stages of the disease before the abnormal heart T2 * is detected. Homozygous beta-thalassemia and splenectomy were strong predictors of LVDD. These data may increase awareness of the disease, especially in the high risk groups. The leading cause of mortality of thalassemia major patients is iron overload cardiomyopathy. Since current diagnosis is made by evaluation of T2* by cardiac magnetic resonance (CMR) imaging , the myoPrevious studies of left ventricular diastolic function in thalassemia major patients have various in results in prevalence and associated factors –10. The The study protocol was approved by the Siriraj Institutional Review Board of the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok Thailand, and all patients provided written informed consent to participate. Adult thalassemia major patients (age ≥ 18 years) with normal LV systolic function (LV ejection fraction ≥55% by CMR ) who werCardiac function and T2* were performed on a 1.5 T Achieva-XR Quasar-Dual-Gradient System  with a 5-element cardiac coil. Steady-state free precession, breath-hold technique was performed for cardiac function evaluation. For cardiac function, cines of 4-chamber, 2-chamber, and short-axis views from apex to atrioventricular valve ring were acquired. The CMR parameters were, as follows: 8 mm slice thickness for 10–12 slices without gap in between, repetition time (TR) 3.4 ms, echo time (TE) 1.7 ms, and flip angle 60°. LV ejection fraction (LVEF), LV end-diastolic volume (LVEDV), LV systolic volume (LVSV), and LV mass were calculated as previously described by Krittayaphong, et al. . Left atLA volume = 0.85 x LA area (2-chamber view) x LA area (4-chamber view)/LA length.(The shortest LA length from the 2-chamber view and 4-chamber view was used in the formula)3. The 8 images were quantitatively analyzed by pixel-wise method for all segments, and then the septum was chosen. The median T2* value was reported as abnormal if the T2* was ≤20 ms. This method was well-validated and had good reproducibility [Cardiac T2* was performed with gradient-echo, black blood technique. Single short-axis mid-ventricular slice with 8 TE from 2.60–16.74 ms with each step of 2.02 ms was used. CMR parameters for T2* were, as follows: slice thickness 10 mm, TR 18 ms, flip angle 20°, field of view (FOV) 400 × 400 mm, and voxel size 3.0 × 1.5 × 10 mmcibility –23. Diascibility –27.2 in female, and > 71 ml/m2 in male) [We use CMR, not echocardiography, to determine LVDD in the present study. The criteria for LVDD was modified from the ASE 2009 criteria , which fin male) , pseudont-test or Mann-Whitney U test. For categorical variables, chi-square test or Fisher’s exact test was used to test for association. Variables associated with LVDD at a p-value less than 0.2 were entered into univariate analysis. Variables found to be significantly associated with LVDD in univariate analysis were entered into multivariate analysis using forward stepwise multiple logistic regression.Baseline characteristics were summarized using descriptive statistics. Frequency and percentage were used to describe categorical variables. Continuous variables were reported as mean ± standard deviation for normally distributed variables, and as median and interquartile range (25th and 75th percentile) for non-normally distributed variables. Kolmogorov-Smirnov test was used to evaluate for normal distribution. The prevalence of LVDD is described as percentage and 95% confidence interval. Association between continuous variables and LVDD was determined using Student’s p-value < 0.05 was considered to denote statistical significance. The statistical software package SPSS  was employed for all data analyses.Intraclass correlation coefficient (ICC) was performed in 10 patients to measure intraobserver and interobserver reliability. For all tests performed, a two-tailed There were 123 patients eligible for inclusion during the 1 October 2014 to 4 January 2017 study period. However, diastolic function could not be interpreted in 7 patients due to absence of A or fusion of E and A waves, so those patients were excluded from the study. The remaining 116 patients were enrolled. Baseline characteristics of study population were shown in Table The prevalence of LVDD was 20.7% (95% CI: 13.7–29.2%). Abnormal relaxation, pseudonormalization, and restrictive filling pattern was found in 3 (13%), 7 (29%), and 14 (58%) patients, respectively. Cardiac T2* was abnormal in 8.3% (95% CI: 1.0–27.0%) of patients in the LVDD group, and in 7.8% (95% CI: 3.6–14.2%) of all patients enrolled in this study.p = 0.004), BSA , homozygous beta-thalassemia , splenectomy , HR , and DBP .Univariate and multivariate analysis was performed to assess for factors significantly associated with LVDD Table . Age, boIntraobserver and interobserver reliability was demonstrated by ICC, and the parameters were found to be well correlated Table . All parWe used CMR, not TTE, to determine LVDD in the present study. Therefore, we did not have data on TTE in every case. Additional analysis was performed to correlate CMR finding on diastolic dysfunction with TTE data among those who had available TTE results within 6 months prior to CMR examination. We demonstrated a good agreement of echocardiogram and CMR for the detection and correctly classified the grading of diastolic dysfunction in 17 patients (89.5%). Restrictive filling which is the most severe form of diastolic dysfunction was correctly detected in 100%.The prevalence of LVDD in thalassemia major patients with normal LV systolic function in this study was 21.4%. Factors associated with LV diastolic dysfunction in multivariate analysis were age, BSA, DBP, HR, homozygous beta thalassemia, and splenectomy.Previous studies showed that LVDD may relate to changes that occur in the early stage of cardiac iron overload, and that these changes may be detectable before systolic failure. The prevalence of LVDD in our study was more than that reported by Kremastinos, et al. , but lesSpirito, et al.  reportedThe relationship between CMR estimation of myocardial iron and LV systolic and diastolic function was studied in 24 transfusion-dependent thalassemia (TDT) patients with 47 paired TTE and CMR by Leonardi, et al. . In thatWe proposed a CMR criteria for the detection of LVDD modified from the ASE 2009 criteria , which fPrevious data on prevalence of LVDD in patients with normal left ventricular systolic function showed that if we used the stringent criteria , the prevalence would be 7.9% but is we used the loose criteria , the prevalence would be 50%. The prevalence in our report should be more reasonable because our proposed CMR criteria are not too stringent and not too loose.p = 0.004). Patients with splenectomy had the risk of LVDD of 36% compared to the risk of 16% for those without splenectomy (p = 0.024). When we combined the effect of these 2 factors, patients with beta-thalassemia/Hb E and no splenectomy had the risk of LVDD of 12%. The risk increased to 30, 44 and 60% for beta-thalassemia/Hb E with splenectomy, homozygous beta-thalassemia without splenectomy and homozygous beta-thalassemia with splenectomy respectively. The effect of splenectomy and homozygous beta-thalassemia on the prevalence of LVDD was mainly in population without cardiac iron overload since our population were well treated with chelation therapy. No correlation was found between LV diastolic function and cardiac T2*. The prevalence of abnormal cardiac T2* defined as lower than 20 ms was only 8% which is too small to make conclusion for the relation of cardiac iron overload and LVDD and other factors. However, when we looked at data of 4 patients with severe cardiac iron overload (defined as cardiac T2* less than 10 ms), the prevalence of LVDD in this group was 50% which is much higher than 20% those with cardiac T2* less than 10 ms but the number is too small to achieve statistical significance. Patients with homozygous beta-thalassemia had an increased risk of cardiac iron overload compared to beta-thalassemia/Hb E . Patients with splenectomy also had an increased trend of cardiac iron overload . Splenectomy results in loss of function to remove hematologic debris and to suppress intravascular hemolysis, which leads to increased oxidation, procoagulative state, vasoconstricting substances, smooth muscle proliferation, and endothelial dysfunction [Age, obesity, and hypertension are known to be associated with diastolic dysfunction , 35; howDecrease in BSA associated with more LVDD. BSA may reflect a patient’s nutritional status, since vitamin B, vitamin D, and carnitine deficiency were reported to be associated with impaired cardiac function . HR and This study has some limitations. First, we cannot extrapolate the results since we enrolled only thalassemia patients who were requested for cardiac T2*. Second, we did not have echocardiographic data to correlate with CMR findings in every patient. We have echocardiographic data in only 19 patients. Although echocardiogram and CMR was not performed on the same day, we could demonstrate a good correlation with CMR for the detection and grading of diastolic dysfunction in 89.5%. E and A values had moderate positive relationship between TTE and CMR methods. Both E and A values from TTE were higher than those measured by CMR, which is similar to previous study . A stronFurther prospective studies of the left ventricular diastolic function by CMR and the cardiac iron overload in various stages of the disease since diagnosis and follow up during treatment may yield more understanding of the disease physiology. Future research for the effect of early detection and treatment on the progression of disease and prognosis is encouraged.LVDD already exists from the early stages of the disease before the abnormal heart T2 * is detected. Homozygous beta-thalassemia and splenectomy were strong predictors of LVDD. These data may increase awareness of the disease, especially in the high risk groups.


To describe the use of speckle tracking echocardiography (STE) in the biventricular assessment of athletes’ heart (AH). Can STE aid differential diagnosis during pre-participation cardiac screening (PCS) of athletes?Data from recent patient, population and athlete studies suggest potential discriminatory value of STE, alongside standard echocardiographic measurements, in the early detection of clinically relevant systolic dysfunction. STE can also contribute to subsequent prognosis and risk stratification.Despite some heterogeneity in STE data in athletes, left ventricular global longitudinal strain (GLS) and right ventricular longitudinal strain (RV ɛ) indices can add to differential diagnostic protocols in PCS. STE should be used in addition to standard echocardiographic tools and be conducted by an experienced operator with significant knowledge of the AH. Other indices, including left ventricular circumferential strain and twist, may provide insight, but further research in clinical and athletic populations is warranted. This review also raises the potential role for STE measures performed during exercise as well as in serial follow-up as a method to improve diagnostic yield. Echocardiography has advanced our knowledge and understanding of the structural and functional adaptation that occurs in the athletes’ heart (AH) in response to chronic training –4. WhilsCardiac chamber enlargement and impaired functional parameters can be also associated with pathological disease, more specifically, cardiomyopathy including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC) . This crCurrently, the most commonly used ɛ imaging modality for assessment of cardiac mechanics is STE. This technique allows for estimation of myocardial ɛ to identify local shortening, thickening and lengthening of the myocardium and provides quantitative measurements of LV regional and global function . ɛ descrRV function assessment by conventional 2D echocardiography is challenging due to the complex RV geometry and the heavily trabeculated inner wall contour . Due to Standard echocardiographic global functional assessment of the LV is limited by the perception that normal LV ejection fraction (LVEF) equals normal systolic function and that abnormal LVEF equals abnormal function. This may not always be the case and is highlighted by recent studies of pathological hypertension in patients diagnosed with heart failure with normal ejection fraction (HFNEF). HFNEF patients have by definition normal LVEF were systolic function and contractility have been assumed to be normal  but therLV global longitudinal strain (GLS) is the most frequently reported deformation parameter in clinical and AH studies •• and itWhen considering the differentiation from pathology, a study of athletes, controls and hypertensive patients  demonstrFewer studies report global circumferential strain (GCS) and global radial strain (GRS) in the AH. Nottin et al.  reportedGCS and GRS were similar between controls, rowers and early hypertensive patients . PatientFew studies have reported comprehensive data sets for regional STE data across longitudinal, circumferential and radial planes , 48 but LV twist from helically orientated fibres is a key component of myocardial performance and can be determined by STE. LV twist data from STE is concordant with torsion measurements from tagged MRI studies in patients with a variety of cardiac pathologies . In a nuHigher LV twist has been reported in hypertensive patients compared to controls with no difference between athletes and controls , but in Resting RV ɛ parameters in endurance athletes  have beePeak systolic RV ɛ is significantly reduced in ARVC patients compared to controls and RV STE has been used to identify regional wall abnormalities in patients  making SHCM patients have been found to have lower LV longitudinal and radial ɛ at rest with higher circumferential ɛ and twist compared to controls. Exercise has induced a modest increase in longitudinal ɛ in HCM patients but not in twist whilst both parameters increased in controls . In pre-La Gerche et al.  reportedAs reduced RV ɛ can be an early marker of disease in ARVC , 72, 73,A major limitation of the clinical application of STE is the lack of universally accepted STE data collection protocols and normative data for healthy adults, athletes and cardiac patients. Variability in specific data can be related to vendor-to-vendor differences , softwarDespite some technical limitations, we suggest on current evidence that LV GLS and RV ɛ should now be included in the standard echocardiographic assessment of AH. We are not suggesting that STE parameters are used solely for differentiation of physiology and pathology in AH, but used together with standard structural and functional echocardiographic parameters they may aid in the differentiation of AH from HCM, DCM and ARVC. STE has the potential to reveal early cardiac dysfunction and may corroborate conventional findings, increasing sensitivity and specificity of PCS. There is an increasing evidence base for normative GCS, GRS, regional and twist parameters in athletes, and whilst the potential of these parameters has been eluded to in this review, it is our opinion that much more work is needed before these parameters can be routinely used in AH assessment.In conclusion, clinicians should be familiar with the assessment of AH and STE analysis should be performed by experienced operators. If STE is applied with caution and with an understanding of the limitations, global and regional ɛ, LV GLS and RV ɛ will provide additional information to aid the differential diagnosis of physiological and pathological adaptation.


Chemotherapeutic agents of anthracyclines class and humanized monoclonalantibodies are effective treatments for breast cancer, however, they presenta potential risk of cardiotoxicity. Several predictors have been recognizedas predictors in the development of cardiac toxicity, and the evaluation ofleft ventricular segmental wall motion abnormalities (LVSWMA) has not beenstudied.To analyze prospectively the role of LVSWMA among echocardiographicparameters in the prediction of development of cardiotoxicity in breastcancer patients undergoing treatment with chemotherapy.Prospective cohort of patients diagnosed with breast cancer and inchemotherapy treatment with potential cardiotoxicity medications includingdoxorubicin and trastuzumab. Transthoracic echocardiograms including speckletracking strain echocardiography were performed at standard times before,during and after the treatment to assess the presence (or lack thereof) ofcardiotoxicity. Cardiotoxicity was defined by a 10% decrease in the leftventricular ejection fraction, on at least one echocardiogram. Multivariatelogistic regression models were used to verify the predictors related to theoccurrence of cardiotoxicity over time.Of the 112 patients selected , 18participants (16.1%) had cardiotoxicity. In the multivariate analysis usingthe logistic regression model, those with LVWMA , LV systolic dimension  and global longitudinal strain by speckle tracking  were strongly associated with cardiotoxicity.In the present study, we showed that LVWMA, in addition to globallongitudinal strains, were strong predictors of cardiotoxicity and could beuseful in the risk stratification of these patients. Thus, the need for optimal cardiac care in the cancer population has becomeevident. Early detection of cardiac dysfunction may allow implementation ofcardioprotective strategies before potentially irreversible myocardial damage hasoccured.5Chemotherapy may induce numerous cardiovascular complications, includinghypertension, congestive heart failure, thromboembolic diseases, ischemic heartdisease, QT prolongation, and bradycardia.6The definition of cancer therapy-related cardiac dysfunction (CTRCD) is based on aserial decline in left ventricular (LV) ejection fraction (EF). Two-dimensionalechocardiography (2DE) is increasingly used for monitoring cardiac function duringcancer treatment due to its widespread availability and safety. Echocardiographyallows assessment of systolic and diastolic function, pulmonary pressures, valvularfunction, right ventricular function, and the pericardium.6-8Previous studies have demonstrated the presence of regional myocardial dysfunctionin patients with CTRCD,9-11however its role as a risk predictor has not been established. The purpose of thisstudy is to verify the association between the occurrence of LV segmental wallmotion abnormality and the development of cardiotoxicity in patients with breastcancer undergoing chemotherapy.Reduction in LV EF likely occurs late in the natural history of CTRCD patients asreduction in LV EF may not be overt until a substantial amount of myocardial reservehas been exhausted, therefore more sensitive screening modalities for LV dysfunctionare needed. Despite the recognition of several echocardiographic parametersassociated with CTRCD, including novel echocardiography-derived parameters ofmyocardial mechanics, such as strain and strain rate, currently there is noconsensus in the medical practice to fully predict which patients are prone todevelop cardiotoxicity.12This study is part of a prospective cohort study of patients with breast cancerrecruited from the Mater Dei Hospital in the city of Belo Horizonte - MG fromJanuary 2010 through December 2016. Inclusion criteria were, age above 18 years,histologically confirmed breast cancer diagnosis, treatment with doxorubicinand/or trastuzumab, and who underwent echocardiography, according to the rulesof the hospital protocol. Exclusion criteria were patients with previousdiagnosis of ventricular dysfunction including regional wall motion abnormality,significant valve disease, congenital heart disease, arrhythmias, chroniccoronary artery disease and left bundle branch block by electrocardiography.Treatment regimens were at the discretion of the oncologist and consisted of theuse of the following drugs alone or in combination: 1) doxorubicin andcyclophosphamide; 2) paclitaxel; 3) trastuzumab. The dosages of the medicationswere prescribed according to guidelines.Clinical  laboratorial  and transthoracicechocardiograms were collected at baseline and standardized time intervals foreach treatment regimen, 6 months after treatment completion and annuallythereafter.13 Abnormal septal motion was characterized as aatypical movement of the interventricular septum during cardiac cycle with atwo-dimensional echocardiography-guided M-mode approach. Diastolic function wasassessed and classified using published criteria.14 LV diastolic dysfunction was stratified intofour grades as normal, impaired relaxation, pseudo normal filling orrestrictive.All patients were referred to a transthoracic echocardiogram, includinglongitudinal strain assessment with two-dimensional speckle-trackingechocardiography (2D STE). The echocardiographic studies and analyses wereperformed by an experienced cardiologist (M.V.L.B.). The followingechocardiographic parameters were assessed: LV end-systolic and end-diastolicdiameters and left atrial diameter. LV ejection fraction was assessed usingSimpson's biplane method. Visual assessment of regional myocardial function wasassessed on the basis of the observed wall thickening and endocardial motion ofthe myocardial segment, as described previously.15Longitudinal strain by 2D STE was obtained from apical four-chamber, two-chamber, and long-axis views. Three cardiac cycles from each view were recordedfor offline analyses with a frame rate > 50 frames/sec. Peak negativelongitudinal strain was assessed in 16 LV segments, defined as the peak negativevalue during the entire cardiac cycle, hence including post systolic shortening,and was averaged to global longitudinal strain (GLS). CTRCD was defined as adecrease in LVEF of > 10 percentage points, to a value < 53% at repeatedcardiac imaging studies during follow-up after chemotherapy.The echocardiographic studies were performed at standardized intervals accordingto the treatment regimen. 1) Patients treated with anthracyclines withouttrastuzumab underwent an echocardiographic study at baseline, at completion ofchemotherapy, and every six months after completed treatment. 2) Patientstreated with anthracyclines and trastuzumab underwent an echocardiographic studyat baseline, after completion of the anthracycline treatment regimen, every 3months during trastuzumab therapy, and every six months after completedtreatment. 3) Patients treated with trastuzumab without anthracyclines underwentan echocardiographic study at baseline, every 3 months during trastuzumabtherapy, and every six months after completed treatment.Echocardiographic assessment was completed in patients with at least threeechocardiographic studies performed during the research period.To describe the qualitative variables, the absolute and relative frequencies wereused, while to describe the quantitative variables, measures of centraltendency, dispersion and position were used.In order to identify the factors that influenced the occurrence of cardiotoxicityover time, the Generalized Estimation Equations (GEE) approach was used. Anexchangeable correlation structure was assumed for the repeated observations ofthe same individual. Univariable and multivariable models with a logit linkfunction were considered. There was no occurrence of cardiotoxicity at the firstmeasurement occasion and therefore we also included the baseline values of thetime-dependent predictors. Missing values were excluded from the analyses.Variables that were statistically significant at the 0.20 level were included inthe multivariable model. For this final model, a level of significance of 0.05was adopted. Reproducibility of visual assessment of abnormal regionalmyocardial function was evaluated by the kappa statistics.ROC curves were built and the discrimination ability of the model was assessed bythe area under the ROC curve. All statistical analysis was performed using RStatistical Software 3.4.1 and the R packages gee, pROC and PredictABEL.The study complies with the Declaration of Helsinki and was approved by theResearch and Ethical Council of the Mater Dei Hospital.A total of 112 patients were included. Mean follow-up time was 491 days. Thecharacteristics of the population studied are summarized in Of the 112 patients followed up, 18 (16.1%) presented CTRCD.The characteristics of the patients with abnormal LV segmental wall motion aresummarized in Among the variables studied, it was observed at multivariable analysis that GLSmeasurements as well as LV systolic dimensions and the presence of LV regionalwall motion abnormalities at the baseline study could predict development ofcardiotoxicity  and 4. TIn this prospective, longitudinal cohort study, we showed that the presence ofregional wall motion disturbance and decreased GLS are strong predictors ofCTRCD.16 As the toxicity evolves, the frequency of thesealterations increases, leading to significant myocardial damage and later on todiffuse myocardial fibrosis. Thus, segmental contractile dysfunction may precede theintense and diffuse involvement of the heart seen in CTRCD. In this context,interventricular septum dyssynchrony, as well as segmental hypokinesia may bepresent due to tissue edema and/or focal cellular damage.17Earlier histopathological studies performed from endomyocardial biopsies havedemonstrated an initially focal and dispersed involvement of myocytes, surrounded bynormal cells in patients treated with anthracyclines.9demonstrated that in 60.9% of patients with LV systolic dysfunction regional wallmotion abnormalities were observed in the first echocardiography that revealed asignificant drop of LVEF. In the majority of these cases (64%), regional hypokinesisinvolved the interventricular septum.9 Previous studies using tissue Doppler and 2D strain have alsoshown regional contractile alterations in patients treated withchemotherapy.10,11Boyd et al.18 demonstrated that inthe group with subclinical LV dysfunction (> 11% reduction in GLS compared tobefore therapy) 58% of regional segments had a reduction in strain by > 11%,compared to 29% of regional segments in the group without subclinical LV dysfunction(p < 0.001).18Indeed, Piotrowsk et al.19 In another study, Sawaya el al.20 showed that in patients with breast cancer treatedwith chemotherapy, GLS measured at the completion of anthracycline therapy wasuseful in the prediction of subsequent cardiotoxicity.20It is well known that reduction of longitudinal strain is an early predictive factorof cardiotoxicity induced by treatment with anthracyclines and trastuzumab, asconfirmed by our results. Negishi et al. showed that GLS was an independentpredictor of subsequent reductions in EF, with a discrimination improvement byadding GLS of -18.6% to traditional parameters by echocardiography in patients atrisk for trastuzumab-induced cardiotoxicity.21 A small cohort study was associated withsubclinical LV dysfunction as early as 1 week after treatment, showing a significantdecrease in GLS and annular systolic velocity of the lateral LV wall 7 days after bytrastuzumab treatment.22 Fei etal.23 found, in a cohortof 95 patients treated with anthracycline and trastuzumab, and followed for a meantime of 17 months, 20% with cardiotoxicity, demonstrating a significant associationbetween GLS reduction and LVEF decline.23It was shown in a systematic review that an early reduction of 10% to 15% in GLS wasa useful parameter for the prediction of cardiotoxicity.24,25and transtuzumab26,27 induced cardiotoxicity. Development of diastolic dysfunctionhas been reported in up to 57% of patients after treatment with anthracyclines oranthracyclines plus trastuzumab.28Cochet et al.28 Serrano etal.29 evaluatedMUGA-derived diastolic parameters and found that impaired LV diastolic functionbefore treatment was an independent predictor of trastuzumab-mediatedcardiotoxicity. Boyd at al.18showed in a cohort involving 140 patients followed for seven days that LV diastolicdysfunction grade significantly increased from 46% to 57% (p < 0.001) aftertreatment with anthracyclines. Importantly, diastolic dysfunction was more prevalentin the subgroup with a significant reduction in GLS, demonstrating the closeassociation between systolic and diastolic dysfunction.18 A study using MUGA-derived diastolic functionparameters investigated whether impairment of systolic function was preceded bydiastolic dysfunction in a group of 77 female breast cancer patients undergoingtrastuzumab therapy. The results of this study showed a nearly even number ofpatients with diastolic dysfunction preceding systolic dysfunction (54%), ascompared to the number of patients with the opposite order (42%).30 Discrepancy among those studies isprobably related to the different designs and interpretation of the results.The presence of diastolic dysfunction was not an independent predictor of CTRCD inour study. The use of diastolic dysfunction as a surrogate marker for predictingtrastuzmab-induced cardiotoxicity is controversial. Earlier studies have shown thatdiastolic impairment of the LV occurs before deterioration in LV EF inanthracyclineAll patients were recruited from one center and the study consisted of a limitednumber of patients. The study was limited by a short duration of patientfollow-up, and therefore any possible long term impact of the earlyechocardiography abnormalities are uncertain. Long term follow up is thereforenecessary to determine the significance of these early observations. Theproposed treatment was individually defined, including the use ofcardio-protective drugs, which may have influenced our results.In this prospective cohort of 112 patients undergoing treatment with chemotherapy forbreast cancer, we found segmental wall motion abnormality to be a strong predictorof cardiotoxicity. Therefore, assessment of segmental wall motion might be a usefultool in the evaluation of patients at risk of developing CTRCT, resulting in earlydetection of myocardial dysfunction and potential reduction in morbidity andmortality in these patients.


Manganese-enhanced MRI (MEMRI) has the potential to identify viable myocardium and quantify calcium influx and handling. Two distinct manganese contrast media have been developed for clinical application, mangafodipir and EVP1001-1, employing different strategies to mitigate against adverse effects resulting from calcium-channel agonism. Mangafodipir delivers manganese ions as a chelate, and EVP1001-1 coadministers calcium gluconate. Using myocardial T1 mapping was performed in healthy adult male Sprague-Dawley rats using a 7T MRI scanner before and after nonchelated (EVP1001-1 or MnCl2 (22 μmol/kg)) or chelated (mangafodipir (22–44 μmol/kg)) manganese-based contrast media in the presence of calcium channel blockade (diltiazem (100–200 μmol/kg/min)) or sodium chloride (0.9%). A second cohort of rats underwent surgery to induce anterior myocardial infarction by permanent coronary artery ligation or sham surgery. Infarcted rats were imaged with standard gadolinium delayed enhancement MRI (DEMRI) with inversion recovery techniques (DEMRI inversion recovery) as well as DEMRI T1 mapping. A subsequent MEMRI scan was performed 48 h later using either nonchelated or chelated manganese and T1 mapping. Finally, animals were culled at 12 weeks, and infarct size was quantified histologically with Masson's trichrome (MTC).  T1 values. This was greatest with nonchelated manganese, and could be inhibited by 30–43% with calcium-channel blockade. Manganese imaging successfully delineated the area of myocardial infarction. Indeed, irrespective of the manganese agent, there was good agreement between infarct size on MEMRI T1 mapping and histology . In contrast, DEMRI inversion recovery overestimated infarct size , as did DEMRI T1 mapping . Increased manganese uptake was also observed in the remote myocardium, with remote myocardial ∆T1 inversely correlating with left ventricular ejection fraction after myocardial infarction .  Both manganese agents induced concentration-dependent shortening of myocardial T1 shortening. MEMRI with T1 mapping provides an accurate assessment of infarct size and can also identify changes in calcium handling in the remote myocardium. This technique has potential applications for the assessment of myocardial viability, remodelling, and regeneration. MEMRI causes concentration and calcium channel-dependent myocardial T With major and sustained advances in imaging techniques over the past 3 decades, magnetic resonance imaging (MRI), along with other advanced modalities such as positron emission tomography (PET), has become an essential element to noninvasive structural and functional cardiac imaging –3. Curre1 relaxivity [There has been increasing interest in a range of alternative contrast media  to broadlaxivity . As suchlaxivity ), and thlaxivity –17. The 2 has been suggested in a pilot study of 15 healthy volunteers at an equivalent molar dose [2 be developed further for clinical utility. However, given that established clinical safety has been demonstrated for both EVP1001-1 and mangafodipir at doses required for cardiac MRI, there is widespread application for both mangafodipir and EVP1001-1, notably for cardiac imaging. Preclinical studies with mangafodipir and MnCl2 in healthy myocardium and EVP1001-1 in myocardial infarction models have described myocardial T1 shortening properties [Whilst short-term cardiac safety of intravenous MnCllar dose , due to operties  and demooperties . Moreoveoperties  as well operties .Despite longstanding knowledge of the paramagnetic properties of manganese, the development and clinical translation of manganese contrast agents have been limited by early issues with toxicity and the subsequent widespread utility of gadolinium agents which have since dominated clinical use in the field. More recently, with concerns about neurological accumulation of some gadolinium agents –25, and 1 mapping to assess and compare their respective T1 shortening properties, utility and accuracy in quantifying myocardial infarction as compared with DEMRI (inversion recovery and T1 mapping) and histological analysis, and explore altered calcium handling in remodelling myocardium. This preclinical work is crucial to inform clinical translation and further development of the potential of MEMRI in myocardial viability assessment.Given the potential benefits of MEMRI, we aimed to compare myocardial enhancement using chelated and nonchelated manganese-based contrast media and to determine the contribution of calcium channels to their uptake. This study represents a novel head-to-head comparison of three manganese contrast agents, using Tn=55) were purchased from Charles River Ltd  and housed, with free access to food and water, in the Central Bioresearch Services, University of Edinburgh for 7 days prior to use in the study.All studies were approved by the University of Edinburgh Animal Welfare and Ethical Review Body and were carried out in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986. Male Sprague-Dawley rats , equipped with a high-performance gradient insert (120 mm inner diameter), maximum gradient strength 400 mT/m. Rats were anaesthetised with 1.5–2% isoflurane  in oxygen/air  with subsequent cannulation of the tail vein for drug/contrast agent administration. The animals were secured in a cradle . The heart rate, respiration rate, and rectal temperature were monitored , with body temperature maintained at 37°C by a heat fan. A 72 mm quadrature volume coil was used for transmission with signal reception by a four-channel phased-array coil .1 mapping sequence. Long-axis cines and a short-axis stack were acquired , with cardiac-gated gradient echo imaging .Scout images were taken to confirm correct positioning and to orientate 9 × 2 mm axial slices from the aortic valve annulus to the apex, perpendicular to the interventricular septum (short axis slices). The slice plan was carefully replicated between scans by ensuring the same slice plan methodology, which was agreed by two operators at the time of scanning to agree adequate orientation. Selection was then made of the mid-ventricle short axis slice for further interrogation with cine and the T1 mapping for calculation of regional left ventricular myocardial T1 relaxation times was accomplished using a gradient-echo, cardiac-gated Modified Look-Locker Inversion recovery sequence (MoLLI) [inversion > 3 × T1 of myocardium (TRinversion > 4.50 s). Imaging readout was with a cardiac fast gradient echo  to compensate for respiratory motion.T (MoLLI)  whereby μmol/kg manganese over 3-4 min. Mangafodipir  was similarly administered as a bolus of 22 or 44 μmol/kg manganese over 3-4 min. Manganese chloride solution (MnCl2) was prepared using MnCl2·4H2O  and sterile water , and 22 μmol/kg manganese was administered over 3-4 min. All manganese contrast media were delivered in volumes of 2.2 mL/kg, diluted with 0.9% saline solution , to maintain the rate of manganese delivery constant between agents.EVP1001-1  was administered as an intravenous bolus at the manufacturer's recommended dosage of 22 μmol/kg/min intravenously, increased to approximately 120–200 μmol/kg/min until a satisfactory chronotropic response was achieved (reduction of >10% in heart rate) or the upper limit was reached. Infusion was commenced approximately 10 min prior to T1 mapping to ensure stable and adequate heart rate response. Control administrations consisted of a similar volume (8 mL/kg) of 0.9% saline over 180 min. Administration of all agents was followed by a further saline flush of 0.4 mL to ensure complete delivery to the circulation accounting for dead space in the fine-bore intravenous line. T1 mapping was performed in all animals at baseline and then at approximately 5, 20, 40, and 60 min after manganese contrast media administration, while cine imaging was performed in a cohort of animals at approximately 15, 30, and 50 min after contrast. The time intervals were determined by technical considerations relating to the length of time required for the T1 mapping sequences.Diltiazem  diluted with 0.9% saline solution was infused at 100 3, respiratory rate 60/min).Rats were anaesthetised with isoflurane (5% in 1.5 L/min oxygen for induction), followed by intraperitoneal ketamine 100 mg/kg  and medetomidine 1 mg/kg  for maintenance anaesthesia. Buprenorphine 0.05 mg/kg  was administered immediately before and 24 hours postoperatively for analgesia. Tracheal intubation was achieved under direct vision, and ventilation was maintained with a rodent ventilator . Due to information gained from the healthy animal data with the manganese contrast agent experiments . Animals were allowed to recover following the scan.Three weeks postoperatively, rats first underwent DEMRI scanning, under isoflurane anaesthesia as described above. Scout images were taken to confirm correct positioning and to orientate 9 × 2 mm axial slices from the aortic valve annulus to the apex, perpendicular to the interventricular septum. Cine images were then acquired in long- and short-axis views as outlined above. Standard DEMRI inversion recovery was performed using gadolinium complex  with 0.5 mmol/kg administered intravenously via slow injection into the tail vein over 1-2 min. Standard inversion recovery prepared imaging with myocardial nulling was performed 10 min following injection , TR ≈ 500 ms; TE = 1.6 ms; flip angle = 90°; FOV = 50 × 50 mmeriments  as well 1 mapping was then achieved using one of the two manganese-based MRI contrast media being developed for clinical use; EVP1001-1 (n=8) and mangafodipir (n=9) at doses of 22 and 44 μmol/kg, respectively, administered via slow intravenous injection into the tail vein over 1-2 min. T1 mapping was performed at the maximal infarct slice  defined by the DEMRI scan acquired in the first imaging session. The doses selected of EVP1001-1 and mangafodipir and the timings of the T1 mapping sequences after manganese contrast agent administration were informed by the healthy animal data to ensure similar degrees of T1 enhancement and therefore sensitivity to detect myocardial viability. Finally, a cine acquisition in the short axis at the maximal infarct slice was repeated following contrast injection. Animals were allowed to recover following the scan.MEMRI was performed 48 h after the DEMRI protocol. Scout images were taken followed by a single short-axis cine at the maximal infarct slice, using the method outlined above. MEMRI TAt 12 weeks after surgery, DEMRI and MEMRI (48 h apart) were repeated using the identical protocols described above. Animals received the same manganese contrast agent at both time points. MRI parameters were unchanged with the exception of an increased FOV (55 × 55 mm) on account of growth of the animals. After the second MEMRI scan, animals were culled by exsanguination by femoral puncture under anaesthesia for tissue harvest. µm sections were taken at intervals in the short axis from apex to base, corresponding to MRI short axis T1 mapping data. Staining was performed with Masson's trichrome (MTC) to delineate areas of collagenous fibrosis, staining infarct blue, and noninfarct purple, before mounting for computer-aided analysis. Slides were scanned at 20x magnification on a Zeiss Axio Scan Z1  with infarct size calculated as a percentage of total left ventricular area at the comparable maximal infarct slice defined by MRI. Automated tissue detection was conducted using Tissue Studio v2.4  as follows: a training set of 4 images was automatically segmented, and segments within three 50 × 50 µm regions comprising remote myocardium, infarct, and cross-over regions from each training image  were manually classified as “Normal myocardium,” “Collagen,” and “nontissue.” These manual classification samples were used to train the software's automated classification algorithm which was then applied to all images in the dataset. Automated detection produced pixel counts and areas for the ROIs within the left ventricle, from which percentage infarct area at maximal infarct slice was calculated.Hearts were fixed by immersion in 4% paraformaldehyde for 24 hours before being transferred to 70% ethanol and processed to paraffin wax for sectioning thereafter. Serial 5 1 relaxation times before and after administration of manganese contrast media. The 14–20 images at unique inversion times were exported offline and combined to generate T1 maps using commercially available software  using three-parameter nonlinear curve fitting as previously described [Quantitative analysis of manganese accumulation was achieved by calculation of regional Tescribed .in vivo experiments, it was noted that there was an approximate ±10% variation in the measurement of T1 values in healthy myocardium and skeletal muscle before the administration of manganese contrast agents. In an attempt to compensate for these fluctuations in T1 measurements, myocardial T1 values were normalized to the T1 of skeletal muscle, where it was noted that the T1 values did not vary over the time course of the contrast agent infusion  with normalized myocardial T1 values obtained from regions of interest (ROIs) drawn on the left ventricle. Change in T1 between the baseline myocardial T1 and the myocardial T1 at each time point was then calculated (ΔT1). Infarct was defined as 2x standard deviations of infarcted and remote myocardial T1 with an averaged intermediate value representing borderzone myocardium.During the lengthy μmol/kg MnCl2, 22 μmol/kg EVP1001-1, and 44 μmol/kg mangafodipir was performed using a two-way analysis of variance (ANOVA) with post hoc multiple comparison Bonferroni tests for individual time points (compared to baseline). For infarct quantification, DEMRI inversion recovery, DEMRI T1 mapping, MEMRI T1 mapping, and MTC assessments were compared using Wilcoxon signed-rank test (2-tailed), with post hoc multiple comparison Bonferroni testing. Bland–Altman plots were used to assess agreement between DEMRI inversion recovery, DEMRI T1 mapping, MEMRI T1 mapping and MTC, and Pearson correlation tested for relationship between changes in remote T1 between early and late time points  and ejection fraction (independent variable). Statistical analysis was performed using GraphPad PRISM . Statistical significance was taken as two-sided P<0.05.Data are presented as mean ± standard deviation unless otherwise stated. Comparison of the time course curves between the saline (control) and diltiazem-infused groups for each of the 22 2, EVP1001-1, and mangafodipir altered T1 relaxivity values evident with T1 mapping  at 20 min were 29.4 ± 5.1%, 28.0 ± 4.4%, and 8.5 ± 4.2%, respectively. To improve the degree of T1 shortening with mangafodipir, the dosage of manganese administered was increased to 44 μmol/kg resulting in a T1 shortening of 12.8 ± 3.4% at 20 min which increased to 15.0 ± 2.9% at 40 min. MnCl2 and EVP1001-1 values at 40 min after administration were unchanged.Thirty-one animals underwent experiments with the manganese contrast agent with a concurrent infusion of either 0.9% saline or diltiazem at a median age of 84 ± 19 days, with a median weight of 356 ± 45 g. Four animals (two control and two with diltiazem) were excluded as venous access was compromised resulting in unpredictable contrast agent administration. MnCl mapping . Mean shth MnCl2 . Peak che points . Overall1 shortening .Pre-treatment with a calcium-channel antagonist (diltiazem) inhibited MEMRI-induced Tortening . This inAll contrast agents were well tolerated across all animal groups. Following administration of gadolinium and EVP1001-1, a transient increase in the respiratory rate was observed, which was self-limiting and resolved within 1 minute. This was seen in healthy animals, shams, and infarcted animals. The present study was not designed to assess safety and tolerability, and no further adverse effects were observed.Healthy myocardial left ventricular ejection fraction was measured before and after manganese contrast agent administration to assess for discernible myocardial depression. The mean LVEF (±standard deviation) and the mean difference in LVEF at each time point from the cohorts of healthy rats administered with each of the manganese contrast agents with concurrent diltiazem  infusion are shown in μmol/kg and mangafodipir 44 μmol/kg (P=0.78) by two-way ANOVA indicating that the higher mangafodipir dosage was well tolerated. There were significant differences detected between the mean difference in LVEF with each of the contrast agents in the presence of diltiazem: MnCl2 22 μmol/kg (P<0.001 and significant (P<0.05) at the 15 min and 50 min time points on multiple comparison); EVP1001-1 (P=0.02) and mangafodipir 44 μmol/kg (P=0.04). This may potentially indicate that in the presence of depressed LVEF the manganese contrast agents at the dosages used may further compromise myocardial contractility; however, further study would be required beyond this preclinical study with low numbers of animals. There was no change in left ventricular ejection fraction following manganese administration with any of the agents in the saline control animals. These data support the assertion that there is minimal change in left ventricular myocardial function in healthy animals with the dosages used in this study. In the infarct group, left ventricular function was assessed at the maximal infarct slice by fractional area change (single slice only due to concern over prolonged anaesthetic risk) before and after manganese-based contrast media administration to assess for discernible myocardial depression. There was no change in fractional area change at this slice following administration of either EVP1001-1 or mangafodipir at early  or late  imaging time points, respectively.No difference was noted between the mean difference in LVEF between baseline and the time points between mangafodipir 22 1 relaxivity, the difference in mean T1 between infarcted and remote myocardium was highly significant across all contrast agents as well as native T1 mapping. Remote myocardial mean T1 was similar between both mangafodipir and EVP1001-1  at a median age of 58 ± 7 days, with a median weight of 262 ± 52 g. One animal in the surgical cohort died unexpectedly 11 days after surgery, resulting in 17 animals completing the experimental imaging protocol. Another animal died unexpectedly 31 days after surgery, with large myocardial infarction, a likely substrate for ventricular arrhythmia as previously observed , and 2 aP<0.001). Myocardial infarction was also associated with higher left ventricular end diastolic volume  and mass . There were no differences in left ventricular function nor volume between animals administered mangafodipir or EVP1001-1 .Three weeks following surgery, all animals in the infarct cohort had left ventricular impairment with anterior wall akinesis and wall thinning associated with a reduced left ventricular ejection fraction , although the differences were less marked by 12 weeks . However, unlike manganese, gadolinium-based assessments tended to overestimate infarct size by around 10%  appeared to have lower mean MEMRI T1 mapping values at late (12 week) compared with early (3 week) time points in animals with the largest infarcts by ejection fraction, but this was not statistically significant .There was an inverse correlation between ejection fraction and remote myocardial ∆T1 mapping to manganese-enhanced MRI, in healthy myocardium in addition to remote myocardium after infarction. This novel combination of imaging techniques has been employed to directly compare two distinct manganese contrast agents with conventional DEMRI in the assessment of viability by infarct size, as well as examine altered calcium handling in remodelling myocardium over time, building on previous pilot data in myocardial infarction. This work was designed as a precursor to clinical translation of intramyocardial contrast imaging, for development of this promising field within cardiac MRI which has potential to improve accuracy of myocardial viability assessment, improve understanding of pathophysiology, and monitor response to therapy in different forms of cardiomyopathy.The present study applies myocardial T1 values. We have further shown that MEMRI T1 mapping provides a better estimate of infarct size than DEMRI using both inversion recovery and T1 mapping and correlates with left ventricular remodelling within the remote myocardium following myocardial infarction. We conclude that MEMRI holds major potential for the assessment of myocardial viability, dysfunction, and regeneration with a wide range of clinical applicability.We have demonstrated that MEMRI causes an ionic, concentration, and calcium channel-dependent shortening of myocardial Tin vitro where transmetallation with zinc occurs rapidly, almost to completion, within 1 minute of incubation in human serum [2 and EVP1001-1) manganese contrast media were compared in healthy myocardium. Intracellular T1 shortening properties of manganese were clearly demonstrated with a reduction in myocardial T1 values of 29.4 ± 5.1%, 28.0 ± 4.4% and 12.8 ± 3.4%, compared with baseline values, with 22 μmol/kg MnCl2 and EVP1001-1 at 20 min, and 44 μmol/kg mangafodipir at 40 min, respectively. The paramagnetic performance of manganese when administered as EVP1001-1 was highly comparable to that of MnCl2, with rapid increase in T1 relaxivity over time achieving close to maximal relaxivity by 5 min  and histological infarct size at 6 h, resolving at 7 days [1 mapping as a technique to define viability in this way using mangafodipir, demonstrating manganese enhancement in myocardium remote to the infarcted region, 3-4 weeks after infarction [2 carried significant risk of adverse events in cardiac patients which prohibits further clinical development, mangafodipir and EVP1001-1, both having established clinical safety and tolerability, are now primed for clinical applications in cardiac imaging. The clinical significance of viable myocardium defined in this way is as yet unknown and underscores the need for robust clinical trials in this field.In myocardial infarction, both DEMRI inversion recovery and Tt 7 days . In clint 7 days  and somet 7 days . MEMRI mt 7 days . In the farction . Whilst 1 mapping of remote myocardium was compared over time following myocardial infarction allowing time for left ventricular remodelling. Changes in T1 values between early and late time points were compared for each animal. Despite variability between animals, an inverse correlation was observed between ejection fraction and change in T1 value for all animals, including shams. The significance and precise mechanisms underlying this preliminary finding are unconfirmed as there are contradictory data on activity of voltage-gated L-type calcium channels in heart failure. L-type calcium channels in remodelling remote myocardium may have a greater propensity to remain open for longer, on account of prolongation of the plateau phase of the cardiac action potential, as compared with uninjured myocardium resulting in greater relative manganese uptake [Te uptake . A studye uptake . The dat1 mapping sequence used in this study, the small myocardial volume in conjunction with heart rates in excess of 350 beats per minute, obligate free-breathing acquisition with respiratory rates in excess of 40 breaths per minute (with a high degree of variability in both these parameters) provide significant challenges resulting in prolonged T1 mapping sequences (11–13 minutes) and the margin for error is consequently much narrower than in a clinical equivalent. This constraint resulted in the practical requirement to select one slice representative of the myocardial infarction, rather than acquire a full T1 map short-axis stack. The potential for sampling error between scans was minimized by careful adjudication by two experienced operators at the time of scanning to agree adequate orientation and replicate slice planning methodology, as described above. Whilst unable to guarantee maximal infarct slice, this ensures equivalent slice comparison between scans.The present study has been designed as a proof-of-concept study of MEMRI in ischaemic cardiomyopathy to inform clinical translation of this imaging modality in ischaemic heart disease as well as other forms of cardiomyopathy. The aim was to assess the application of manganese in noninfarcted myocardium undergoing remodelling following infarction rather than specific dynamics of the infarct region acutely after infarction. Therefore, a permanent coronary artery ligation model was used over an ischaemia-reperfusion model. There are several aspects that are specific to preclinical MR imaging of rodents which do not apply to clinical imaging which are relevant. Despite the excellent spatial resolution of the T1 mapping removes the issue of timing in correct nulling, which is made problematic due to the animal-specific issues above. In the imaging of an infarct, it is possible to select an inversion time following manganese administration whereby the infarct is nulled and the myocardium enhances, in an opposite fashion to DEMRI with gadolinium. Inversion recovery imaging with these nulling techniques is easily degraded by artefact of irregular heart rate or breathing and accurate conclusions are highly dependent on the quality of the nulling. Moreover, where more diffuse myocardial processes are concerned, such as remote remodelling in ischaemic cardiomyopathy, T1 mapping offers the ability to quantify the graduation of T1 across all regions of myocardium. Finally, in clinical MR imaging, a motion correction algorithm is applied to the T1 mapping sequence. This algorithm is not available to us in the preclinical setting. These technical factors, unique to the preclinical nature of this study, necessitated the use of normalization of T1 values and underscore the need for clinical translation.The use of TWhat future diagnostic and therapeutic possibilities does manganese-enhanced cardiac MRI offer the clinician? The prospect of intracellular myocardial tissue characterisation is novel and has far reaching potential. The present study demonstrates unique description of myocardial infarction and viability through calcium handling which has exciting applications in development of novel therapies in myocardial infarction, recently explored in preclinical ischaemia-reperfusion assessing myocardial regeneration with stem cell therapy  and clinThe present study demonstrates the utility of MEMRI with two distinct agents, chelated and nonchelated manganese, as a noninvasive imaging modality which can accurately quantify viable myocardium. Furthermore, these data indicate an ability to detect and quantify altered calcium handling in the remodelling remote myocardium. This novel technique has potential to actively quantify viable myocardium, rather than inferring viability using infarct extent as a surrogate. Furthermore, calcium-handling dysfunction observed in a wide range of cardiomyopathies and heart failure syndromes eludes current noninvasive investigation, an application where MEMRI holds great promise. Clinical translation of the work presented here is an essential next step.


Infarct evolution rate and response to acute reperfusion therapy may differ between patients, which is important to consider for accurate management and treatment of patients with ST-elevation myocardial infarction (STEMI).The aim of this study was therefore to investigate the association of infarct size and myocardial salvage with gender, smoking status, presence of diabetes or history of hypertension in a cohort of STEMI-patients.n = 301) with first-time STEMI from the three recent multi-center trials  underwent cardiac magnetic resonance (CMR) imaging to determine myocardium at risk (MaR) and infarct size (IS). Myocardial salvage index (MSI) was calculated as MSI = 1-IS/MaR. Pain to balloon time, culprit vessel, trial treatments, age, TIMI grade flow and collateral flow by Rentrop grading were included as explanatory variables in the statistical model.Patients (n = 66) had significantly smaller MaR (mean difference: 5.0 ± 1.5% of left ventricle (LV), p < 0.01), smaller IS , and larger MSI  compared to men (n = 238). These differences remained significant when adjusting for other explanatory variables. There were no significant effects on MaR, IS or MSI for diabetes, hypertension or smoking.Women (Female gender is associated with higher myocardial salvage and smaller infarct size suggesting a pathophysiological difference in infarct evolution between men and women. Ischemic heart disease (IHD) is a major cause of death worldwide. In the acute setting of IHD, a coronary occlusion may cause ischemia which gradually develops into myocardial infarction unless the myocardium is reperfused –3. ReperFactors such as gender, smoking, history of hypertension and diabetes have all been implicated to affect risk after acute myocardial infarction. It has been shown that women have higher mortality compared to men after acute myocardial infarction  althoughIt is not clear what mechanisms could explain differences in prognosis and pathophysiology or how they relate to the development of acute ischemic injury during coronary occlusion. Quantifying the extent of injury is therefore an important step towards better understanding of the pathophysiology associated with acute ischemic myocardial injury. Cardiovascular magnetic resonance (CMR) enables quantification of both irreversibly damaged myocardium, infarct size , 20, andOur aim was therefore to investigate to what extent gender, smoking status, presence of diabetes or history of hypertension affects the rate at which infarct evolves by assessing the amount of infarction and myocardial salvage observed using CMR in a cohort of ST elevation myocardial infarction (STEMI) patients from three recent multi-center trials.Patients from the CHILL-MI NCT01379261), MITOCARE (EudraCT Number 2010–024616-33) and SOCCER NCT01423929) trials underwent CMR imaging 1–8 days after primary percutaneous coronary intervention (PCI) for first-time STEMI using previously published inclusion and exclusion criteria [1, MITOCA trials uHypothermia was induced prior to PCI for patients in the CHILL-MI trial randomized to treatment group as previously described . The SOCCulprit vessel was determined by coronary angiography and the ischemic injury was designated as anterior (left anterior descending artery) or non-anterior (right coronary artery or left circumflex artery). Coronary artery flow before PCI was graded according to the thrombolysis in myocardial infarction (TIMI) grading system and coronary collateral flow was graded according to the Rentrop grading system .The CMR examinations were undertaken on scanners of 1.5 T field strenght from Philips , Siemens  or General Electrics . All subjects were imaged in a supine position. ECG gating was used and images were acquired at end-expiratory breath hold. Black blood triple inversion recovery T2-weighted (T2-STIR) images were acquired including full coverage of the left ventricle from base to apex prior to administration of an intravenous gadolinium-based extracellular contrast agent (0.2 mmol/kg). Contrast-enhanced steady state free precession (CE-SSFP) images were acquired approximately 5 min after contrast injection including short-axis images corresponding to the T2-STIR images. Slice thickness was 8 mm with no slice gap. In-plane resolution was typically 1.5 × 1.5 mm.http://segment.heiberg.se), was used to analyze images [The software Segment, version 1.9R3314 . Continuous parameters are expressed as mean ± standard deviation and categorical parameters are expressed as per cent of total. Independent samples t-tests were used to compare population characteristics between men and women for continuous variables and Fisher’s exact test was used for categorical variables. To explain infarct development, myocardium at risk, infarct and myocardial salvage multivariable linear regressions were used. MaR and infarct were described as % of left ventricular mass while MSI was described as % of MaR. Univariable linear regression analysis was used to determine which parameters to include in the multivariable analysis. The parameters included in the univariable analysis were: age (continuous variable), pain to balloon time (continuous variable), female gender (yes/no), troponin T levels of < 15 ng/L (yes/no), left ventricular mass , LVM per body surface area , current smoker (yes/no), current smoker or ex-smoker (yes/no), diabetes (yes/no), hypertension (yes/no), treated with hypothermia (yes/no), treated with oxygen (yes/no), treated with TRO40303 (yes/no), anterior injury (yes/no), TIMI 0 flow before reperfusion (yes/no), Rentrop grade 0 of the culprit vessel (yes/no). BSA was calculated by the Du Bois Formula . MultivaDiagnostic CMR data of MaR was available from 298 subjects, of infarct from 285 subjects and of MSI from 282 subjects. Population characteristics are summarized in Table Gender, anterior injury, LVM, and hypothermia were found to be variables associated with MaR in the univariable analysis Table . Female Gender, current smoker, smoker or ex-smoker, anterior injury, age, LVM, LVM/BSA, and TIMI grade flow were variables found to be associated with infarct size in the univariable analysis Table Gender, smoker or ex-smoker, anterior injury, age, LVM, LVM/BSA, and TIMI grade flow were variables found to be associated with MSI in the univariable analysis Table . Female This study shows that in the highly controlled setting of three multi-center trials in STEMI patients, women had smaller myocardium at risk, smaller infarct size, and larger myocardial salvage index. Myocardium at risk, infarct, and MSI were unaffected by smoking, presence of diabetes, and history of hypertension.In previous studies women had poorer prognosis than men following STEMI , 36 and In summary, previous results have been mixed with some studies showing that women have higher MSI and smaller infarcts, both in selected STEMI-populations and in less selected populations, while others show no difference. Our study adds evidence supporting higher MSI and smaller infarcts in women in a well-defined population with similar characteristics for men and women compared to previous results.This difference has implications when designing cardioprotection trials, both regarding the importance of balancing men and women in the study arms and regarding how ratio of women in the trial might affect sample size .As infarct size is one of the strongest predictors of long-term outcome and as high myocardial salvage show good treatment efficacy, it therefore seems that women would have the potential for better outcomes compared to men. Why this does not seem to be the case in bigger, less selected, materials needs to be further elucidated.The expression “smokers’ paradox” comes from the notion that smokers may have lower mortality compared to non-smokers after a myocardial infarction , 43. TheDiabetic patients have been shown to have worse outcomes after STEMI , 45, 46.As expected, anterior injuries had larger MaR and infarct compared to non-anterior injuries. Note, however, that MSI was lower for anterior injuries suggesting that infarct develops faster for this group. Thus, larger MaR in the patients with anterior injuries might contribute to a faster infarct development. Van der Pals et al.  have shoAt least one earlier study, on 100 patients, has reported a relation between the LVM and infarct size as measured by biomarkers which could not be seen in the current study . It shouThe present study uses data from three different trials and pools data from both treatment and control groups which is a potential limitation. All three trials were negative however and the data was controlled for differences between treatment groups and controls. Reliable data on whether oxygen was administered or not during the acute phase of ischemia was available only for patients included in the SOCCER trial. However, as the trial was designed to detect differences in myocardial salvage, MaR, and infarct size, and did not detect any such differences, it is unlikely that this would affect the results in the present study. Data on pain to balloon time was missing in one of the studies. Pain to balloon time did not show any trend towards significance in the remaining data and it is thus unlikely that this will affect results.Female gender, but not diabetes, hypertension or smoking, was associated with smaller infarct size and higher myocardial salvage when adjusting for confounders, suggesting a pathophysiological difference in infarct evolution between men and women.