Ex vivo, under physiological conditions, MLC901 did not show any modification in heart rate and contraction amplitude. However, upon an ischemic insult, MLC901 administration during reperfusion, improved coronary flow in perfused hearts. In vivo, MLC901 (4 µg/kg) intravenous injection 5 minutes before reperfusion provided a decrease in both infarct size (49.8%) and apoptosis (49.9%) after 1 hour of reperfusion. Akt and ERK1/2 survival pathways were significantly activated in the myocardium of those mice. In the 4-month clinical follow-up upon an additional continuous per os administration, MLC901 treatment decreased cardiac injury as revealed by a 45%-decrease in cTnI plasmatic concentrations and an improved cardiac performance assessed by echocardiography. A histological analysis revealed a 64%-decreased residual scar fibrosis and a 44%-increased vascular density in the infarct region. This paper demonstrates that MLC901 treatment was able to provide acute and long-term cardioprotective effects in a murine model of myocardial ischemia-reperfusion injury in vivo.MLC901, a traditional Chinese medicine containing a cocktail of active molecules, both reduces cerebral infarction and improves recovery in patients with ischemic stroke. The aim of this study was to evaluate the acute and long-term benefits of MLC901 in ischemic and reperfused mouse hearts.  The sudden return of blood flow precipitates the death of ischemic myocardial cells by activation of a cascade of complex intracellular events including the release of oxygen free radicals, loss of intracellular and mitochondrial calcium homeostasis, dysfunction of microcirculation, influx of inflammatory cells, edema and apoptotic processes. The damage attributable to IR injury is estimated between 25 and 50% of the total infarct.Cardiovascular diseases are still the leading cause of morbidity and mortality worldwide, especially in industrialized countries. Acute myocardial infarction (AMI) is mainly due to the occlusion of a coronary artery inducing an ischemic insult and tissue necrosis. The prognosis of myocardial infarction, complicated by severe arrhythmias and heart failure episodes, is directly related to infarct size. The extent of scar depends mainly on the duration of ischemia. The challenge therefore is the earliest reopening of the culprit artery. Myocardial reperfusion, either by angioplasty or thrombolysis, is so far the only approved treatment able of saving the myocardium subjected to ischemia and to improve the prognosis of patients. However, this abrupt reperfusion induces deleterious secondary effects called ischemia-reperfusion (IR) injury that add to ischemic damage and further increase infarct size8 the only treatment providing efficient cardioprotective effects for AMI patients is early revascularization of the culprit artery. Numerous drugs identified as cardioprotective in animal models have been evaluated in clinical trials and not one drug or product of potential clinical utility has emerged10. This difficulty of discovering new treatments for patients suffering from myocardial ischemia comes from the failure to translate to humans the strategies developed in animal models. This suggests that other strategies of translation are clearly needed11 and that the concept of a “magic bullet” for a “magic single target” is exceeded.Despite an extensive research to target specifically IR injury12. Combination therapy has been used for centuries. Multiple drug-treatment targeting multi-pathways would possess stronger therapeutic efficacies for complex multifactorial diseases13.A new hope for targeting myocardial reperfusion injury could emerge from compounds already used in humans as a cocktail of molecules active on different harmful pathways such as traditional Chinese medicine (TCM). Evidence from clinical trials has promoted acceptance of TCM for the treatment of cardiovascular disease14. MLC601 combines 9 plants and 5 animal components. The beneficial effects of MLC601 in cerebral infarction have been well documented both in rodent models and humans16. The clinical trial CHIMES, which compared MLC601 with placebo in 1099 patients with acute ischemic stroke of intermediate severity, reported a reduction of early recurrent vascular events and vascular deaths in post-stroke patients18. Interestingly, in the Philippine cohort of the CHIMES trial including more patients with predictors of poorer prognosis, functional and neurological outcomes were improved in favor of MLC60119. A recent extension study of the CHIMES trial (CHIMES-E) revealed that a 3-month treatment with MLC601 after stroke improves the functional outcome for up to 2 years among patients with cerebral infarction of intermediate severity20. A simplified formula called NurAiDII™ (MLC901) composed of only the 9 ingredients of plant origin also displays the same beneficial effects as MLC601 in animal models of focal and global ischemia22. Clinical assays, including large trials (>1000 patients) on the use of MLC601, have shown that the compound has a good safety profile and a marked efficacy in specific cohorts of patients23. Safety for MLC901 was recently demonstrated in a recent pilot study in patients with mild and severe brain trauma injury24. Pharmacological data obtained from rodents have demonstrated that, in addition to infarct volume reduction, MLC601 and MLC901 drastically improve recovery of both motor and cognitive functions in focal and global ischemia21. These beneficial effects have also been observed in an in vitro assay of oxygen-glucose deprivation that mimics brain ischemic conditions25. Both MLC601 and MLC901 are cocktail of molecules acting on many different targets such as free radical production (inhibition), ATP-sensitive K+ channels (activation)25 or HIF1α , EPO (erythropoietin), VEGF  expression26.NeuroAiD™ (MLC601) is a TCM, that is used in China to facilitate recovery after stroke27, (ii) that the interesting result of the large clinical study (CHIMES) has shown that MLC601 provides a reduction of early recurrent vascular effects and cardiovascular death in post-stroke patients28 and (iii) that MLC901 contains molecules  previously shown to be cardio-beneficial33, we decided to explore the efficacy of MLC901 in vivo and ex vivo in mice, at the acute phase of myocardial ischemia-reperfusion injury as well as in a long-term study after 4 months following myocardial ischemia in mouse. This paper reports very encouraging beneficial effects of MLC901. The result of such a work is potentially of a great interest since both MLC901 and MLC601 are currently used in patients with stroke, facilitating extension of its use from stroke to cardiac ischemia in humans.Considering (i) that cardiac and brain ischemia share many of the mechanisms that lead to cell death in the two different organs24. We showed that MLC901 administration does not affect the basic cardiac physiological parameters. To do this, heart rate and contraction amplitude were recorded on isolated mouse hearts perfused with MLC901 ex vivo. In addition, in vitro experiments on isolated mouse ventricular cells were performed in order to measure the changes in sarcomere length as an index of contractile performance  and by 49.8% for the 4 µg/kg dose  . When the duration of reperfusion was prolonged to 24 hours  in infarct size was observed when mice were treated at 4 µg/kg, the dose providing the maximal cardioprotective effects  Fig. . There wurs Fig.  a signifcts Fig. . No statcts Fig. .In order to evaluate the long-term cardioprotective effects of MLC901, we used a mouse model of ischemia (50 min)-reperfusion followed by a 4-month period of clinical follow-up Fig. .Figure 235 as well as in mouse animal models36. A significant increase in cTnI concentration was observed in the IR group versus SHAM  evaluated at 24h-post-infarction . At 1-month post-infarction, plasmatic cTnI concentrations were close to zero in all groups as expected. In parallel, we observed a decrease in mortality for mice treated with MLC901 during the 4-month period corresponding to a 33.33% total mortality rate for the MLC901 group versus 55% for the IR group  and T isoforms are well established biomarkers used in the clinical setting for the diagnosis and prognosis of cardiac injury and they play a key role in the risk stratification of acute coronary syndromesion Fig. . The tre37. For this reason, LV systolic function was evaluated using non-invasive echocardiography in mice that survive during the post-infarction period   Fig. . These r75) Fig. .Figure 338. In particular, global longitudinal strain, which represents the myocardial deformation from the base to apex, detects the percent change in length of LV and was reported in mice to be closely correlated with infarct size39. MLC901 treatment was able to fully prevent the IR-induced decrease in the global longitudinal strain  permits the identification of beneficial effects following rescue therapy on myocardial function before changes in global LV function40. Cardiac fibrosis is a critical problem of post-MI ventricular remodeling41. In the group of mice subjected to IR, the percentage of fibrosis assessed by Picrosirius staining was 8-fold higher than in the SHAM group (*p = 0.01) . No significant difference was observed between the SHAM and the MLC901 groups .Literature describes that infarct size is correlated with cardiac remodelling, which strongly impairs cardiac function after myocardial infarction01) Fig. . In the 30R control, n = 7; Fig. in vivo doses . A significant 33.8%-decrease  in infarct size versus I30R was observed in the group of hearts reperfused with the 50 ng/ml MLC901 solution showing that the cardioprotective is really related to a cardiac mechanism. MLC901 perfused at 5 ng/ml did not induce a cardioprotective effect compared to I30R . The highest dose of MLC901 (500 ng/ml) induced an increase in infarct size compared to I30R control  followed by reperfusion (60 minutes) on a Langendorff system perfused with a Krebs solution (Iin vivo. MLC901 at 4 µg/kg was able to decrease significantly (by 49.9%) specific DNA fragmentation at 1-hour reperfusion  in DNA fragmentation was observed when mice were treated at 4 µg/kg, the dose providing the maximal cardioprotective effects both in vivo and ex vivo . This increase in coronary flow was not accompanied with changes in cardiac frequency . The situation was very different for MLC901-treated mice. In that case, the vascular density was similar in the ischemic zone and in the non-ischemic region .Since it was previously observed that MLC901 treatment was associated with a pro-angiogenic protective effect after strokein vivo, which was correlated with decreased apoptosis in left ventricular tissues. Second, based on a 4-month follow-up study in mice, we have shown that MLC901 provides long-term cardioprotective effects characterized by a preserved cardiac function and a decreased extent in fibrosis. This cardioprotection was confirmed in ex vivo experiments indicating that it was mediated by non-neurogenic pathways. Cardioprotection was shown to be related to an anti-apoptotic effect, an improvement of the coronary flow at reperfusion and to a full preservation of the vessel density in the infarct area at 4 months.This work demonstrates for the first time acute and long-term cardioprotective effects of MLC901 during acute myocardial infarction in mice. First, using a mouse model of myocardial ischemia-reperfusion, our results show that a single dose of MLC901 (4 µg/kg) administered at the onset of reperfusion reduced infarct size by 49.8% ex vivo suggesting that the mechanisms underlying cardioprotection originate in the cardiovascular tissue.Short-term cardioprotective effects (49.8% decrease in infarct size) were shown to occur at the optimal dose of 4 µg/kg when MLC901 was administered intravenously as a single bolus at the onset of reperfusion. The reduction in infarct size was also seen in isolated perfused hearts 45. In this study, we show that infarct size, specific DNA fragmentation and phosphorylation of JNK1/2 are decreased in MLC901 treated animals after ischemia-reperfusion. These results are to be put in parallel with those previously obtained for brain ischemia. MLC901 is known to also drastically decrease infarct size as well as apoptosis (e.g. DNA fragmentation) in the brain of mice subjected to focal or global ischemia21.During myocardial reperfusion, the ischemic myocardium is subjected to several abrupt biochemical and metabolic changes. A plethora of studies have shown apoptotic cell death in different models of animal and human ischemic heart disease3kinase/Akt and ERK1/2 are part of the RISK  pathway which plays a crucial role in preventing reperfusion injury in the myocardium46. We examined the expression of protein kinases involved in the RISK pathway including ERK1/2 and PI3kinase-Akt. ERK1/2 activation is associated with protection against apoptosis in cardiac myocytes and from ischemia-reperfusion injury in the heart in vivo47. ERK1/2 phosphorylation was increased in LV tissues from MLC901-treated ischemic mice indicating that TCM compounds protect against ischemic/reperfusion-induced by activating both anti-apoptotic and pro-survival mechanisms as already observed in brain in models of stroke, global ischemia and traumatic brain injury48.The PI35. The cTnI plasma level at 24 h was recently identified as an early marker of infarct size in mouse models with permanent ligation of the left ascendant coronary artery49 and for ischemia-reperfusion injury50. Our results showing a decrease in cTnI plasma level at 24 h in MLC901-treated mice clearly indicate that cardiac injury is decreased at this time point. These results are corroborated by results obtained in our murine model of acute myocardial ischemia-reperfusion, where a 45.5%-decrease in infarct size in the MLC901 treated group was measured in the left ventricle at 24 h of reperfusion.In our 4-month follow-up study, the cTnI level was assessed in all mice as early as 24 h post-MI. A 45%-decrease was observed in mice treated by MLC901 compared to non-treated IR. cTnI is a biomarker for the diagnosis and prognosis of cardiac injury in humans40. Cardiac fibrosis is a critical character of post-MI ventricular remodeling41. Our results show a drastic decrease in fibrosis in the left ventricle of mice treated by MLC901 versus the non-treated IR group when evaluated 4 months after cardiac injury. This remodeling involves pathological changes that include chamber dilation, which ultimately progresses to heart failure. Echocardiographical evaluation of the end-systolic and end-diastolic volumes during the 4-month period of follow-up revealed LV dilatation in the IR group. Increased end-systolic and end-diastolic volumes are correlated with a bad prognosis and more specifically, LV end-systolic volume, which is a strong predictor of adverse outcomes51. In our study, the end-systolic volume was also increased in the IR group and there was a marked beneficial effect of MLC901 to prevent this increase. This beneficial effect was also observed with measurements of the end-systolic area of the left ventricle. Here again, there was a very significant compensative effect of MLC901 to prevent the increase observed in the IR group one month after infarction.The infarct size is strongly correlated with cardiac remodelling, which, in turn, impairs cardiac function after myocardial infarction26. Our results suggest that MLC901 could stimulate revascularization after myocardial ischemia-reperfusion. Recent developments have shown that alteration of the coronary microcirculation52 due to haemorrhage and microvascular obstruction was associated with bad prognosis for AMI patient’s mortality53. In addition, following the failure of clinical trials in cardioprotection, recent recommendations from the clinicians have been made to take into account the coronary circulation as a target of cardioprotection42. MLC901 in this context could reduce mortality rates by decreasing infarct size and inducing vascular remodelling of the infarcted area.All these results obtained from fibrosis quantification and echocardiography indicate that MLC901 is a potent treatment against post-MI LV remodelling. These data are supported also by the significant increase in the vessel density evaluated after a 4-month treatment in the infarcted area in MLC901 treated compared to non-treated IR hearts, suggesting that MLC901 is able to induce a vascular remodelling and repair as already reported after ischemic stroke in a mouse model of middle cerebral artery occlusion54. EF is the main indicator of cardiac performance and remains the simplest and most widely used parameter for the global assessment of LV function. Ejection fraction is a strong predictor of mortality and retains a central role in clinical decisions, including guidelines regarding valve surgery and device implantation55.EF was measured from the bidimensional mode (B-mode) after evaluation of the systolo-diastolic cavity volumes and from the area-length methodIn our study, the reduction in cardiac injury with MLC901 was paralleled with an improvement in LV systolic performance. MLC901-treated mice showed higher LV ejection fraction (LVEF) from 1 month to 4 months after MI compared to non-treated IR mice. These findings are important because better LVEF is definitely associated with improved outcomes.ex vivo study, we observed an increase in infarct size for the highest dose tested at 500 ng/ml. However, we did not observe any toxicity for MLC901 at the corresponding dose of 40 µg/kg in vivo. This difference could be related to the fact that ex vivo, the concentration of MLC901 is artificially maintained constant at this high dose during the perfusion duration, i.e. one hour. This is not the case in vivo where MLC901 (intravenous or per os administration) diffuses in all the different compartments of the internal environment. Our mouse in vivo study corroborates data obtained in clinical trials for MLC601 and MLC90160.An important effect of the MLC901 treatment is that it seems to reduce mortality after myocardial infarction. This positive effect is associated with a trend in the improvement of survival rates in the group of mice treated by MLC901 compared to the non-treated IR during the 4 following months. In our 61. Stroke volume, an important determinant of cardiac output, was evaluated by echocardiography as the difference between the diastolic and systolic volumes. MLC901 was found to significantly prevent the IR-induced decrease in stroke volume observed at 4 months. MLC901 definitely improved cardiac performance after 4 months of treatment.LV stroke volume is an important index of cardiac performance that has been used to gauge therapeutic response and predict adverse clinical event risk62. In mice, our data clearly indicate that MLC901 treatment during AMI improved 2D longitudinal strain. Changes observed in our study were more consistently significant for global longitudinal strain compared to radial and circumferential strains, in accordance with greater vulnerability of endocardial and subendocardial tissue  to ischemia after coronary occlusion38. A recent study performed in mice using STI-methods for echocardiographical analysis of cardiac performance revealed that global longitudinal strain was closely correlated with infarct size and this correlation was better than that between radial strain and infarct size39. These observations are related to the clinical situation where longitudinal strain value is considered as an independent predictor of mortality in high-risk patients with AMI63. Global longitudinal strain was also reported to be highly sensitive in identifying myocardial dysfunction early in the time course of myocardial infarction and subsequently in the follow-up period with more clear evidence of detrimental effect of AMI38.Strain echocardiography provides a detailed evaluation of myocardial mechanics. It predicts clinical outcomes better than LVEF in patients with previous MIMeasurements of conventional parameters for the study of systolic function  and also advanced speckle-tracking based strain measures of cardiac performance, in particular global longitudinal strain show that global left ventricular function was improved in the group of mice treated by MLC901 compared to the non-treated IR group.17. MLC901 and MLC601 contain the same combination of plant extracts. Both compounds have been shown to be safe in clinical trials and both provide the same type of neurological recovery after brain injuries in rodents26.In conclusion, this paper clearly demonstrates that MLC901 treatment administered early at reperfusion and administrated continuously during 4 months post-infarction provides long-term cardioprotective effects in a murine model of myocardial ischemia-reperfusion. MLC901 treatment is characterized by a reduced cardiac injury, a preserved vessel density in the infarcted area and an improved cardiac performance. These beneficial effects seem to be associated with reduced mortality rates. These results also probably explain the protective cardiovascular effects of the parent TCM compound MLC601 previously observed in stroke patients23, results presented here highlight a new potential application of MLC901 as a therapeutic tool against myocardial ischemia-reperfusion injury.In addition to its use for cerebrovascular diseases and particularly for strokeAll experiments were carried out on C57BL/6J mice  in accordance with the European Communities Council directive of November 1986 and conformed to the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health . The surgical protocols were approved by the “Comité d’éthique pour l’expérimentation animale Languedoc-Roussillon” (CEEA-LR) with the authorization number CE-LR-0814.Expanded methods are described in the Supplementary Information file.IR: 40 minutes ischemia–60 minutes reperfusion;24h: 40 minutes ischemia–24 hours reperfusion;IR4m: 50 minutes ischemia–4 months reperfusion;IRSHAM: sham operation without coronary artery ligation (surgery placebo).Investigation conforms to the Directive 2010/63/EU of the European Parliament. Acute myocardial ischemia and reperfusion were performed on male mice as described in details in the Supplementary Information file. Various protocols were applied:30R) or regional ischemia models on EMKA system allowing coronary blood flow and ECG recordings.The heart was mounted on a Langendorff system and retrogradely perfused at pressure- and temperature-constant (37 °C) saline solutions. Longitudinal contraction was measured through a small hook attached to the apex of the heart and connected to a force transducer connected via the DMT amplifier. The recordings were analyzed using Clampfit 9.2 sofware (Axon Instruments). Ischemia-reperfusion was achieved by either using global (ISigma-Aldrich). The ischemic risk area and the infarcted area were measured by planimetry with Image J software (Scion Corp).Left ventricles (LV) were sliced transversally into 1-mm-thick sections and incubated in a 1% solution of 2,3,5-triphenyltetrazolium chloride  designed to measure the amount of cytosolic oligonucleosome-bound DNA, as previously described64.Specific DNA fragmentation was quantified in transmural samples of non-ischemic or ischemic areas of the LV with an enzyme-linked immunosorbent assay kit (Roche Diagnostics). Single rod-shape myocytes were plated on laminin-coated glass bottom chambers (for Ionoptix experiments).Isolated cardiomyocytes were obtained from adult mouse hearts enzymatically dissociated. To do this, hearts were mounted on a Langendorff apparatus and subjected to 1-hour global ischemia followed by 1-hour reperfusion (IR group). At the end of reperfusion, hearts were perfused with an enzymatic solution  was collected from the retro-orbitary sinus in anesthetised mice (ketamine-xylazine) in heparin tubes at various time points (24 hours and 1 month post-surgery). The blood was centrifuged at 4 °C and supernatants were stored at −80 °C.Life Diagnostics) using a Tecan Infinite M 200 system.The amount of plasmatic cardiac troponin I (cTnI) at various time points was quantified with the high sensitivity mouse cardiac Troponin-I Elisa immunoassay kit  was tested at three doses in vivo: 0.4, 4 and 40 mg/kg. For ex vivo and in vitro experiments, MLC901 was tested at 5, 50 and 500 ng/ml corresponding to the in vivo doses taking into account a weight of 25 g per mouse.MLC901 powder .For the long-term follow-up, a MLC901 solution at 10 mg/ml was administered Pierce). Samples of protein were resolved by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose. Anti-AKT, anti-pAKT, anti-ERK1/2, anti-pERK1/2, anti-JNK, anti-pJNK were used. Anti-α actinin antibody was used as internal control for equal loading of total protein extract. Protein bands were visualized by enhanced chemiluminescence method and densitometry analysis was performed using ChemidocTM (Biorad) and ImageJ software.Tissue samples (left ventricle) were rapidly frozen in liquid nitrogen after the end of surgery, homogenized with a grinder in RIPA buffer supplemented with EDTA free protease and phosphatase inhibitor. Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay kit  equipped with a 22–55 MHz bifrequencial transducer . High-resolution images were obtained in the parasternal long and short axes, apical and supra-sternal orientations.Anaesthetized mice (1–1.2% isoflurane) underwent transthoracic echocardiographic examination at 1, 2, and 4 months after coronary artery ligation as previously describedUsing the Vevostrain software and the speckle tracking imaging, global longitudinal and radial strains were measured from the bidimensional long axis view of the left ventricle. Circumferential and radial strains were measured from the bidimensional short axis view.LV tissue specimens were fixated in 4%-PFA and embedded in paraffin, cut from the apex through the base and stained with Picrosirius red. Slides were scanned to acquire whole slide imaging at × 20 magnification. Total fibrosis area was reported to the total area for each slice.Sigma-Aldrich) staining was performed on paraffin-embedded LV sections of the middle part of the LV. Nuclei were stained with DAPI (Sigma-Aldrich). Capillary density was calculated in a blind fashion by two experimenters as the average number of IB4 + vessels per mm2. For counting, n = 3 areas were randomly taken both in the border zone of the infarcted remote areas and in the remote zone of one section per left ventricle. Images obtained with a Zeiss Axioimager Z3 fluorescent microscope were analyzed using ImageJ.For the detection of capillary vessels, Isolectin B4 FITC Conjugate .Data were expressed as mean ± standard deviation. The statistical analysis was performed using GraphPad Prism (One-way ANOVA for parameters measured at one time point and two-way ANOVA for parameters measured at multiple time points (repeated measures) were used. A Tukey’s post-test for multiple comparisons was applied when appropriate. When only two groups were compared, the Mann-Whitney test was used.For Kaplan-Meier analysis, comparison of the curves was performed using the Logrank (Mantel-Cox) test. Statistical significance was noted as ns for p > 0.05,* for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001.Materials, data and associated protocols are available to readers.supplementary information


No evidence for a minimum BHL was found, suggesting that all patients who can tolerate DIBH may yield a benefit from it.A low‐resource visually monitored deep inspiration breath‐hold (VM‐DIBH) technique was successfully implemented in our clinic to reduce cardiac dose in left‐sided breast radiotherapy. In this study, we retrospectively characterized the chest wall and heart positioning accuracy of VM‐DIBH using cine portal images from 42 patients. Central chest wall position from field edge and in‐field maximum heart distance (MHD) were manually measured on cine images and compared to the planned positions based on the digitally reconstructed radiographs (DRRs). An in‐house program was designed to measure left anterior descending artery (LAD) and chest wall separation on the planning DIBH CT scan with respect to breath‐hold level (BHL) during simulation to determine a minimum BHL for VM‐DIBH eligibility. Systematic and random setup uncertainties of 3.0 mm and 2.6 mm, respectively, were found for VM‐DIBH treatment from the chest wall measurements. Intrabeam breath‐hold stability was found to be good, with over 96% of delivered fields within 3 mm. Average treatment MHD was significantly larger for those patients where some of the heart was planned in the field compared to patients whose heart was completely shielded in the plan (PACS number(s): 87.53.Jw, 87.53.Kn, 87.55.D‐ Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


Diffuse myocardial fibrosis is a key pathophysiologic feature in heart failure and can be quantified by cardiac magnetic resonance (CMR) T1 mapping. However, increases in myocardial free water also prolong native T1 times and may impact fibrosis quantification. Thus far, the impact of systemic patient volume status remains unclear. In this study, native T1 time by CMR was investigated in hemodialysis (HD) patients (n = 37) and compared with healthy controls (n = 35). Volume status was quantified by bioimpedance spectroscopy and correlated with CMR T1 time. While no differences between HD patients and controls were present with regard to age (p = 0.180), height (p = 0.535), weight (p = 0.559) and left ventricular (LV) ejection fraction (p = 0.273), cardiac size was significantly larger in HD patients . Fluid overloaded HD patients had significantly longer native T1 times than normovolemic HD patients and healthy controls . By regression analysis, T1 time was significantly associated with fluid status . Our data strongly indicate that native CMR T1 time is significantly influenced by systemic volume status. As fluid overload is common in patients with cardiovascular diseases, this finding is important and requires further study. Native T1 values are a composite signal from myocytes and extracellular volume (ECV). The two most important determinants of an increase in native T1 are edema and fibrosis or amyloid. Native T1 time has been studied as a surrogate marker of diffuse myocardial fibrosis in heart failure with preserved and reduced ejection fraction (HFpEF and HFrEF)4, and also in myocardial inflammation and subsequent edema6. Especially in heart failure, fluid overload is a frequently encountered clinical problem. However, it currently remains unclear to what extent systemic fluid overload influences CMR T1 times and whether a differentiation between fibrosis and overhydration is possible in affected patients. Patients with end-stage renal disease on maintenance hemodialysis (HD), who are closely followed with regard to their fluid status, frequently develop left ventricular hypertrophy and diastolic dysfunction7. This has been linked to a high prevalence of risk factors such as hypertension, coronary artery disease, chronic inflammation and diabetes8. Chronic kidney disease patients frequently develop left ventricular hypertrophy as well as diastolic dysfunction with the most extreme forms typically found in dialysis patients9. The combined effects of pre-existing comorbidities, the continuous strain put on the myocardium through hemodialysis and ultrafiltration and the effects of chronic fluid overload, which remain even after ultrafiltration, lead to a particularly high HFpEF prevalence in HD patients10.Cardiac magnetic resonance imaging (CMR) including T1 mapping is increasingly used to characterize myocardial disease12. In an intricate piece of work, Buchanan et al. were able to show stable T1 times by repeated intradialytic CMR during a HD session, suggesting unchanged myocardial water content during the ultrafiltration process13. Yet, fluid status was not specified and it was acknowledged that additional studies are needed to clarify this issue. Recently, bioimpedance methods, which objectively and quantitatively measure patients’ volume status have been implemented in the care of HD patients and were also used to measure volume status in heart failure patients15. Additionally, it remains currently unclear whether other morphological myocardial changes found in HD patients exert an influence on the measurement of native T1 time.Recently, CMR was applied as a novel tool in HD patients and significantly prolonged native T1 values were foundIn the present study, the potential association of volume status and native T1 time by CMR was investigated in consecutive HD patients and compared with results from healthy controls.16. Further exclusion criteria were defined as dialysis treatment <3 months, prior heart or lung transplantation, known significant untreated coronary artery or significant aortic/mitral valve disease, congenital heart disease, and chronic obstructive pulmonary disease GOLD IV.37 patients undergoing maintenance HD at the Medical University of Vienna were consecutively enrolled. Medical history, information on residual urinary output, and dialysis-associated parameters were collected at study entry. All patients underwent a comprehensive echocardiographic evaluation by board certified cardiologists using scanners such as GE Vivid 7 and Vivid S70 . History of coronary artery disease was defined as prior need for revascularization (percutaneous coronary intervention or coronary artery bypass surgery). Patients diagnosed with HFrEF according to the 2016 guidelines of the European Society of Cardiology (defined by a left ventricular ejection fraction <40% on echocardiography) were excludedAll participants gave written informed consent. The study was approved by the ethics committee of the Medical University of Vienna (EC#1036/2013) and performed according to the Declaration of Helsinki. Native T1 reference values were derived from 35 healthy controls. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.All CMR studies were performed by board-certified physicians on a 1.5 Tesla cardiac-dedicated clinical magnetic resonance system  post-dialysis or on an interdialytic day. The CMR protocol consisted of a functional study without late gadolinium enhancement imaging. 3 standard long-axis slices and a stack of contiguous short-axis slices  were acquired with electrocardiography-gated steady-state free-precession cine-images  in breath-hold technique. For quantification of left and right ventricular ejection fraction and chamber volumes, the endocardial and epicardial contours were traced manually in end-systole and -diastole using dedicated software. The left ventricular (LV) end-diastolic volume was indexed to height in cm. Interventricular septum thickness was measured in the 4-chamber view. For native T1 mapping, a modified Look-Locker inversion recovery (MOLLI) with a 5(3)3 prototype was used. This technique allows an inline, pixel-by-pixel based T1-map within one breath-hold. Motion correction was used for all series of images acquired. Parameters for T1 sequences were: inversion time 120 ms with an increment of 80 ms, measured matrix size 256 × 144, reconstructed matrix size 256 × 218, phase encoding resolution 66%, and field of view 85%. T1 maps were acquired in a mid-ventricular short axis as well as a 4-chamber view. No systematic base-to-apex native T1 variation was found. Left ventricular myocardium was defined as region of interest with a clear distance from the endomyocardial border to avoid blood pool T1 signals  was used. Based on a fluid model using body compartment resistance, extracellular water, intracellular water and total body water as well as adipose and lean tissue mass were measured18. All patients classified as fluid overloaded had both a percental expansion of the extracellular volume above 15% as well as an absolute expansion above 2.5 liters.Extracellular fluid overload was then determined through a BCM algorithm. First, normohydration status was calculated, i.e. the expected normal value for extracellular volume for the patient’s weight. Subsequently, the tissue hydration status was determined as the difference between the actual extracellular volume and the ideal normohydration volume. Fluid overload was calculated as an absolute value in liters and in percent above the normal extracellular volume. In previous studies, the cut-off threshold for pre-HD fluid overload was defined as >15% relative to the extracellular volume, largely corresponding to an absolute value of 2.5 litersFurther, ultrafiltration volume was included in the measurement to calculate pre- and post-dialysis fluid overload. Post-HD hypervolemia was defined as volume expansion >0% extracellular volume.t-test was performed to determine differences between groups. Non-normally distributed values are described as medians [interquartile range] and Mann-Whitney-U-test was applied. Categorical data were assessed using the χ2 test. Group-wise comparison of T1 time was carried out with ANOVA. Correlation calculations were performed using Pearson’s correlation coefficient. A multiple regression analysis was run to predict native T1 time from factors, which were suspected to affect native T1 time , fluid overload, LV end-diastolic volume and LV mass). NT pro-BNP was logarithmized for multiple regression analysis. All statistical analyses were conducted using the software packages SPSS System for Mac version 22.0.0 .Demographic data are presented as mean values with standard deviation (SD) if normally distributed. Paired Student’s Table On average, a pre-HD fluid overload of 2.6 ± 2.3 L above the ideal extracellular volume was found, corresponding to a relative value of 13.9 ± 12.0% above ideal extracellular volume. Overall, 43% of all HD patients were fluid overloaded.20.Table Native myocardial T1 time was significantly longer in the HD group  were analyzed to discriminate between normohydrated (n = 17) and fluid overloaded (n = 13) HD patients  = 5,207, r = 0.422, p = 0.032).By multiple regression analysis including serum NT pro-BNP, LV end-diastolic volume and LV mass, fluid status was the only parameter that was significantly associated with myocardial T1 time  causes an increase in extracellular volume24, and diffuse myocardial fibrosis in cardiomyopathy or heart failure can also increase native T1 times3.Recently, T1 mapping has gained increasing importance in the diagnosis and prognostic evaluation of various myocardial pathologies26. Lately, bioimpedance techniques stemming from HD research have become available to quickly and non-invasively determine a patient’s fluid status as well as other body composition parameters27. Patients with end-stage renal failure are usually in a highly controlled state of fluid gain and removal. In the present study patients on maintenance HD were invited to undergo CMR imaging including T1 mapping. The respective data were related to their fluid status and, in addition, compared with data from healthy controls.However, the impact of systemic fluid status on native myocardial T1 time by CMR has not been addressed so far, although fluid overload is common both among patients suffering from renal failure and/or cardiovascular disease30. Furthermore, it is known that cardiac output increases by 10–17% after the creation of an arterio-venous fistula32. Cases of high-output heart failure due to the overload caused by arterio-venous fistulas or grafts have previously been reported34.Overall, cardiac size was significantly increased in HD patients compared to controls. This might potentially be due to the increased myocardial demand through long-standing arterial hypertension and vascular stiffening caused by mineral-bone disease and chronic inflammation36. Even though fluid overloaded HD patients had significantly lower body mass indices than normovolemic patients, their cardiac mass and size were larger. This stands in contrast with Pfaffenberger et al., who described that height and weight directly affect cardiac size37. Nevertheless, our finding fits in the “reverse epidemiology” concept of HD patients36, in which obesity is regarded a protective survival feature as well as the “obesity paradox” for HF patients38, where a survival advantage was proven for moderately obese patients.Furthermore, those patients who proved to be fluid overloaded differed clinically from normohydrated HD patients: they had a significantly lower body mass index, higher NT pro-BNP serum levels and a higher rate of previously documented coronary artery disease, which are findings that have previously been reported in the literature39. They showed that myocardial T2 times of congested heart failure patients significantly decreased after decongestion and under optimal heart failure treatment. The effect of treatment was monitored by invasive hemodynamics. Interestingly, psoas muscle T2 times also decreased after treatment, but not significantly. Furthermore, the correlation between quantitative myocardial T2 value change and net fluid balance was poor and non-significant. Thus, it is not clear from these data whether changes in T2 times were solely caused by an isolated loss of myocardial water or by improved cardiac function, as reflected by significantly higher mixed venous saturation and cardiac output after treatment. However, the current mainstay for the assessment of extracellular matrix expansion by CMR is the assessment of extracellular volume by T1 mapping. To our knowledge, fluid overload has so far not been related with T1 time prolongation.Importantly, native myocardial T1 time was significantly longer in fluid overloaded HD patients, while the normohydrated group exhibited values comparable to healthy controls, suggesting an influence of hypervolemia on this measure. By multiple regression analysis, only fluid overload remained significantly associated with native T1 time, while other parameters, which are frequently related with myocardial disease in HD patients  were not significantly correlated with T1 time on multiple regression analysis. In line with our results, Verbrugge and coworkers recently published their study on “Global myocardial oedema in advanced decompensated heart failure“ based on T2 measurementset al. showed in a large patient cohort that pre-HD normovolemia as well as post-HD fluid depletion yield the best results with regard to overall survival40. It can therefore be concluded that keeping HD patients “dry” and even dialyzing them slightly below their dry weight is crucial with regard to long-term outcomes. In line with this report, fluid overload was shown to be associated with inferior event-free survival in HFpEF patients, who frequently suffer from additional renal impairment and systemic congestion15. Miller et al. determined total blood volume in these patients in a small pilot trial and found significantly elevated levels41. Closing the circle of these findings, it appears that diastolic dysfunction, which is frequently found in HD patients, is not only associated with interstitial fibrosis but also extracellular fluid volume expansion. As this condition seems to impact on CMR T1 time measurements, it requires careful evaluation.Recently, Dekker Some limitations need to be discussed: the analyzed cohort was small and the results should therefore be interpreted with caution; however, so far this is the only study that systematically evaluated the association of fluid status objectively determined by bioimpedance measurements and native T1 time. Additionally, only 30 out of 37 bioimpedance measurements were technically sound and could be included in the analysis. This number was reached by excluding measurements with a data quality <90  and measurements that did not yield any results at all through the bioimpedance device. Furthermore, fluid overload was accompanied by other patient-specific factors that distinguished normovolemic from hypervolemic patients. Nevertheless, on multiple regression analysis only fluid status remained significantly associated with native T1 time. T2 times were not analyzed in the present study. More interventional studies using bioimpedance-controlled dry weight reduction are needed to clarify to what extent systemic volume overload can influence CMR T1 times and the assessment of extracellular volume.In conclusion, we demonstrate that native T1 time by CMR is influenced by patients’ systemic fluid status. Native T1 time was profoundly prolonged in fluid overloaded HD patients, while normohydrated HD patients had T1 times comparable to healthy controls. As fluid overload is common both in patients on dialysis as well as patients with myocardial pathologies, further studies assessing the impact of fluid status on CMR T1 mapping are urgently required.Supplementary Figures


The effects of left ventricular (LV) cavity size on cardiac function and overload have not yet been fully elucidated. We performed a covariance structure analysis and drew theoretical path models to clarify the effects of hemodynamic parameters on the stroke volume index (SVI) as a marker of cardiac function and on the plasma B-type natriuretic peptide (BNP) level as a marker of cardiac overload. We simultaneously measured various hemodynamic parameters and the BNP levels during cardiac catheterization in 1,715 inpatients of our institution. The current path models tested the validity of the Frank-Starling law in patients with heart failure using the SVI, the LV end-systolic volume index (LVESVI) and the LV end-diastolic volume index (LVEDVI). Using the BNP levels, the path models clearly demonstrated that LVESVI substantially augmented cardiac overload, whereas LVEDVI palliated this parameter. These volume indices exerted opposite effects on cardiac function and overload. These results advance the understanding of the relationships between LV cavity size and both cardiac function and overload and indicate the increasing importance of LV diastolic volume in heart failure and the utility of LVESVI as an important marker of cardiac remodeling for further relevant studies. The relationships between left ventricular (LV) cavity size and both cardiac function and overload in patients with heart failure are important research questions. The Frank-Starling law explains the significance of LV diastolic cavity size in maintaining cardiac output. Increases in LV end-diastolic volume are known to be compensatory and favorable phenomena for the preservation of cardiac output in some cases of cardiac dysfunction.1. If the wall stress exceeds the physiological range, further stress due to neurohumoral activation and oxidative stress can occur in the myocardium4. Furthermore, it is widely believed that cardiac remodeling occurs both histogenetically and morphologically in most patients with cardiovascular diseases6, although the extent of cardiac remodeling that occurs in individual patients remains unclear.However, an association between cardiac function and cardiac overload has not been clearly identified in patients with heart failure, and this remains a pressing issue in cardiac research. Cardiac stress might increase in relation to an increase in LV cavity size, but it remains unclear whether myocardial stress increases within or outside the physiological range when the left ventricle enlarges according to the Frank-Starling lawper se should always react adversely to maintain cardiac output. In contrast, the association between cardiac function and cardiac overload during the diastolic phase is difficult to understand and still requires further research. It is unclear whether increases in LV diastolic volume are harmful in terms of myocardial stress in any practical sense. A more complete evaluation and understanding of LV function in patients with heart failure requires more comprehensive assessments, including assessments of LV geometry, LV cavity size, and left atrial size7.The concept of systolic volume is relatively easy to understand in terms of general morphology because the end-systolic left ventricle enlarges as a direct result of systolic dysfunctions caused by some underlying cardiac disorders. Furthermore, the end-systolic left ventricle volume increased because of cardiac remodeling. In both cases, increases in the end-systolic left ventricle volume As described above, the significance of diastolic volume remains a controversial subject of discussion, and thus we require a more precise examination of the associations between cavity size, especially the end-diastolic LV volume, and both cardiac performance and overload. This apparently straightforward question is actually very difficult to answer in relation to heart failure. One reason for this difficulty is that there is no suitable and sensitive index that represents cardiac overload.10. BNP is primarily secreted in response to mechanical stretching of the myocardium, although neurohumoral factors can also induce its secretion13. Plasma BNP levels are believed to be a sensitive and reliable biomarker of the degree of heart failure in clinical practice17. It is widely accepted that the plasma BNP levels increase in proportion to the severity of not only systolic dysfunction but also diastolic dysfunction18. We believe that BNP can be applied as a sensitive biomarker of cardiac overload and thus examined this issue in the present study.B-type natriuretic peptide (BNP) is a cardiac hormone that is secreted mainly from the ventricle in patients with heart failure10. It would be ideal to answer these questions simultaneously by using one equation model. However, the relevant statistics can be rigid and challenging. Such an analysis cannot be performed with commonly used statistical procedures such as multivariate analysis. Herein, we propose that covariance structure analysis is appropriate for addressing this question. In many areas, covariance structure analysis plays an important role in understanding how the relationships between the observed variables might be generated by an interaction effect and/or hypothesized latent variables. Recently, we successfully analyzed complex paths with covariance structure analysis19.Nonetheless, it would be statistically difficult to determine the individual effect of each parameter among the many factors in actual patients with heart failure because, in general, most hemodynamic parameters are significant confounding factors. Additionally, the plasma BNP levels correlate with the relevant hemodynamic parameters to various degreesIn the present study, we examined the effects of individual hemodynamic parameters, particularly the LV end-diastolic volume, on cardiac function and overload in patients with heart failure using a novel combination of plasma BNP levels and covariance structure analysis.Table We examined the correlations between the LV end-systolic and end-diastolic volume indices , the LV end-diastolic pressure (LVEDP), the stroke volume index (SVI), and the log-transformed BNP (Log BNP). Supplemental Table β: −0.095, 95% confidence interval [CI], , P = 0.072) nor LVEDVI influenced SVI .The analysis was initiated using Group I. The first path models, A and B, were proposed as illustrated in Fig. β: −0.724, 95% CI , P < 0.001) and LVEDVI . We noted that LVESVI and LVEDVI were inversely and positively associated with SVI, respectively. We believe that this model dovetails with the Frank-Starling law.The next theoretical path model proposed is illustrated in Fig. β: 0.501, 95% CI , P < 0.001) and LVEDVI were causatively related to Log BNP .Simple path models D and E were proposed as illustrated in Fig. β: 0.669, 95% CI , P < 0.001) and LVEDVI . We noted that LVESVI and LVEDVI produced precisely opposite effects on cardiac overload as estimated with the Log BNP.Next, the theoretical path model illustrated in Fig. β: 0.647, 95% CI , P < 0.001), LVEDVI  and LVEDP . Importantly, we noted that LVESVI and LVEDVI were positively and inversely associated with Log BNP, respectively. This result was similar to that obtained from path model F .Figure Among the hemodynamic factors that are commonly measured during cardiac catheterization, we proposed that LVESVI and LVEDVI are especially important in terms of cardiac performance because these factors directly determine the stroke volume. In the real world, the extent of LV volume enlargement differs between the systolic and diastolic phases in patients with heart failure. The detection of possible associations between LV cavity size and both cardiac function and overload is very difficult, but it is important for improving our understanding of the basic mechanism of heart failure. Naturally, the Frank-Starling law plays a pivotal role in maintaining cardiac output in patients with heart failure, and myocardial stress or overload should theoretically be related to cardiac volume in the systolic and/or diastolic phase.In this study, we first planned to examine the effect of LVESVI and LVEDVI on SVI, that latter of which was used as a marker of cardiac function. The results of path models A and B both suggested that neither LVESVI nor LVEDVI significantly influenced SVI. Clear distinctions were drawn between the parameters in the next proposed path model (model C). It is important to note that LVESVI and LVEDVI were inversely and positively associated with SVI, respectively. We believe that these results are in absolute accordance with the Frank-Starling law and that increases in LVEDVI improve cardiac function. This study represents the first trial to successfully create an image of the Frank-Starling law on a path diagram based on covariance structure analysis.Next, we examined the possible association between LV cavity size and cardiac overload as estimated by the plasma BNP level. Path models D and E both suggested that both LVESVI and LVEDVI augmented Log BNP. We subsequently drew the theoretical path model F and successfully demonstrated that LVESVI directly caused increases in the plasma BNP levels and that LVEDVI adaptably reduced the plasma BNP levels. The outcome for LVEDVI in path model F was the opposite of that observed in path model E. Importantly, an increase in LVEDVI decreased cardiac overload as estimated by the plasma BNP level.P-values were significant, but the R2 values were very low in some of the models for the determination of the plasma BNP. As previously reported, there are actually many factors that are associated with plasma BNP, including aging, gender, renal dysfunction and obesity19. It is thus logical that the R2 values were relatively small in this study design. Nonetheless, this analysis was planned to focus only on the effects of LVESVI and LVEDVI, and the other factors were essentially irrelevant. Moreover, this study clearly demonstrated that LVESVI is a major component in the determination of Log BNP and that LVEDVI palliated the Log BNP. Therefore, the contribution of SVI to plasma BNP logically cannot be too extensive. Indeed, understandably, the correlation of −0.137 between SVI and Log BNP was significant but relatively small  using path models. Although the pathology and contributing factors for HFpEF have not been fully clarified, covariance structure analysis should help answer the remaining questions.21. There is probably no room for doubt about the effects of volume reduction therapy in patients with significantly enlarged ventricular aneurysms owing to myocardial infarction. However, the over-indication of surgical resection for reducing systolic volume might be undesirable if the procedure results in a reduction in diastolic volume. Furthermore, such surgical methods should not be recommended without caution simply because the diastolic volume is elevated. Based on the current information, when considering surgical therapies, the portion with inadequacy in both contraction and dilatation should theoretically be the only area that is considered for surgical excision.LV volume reduction has become a potential surgical therapy for heart failure patients with enlarged left ventricles. Cardiac remodeling is thought to be repaired by this procedure19. Path models enable us to draw conclusions about which factors we believe to underlie examined measures. This method provides more credible information than multiple regression analysis. However, the basic concept of drawing a path model is important. In this study, we successfully proposed concise and clear path models based on accumulated scientific information and our past experiences.In this study, we used covariance structure analysis to test a complicated hypothesis. This statistical method is currently used in many fields because of the widespread availability of computer processing facilities. Recently, we successfully used this method in a practical mannerIn this study, the technique used to calculate the LV volumes from single-plane cineangiograms of the LVs is associated with significant limitations.The path model described in this report was designed to test the validity of the Frank-Starling law in patients with heart failure. When viewing plasma BNP levels as a marker of cardiac overload, LVESVI substantially augmented cardiac overload, whereas LVEDVI palliated it. The current results advance the understanding of the relationships between LV cavity size and both cardiac function and overload. The results of this study are suggestive of the increasing importance of the LV diastolic volume in patients with heart failure and the utility of the LVESVI as an important marker of cardiac remodeling for further relevant studies.The study patients consisted of 1,715 subjects who were consecutively admitted to our institution with heart disorders from 2012–2015. We simultaneously measured their plasma BNP levels and hemodynamic parameters, which included LVESVI, LVEDVI, LVEDP, and SVI, during cardiac catheterization. The LVESVI and LVEDVI were obtained from left ventriculography (LVG) traces during the end-systolic and end-diastolic phases. The contrast LVG images were acquired at a frame rate of 30 frames per second in the right anterior oblique 30-degree projection. The LVESVI and LVEDVI and the left ventricular ejection fraction were calculated from single-plane cineangiograms by means of the area-length formula using a semi-automated trace method with QAngio XA version 7.1 . This technique for calculating LV volumes might include methodological limitations. The SVI was calculated using the thermal dilution method and a Swan-Ganz catheter. To evaluate the contributions of LV cavity size to both cardiac function and overload, we divided the patients into different groups. We performed the analyses for each group, and the groups were classified based on the following criteria: Group I consisted of all 1,715 patients who were recruited for this study, and all patients underwent left ventricular catheterization. Among these patients, right ventricular catheterization was performed in 353 patients. Group II consisted of 1,363 patients with all types of IHD. Among these patients, right ventricular catheterization was performed in 135 patients. Group III consisted of 1,080 patients with chronic stable IHD after the exclusion of those with acute coronary syndrome. Among these patients, right ventricular catheterization was performed in 133 patients. Finally, Group IV consisted of 352 patients without IHD. Among these patients, right ventricular catheterization was performed in 218 patients. The study protocol (24-355[7121]) was approved by the Ethics Committee of the Jikei University School of Medicine, and we complied with the routine ethical regulations of our institution. This was a retrospective study, and informed consent could not be obtained from each patient. Instead of obtaining informed consent from each patient, we posted a notice about the study design and contact information at a public location in our institution.22.The definitions of the diseases were as follows: In brief, IHD was diagnosed based on symptoms, electrocardiography results, blood sampling, and the morphology of the coronary arteries. The patients with IHD included those with clinically stable IHD. Organic stenosis was defined by a ≥ 75% occlusion of the coronary arteries on coronary angiography. Patients with coronary spastic angina were included in the IHD group if the disease activity was stable and a provocation test was planned during hospitalization. Valvular diseases included heart failure caused by moderate valvular disease, and patients who were scheduled for surgery were included in this group. Arrhythmia was defined by the need for catheter ablation, an implantable cardioverter-defibrillator, or cardiac resynchronization therapy, and patients with a pacemaker or syncope were included in this group. Cardiomyopathy was defined when a patient was diagnosed before admission and underwent treatment or if a patient was diagnosed after admission (excluding cases of ischemic cardiomyopathy). Infectious heart disease included pericarditis, myocarditis, and infectious endocarditis. Hypertension, diabetes mellitus, and dyslipidemia were defined as described previously19. In brief, whole blood (5 mL) was collected in tubes containing potassium ethylenediaminetetraacetic acid (1 mg/mL blood). The plasma BNP level was then measured with a rapid enzyme-linked immunosorbent assay (non-extracted) kit using an antibody to human BNP . Group III consisted of patients with chronic and stable IHD after excluding patients with acute coronary syndrome because plasma BNP levels noticeably and rapidly increase during the 24 h after the onset of acute myocardial infarction in a monophasic manner and then transiently decrease; moreover, the latter decrease is potentially followed by another increase 2–3 days after onset (depending on the degree of ventricular remodeling), which results in a biphasic profile23.We used blood sampling and hemodynamic data during the cardiac catheterization. The serum biochemical analyses and the measurements of the plasma BNP levels were performed in a central laboratory at our hospital during the study period. The plasma BNP level was measured as described in previous reportsContinuous variables are expressed as the means ± the standard deviations (SDs) or the medians with the ranges. Categorical variables are expressed as percentages. Comparisons between two continuous variables were performed using Pearson’s product-moment correlation coefficient analysis. The Kolmogorov-Smirnov test was used to determine whether the BNP values were normally distributed. Subsequently, the BNP data were log-transformed (Log BNP) to achieve a normal distribution for the analysis. All statistical analyses were performed using SPSS Statistics version 23.0 .P < 0.05. The causality model defined some hierarchical regressions between the hemodynamic parameters and cardiac function/overload. The paths between the variables were drawn from the independent to the dependent variables with directional arrows that were used for every regression model . A two-way arrow between two variables indicated a correlation between these variables. For every regression, the total variance in the dependent variable was theorized to be caused either by the independent variables in the model or by extraneous variables (e). Each path had a coefficient that indicated the standardized coefficient of the regressing independent variable on the dependent variable of the relevant path. When obtaining the critical ratios of the differences between parameters, we used AMOS to display the matrix, which included a row and a column for each parameter of the model. Each off-diagonal entry in the matrix provided a statistic for testing the hypothesis that the two-model parameters were equal in the population. The corresponding author had full access to all of the data in the study and takes responsibility for both its integrity and the data analysis.A path model based on covariance structure analysis was proposed to investigate the relationships between clinical factors in the study population and specifically to identify the factors that most likely exerted causal effects on IHD. The path analyses were performed with IBM SPSS AMOS version 23 . The obtained structural equation models were tested and confirmed at a significance level of Supplementary Table 1-14


Patients with Tetralogy of Fallot (TOF) and pulmonary insufficiency and a dilated right ventricle (RV) may suffer from a reduction in left ventricular (LV) performance. It is not clear whether timely pulmonary valve replacement (PVR) preserves LV mechanics.Ten TOF patients who underwent PVR were identified from hospital records, and pre- and postoperative cardiac magnetic resonance images were post-processed with a semi-automatic tissue tracking software. LV circumferential strain, time to peak strain, and torsion were compared before and after PVR. A control group of 10 age-matched normal volunteers was assessed as a comparison.p = 0.082; mid-ventricular −18.4 ± 3.6 vs. −19.1 ± 2%, p = 0.571; apical −22.7 ± 5.2 vs. −22.1 ± 4%; p = 0.703). There was also no difference seen between the baseline strain and normal controls . LV torsion remained unchanged from baseline to post PVR . There was no difference in time to measured peak LV circumferential strain before vs. after PVR . At the same time, pulmonary regurgitation and RV end-diastolic and end-systolic volume indices decreased and LV end-diastolic volume increased after PVR. RV and LV ejection fractions remained constant.LV circumferential strain did not change before vs. after PVR (basal −18.3 ± 3.7 vs. −20.5 ± 3%, PVR allows for favorable remodeling of both ventricular volumes for TOF patients with significant pulmonary regurgitation. In this cohort, LV myocardial functional parameters such as circumferential strain, time to peak strain, and LV torsion were normal at baseline and remain unchanged after PVR. The right ventricle (RV) in patients with Tetralogy of Fallot (TOF) is at risk for progressive dilation secondary to pulmonary regurgitation. This may lead to an impairment of left ventricular (LV) mechanics, especially to a reduction in LV circumferential and radial strain, even in asymptomatic children and adolescents . ReducedThe indication for treatment by pulmonary valve replacement (PVR) is subject to much discussion currently, as both surgical and transcatheter PVR are being evaluated for their long-term benefit . The effCardiac magnetic resonance (CMR) is recommended in TOF patients for follow-up of RV volume and function . With thGlobal circumferential strain has been shown to be the most reproducible measure of strain on CMR feature tracking . LV circWe hypothesized that LV circumferential strain and time to peak strain as well as LV torsion improve after PVR in TOF patients with dilated RV.The retrospective, anonymized use of data was approved by the St. Thomas’ Hospital Research Ethics Committee  08/H0810/058. Written informed consent was not required.Any patient who had undergone surgical PVR for pulmonary regurgitation after repair of TOF was included, if they had undergone pre- and post-procedure cine CMR including a short-axis cine stack of the left ventricle between July 2004 and August 2015, and the MRI study was available in the digital archive. Patients were excluded if other significant hemodynamic lesions were present . Sixty-four surgical cases were performed in this time. Of these, only 14 met the inclusion criteria. Of these, four were excluded due to inadequate images.2), or any reduction in ventricular ejection fractions.Patients underwent surgical PVR 19 ± 9 years after initial tetralogy repair due to moderate to severe pulmonary regurgitation with significant RV dilation. The institutional policy at this time was for elective PVR if there was progressive dilation of the RV, significant RV diastolic chamber enlargement . ECG gated balanced cine steady-state free precession images were obtained in a short-axis stack of 9–13 slices from the atrioventricular valves to the apex, in 20–30 phases per cardiac cycle with a slice thickness of 8–10 mm, gap 0–2 mm, field of view between 272 mm × 272 mm and 390 mm × 390 mm, echo time 1.11–1.68 ms, temporal resolution at a median of 34.5 ms (25.3–50 ms), in breath-holding technique. CMR images of age-matched healthy volunteers were used as a control group.Off-line post-processing was performed using cmr42 Release 5.3.4 . Ventricular volumes and ejection fractions of both ventricles were obtained from systolic and diastolic tracings as described elsewhere , and volFor tissue tracking analysis, basal, mid-ventricular, and apical slices were identified, which displayed myocardium along the entire LV circumference in all phases, avoiding the most basal and the most apical slices. End-diastole and end-systole were manually defined by comparing mid-ventricular slices in all phases. Endocardial and epicardial contours of the left ventricle were drawn manually, starting in end-diastole Figure , trackedt-tests were used for normally distributed variables. Independent sample t-tests were used for comparisons between patient groups. Coefficients of variation  and intraclass correlation coefficients were calculated to describe intra- and interobserver variability of measurements.Ventricular volumes were indexed to body surface area. Statistical analyses were performed with IBM SPSS Statistics 24 . Continuous data are expressed as mean ± SD or as median (range) as appropriate. The one-sample Kolmogorov–Smirnov test was used to test for normal distribution. Pre- and post-PVR conditions were compared by Wilcoxon signed-rank tests for non-normally distributed variables, and paired sample Ten patients qualified for pre- and postsurgical CMR analysis . Patient and control subject ages, weights, and heart rates are summarized in Table Pulmonary regurgitation and RV end-diastolic and end-systolic volumes were significantly reduced after PVR compared to baseline. LV end-diastolic volume showed an increase, although it was not significantly abnormal before PVR. There were no changes in LV end-systolic volume, RV or LV ejection fractions Table .Left ventricular circumferential strain or torsion did not change at the basal, the mid-ventricular, or the apical level after PVR compared to before PVR Table . No diffThe fraction of phases to peak LV circumferential strain based on the total number of phases per cardiac cycle did not differ before vs. after PVR Table .(a)n = 5; control n = 5) are presented in Table Intraobserver variability results of 10 subjects (TOF (b)n = 5; control n = 5) are presented in Table Interobserver variability results of 10 subjects  in apical slices. We found that our TOF patients had strain values largely in this range. Our findings are supported by Kempny et al., whose TOF patients had similar circumferential strain in a single mid-ventricular slice compared to their normal controls . A reduction in circumferential strain may therefore be a late sign of adverse LV myocardial condition.Although Padiyath et al.  reportedYoung adults with repaired TOF with and without PVR both show decreased LV twist on 3D echocardiography . There iTime to peak strain is easy to measure from strain curves and was prolonged for longitudinal strain in the RV of TOF patients in an echocardiographic speckle tracking analysis by Mueller et al.  but not All patients in this study had PVR surgery. In a similarly small cohort of 13 patients with pulmonary regurgitation undergoing transcatheter PVR, most of them with an underlying diagnosis of TOF, Harrild et al.  found anIn our cohort of patients with repaired TOF, LV myocardial deformation parameters were in normal ranges both before and after PVR. Again, this may be due to an institutional bias to replace a dysfunctional pulmonary valve early, before RV and LV function might suffer.post hoc analysis showed that at a significance level of 0.05 and 80% power, the study was empowered to show a 3% difference in circumferential strain at the mid-ventricular level.Patient recruitment for this analysis was retrospective. It is possible that statistical significance for some parameters was not reached because of the number of patients being too small. However, Pulmonary valve replacement improves the interventricular interaction of TOF patients to volume unloading, but intrinsic LV myocardial function parameters such as LV circumferential strain and LV torsion, as measured by CMR tissue tracking, are normal in the majority of TOF patients and remain unchanged after PVR. The literature suggests that there is a small subgroup of patients that have reduced circumferential strain and have adverse outcomes, whereas torsion requires further study in this context.This study was carried out in accordance with the recommendations of the St. Thomas’ Hospital Research Ethics Committee  with a waiver of written informed consent from all subjects in accordance with the Declaration of Helsinki, for the retrospective use of anonymized data. The protocol was approved by the St. Thomas’ Hospital Research Ethics Committee  under 08/H0810/058.BB contributed to research question, measured and analyzed data, interpreted data, and wrote the manuscript. MF contributed to research question, acquired data, and revised manuscript for intellectual content. SD and IR acquired data and revised manuscript for intellectual content. AT performed measurements and revised manuscript for intellectual content. JS revised manuscript for intellectual content. TH conceived research question, identified cases, analyzed data, interpreted data, and revised manuscript for intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Left ventricular systole involves coordinated contraction of longitudinal, circumferential, and radial myocardial fibers. Longitudinal fiber dysfunction appears to be an early marker for a number of pathological states. We hypothesized that reduced mitral annular plane systolic excursion (MAPSE) measured during cine-Cardiac Magnetic Resonance (CMR) imaging reflects changes in longitudinal fiber function and may be an early marker for adverse cardiovascular outcomes.400 consecutive patients with known or suspected coronary artery disease undergoing CMR were prospectively enrolled. Lateral MAPSE was measured in the 4-chamber cine view by two independent observers. Patients were prospectively followed for major adverse cardiac events (MACE) - death, non-fatal myocardial infarction, hospitalization for heart failure or chest pain, and late revascularization. Cox proportional hazards regression modeling was used to identify factors independently associated with MACE.The mean age of the study population was 58± 15) years, with a mean ejection fraction of 59(± 14%). 31% of the individuals had known coronary artery disease and 33% were diabetic. 72 MACE occurred during a median follow-up of 14.5 months. By Kaplan-Meier analysis, patients with lateral MAPSE ≤1.11 cm (median) experienced significantly higher incidence of MACE than patients with a MAPSE >1.11 cm (p = 0.0270) (Figure 5 years, Longitudinal fiber dysfunction assessed with lateral MAPSE during cine-CMR is an independent predictor of MACE in patients with known or suspected coronary artery disease.


Coexistence of left ventricular (LV) longitudinal myocardial systolic dysfunction with LV diastolic dysfunction could lead to heart failure with preserved ejection fraction (HFpEF). Diabetes mellitus (DM) is known as a significant factor associated with HFpEF. Although the mechanisms of DM-related LV myocardial injury are complex, it has been postulated that overweight contributes to the development of LV myocardial injury in type 2 diabetes mellitus (T2DM) patients. However, the precise impact of overweight on LV longitudinal myocardial systolic function in T2DM patients remains unclear.2. Ninety age-, gender- and LVEF-matched healthy volunteers served as controls.We studied 145 asymptomatic T2DM patients with preserved LV ejection fraction (LVEF) without coronary artery disease. LV longitudinal myocardial systolic function was assessed by global longitudinal strain (GLS), which was defined as the average peak strain of 18-segments obtained from standard apical views. Overweight was defined as body mass index (BMI) ≥ 25 kg/mGLS of overweight T2DM patients was significantly lower than that of non-overweight patients , whereas GLS of both overweight and non-overweight controls was similar . Furthermore, multiple regression analysis revealed that for T2DM patients, BMI was the independent determinant parameters for GLS as well as LV mass index.Overweight has a greater effect on LV longitudinal myocardial systolic function in T2DM patients than on that in non-DM healthy subjects. Our finding further suggests that the strict control of overweight in T2DM patients may be associated with prevention of the development of HFpEF. Heart failure (HF) with preserved ejection fraction (HFpEF), which is determined as the presence of HF symptoms and signs with left ventricular ejection fraction (LVEF) ≥ 50% , current2; (6) uncontrolled hypertension > 180/100 mmHg; (7) more than moderate valvular heart disease; and (8) atrial fibrillation. All patients underwent exercise stress or pharmacological testing such as treadmill exercise or stress myocardial perfusion scintigraphy > 2 weeks after admission, and none of them showed an ischemic response. The diagnosis of T2DM was based on the World Health Organization criteria [Between July 2013 and September 2015, 155 asymptomatic T2DM patients admitted to Kobe University Hospital were prospectively enrolled in this study. Patients were excluded from enrolment study if they met any of the following criteria: (1) previous or current history of HF; (2) previous history or suspicion of coronary artery disease; (3) LVEF < 55%; (4) previous history of open-heart surgery and congenital heart disease; (5) serious renal dysfunction defined as glomerular filtration rate < 30 mL/min/1.73 mcriteria . For comAll patients underwent a resting standard echocardiographic examination less than 2 weeks after admission by means of a 3.5 MHz transducer on a single commercially available echocardiographic system . Digital routine grayscale two-dimensional cine loops from three consecutive heart beats were obtained at end-expiratory apnea from standard parasternal and apical views used for speckle-tracking strain analysis. Sector width was optimized to allow complete myocardial visualization while maximizing the frame rate. Standard echocardiographic measurements were obtained according to the current guidelines of the American society of echocardiography/European association of cardiovascular imaging . ConventTwo-dimensional speckle-tracking strain analysis was semi-automatically performed with dedicated software . Briefly, the first region of interest was manually traced with the point-and-click approach on the endocardium of LV at the end-systole phase. The second larger region of interest was then generated outside and carefully adjusted near the epicardium. Finally, six strain segments and corresponding time–strain curves were generated. We used the onset point of the QRS complex as a reference for LV strain analysis. Global longitudinal strain (GLS) was then determined as the averaged peak strain from three standard apical views in accordance with current guidelines , and the remaining 70 (48%) were classified as T2DM patients without overweight. Clinical data showed that the prevalence of dyslipidemia in T2DM patients with overweight was higher than in those without overweight, while echocardiographic data showed that T2DM patients with overweight had a higher LV mass index and lower GLS compared to those without overweight. Intra- and inter-observer reproducibility for GLS for DM patients in our study group was previously reported [Of the total of 155 T2DM patients enrolled in this study, 10 patients (6%) were excluded from all subsequent analyses because of suboptimal quality of echocardiographic images, so that the final study population consisted of 145 T2DM patients. The baseline characteristics of the latter and the 90 controls are summarized in Table reported , 24.TablLV longitudinal myocardial systolic function as assessed in terms of GLS was similar for controls with and without overweight , whereas GLS for T2DM patients with overweight was significantly lower than that for those without overweight   who underwent a 12-week low-calorie fasting phase of a formula die. Furthermore, in eight obese patients with T2DM (BMI: 44 ± 9 kg/m2) who underwent sleeve gastrectomy, GLS improved from 13.2  ±  3.7% to 19.7  ±  2.2% (p  <  0.001) and E/e′ also improved from 12 ± 4 to 9 ± 3 (p = 0.028) after surgery [Several investigators have reported the association of weight loss with LV diastolic function in obese patients. Karimian et al.  reported surgery .2 vs. 71 ± 19 mg/m2, p < 0.05; 30 ± 9 mL/m2 vs. 30 ± 9 mL/m2, p < 0.05, respectively). On the other hand, LV mass index and left atrial volume index in controls were not different between with overweight and without overweight. . These differences were thought to be due to the presence of LV subclinical dysfunction as assessed by lower GLS.De Jong et al.  used 353Unlike findings for HFrEF, large trials testing neurohumoral inhibition have consistently failed to attain positive primary outcomes for patients with HFpEF by using, among others, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, β-blockers and mineralocorticoid receptor antagonists, thus leading to a poor prognosis for patients with HFpEF . In our 2 was calculated as 2.5- and 3.9-fold, respectively, of the risk of those with a BMI < 25 kg/m2 [This study was a single-center cross-sectional study, so that further longitudinal-based cohort studies are required to validate our results. Another limitation is that no long-term clinical outcome data was not available for this study. For a follow-up of this study, however, long-term clinical data is now being collected for validation of our findings through long-term follow-up studies of the associations of T2DM and overweight with the development of HFpEF in patients with T2DM patients. In addition, the blood examination in a control group such as plasma glucose or lipid data were not part of this study. Finally, the prevalence and degree of obesity in our study was mild compared to that in previous studies from Western countries. This is accounted for by the fact that many obese Asian subjects, including Japanese, show a lesser degree of adiposity than that observed in Western countries , 44. How25 kg/m2 .Overweight has a greater effect on LV longitudinal myocardial systolic function in T2DM patients than on that in non-DM healthy subjects. Our finding further suggests that the strict control of overweight in T2DM patients may be associated with prevention of the development of HFpEF.


Large myocardial infarction (MI) is associated with adverse left ventricular (LV) remodeling (LVR). We studied the nature of LVR, with specific attention to non-transmural MIs, and the association of peak CK-MB with recovery and chronic phase scar size and LVR.Altogether 41 patients underwent prospectively repeated cardiovascular magnetic resonance at a median of 22 (interquartile range 9–29) days and 10 (8–16) months after the first revascularized MI. Transmural MI was defined as ≥75% enhancement in at least one myocardial segment.r ≥ 0.80 for all, r ≥ 0.74 for non-transmural MIs; p < 0.001). Peak CK-MB, recovery scar size, and chronic scar size, were all strongly correlated with chronic wall motion abnormality index (WMAi) . There was proportional scar size and LV mass resorption of 26% (0–50%) and 6% (− 2–14%) in median. Young age  was associated with greater LV mass resorption .Peak CK-MB was 86 (40–216) μg/L in median, while recovery and chronic phase scar size were 13 (3–23) % and 8 (2–19) %. Altogether 33 patients (81%) had a non-transmural MI. Peak CK-MB had a strong correlation with recovery and chronic scar size (Peak CK-MB has a strong association with chronic scar size and wall motion abnormalities after revascularized non-transmural MI. Considerable infarct resorption happens after the first-month recovery phase. LV mass resorption is related to age, being more common in younger patients. Left ventricular (LV) remodeling after acute myocardial infarction (MI) is a major precursor of heart failure , 2 and pThe best predictor of LV dysfunction after MI is the infarct size , 7. MostThe purpose of this study was therefore, using repeated CMR imaging, to study 1) the course of LV remodeling after the first revascularized spontaneous acute MI, 2) the more subtle remodeling process after non-transmural MI, and 3) the association of peak CK-MB with recovery phase (1–4 weeks) and chronic phase (> 6 months) scar size, and global and local LV dysfunction. We also made a post-hoc analysis of long-term survival.This is a CMR substudy of the prospectively conducted ISKE project  . In brieIn this CMR substudy, all eligible patients were diagnosed with acute spontaneous type 1 MI , with elAll patients were treated with successful PCI, CABG or thrombolysis in less than 3 days after the admission except for one patient treated successfully with CABG after 9 days during the same hospital stay, and received optimal secondary preventive medication for coronary artery disease.All patients underwent two CMR examinations after MI to assess LV and infarct remodeling. The first CMR study occurred during the recovery phase 7–30 days after MI, and the second at the chronic infarct phase ≥6 months after MI. Revascularization was always performed before the first CMR.CK-MB was measured according to routine clinical praxis: at arrival at the hospital, the following evening or morning, or both. The maximum value of these measurements, peak CK-MB, served as a measure of infarct size. In most patients, measurement of peak CK-MB occurred 12–24 h after the onset of chest pain.CMR imaging was performed with a 1.5-T imager  using body-array coil as a receiver. Breath-hold cine CMR was performed using retrospectively electrocardiographically gated segmented imaging with steady-state free-precession (SSFP). Cine CMR images were acquired in vertical, horizontal long-axis and short-axis planes covering the whole LV. Typical imaging parameters were TR/TE 3.0/1.51 ms, flip angle 52 degrees, 256 × 256 matrix and 240 × 340 mm field of view. Slice thickness was 6 mm. The temporal resolution was 42–47 ms.Ten to fifteen minutes after intravenous injection of a contrast agent , late gadolinium enhancement (LGE) images were acquired in the same views as for cine images, using inversion-recovery turbo fast-low angle shot (FLASH). Typical imaging parameters were TR/TE 8.6/4.3 ms, 256 × 256 matrix, slice thickness 8 mm and interslice gap 20%. Inversion times were optimized to null the signal intensity of normal myocardium (250–300 ms).CMR images were analyzed by two experienced observers blinded to clinical outcome. LV mass, end-diastolic (EDV) and end-systolic (ESV) volumes were evaluated by manually tracing the epicardial border  and endocardial borders (excluding papillary muscles) at end-diastole and -systole for short-axis slices. Volumetric indices were obtained by dividing values by body surface area.LGE images were analyzed using the standard 17-segment model of the LV, leaving out the apex . Scar arRepeatability of LGE analyses was also studied: 384 segments were analyzed for the presence of scar by two independent observers, and results were concordant for 366 (95%) of the segments (kappa = 0.86).Segmental wall motion was visually estimated, blinded to LGE results. The degree of wall motion abnormality in each segment was scored as 0 (normokinesia), 1 (hypokinesia), 2 (akinesia) or 3 (dyskinesia). Wall motion abnormality index (WMAi) of the LV was then calculated as the sum of all segmental scores.Follow-up data of major adverse cardiac events (MACEs) was post-hoc collected until the end of year 2012 based on local and national registries, including university and local hospital records, general practitioners’ records and the mortality information from the public organization Statistics Finland, without direct contact to patients. The survival time was calculated from the first CMR and only one event was tabulated per subject. A MACE was defined as cardiovascular death, aborted sudden death, heart failure hospitalization, or recurrent MI. Aborted sudden death was defined as documented resuscitation from cardiac arrest or appropriate implantable cardioverter defibrillator therapy, i.e. antitachycardia pacing or shock, for ventricular tachycardia or fibrillation.p-value of < 0.05 was considered statistically significant and all tests were 2-sided. Statistical analysis was performed on SPSS 21 statistical package .Continuous variables are presented as median (interquartile range [IQR]) and categorical variables as frequency (%). Comparison between continuous variables was performed with Mann-Whitney U test. CMR variables were compared between recovery and chronic phase studies using related samples Wilcoxon signed rank test. Correlation between variables was calculated using Spearman’s method. Linear regression was performed to identify predictors of chronic phase ejection fraction (EF), reaching sufficient normality of residuals. In survival analysis, Cox regression analysis was performed to identify predictors of MACEs. A From June 2003 to June 2005, altogether 85 patients were enrolled as a part of the ISKE project. Derivation of the CMR study group of 41 patients with their first revascularized MI and complete CMRs data are presented in Fig. Recovery phase CMR study was performed in a median of 22 (9–29) days after hospital admission. Median scar size was 13%  of the LV (Table n = 41), peak CK-MB had a strong correlation with recovery phase scar size   (Table In the whole study group (01) Fig. , moderat1) Table . There wChronic phase CMR study was performed in a median of 10 (8–16) months after hospital admission.r = 0.83, p < 0.001)  . Similar, but weaker correlations with ESV and EF, were found in non-transmural MI group.Peak CK-MB had a strong correlation with chronic scar size (01) Fig.  and chrop < 0.001 for both)  Fig. , and simp = 0.001), LV mass , and WMAi  , meaning that younger age was associated with greater LV mass resorption .Peak CK-MB or recovery scar size were not associated with late remodeling, i.e. change in LV mass or WMAi between CMRs. Considering the association of all continuous baseline and recovery phase CMR variables with late remodeling parameters, the only significant correlation was found between age and LV mass change   and 26% (0–50%). Absolute scar change correlated with recovery phase scar size (01) Fig. , i.e. lap < 0.001) and peak CK-MB .Univariate predictors of chronic EF included peak CK-MB, recovery phase scar size, EDV, ESV, EF, LV mass and WMAi. By multivariate regression (stepwise forward), the significant predictors of chronic phase EF were recovery EF . Multiple vessel disease , EDV late remodelling  and WMAi late remodeling  showed a trend in predicting MACEs.Nine (21%) patients reached a MACE during a median follow-up of 7.7 (4.1–8.5) years. There were one cardiovascular death, one aborted sudden death, two heart failure hospitalizations and five recurrent MIs. In univariate Cox regression analysis of all baseline, recovery and chronic phase CMR indices, and late remodelling variables, the only significant predictor of MACEs was WMAi at chronic phase  at 1 month follow-up CMR [EF and ESV have been established as strong predictors of cardiac outcome after MI , while iw-up CMR . In anotw-up CMR . Earlierw-up CMR . In a stw-up CMR . Our patr ≥ 0.75, p < 0.001), and similarly in non-transmural MIs only , is in accordance with a recent retrospective analysis of patients with previous (> 6 months) MI, in which infarct size correlated with WMAi but the association was more significant in transmural compared to non-transmural MIs [Few prospective data is available on the association between infarct size and chronic local segmental wall motion abnormalities after revascularized acute MI. Our finding that peak CK-MB, recovery scar size, and chronic scar size, all correlate strongly with chronic WMAi . Left ventricular hypertrophy is an important compensatory mechanism after MI, but prolonged progression of cardiac hypertrophy leads to heart failure. It has been presented that post-infarction LV remodeling is more pronounced in elderly patients due to inability to cope with increased myocardial stress [Large infarct size has been previously associated with progressive LV remodeling . In our l stress , explainIn our patients, chronic WMAi was the only predictor of MACEs in the long-term, while multiple vessel disease, late increase in EDV or WMAi, showed tendency toward adverse prognosis. Predictors of long-term outcome in our study may be more related to severity of coronary artery disease than infarct size, since the median scar size was relatively small and most MACEs were recurrent MIs. Earlier, both WMAi and scar size have been shown to be independent predictors of MACEs after large reperfused ST-elevation myocardial infarction  .The number of patients was limited. Infarct related artery patency was not verified after baseline. Our data included patients treated with different revascularization strategies, such as thrombolysis, PCI or CABG, and cannot be generalized to solely one of these groups. Tamaki et al. have shown that acute coronary reperfusion alters kinetics of CK-MB, resulting in greater and earlier CK-MB release in the serum with equivalent infarct volume . In our Peak CK-MB has a strong association with chronic scar size and WMAi after revascularized non-transmural MI. Considerable infarct resorption happens after the first-month recovery phase, independently of infarct size, and LV mass resorption is related to age, being more common in younger patients. At the era of increasing imaging modalities and costs, peak CK-MB provides robust estimation of infarct size, predicting chronic local and global LV function after revascularized MI.