At a median follow-up of ~1.2 years, 42% vs. 11% of the event/no-event group respectively reached a combined endpoint of event occurrence/recurrence or cardiovascular mortality. Of the independent predictors resulting from logistic regression analyses, only LGE (hazard ratio (95% CI): 1.20 (1.11–1.30), p < 0.001), T2-mapping (1.07 (1.01–1.14), p = 0.025) and native T1-mapping (1.08 (1.01–1.15), p = 0.017) independently predicted the combined endpoint. Conclusions: A normal echocardiogram does not preclude myocardial lesions in diffuse SSc patients, which can be detected by CMR especially in symptomatic patients.Background: Acute cardiac events are a significant contributor to mortality in systemic sclerosis (SSc). However, echocardiographic evaluation may be deceptively normal during an acute presentation. We hypothesized that in diffuse SSc patients presenting with acute cardiac events and a normal echocardiogram, cardiovascular magnetic resonance (CMR) would have incremental diagnostic/prognostic value. Methods. 50 consecutive diffuse SSc patients with normal echocardiograms were evaluated using a 1.5T system. A total of 27 (63%) had experienced an acute cardiac event three to tendays before CMR evaluation . Left/right ventricular (LV/RV) volumes and ejection fractions (EF), as well as LV mass, the T2-signal ratio, early/late gadolinium enhancement (EGE/LGE), native/post-contrast T1-mapping, T2-mapping and extracellular volume fraction (ECV) were compared between the event and no-event groups. Results: No differences were identified in LV/RV volumes/EF/mass. In logistic regression analyses, independent predictors of belonging to the event group were EGE (odds ratio (95% CI): 1.55 (1.06–2.26),  Systemic sclerosis (SSc) is an autoimmune disease (ARD) of unknown etiology, characterized by fibrosis of the skin and internal organs, typically microvascular vasculopathy and systemic inflammation with presence of circulating autoantibodies and pro-inflammatory cytokines ,2. DespiSSc has an estimated 10-year survival rate of 66–82% ,5,6,7, wThe first non-invasive imaging modality usually employed for the cardiovascular assessment of SSc patients is echocardiography, which has the advantage of being inexpensive, fast and widely available . HoweverCardiovascular magnetic resonance (CMR) is a non-invasive imaging modality that provides detailed information regarding various cardiovascular pathologies without employing ionizing radiation. CMR can make up for many of the limitations of echocardiography and is considered the reference standard for quantitative evaluation of ventricular volumes, mass, function and cardiac tissue characterization . We hypoRecent-onset supraventricular arrhythmia;Recent-onset angina pectoris (AP);Sustained or non-sustained ventricular tachycardia (sVT/nsVT);Other ventricular rhythm disturbances (VRDs) or;Recent onset shortness of breath (SOB).We prospectively recruited 50 consecutive diffuse SSc (dSSc) patients diagnosed based on the 1980 American College of Rheumatology criteria . Of thesThe remaining 19 (38%) patients experienced no such symptoms/events during that time (the ”no-event group”). All patients had undergone standardized echocardiographic evaluation  within oAll participants were followed-up for a median duration of 1.23 (interquartile range 0.71–2.00) years after undergoing CMR evaluation and cardiovascular mortality as well as recurrence or first occurrence of adverse events as defined previously was determined. A combined endpoint was generated, which incorporated the aforementioned adverse outcomes as events.Exclusion criteria included contraindications to CMR, renal/liver impairment, pregnancy and hypersensitivity to gadolinium-based paramagnetic contrast agent. This study was approved by the Onassis Cardiac Surgery Center medical ethics committee  and all participants provided written informed consent before inclusion.Plasma levels of high-sensitivity cardiac troponin-T (hs-cTnT) were determined using sandwich enzyme-linked immunosorbent assays. CMR was performed using a 1.5-T scanner . The CMR protocol included standard steady-state free-precession cine CMR for the determination of left/right ventricular (LV/RV) end-systolic/end-diastolic volumes (ESV/EDV) and ejection fractions (EF), black-blood T2-weighted short tau inversion recovery images, T1-weighted spin-echo early gadolinium enhancement (EGE) CMR, and phase-sensitive inversion recovery late gadolinium enhancement (LGE) CMR as described previously . A dose T1-mapping was performed using a modified Look–Locker inversion recovery (MOLLI) sequence with a 3(3)5 scheme on 3 representative short-axis positions before and 15 min after contrast-medium administration. ECV was calculated from native and post contrast T1 mapping using the previously described equation . T2-mappGlobal myocardial inflammation was assessed on T2-weighted images by calculating the T2 signal intensity ratio as the ratio of the signal intensity of myocardium divided by the signal intensity of skeletal muscle . Global The accuracy of the T1 and T2 mapping methods was evaluated by a relaxometry study in a Eurospin Gel-Phantom : the comparison of T1 values obtained by the MOLLI 3(3)5 and a reference scan has been previously reported . T2 valun (%). Normally distributed, continuous not-normally distributed and binary/categorical variables were compared between the event and no-event groups using independent-sample t-tests, Mann-Whitney U tests and chi-square tests respectively. Logistic regression analysis was used to create univariable and multivariable prediction models for discriminating between the event and no-event groups based on a backwards stepwise approach. To investigate the utility of CMR indices as predictors of the combined endpoint as defined above, univariable and multivariable Cox regression analysis was employed. Multivariable corrections for both logistic and proportional hazard models were for age, sex and disease duration (time since diagnosis at inclusion). Proportionality of hazards was determined using standardised Schoenfeld residuals. Additionally, Kaplan–Meier curves combined with the log-rank test were used for presenting the prognostic value of CMR variables visually. For the dichotomisation of predictors, we used locally established cut-off points for normal values at our center. A Benjamini–Hochberg correction for multiple comparisons was applied to account for the various logistic and Cox regression analyses that were carried out.Statistical analyses were carried out using Stata v15.0SE. Normality of continuous variables was determined by visual assessment of histograms or Q-Q plots when necessary. Normally distributed variables are presented as mean (SD), not-normally distributed continuous variables are presented as median (interquartile range-IQR) and binary/categorical variables are presented as p = 0.10), age (median (IQR): 55.0 (40.0–61.0) vs. 56.0 (47.0–64.0), p = 0.51) or median time since diagnosis  vs. 3.0 (1.0–5.0), p = 0.44). Of the 31 patients in the event group, 17 (54.8%) experienced AP, seven (22.6%) experienced SOB, six (19.4%) experienced sVT/nsVT, three (9.7%) experienced other VRDs, and one (3.1%) experienced paroxysmal supraventricular tachycardia.Baseline characteristics and CMR variables are compared between the event and no-event groups in p = 0.065) (p = 0.009). Patients in the event group had noticeably higher LGE values compared to the no-event group (median (IQR): 6.0 (5.0–12.0) vs. 3.0 (0.0–5.0), p < 0.001) with similar differences being observed in T2-mapping (median (IQR): 63.0 (55.0–65.0) vs. 55.0 (49.00–58.0), p < 0.001) and native T1-mapping (median (IQR): 1135.0 (1117.0–1202.0) vs. 1065.0 (1018.0–1126.0), p < 0.001). Smaller but significant differences between groups were identified in T2 signal ratio, EGE, post-contrast T1-mapping and ECV (p = 0.004 and 1.19 (1.03–1.38), p = 0.019 respectively).The two groups had largely similar LV and RV systolic function as well as systolic and diastolic volumes with only a trend towards significantly lower median RVEF in the event group (60.0 (50.0–65.0) vs. 65.0 (59.0–68.0), = 0.065) . No pati and ECV . Resultsp = 0.071 and 0.082 respectively). However, these became significant predictors when corrected for age, sex and disease duration (p = 0.022), this was not the case for ECV (p = 0.144).During a median follow-up period of 1.23 (0.71–2.00) years, 13 (41.9%) of the 31 patients in the event group reached the combined endpoint by experiencing either ventricular tachycardia (5/13), sudden cardiac death (2/13), heart failure (7/13) or pulmonary hypertension (2/13), while in the no-event group only two (10.5%) of the 19 patients experienced adverse events, those being atrial arrhythmias. Univariable Cox regression analyses demonstrated that biventricular volumes and ejection fractions, LV mass, LGE and ECV at baseline were all significant predictors of the combined endpoint . These rduration . All resThis study examined a cohort of SSc patients with and without an acute presentation of cardiovascular symptoms/adverse events and a normal echocardiographic evaluation according to currently employed SSc-specific protocols. At presentation, only 29% of patients in the event group had elevated hs-cTnT levels and cardiac functional indices did not differ significantly between the event and no-event groups. At a median follow-up duration of ~1.2 years, approximately 40% of the event group vs. 10% of the no-event group reached a combined endpoint of adverse event occurrence/recurrence or cardiovascular mortality. LGE, T2 mapping and native T1 mapping were the only variables that both independently discriminated between the event and no-event groups at baseline and independently predicted prognosis at follow-up.Few studies have previously investigated SSc patients presenting with acute-onset cardiac symptoms/events and to our knowledge none have thus far incorporated CMR evaluation specifically in patients with cardiac events and normal echocardiograms. One recent study reported that myocardial fibrosis, elevated high-sensitivity c-reactive protein, and higher maximum modified Rodnan skin scores were independent predictors of cardiovascular outcomes in a cohort of 62 SSc patients presenting with cardiac symptoms . HoweverTo our knowledge, our study is the first in the literature providing a detailed comparison of CMR findings in dSSc patients with and without acute cardiac events. We demonstrate that ventricular function indices did not differ significantly between patients with and without events at baseline although they did have prognostic value at follow-up. In contrast, tissue characterization indices in the form of LGE, T2 mapping and native T1 mapping were the only variables that could both discriminate between the event and no-event groups at baseline and independently predict prognosis at follow-up. Considering that only 29% of patients with events had elevated hs-cTnT and all had normal echocardiograms, our findings suggest that the latter indices are important for the identification of high-risk SSc patients that may necessitate prompt initiation of treatment but may otherwise be diagnostically ambiguous. Additionally, our findings suggest that hs-cTnT testing may underestimate the presence of cardiac pathology in SSc. In spite of the diagnostic/ prognostic value of hs-cTnT measurements in coronary artery disease, the broad range of conditions associated with elevated hs-cTnT values may further confound prompt and accurate diagnosis and generate additional clinical dilemmas in SSc patients .An additional innovation of our study is the evaluation of both classic and novel CMR indices. According to the modified Lake Louise criteria for inflammatory cardiomyopathies , a diagnp-value. Similarly, ECV was marginally not-significant as a discriminatory parameter for distinguishing between the two groups at baseline. The same is the case for RVEF, which had a trend towards significant difference between groups at baseline.This study was a single-center investigation that included a moderate amount of SSc patients considering the prevalence of the disease. Although clear CMR variable cut-off points for the identification of at-risk patients would be of greater relevance for the clinician managing SSc patients, their determination was ultimately constrained due to lack of statistical power and a broad representative sample in this cohort and thus locally developed cut-off points were used. The accurate identification of such cut-off points in a larger, more diverse and representative population should be the subject of future investigations on SSc patients. Furthermore, the role of ECV in this study might have been underestimated due to a lack of statistical power. This was especially obvious for the cut-off of 28%, which perfectly identified all events at follow-up, despite ultimately not yielding a significant This investigation demonstrates for the first time that in dSSc patients with suspected cardiac involvement and a normal echocardiographic evaluation according to current standards, only tissue characterization indices  but not cardiac functional indices offer incremental diagnostic value in the identification of high risk patients at baseline investigation, in addition to independently predicting adverse outcomes at follow-up. Thus, CMR may be considered in cases where echocardiographic results are equivocal but the clinical picture warrants a higher index of suspicion, in order to identify high-risk patients early on and to institute prompt changes in the therapeutic management of SSc-related cardiac involvement.


Cardiac positron emission tomography (PET) and positron emission tomography/computed tomography (PET/CT) are encouraging precise non-invasive imaging modalities that allow imaging of the cellular function of the heart, while other non-invasive cardiovascular imaging modalities are considered to be techniques for imaging the anatomy, morphology, structure, function and tissue characteristics. The role of cardiac PET has been growing rapidly and providing high diagnostic accuracy of coronary artery disease (CAD). Clinical cardiology has established PET as a criterion for the assessment of myocardial viability and is recommended for the proper management of reduced left ventricle (LV) function and ischemic cardiomyopathy. Hybrid PET/CT imaging has enabled simultaneous integration of the coronary anatomy with myocardial perfusion and metabolism and has improved characterization of dysfunctional areas in chronic CAD. Also, the availability of quantitative myocardial blood flow (MBF) evaluation with various PET perfusion tracers provides additional prognostic information and enhances the diagnostic performance of nuclear imaging. Despite considerable advances in prevention and management of atherosclerotic heart disease and its devastating consequences, the morbidity and mortality of coronary artery disease (CAD)  in develSince the first PET scanners were introduced into a daily routine more than thirty years ago , they haPET is a non-invasive method that produces images of a radionuclide-labelled tracer distribution in the body . PET imaThe dominant applications of radiopharmaceuticals in clinical cardiology can be classified into two main categories: studies of myocardial metabolism (viability) and the assessment of regional myocardial blood flow (MBF). 18F-FDG (fludeoxyglucose F) is established as the myocardial viability tracer [18F nuclide has a rather long physical half-life  [13N-labelled ammonia (13NH3) [82Rubidium (82Rb) [18F-labelled PET perfusion tracer 18F-BMS747158-02 (Flurpiridaz F18) that has been recently validated in animals [Assessment of myocardial perfusion is one of the major PET clinical applications. For the evaluation of MBF mainly three tracers are used: 15O-H2O) ,18 13N-l (13NH3) ,20 and tm (82Rb) . In addi animals  and huma animals ,24.15O-H2O) has been widely used for the experimental issues [15O-water is metabolically inert and freely diffusible perfusion tracer  [82Rb can be produced by the 82Sr/82Rb generator that generally needs replacement once a month  , however a month . Neverth and MBF , Table 2erfusion ,38,39,4013N-NH3 is taken up by myocardium by passive free diffusion across cell membranes as ammonia (NH3), where it equilibrates with its charged form ammonium (NH4)  [13N-NH3 has linear myocardial uptake relationship with MBF, except the very high MBF rates   and getslutamine . 13N-amm.96 min)   is an analogue of the insecticide pyridaben with a good uptake in the heart because of high affinity for complex I of the mitochondrial electron transport chain [18F-flurpiridaz for perfusion imaging. The half-life of this tracer is relatively long (109 min), therefore, the on-site cyclotron is not necessary. In pre-clinical and clinical trials, 18F-flurpiridaz has exhibited properties of a perfect PET perfusion radiotracer: a high myocardial extraction fraction , 4-fluorophenyl triphenyl phosphonium ion (18F-FTPP) and fluorodihydrorotenone (18F-FDHR) [18F-flurpiridaz has reached advanced clinical evaluation with promising results [rt chain . The avafraction , Table 28F-FDHR) ,47,48; h results ,50,51.There are two ways that dysfunctional but viable myocardium can exist . Myocard201TI SPECT, nitrate augmented MPI, FDG PET and delayed enhancement MRI [13N-NH3 PET, 99mTc-MIBI, 99mTc-Tetrofosmin or 201TI SPECT), it is possible to distinguish viable myocardium from a fibrotic scar . Regions with reduced perfusion and FDG uptake are considered as irreversibly injured and without viability (“flow-metabolism match”), whereas reduced perfusion regions with relatively preserved FDG uptake (“flow-metabolism mismatch”) are considered to be viable , three prospective randomized trials, the PET and recovery following revascularization (PARR-2) trial , the heaAfter decades of experience with scintigraphic assessment of myocardial perfusion, myocardial perfusion imaging (MPI) has been validated for the prognosis of cardiac disease and the technique embedded in national and international guidelines. PET has been widely used for myocardial viability assessment but now it is being increasingly applied to detect flow-limiting CAD.Clinical manifestation of CAD is symptoms caused by myocardial ischemia. Nevertheless, significant part of CAD includes asymptomatic subclinical stages, such as functional vascular alteration , which PET allows to detect in patients at risk of developing CAD . PET leaPET appears to be a superior myocardial perfusion technique compared to the widely used SPECT, as its spatial resolution is higher and there are methods of attenuation correction available. The average sensitivity and specificity, comparing PET with invasive coronary angiography, was 93% and 92%, respectively . PET hasSymptomatic CAD at early stages and/or asymptomatic CAD can be evaluated with PET assessing an absolute MBF in millilitres per gram per minute . For corIdentification of subclinical CAD. SPECT myocardial perfusion imaging is a traditional evaluation of symptomatic patients with known or suspected CAD ,72,73. HEnhanced characterization of CAD burden and identification of “balanced” decrease of MBF in all vascular territories. Patients with multivessel CAD may show normal SPECT results due to an absolute decrease of radiotracer uptake in the left ventricle (LV) myocardium. Previous studies have disclosed that SPECT perfusion imaging can detect approximately only 10% of patients with severe three-vessel CAD or significant left main coronary artery (LMA) stenosis (≥50%) ,78. If tPET assessment of MBF allows :Identifi13N-NH3 PET) is a strong and independent prognostic factor of cardiac mortality  [The coronary flow reserve (CFR) is a ratio of near-maximal coronary vasodilatation MBF to basal MBF and is known as an indirect parameter to evaluate the function of the coronary circulation . Additionally, endothelium-dependent vasoreactivity  also can be evaluated by PET, providing an estimate of endothelial integrity, which can be reduced in patients with coronary risk factors , such as of CFR) . This anOver the past several years, there has been a substantial development in combination of PET with computed tomography (CT) or MRI. Such fusion of different imaging modalities can supply complementary data in a single examination  and allo13N-ammonia was used to assess different variables of myocardial blood flow: flow reserve and flow gradients, which were related with computed tomography coronary angiography (CTCA)) [13N-ammonia PET) [Several studies have investigated PET/CT benefit in suspected CAD patients ,88. Vale (CTCA)) . Dey et PET/MRI was originally developed for neuroimaging ,90 and, Cardiac PET and PET/CT are encouraging and precise non-invasive imaging modalities that allow functional imaging of the heart, while other non-invasive cardiovascular imaging modalities are considered to be techniques of anatomical imaging. The role of cardiac PET is growing rapidly as it allows diagnosing CAD with high accuracy. In clinical cardiology, PET is established as the best available technique for evaluation of myocardial viability and is recommended for the proper management of reduced LV function and ischemic cardiomyopathy.Hybrid PET/CT imaging has enabled simultaneous integration of coronary anatomy and function with myocardial perfusion and metabolism in the same examination and has improved characterization of the dysfunctional areas in chronic CAD. Also, the availability of quantitative MBF evaluation with various PET perfusion tracers provides additional prognostic information and increases diagnostic performance in CAD management. However, there are no ideal imaging modalities yet that could offer an accurate picture of complex pathophysiological substrate of LV dysfunction and the potential for its reversibility. Revascularization decisions in cases of ischemic LV dysfunction continue to be individual and complicated, demanding integration of different clinical and anatomic imaging modalities. This leads to a conclusion that the future cardiac PET research lies in the further development and understanding of molecular imaging, as well as in the investigation and establishment of new perfusion tracers suitable for PET imaging in diagnostic centres without an on-site cyclotron facility.


The technique possesses a powerful combination between high imaging depth, fast volumetric imaging speed, functional and molecular imaging capacities not available with other imaging modalities currently used in cardiac research.The Langendorff-perfused heart technique has become the model of choice for multiparametric optical mapping of cardiac function and electrophysiology. However, photon scattering in tissues represents a significant drawback of the optical imaging approach, fundamentally limiting its mapping capacity to the heart surface. This work presents the first implementation of the optoacoustic approach for 4D imaging of the entire beating isolated mouse heart. The method combines optical excitation and acoustic detection to simultaneously render rich optical contrast and high spatio-temporal resolution at centimeter-scale depths. We demonstrate volumetric imaging of deeply located cardiac features, including the interventricular septum,  The method involves quick cannulation of the excised heart and its retrograde perfusion via the aorta. In this way, cell viability and heart function are preserved by delivering nutrient-rich buffer solution to the entire heart through the coronary arteries, vascular bed, and veins. With careful maintenance of the pH and temperature of the perfusate, it is possible to maintain the isolated heart beating for several hours. With its relative simplicity as compared to in vivo setups, the Langendorff-perfused heart has become essential in many biomedical studies, providing access to physiologists, biochemists, and clinicians to better study heart biology. Moreover, continuous perfusion makes the method optimal for pharmacological testing of drug delivery further enabling studying the efficacy of genetic alterations and other therapeutic interventions combating ischemia injury5.The technique of Langendorff-perfused heart isolation has led to major advancements in assessing cardiac mechanical and bioelectrical parameters by decoupling the influence of multiple factors complicating in vivo setting. For instance, voltage- and calcium- sensitive dyes have been used to map transmembrane potential and intracellular calcium transients9, popularizing optical imaging as a standard tool for the study of cardiac and epicardial electrophysiology. Advancements in optical imaging technology have enabled the acquisition of full planar views of the heart surface at a frame rate of several kilohertz7. In this way, rotor dynamics found in spiral propagation of potential across the heart has been studied to shed light onto atrial and ventricular arrhythmia and sudden cardiac death10. Most recently, the method has gained major significance due to the ability of simultaneous electro-mechanical optical and ultrasound mapping of membrane potential, intracellular calcium, and mechanical deformation, which provided the first experimental evidence for the existence of co-localized electrical and mechanical rotors during ventricular fibrillation11. However, while valuable information can be gathered from the epicardium, optical visualization of endocardium and other internal structures is prohibited due to intense light scattering in tissues. Chemical tissue clearing can facilitate high-resolution 3D observations through the entire heart with selective-plane microscopy techniques13, which is however not compatible with cell viability. Other modalities, such as magnetic resonance imaging (MRI) or computed tomography (CT), can alternatively be used to image the heart16. However, they mainly reveal anatomical tissue contrast while dynamic imaging in three dimensions further relies on gating approaches, impeding real-time imaging of perfusion and other rapid biological processes.The isolated heart preparation ensures easy access by optical imaging that offers imaging speed, sensitivity, high spatial resolution, as well as a great variety of targeted moieties for the assessment of biophysical and biochemical parameters not available in the in vivo imaging scenario18.In this work, we devised a volumetric optoacoustic tomography (OAT) system to enable real-time functional imaging of the entire isolated Langendorff-perfused heart. The method synergistically combines optical excitation and acoustic detection to simultaneously render rich optical contrast and high resolution at centimeter-scale depths while further maintaining fast 3D imaging capacity crucial for beat-by-beat dynamic visualization of whole beating heart. The isolated heart allows for direct imaging of the heart without the presence of skin, ribs, and strongly absorbing blood, thus significantly improving the depth and image quality with respect to the x, y, and z views are displayed as maximum intensity projections (MIP). The outline of the heart along its long and short axis as well as the apex can be clearly identified. The black suture thread used during cannulation is a strong absorbing source that generates a bright spot in the images. The intensity distribution on the MIPs mainly corresponds to the light fluence distribution on the heart surface. The suspended heart was slightly tilted with the strongest OA signal on its lower right side corresponding to the excitation light spot. Yet, the excitation light was diffused across the heart volume and hence internal structures are clearly distinguished in the cross-sectional planes displayed in Fig. x, y, and z planes respectively. Most of the blood was washed out of the heart. Thus, OA contrast was provided by myoglobin and the small amount of hemoglobin remaining in the heart muscle capillaries.The volumetric OAT setup designed to optimally support imaging of entire isolated beating murine hearts is shown in Fig. trabeculae carneae, which appear as ridge-like structures of the ventricular wall protruding into the lumen of the ventricle. Figure x, y, and z dimensions.Key structures can be resolved in the cross-sectional images, and the heart anatomy was characterized and marked in the side-by-side sketches, including the left (LA) and the right atria (RA), left (LV) and right ventricles (RV), and the interventricular septum. Other internal features that can be recognized include the papillary muscles and Information on the heart beat dynamics could be further extracted from the full 4D OA dataset by analyzing temporal profiles of the reconstructed images shown in Fig. With the retrograde perfusion, where fluid was constantly injected into the aorta, the aortic pressure was maintained, permanently closing the aortic valve. When scrutinizing the time-lapse 4D dataset, repeated motion due to opening and closing egged to the heart-beat cycles could lead to identification of the three other valves. Representative temporal profiles captured at various anatomical locations are displayed in Fig. T to T + 0.03 s) correspond to that of the temporal snapshots shown Fig. y- direction) of the pulmonary, mitral, and tricuspid valves, respectively. The corresponding positions in the whole-heart view are indicated in Fig. chordae tendineae and papillary muscles were also resolved in the mitral valve with Supplementary Movie Furthermore, the high temporal resolution of the OA imaging system was sufficient to resolve the transient states, exemplified by the dotted lines in Fig. 8, rats20, rabbits9, and larger animals21. Herein, the mouse model was adopted taking into consideration the abundance of the available disease models and the effective FOV of the OAT imaging system employed. The entire volume of the mouse heart could be captured in real time while the spatial resolution was adequate to resolve cardiac walls, valves, papillary muscles and even fine structured chordae tendineae.This work presents the first implementation of an OA approach for 4D whole-organ imaging of a beating Langendorff-perfused heart. To this end, a wide range of mammalian species have been used in Langendorff-perfused heart preparations to study cardiac function in health and disease, including micein vivo18. However, the effective penetration depth was limited to about 2–3 mm, mainly compromised by the vast amount of blood in the heart chambers causing strong light attenuation and amplitude decay of the OA signals versus depth. In addition, the lungs and the thorax are responsible for introducing severe distortions along the propagation path of the generated OA responses, subsequently leading to artifacts in the images obtained with standard acoustic inversion methods assuming a homogeneous acoustic medium23. Herein, it was shown that the basic imaging depth advantage of optoacoustics is further augmented by the lack of skin, ribs, lungs, and the whole blood, enabling the entire organ to be imaged accurately. Contrast was mainly provided by the residual hemoglobin in the myocardium, facilitating the visualization of deep tissue and exposing internal anatomy for investigation. For the first time to our knowledge, the interventricular septum could be observed with optical contrast in a Langendorff-perfused heart preparation.Fundamentally, acoustic waves undergo several orders of magnitude less scattering as compared to light in biological tissues. Thus, the OA approach enabled herein resolving deep-tissue optical contrast with high spatial resolution, not attainable with fluorescence-based imaging methods. Volumetric OA tomography was recently employed for non-invasive imaging of the murine heart 24. However, the approach is equally suitable for real-time visualization of targeted or genetically-encoded proteins, such as selectin-targeted probes25 or calcium-sensitive proteins that were recently successfully employed for OA imaging of neuronal activity26. Since calcium and voltage indicators similarly play a significant role in the understanding of myocardial physiology27, the newly introduced OA cardiac imaging approach may enable monitoring changes in these sensors in the entire heart. Calcium-dependent ionic currents are known to be implicated in the mechanisms of cardiac arrhythmias, cardiac myopathy, myocardial infarction and other conditions28. Four dimensional imaging of the entire heart with optical contrast can potentially provide a powerful tool to investigate the role of calcium in these conditions.The proof of concept experiments performed in this work relied on the excellent endogenous optical absorption contrast of the heart muscle, mainly provided by remaining hemoglobin molecules and other intrinsic tissue chromophoresAnother key characteristic of the presented method is its excellent temporal resolution. With the presently demonstrated 100 Hz volumetric acquisition rate determined by the 100 Hz PRF of the laser, real-time imaging on a beat-by-beat basis is possible without the need for cardiac gating. While such a high volumetric imaging speed is beyond reach for other bio-imaging modalities used in cardiac research, much faster 3D frame rates can be readily achieved as it only takes about 20 µs to record all the OA tomographic information from the entire mouse heart generated with a single laser shot. The powerful combination between high imaging depth, fast imaging speed, functional and molecular imaging capacities puts forth unprecedented new capabilities for cardiac research.In conclusion, OA imaging of the entire Langendorff-perfused heart with high spatio-temporal resolution has been demonstrated. This represents an important milestone in the development of OA technology for cardiac imaging applications, which is poised to provide new insights into heart function in health and disease.All experiments were performed in full compliance with European laws on the protection of animals used for scientific purpose and the institutional guidelines of the Helmholtz Center Munich, and with approval from the Government District of Upper Bavaria.8. Tyrode’s solution8 (2 L) was pumped through via coronary artery as well as around the heart (superfusion). The solution was oxygenated by carbogen gas (95%/5% O2 to CO2 ratio) and its pH was maintained at 7.35 ± 0.05. Separate water baths  were used to maintain the Tyrode’s solution at 37 °C for the perfusion and superfusion circuits. CD1 mice were overdosed with ketamine (150 mg/kg) and xylazine (10 mg/kg) to harvest their hearts. Once the animal exhibits no pain reflex, established by pinching the paw, the thoracic cavity was opened to excise the heart. A 21-gauge cannula filled with perfusion buffer was inserted in the ascending aorta and fixed using 4-0 black-braided silk sutures .We adopted a well-established Langendorff method of retrograde perfusion in murine heartsThe cannulated heart was immediately moved into the imaging system containing sample chamber, illumination fiber bundle and a spherical-matrix detection array. The spherical array probe was attached to the sample chamber pointing upwards and tightly sealed at the edges. The volume enclosed in the chamber was superfused with the Tyrode’s solution, which further facilitated acoustic coupling to the detection array. The heart was attached to the output of a heating coil providing the perfusion fluid and suspended into the sample chamber from above. An automatic pump  controlled the perfusion flow rate at 5 mL/min. Superfusion of the heart was maintained at a constant flow of 70 mL/min by a second pump . The perfusion was initiated at least 10 minutes before the measurement to wash out remaining blood and allow the heart to stabilize.2. The broadband optoacoustic (OA) responses generated in the imaged sample were captured by the 512 individual piezocomposite elements  distributed on a concave spherical surface of the custom-made matrix array covering an angle of 140° (1.3 π solid angle)18. The system provides an approximately isotropic resolution of 150 μm around the center of the sphere. Following each laser pulse, the OA signals captured by all the channels were simultaneously sampled by a custom-made multi-channel digitizer . 1000 consecutive volumetric image frames were recorded at a rate of 100 Hz, corresponding to a total acquisition duration of 10 s.Volumetric imaging of the beating heart was performed by illuminating it with ~10 ns duration pulses of light at 800 nm wavelength generated by an optical parametric oscillator (OPO) laser  running at pulse repetition frequency (PRF) of 100 Hz. A custom-made fiber bundle  was used to guide the laser output through the center of the spherical array probe, thus illuminating the sample from the bottom with a fluence of ~20 mJ/cm29. Subsequently, band-pass filtering with a second-order Butterworth filter having cut-off frequencies of 0.1 and 7 MHz was performed to remove low frequency bias and high frequency noise outside the effective transducer bandwidth. 3D OA images on a grid of 150 × 150 × 150 voxels (15 × 15 × 15 mm3), covering the entire mouse heart, were rendered for each laser pulse using a GPU-accelerated back projection algorithm30. The reconstruction time for each 3D frame was approximately 10 ms.The acquired image sequence was processed off-line. No signal averaging was performed. Prior to image reconstruction, the acquired temporal signals were first deconvolved by the electrical impulse response of the detection arraySupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplemental Materials


What is the rate of cardiovascular adverse events among patients with melanoma treated with BRAF and MEK inhibitors compared with patients treated with BRAF inhibitor monotherapy?In this systematic review and meta-analysis of 5 randomized clinical trials including 2317 patients, treatment with BRAF and MEK inhibitors was associated with a higher risk of pulmonary embolism, decrease in left ventricular ejection fraction, and arterial hypertension compared with treatment with BRAF inhibitor monotherapy. The risks of myocardial infarction, atrial fibrillation, and QTc prolongation were similar between groups.These findings demonstrate an association of cardiovascular adverse events with BRAF and MEK inhibitor therapy, which may guide clinical cardio-oncological management. This systematic review and meta-analysis determines the association of BRAF and MEK inhibitor treatment with cardiovascular adverse events in patients with melanoma compared with BRAF inhibitor monotherapy. Cardiovascular adverse events (CVAEs) after treatment with BRAF and MEK inhibitors in patients with melanoma remain incompletely characterized.To determine the association of BRAF and MEK inhibitor treatment with CVAEs in patients with melanoma compared with BRAF inhibitor monotherapy.vemurafenib, dabrafenib, encorafenib, trametinib, binimetinib, and cobinimetinib from database inception through November 30, 2018.PubMed, Cochrane, and Web of Science were systematically searched for keywords Randomized clinical trials reporting on CVAEs in patients with melanoma being treated with BRAF and MEK inhibitors compared with patients with melanoma being treated with BRAF inhibitor monotherapy were selected.Data assessment followed the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. Pooled relative risks (RRs) and 95% CIs were determined using random-effects and fixed-effects analyses. Subgroup analyses were conducted to assess study-level characteristics associated with CVAEs.The selected end points were pulmonary embolism, a decrease in left ventricular ejection fraction, arterial hypertension, myocardial infarction, atrial fibrillation, and QTc interval prolongation. All-grade and high-grade (≥3) CVAEs were recorded.P = .02), a decrease in left ventricular ejection fraction , and arterial hypertension  compared with BRAF inhibitor monotherapy. The RRs for myocardial infarction, atrial fibrillation, and QTc prolongation were similar between the groups. These results were consistent when assessing high-grade CVAEs , but RRs for high-grade pulmonary embolism were similar between groups. A higher risk of a decrease in left ventricular ejection fraction was associated with patients with a mean age younger than 55 years , and the associated risk of pulmonary embolism was higher for patients with a mean follow-up time longer than 15 months .Overall, 5 randomized clinical trials including 2317 patients with melanoma were selected. Treatment with BRAF and MEK inhibitors was associated with an increased risk of pulmonary embolism (RR, 4.36; 95% CI, 1.23-15.44; Therapy with BRAF and MEK inhibitors was associated with a higher risk of CVAEs compared with BRAF inhibitor monotherapy. The findings may help to balance between beneficial melanoma treatment and cardiovascular morbidity and mortality. Subsequent research showed that treatment with BRAF inhibitors alone causes resistance through a paradoxical signaling cascade mediated by MEK, leading to the development of MEK inhibitor therapies.5 The combination of BRAF and MEK inhibitor therapy has emerged as an optimal treatment of metastatic BRAF-mutated melanoma, with improved survival rates compared with monotherapy.10 A 2018 study11 found that the first BRAF and MEK inhibitor combination showed significant improvement in relapse-free survival in treating adjuvant stage III melanoma, which led to global approval. To date, 3 BRAF inhibitors  and 3 MEK inhibitors  have received US Food and Drug Administration and European Medicines Agency approval. Common combination therapies are dabrafenib and trametinib,9 vemurafenib and cobimetinib,15 and encorafenib and binimetinib.17The incidence of melanoma has been increasing, and it now accounts for 55 500 cancer deaths annually worldwide.18Cardiovascular adverse events (CVAEs) associated with BRAF and MEK inhibitors have been reported in several studies, particularly regarding a reduction in left ventricular ejection fraction (LVEF), arterial hypertension, and prolongation of QTc interval. However, data suggest that the spectrum of CVAEs is broader and could have tremendous effects on maintaining these patients’ antitumoral treatment.22 Clinical data document a reduction in LVEF under BRAF and MEK inhibitors in 5% to 11% of all patients, while arterial hypertension occurred in 10% to 15% of patients.17 Although QTc interval prolongation is denoted as a potential CVAE, several studies reported no association of BRAF and MEK inhibitor therapy with QTc intervals.17 Therefore, questions remain about whether this is a salient CVAE.The inhibition of BRAF and MEK negatively interferes with cardiovascular MAPK signaling. This generates oxidative stress and apoptosis of myocytes and impairs angiogenesis, leading to significant cardiovascular diseases.The nature and incidence of CVAEs associated with BRAF and MEK inhibitor therapy are incompletely described. However, cardiovascular complications may affect a patient’s quality of life or may require temporary or permanent cancer therapy termination. With this systematic review and meta-analysis, we set out to clarify the type, incidence, and relative risk of CVAEs in patients with melanoma who are being treated with a combination of BRAF and MEK inhibitors compared with patients receiving BRAF inhibitor monotherapy.PRISMA) reporting guideline23 and followed the Cochrane Handbook for Systematic Reviews of Interventions recommendations.24 The study was registered with PROSPERO (CRD42018108198). A systematic search was conducted through PubMed, Cochrane, and Web of Science databases, the major cardiology websites , and abstracts or presentations from annual meetings of the major cardiovascular and cancer societies to identify relevant studies published from the inception of the databases to November 30, 2018, using the search terms vemurafenib, dabrafenib, encorafenib, trametinib, binimetinib, and cobinimetinib. We made our search specific and sensitive using Medical Subject Heading terms and free text and considered studies published in English only. The search strategy through PubMed is depicted in eTable 1 in the This meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses  in which adult participants received the available combinations of BRAF inhibitors and MEK inhibitors  and were randomly assigned to a treatment or a control group. The meta-analysis excluded abstracts, reviews, animal and in vitro studies, meta-analyses, case reports, single-arm BRAF and MEK inhibitor treatment studies, monotherapy with BRAF inhibitor studies, studies with MEK inhibitor treatment with other therapies for melanoma, nonrandomized clinical trials, studies that did not report on CVAEs, and special population studies .After removing duplicates, 3 of us  independently reviewed the abstracts. Any discrepancies in results between the 3 investigators were solved by discussion with the other investigators. When the inclusion criteria appeared to be met, the full-text publication was reviewed by the 3 authors mentioned above. At the end of the review process, the full texts of the studies considered eligible were reviewed by all investigators.24: (1) random sequence generation , (2) allocation concealment , (3) blinding of participants and personnel , (4) blinding of outcome assessment , (5) incomplete outcome data, (6) selective reporting , and (7) other bias . Authors resolved disagreement by consensus, and a fourth author (M.T.) was consulted to resolve disagreement.Three of us  independently performed data extraction using a standard data extraction form that contained the following fields: (1) publication details , (2) study design, (3) characteristics of study population , (4) therapy dosage, (5) mean follow-up, and (6) study end points. Study-level characteristics were abstracted. The trial quality was assessed by 3 of us  for each study separately against the following criteria according to the Cochrane Risk-of-Bias Tool25 All-grade and high-grade  treatment-emergent CVAEs were abstracted. The selected end points were as follows: (1) pulmonary embolism, (2) a decrease in LVEF, (3) arterial hypertension, (4) myocardial infarction, (5) atrial fibrillation, and (6) QTc interval prolongation. The definitions of study end points are detailed in eTable 2 in the The end points were defined according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.26 Statistical significance was set at P < .05, and all tests were 2-tailed. A 2018 study17 had 3 arms: an encorafenib and binimentinib arm, an encorafenib arm, and a vemurafenib arm. We compared the patients with adverse events from the encorafenib and binimetinib arm with those from the encorafenib arm and the patients with adverse events from the encorafenib and binimentinib arm with those from the vemurafenib arm. For the analysis, we used both random-effects and fixed-effects models. A random-effects model was preferred owing to the assumption that different studies are estimating different yet related intervention effects. In the presence of heterogeneity, the use of the random-effects method will result in wider CIs for the average intervention and corresponding claims of statistical significance will be more conservative.24 We considered both the presence of a P value at 10% of the level of significance (P < .01) derived from Q analysis and a value of inconsistency (I2) of more than 40% to denote heterogeneity between the selected studies. A value of I2 less than 40% denoted that heterogeneity might not be important, I2 from 40% to 60% may have represented moderate heterogeneity, I2 from 50% to 90% may have represented substantial heterogeneity, and I2 from 75% to 100% represented considerable heterogeneity.24 The funnel plot test could not be used to assess publication bias because our analysis included fewer than 10 studies.24 The analyses were conducted using Review Manager version 5.3 (The Cochrane Collaboration) and the Comprehensive Meta-Analysis .The meta-analysis was conducted on eligible studies by dividing the patients into the following 2 groups: (1) the BRAF and MEK inhibitor group, which included patients with melanoma treated with a combination of BRAF inhibitors and MEK inhibitors; and (2) the control group, which included patients with melanoma treated with BRAF inhibitor monotherapy. The proportion of patients with CVAEs receiving BRAF and MEK inhibitors was compared with that of the control group in the same RCT. All studies included in our meta-analysis reported the number of patients with adverse events and not the number of adverse events for a group. The data are expressed as percentage of patients with CVAEs, calculated by dividing the number of each CVAE by the total sample size. Risk ratios (RRs) and 95% CIs are used to express dichotomous outcomes.27 A total of 2317 patients with melanoma were included. The agreement between the authors that performed the search was more than 88%, with a κ of more than 83.3%. General characteristics of the study population are detailed in A total of 1904 eligible articles were identified through database searching . We idenP = .02; I2 = 0%), with 2.2% of patients from the BRAF and MEK group developing pulmonary embolism compared with 0.4% of the BRAF inhibitor monotherapy group. The combination of BRAF and MEK inhibitors was associated with a more than 3-fold increase in risk of a decrease in LVEF and a 1.4-fold increase in risk of arterial hypertension   . Overall2 = 47%) .P = .005; I2 = 29%) and high-grade arterial hypertension   were analyzed . TreatmeI2 = 0%) . In contI2 = 0%) . The RR I2 = 0%) . The proP = .001), while the association with arterial hypertension was not significant for the younger population subgroup  (P = .02). The study-level characteristics for the subgroup analysis are listed in eTable 3 in the A higher risk of decrease in LVEF was associated with patients with a mean age younger than 55 years  . The risThe heterogeneity for each analysis of CVAEs was not statistically significant, except for decrease in LVEF, arterial hypertension, and QTc interval prolongation analyses, where heterogeneity could be rated as moderate. A sensitivity analysis was performed by excluding each study in a stepwise manner from the analysis to determine the relative importance of each study. Treatment with BRAF and MEK inhibitors remained a risk factor for the selected outcomes.The studies were reviewed for publication bias. The risk of bias of the included studies was low, except for the blinding of outcome assessment, as depicted in eFigure 3 in the We performed a meta-analysis of CVAEs for patients with melanoma being treated with BRAF and MEK inhibitors using data from 5 RCTs with 2317 participants. The main results of our study were as follows: (1) BRAF and MEK inhibitor therapy was associated with a higher RR of pulmonary embolism, decrease in LVEF, and arterial hypertension compared with BRAF inhibitor monotherapy; (2) BRAF and MEK inhibitor therapy was not associated with higher rates of myocardial infarction, atrial fibrillation, or QTc interval prolongation compared with BRAF inhibitor monotherapy; (3) the RRs of high-grade decrease in LVEF and high-grade arterial hypertension were higher in the group being treated with BRAF and MEK inhibitors than in the group being treated with BRAF inhibitor monotherapy; (4) a higher risk of a decrease in LVEF was associated with patients with a mean age younger than 55 years; and (5) BRAF and MEK inhibitor therapy was associated with a higher risk of pulmonary embolism in studies with a mean follow-up time of more than 15 months.28 The other mechanism is associated with the reduced bioavailability of nitric oxide. Nitric oxide production is reduced because of an impaired vascular endothelial growth factor pathway, mediated through the MAPK pathway.29 The upregulation of cluster of differentiation 47 induced by BRAF and MEK inhibition also inhibits the signaling of nitric oxide–cyclic guanosine monophosphate, reduces nitric oxide bioavailability, and thus contributes to recurrent vasoconstriction, hypertension, and an imbalance between thrombotic and antithrombotic states.31 This could serve as an explanation for the occurrence of pulmonary embolism and myocardial infarction.The mechanisms of cardiotoxicity for BRAF and MEK inhibitors are incompletely understood. The occurrence of arterial hypertension could be triggered by interference with 2 mechanisms. One is the perturbance of the renin-angiotensin system regulation induced by the inhibition of BRAF and MEK signaling.22 Another hypothesis is that an impairment of angiogenesis relies on the MAPK pathway to activate smooth muscle cell proliferation.20 This may contribute to hypertension, decreased LVEF, occurrence of atrial fibrillation, and, in the long term, the development of heart failure.Evidence for the pathomechanism underlying the association of BRAF and MEK inhibitor therapy with LVEF impairment is particularly scarce. Experimental evidence suggests that the MAPK pathway has a cardioprotective role in the heart. Treatment with BRAF and MEK inhibitors could lead to a perturbance of the physiological mechanisms of hypertrophy, apoptosis, remodeling of myocytes, and subsequently to a reduction in LVEF though this pathway.17 A higher risk of a decrease in LVEF has been reported in other studies, probably owing to a lower number of included patients.33 An analysis concerning the proportion of patients with reduced ejection fraction who developed heart failure was not possible because only 2 studies17 reported a reduced number of events not tailored on the actual cardiological definition of heart failure.34 While QTc interval prolongation during the therapy with BRAF and MEK inhibitors was higher compared with control therapies, it was statistically insignificant, which is concordant with our findings.33 Prolonged QTc of more than 60 milliseconds compared with baseline or more than 500 milliseconds was associated with BRAF inhibitors vemurafenib35 and dabrafenib36 but not with the MEK inhibitor trametinib.27 To our knowledge, the RRs of pulmonary embolism, atrial fibrillation, and myocardial infarction were analyzed for the first time in our study.The decrease in LVEF seems to be a MEK-mediated adverse event. It was higher in the BRAF and MEK inhibitor arms compared with BRAF inhibitor monotherapy arms, concordant with the rates published in the individual studies included in the meta-analysis.17 and it is known to ameliorate BRAF inhibition–related adverse events caused by MEK hyperproduction, like squamous skin cancer and other skin-related toxicities.37 Although the available BRAF and MEK inhibitor combinations have largely overlapping adverse events, pyrexia has been associated with dabrafenib and trametinib9 and photosensitivity with vemurafenib and cobimetinib,10 but both adverse effects were infrequent with encorafenib and binimetinib.17 A direct comparison among the 3 combinations has not been performed until now.It has been shown that the effective inhibition of the MAPK pathway through a combination of BRAF and MEK inhibitor therapy resulted in better clinical outcomes,14 comparing the MEK inhibitor trametinib with chemotherapy showed that 6.6% of patients in the MEK inhibitor group experienced decrease of LVEF with no reported cases of decrease in LVEF in the chemotherapy group. However, the difference between the groups did not reach statistical significance. On the other side, RCT trials of BRAF inhibitors did not report on a decrease in LVEF. Furthermore, the observed association of BRAF and MEK inhibitors with arterial hypertension has not been confirmed in studies of BRAF and MEK inhibitors compared with placebo8 or the MEK inhibitor trametinib compared with chemotherapy.14 On the other side, studies of the BRAF inhibitor vemurafenib compared with dacarbasine38 or with placebo39 showed a significant association with arterial hypertension. The increased rate of pulmonary embolism under dual therapy is not supported by a high rate of pulmonary embolism in patients treated with BRAF inhibitors alone vs chemotherapy38 or placebo.39However, it is difficult to interpret if the association with an increased risk of a decrease in LVEF and arterial hypertension under the BRAF and MEK combination presented here is a MEK-mediated adverse event or if this is subsequent to a more efficient inhibition of the MAPK pathway through dual therapy. A 2012 study40 A single clinical approach algorithm41 for the management of BRAF and MEK inhibitor–related CVAE has been already published. The clinical approach states that therapy with BRAF and MEK inhibitors should be stopped in case of stage 2 hypertension, defined as systolic blood pressure more than 160 mm Hg or diastolic blood pressure more than 100 mm Hg. Arterial hypertension should be treated according to current guidelines, and therapy should be reintroduced at a reduced dosage after the reduction of arterial hypertension to stage 1 or below.42 In cases of an asymptomatic decrease in LVEF of 10% or more from baseline and below institutional lower limits of normal from pretreatment level, the dosage of BRAF inhibitors should not be modified, but MEK inhibitors should be stopped. A follow-up should be made after 4 weeks, and the therapy with MEK inhibitors should be resumed in the event of recovery of LVEF to normal values or permanently discontinued in the event of persistent changes in LVEF. In case of heart failure or a decrease in LVEF of 20% or more from baseline that is below the lower limit of normal, therapy with BRAF inhibitors should be stopped and, in the event of recovery, resumed at the same dosage, while therapy with MEK inhibitors should be permanently discontinued.43 Initiation of treatment with BRAF inhibitors is not recommended in patients with QTc of more than 500 milliseconds, and BRAF inhibitors should be stopped at a QTc of more than 500 milliseconds or at an increase in QTc of more than 60 milliseconds from baseline values at any time during the treatment. In case of increased QTc of less than 60 milliseconds from baseline, the BRAF inhibitors should be discontinued and reintroduced at a reduced dosage in the event of recovery. The measurement of QTc and serum electrolytes is recommended before treatment, after 1 month of treatment, and after any dosage modification made for prolonged QTc.42 There are no specific indications for the management of pulmonary embolism, myocardial infarction, or atrial fibrillation, and these should be managed according to the cardiology guidelines, with BRAF and MEK inhibitor treatment considered separately for every individual.Our findings increase the awareness of CVAEs in patients with melanoma treated with BRAF and MEK inhibitors and may help to determine the appropriate clinical cardio-oncology management. The management of CVAEs is based on expert opinion, and the available guidelines do not give specific indications.17 was analyzed as 2 separate studies, which could induce bias in the final analysis. However, excluding an arm from the final analysis did not influence the conclusion. Sixth, many studies did not report on CVAEs and could not be included in the final analysis.This study has some limitations that need to be addressed. First, the definition of adverse events is based on the oncological recommendations that are not perfectly aligned with cardiological definitions. Second, the double therapy regimen was compared with single therapy, and a perfect delimitation of the adverse events deriving from BRAF inhibitors or MEK inhibitors cannot be done. Third, the treatment regimens were different between the studies; although from the same class of therapies, there are some specific adverse events related to each regimen. Fourth, because this is a study-level meta-analysis, it is not possible to make inferences regarding which individual patients are at higher risk of CVAEs. Fifth, 1 studyIn conclusion, therapy with BRAF and MEK inhibitors was associated with an increased risk of CVAEs, especially pulmonary embolism, a decrease in LVEF, and arterial hypertension, compared with BRAF inhibitor monotherapy. The risks of myocardial infarction, atrial fibrillation, and QTc prolongation were similar between the assessed groups. These adverse events should be carefully approached in cardio-oncology teams for an optimal treatment of patients with melanoma.


The main aim of this study was to characterize changes in the left ventricular (LV) blood flow kinetic energy (KE) using four-dimensional (4D) flow cardiovascular magnetic resonance imaging (CMR) in patients with myocardial infarction (MI) with/without LV thrombus (LVT).ρ = density, V = volume, v = velocity, and was indexed to LV end-diastolic volume. Patient with MI had significantly lower LV KE components than controls (P < 0.05). LVT+ and LVT− patients had comparable infarct size and apical regional wall motion score (P > 0.05). The relative drop in A-wave KE from mid-ventricle to apex and the proportion of in-plane KE were higher in patients with LVT+ compared with LVT− . The time difference of peak E-wave KE demonstrated a significant rise between the two groups . In logistic-regression, the relative drop in A-wave KE  demonstrated the strongest association with LVT.This is a prospective cohort study of 108 subjects . All underwent CMR including whole-heart 4D flow. LV blood flow KE wall calculated using the formula: Patients with MI have reduced global LV flow KE. Additionally, MI patients with LVT have significantly reduced and delayed wash-in of the LV. The relative drop of distal intra-ventricular A-wave KE, which represents the distal late-diastolic wash-in of the LV, is most strongly associated with the presence of LVT. Early echocardiographic studies have demonstrated that abnormal flow patterns are associated with LVT.The development of four-dimensional (4D) flow cardiovascular magnetic resonance imaging (CMR) now allows mapping and quantification of intra-cavity LV flow kinetic energy (KE).The aim of this study was to use 4D flow CMR to map LV flow KE and characterize flow changes in patients with MI associated ischaemic cardiomyopathy with and without LVT. We hypothesized that patients with LVT show a re-distribution of LV flow KE resulting in reduced wash-in and wash-out of the LV. Furthermore, we aimed to investigate if LV flow KE mapping parameters are better associated with presence of LVT than the traditional risk factors for the development of LVT.Figure 1). Controls were recruited from two centres (Leeds and Leiden). They had no history or symptoms of cardiovascular disease, were on no cardiovascular or other relevant medication and had no contraindications to CMR. MI patients were recruited in Leeds and included both acute ST-elevation MI (STEMI) and chronic MI patients (MI > 3 months). Because of the relatively low incidence of LVT, we identified patients with LVT from routine clinical echocardiography lab and clinical CMR lists between January 2015 and April 2017 and in parallel recruited age and gender matched patients with MI but without LVT. Patients identified for this study were offered research CMR bolt-on scans immediately after clinical CMR or full protocol CMR if identified from echocardiography lab. All patients presenting to our CMR lab gave prospective consent prior to their clinical scans for the bolt-on research protocol.This was a prospective cohort study of patients with MI and matched controls (The inclusion criteria for acute STEMI patients were: first-time acute STEMI revascularized by primary percutaneous coronary intervention within 12 h of onset of chest pain. Acute STEMI patients were scheduled for CMR imaging within 72 h of indexed presentation. The inclusion criteria for chronic MI patients were: previous history of MI and presence of scar on late gadolinium enhancement (LGE) imaging. Exclusion criteria for all included the following: cardiomyopathy, atrial fibrillation, haemodynamic instability, and any contraindications to CMR.The study protocol was approved by the National Research Ethics Service (12/YH/0169) in the United Kingdom and the institutional Medical Ethical Committee (P11.136) in Leiden, The Netherlands. The study complied with the Declaration of Helsinki and all patients gave written informed consent.All controls and patients underwent CMR imaging on identical 1.5 T systems at the two study sites  with 28-channel coils and digitization of the magnetic resonance signal in the receiver coil.The CMR protocol included survey, cines, early gadolinium enhancement imaging, LGE imaging, and at the end 4D flow CMR.For 4D flow, the field-of-view was planned in the trans-axial plane ensuring full cardiac coverage by adjusting the number of slices. A free-breathing, non-respiratory navigated, Echo-Planer Imaging (EPI)- accelerated 4D flow sequence was used.Figure 1) and tissue characterisation was done at the main clinical acquisition site (Leeds) and remained blinded to core lab functional/flow analysis. Methods of analysis are descripted in the All images were evaluated offline using in-house developed research software . For functional and flow analysis, anonymised cine/4D flow CMR data were shared with the core lab at Leiden . Correction for translational and rotational misalignment between the short-axis cine and the 4D flow CMR acquisition was performed using automated image registration as previously described.LV was contoured manually in the images of the short-axis cine acquisition (3), EDV).For calculation of LV blood flow KE parameters, the LV volumetric mesh was resliced into short-axis sections of 2 mm thickness and pixel spacing equal to the original reconstructed pixel size of the short-axis cine acquisition (1.0–1.2 mm). For each volumetric element (voxel) the KE was computed as Figure ). The global, wash-in, and wash-out components of the LV KE that were mapped are described in Figure . During diastolic filling, the flow velocity and its associated KE of early (E-wave) and late (A-wave) filling decrease from base to apex. In this study, this drop has been measured as a relative drop in percentage from each level  for both E-wave flow KE and A-wave flow KE. Higher relative-drop of LV in-flow KE signifies reduced wash-in of the LV.In patients with LVT, LVT was excluded from KE mapping to reduce under-estimation of LV flowing blood KE , as measured by M-mode echocardiography is lower. Hence, the transit time of the peak KE from base to mid-ventricle, described as the TD in this study, may represent a novel marker of delayed filling.We also computed the time difference (TD) to peak early mitral in-flow velocity (E-wave) from the base of the LV to mid-ventricle. This transit time or TD should be higher if the mitral valve propagation velocity  with Bonferroni corrections. Imaging data was handled as non-parametric. Step-wise multivariate logistic regression was used for clinical, functional, and KE parameters with statistical significance from one-way analysis (P < 0.1). Diagnostic performance tests were done using the receiver-operator characteristic. To avoid collinearity issues within volumetric parameters, only LV EF was included in the multivariate analysis. A P-value <0.05 was considered statistically significant.Statistical analysis was performed using IBM SPSSSample size calculations are described in the Figure 1). These included 40 controls , 36 LVT− patients and 32 LVT+ patients. From the 32 LVT+ patients, 5 LVT+ patients (16%) were identified at echocardiography lab and the rest were identified at the CMR lab. Patients on anti-coagulation had already been diagnosed with LVT by echocardiography.We identified 135 subjects for this study, 23 did not meet the eligibility criteria and 4 were claustrophobic. Hence, 108 subjects completed the study (P > 0.05) (Table 1). Heart failure status was comparable between the two patient groups. Patients with LVT were more likely to have diabetes (P = 0.01) and to be on anti-coagulation than patients without LVT (P = 0.04).All subjects had comparable heart rates (P = 0.01). Infarct size was comparable between patients with/without LVT (P = 0.6) . Also, for all four apical segments, scar transmurality was not different between patients with/without LVT (P > 0.05) (P = 0.055). EF was significantly lower in patients with LVT and end-diastolic/end-systolic volumes and mass were significantly increased in patients with LVT compared with patients without LVT.Patients with LVT were more likely to have anterior MI than those without LVT  . Apical P = 0.01). However, MR was similar in both patient groups (P = 0.09). No other mitral in-flow diastolic function parameter demonstrated any significant difference between the two MI groups.Patients with infarct were more likely to have mitral regurgitation (MR)  patients had mural thrombus, 6 (17%) had mobile thrombus and 10 (27%) had protruding thrombus. Average indexed volume of thrombus was 4.9 ± 10 mL/mEDV averaged over the complete cardiac cycle was significantly lower in both patient groups vs. healthy controls (P < 0.05) . Similarly, average systolic KEiEDV and peak E-wave KEiEDV were significantly lower in patients than in age-matched controls (Table 3). Peak late filling (A-wave) KEiEDV was not different between the three groups (P > 0.05). The proportion of in-plane KE of the LV was not different between controls and LVT− patients (P = 0.82). However, patients with LVT demonstrated a significantly higher proportion of in-plane KE vs. LVT−  .LV KEiP > 0.05) (Table 3). Peak A-wave KE drop from mid-ventricle to apex was not different in controls and LVT− patients (P = 0.69). However, LVT+ patients demonstrated a significantly higher drop in A-wave KE from mid to apex when compared with LVT− patients  .The relative drop of proximal and distal, intra-ventricular E-wave KE, did not differ across the three groups (P = 0.52); age-matched older controls = 18 ± 28 ms vs. LVT− = 38 ± 38 ms (P = 0.07); LVT− = 38 ± 38 ms vs. LVT+ = 62 ± 56 ms (P = 0.04) with an overall ANOVA between groups of <0.001) .The TD of peak E-wave KE propagation from base to mid-ventricle demonstrated rise between all the four groups of subjects—younger controls = 14 ± 15 ms vs. age-matched older controls = 18 ± 28 ms (Table 4). In multivariate analysis, only distal drop of A-wave KE  and EF  demonstrated independent association with LVT. A combined CMR model of EF and relative drop in A-wave KE demonstrated significantly larger area under the curve than LV EF  and infarct size  .In univariate analysis, the following parameters were associated with the presence of LVT: history of diabetes, anterior MI, EF, drop of A-wave KE from mid-ventricle to apex, proportion of LV in-plane KE, and TD of peak early-filling (E-wave) KE from base to mid-ventricle  and good precision . Inter-rater reliability of main KE parameters thresholds were good . Comprehensive results for individual parameters can be found in the EDV is reduced in MI patients compared with healthy, age-matched controls. Secondly, MI patients with LVT demonstrated reduced wash-in of blood to the distal LV during late diastole, detected by the prominent drop of A-wave KE from the mid-ventricle to the apex. This parameter of LV blood flow disturbance was most strongly associated with the presence of LVT.This study provides mechanistic insights into intra-cavity LV flow disturbances in MI patients with and without LVT. Firstly, we demonstrate that global LV KEiEDV averaged over the complete cardiac cycle, systolic KEiEDV and peak E-wave KEiEDV were all significantly reduced in patients with MI. Even though these global LV flow KE parameters were not significantly different in patients with/without thrombus, they are likely to be the key initial substrates of flow stasis post-MI.Blood stasis in the LV is the hallmark of LVT formation. In the detailed mapping of LV flow KE, we noted that the global LV KE parameters are significantly altered in health vs. MI patients. LV KEiDuring early and late diastole, blood flow into the LV cavity takes very little time due to intra-ventricular pressure gradients. In this study, the TD showed an increase from healthy controls to LVT− and further to LVT+ patients, demonstrating that patients with LVT have significantly delayed wash-in of the LV.Additionally, the relative drop of distal A-wave KE was significantly higher in MI patients with LVT vs. patients without LVT. This finding suggests that there is a reduction in distal intra-ventricular pressure gradients due to a relative increase in distal  pressures within the LV cavity in patients with LVT and that the late filling phase of diastole plays an important role in reduced wash-in of the LV.The in-plane KE of blood flow was significantly higher in MI patients with LVT than those without LVT. Our data support the notion that an increase in the in-plane flow will reduce the proportion of through-plane flow in the LV cavity, and thus less blood will pass through the ventricle per-unit-time resulting in reduced global wash-in and wash-out of the LV. In addition to lower wash-in and wash-out, such non-physiological in-plane flow may exert strain on the LV wall, resulting in more dilatation and increase in endothelial dysfunction in the endocardium, similar to the vascular system.,,P = 0.82). This is possibly because we included patients with chronic infarction in both the MI groups. In chronic infarcts, the infarct size substantially decreases from the acute stage, which may lessen the overall impact of infarct size. In addition, even though there was a trend of apical regional wall motion score to be higher in LVT− patients, this study did not demonstrate any significant changes in LVT+ patients. This may be explained by the fact that in a previous study by Keren et al.,P < 0.01). This finding is likely due to under representation of patients with diabetes in the LVT− MI cohort as observational studies have demonstrated prevalence of diabetes is around 24–36% in MI.Akin to published studies in patients with LVT, our study also demonstrated the association of infarct location and depressed EF to LVT.LVT volume was the only parameter which had some association to flow characterisation . Mobility, produrance, and murality of the thrombus did not demonstrate any significant flow association. We speculate this may be because thrombus characteristics change rapidly after MI and probably depend on the timing of the imaging.This study was a prospective cohort study, hence our results cannot be used to determine the prevalence of thrombus in MI. Additionally, we studied differences in flow patterns in the presence of LVT and not prior to its genesis, and a prospective evaluation of the parameters tested in this study is required. Arrhythmias can introduce errors in 4D flow analysis. To reduce these errors, we performed robust quality checks on all the data. Additionally, we used retrospectively gated acquisition sequence for 4D flow to reduce time blurring.This study provides mechanistic insights into disturbed flow patterns in MI patients with and without thrombus. Patients with MI have reduced global LV KE and MI patients with LVT have evidence of reduced wash-in of the LV. Among all imaging biomarkers, the relative drop of distal intra-ventricular A-wave KE, which represents the distal late-diastolic wash-in of the LV, was most strongly associated with the presence of LVT. Future studies need to evaluate the prognostic significance of blood flow KE changes in the LV in patients with LVT.Supplementary DataClick here for additional data file.


Ageing-related alterations in cardiovascular structure and function are commonly associated with chronic inflammation. A potential blood-based biomarker indicative of a chronic inflammatory state is N-Terminal Pro C-Type Natriuretic Peptide (NTproCNP). We aim to investigate associations between NTproCNP and ageing-related impairments in cardiovascular function. Community-based participants underwent same-day assessment of cardiovascular function and circulating profiles of plasma NTproCNP. Associations between cardiovascular and biomarker profiles were studied in adjusted models including standard covariates. We studied 93 participants , of whom 55 (59%) had impaired myocardial relaxation (ratio of peak velocity flow in early diastole E (m/s) to peak velocity flow in late diastole by atrial contraction A (m/s) <0.84). Participants with impaired myocardial relaxation were also found to have lower peak early phase filling velocity  and higher peak atrial phase filling velocity . NTproCNP levelswere significantly lower among participants with impaired myocardial relaxation . After multivariable adjustments, NTproCNP was independently associated with impaired myocardial relaxation . Community elderly adults with myocardial ageing have lower NTproCNP levels compared to those with preserved myocardial function. Given that impaired myocardial relaxation probably represents early changes within the myocardium with ageing, NTproCNP may be useful as an ‘upstream’ biomarker useful for charting myocardial ageing. Current knowledge suggests that ageing-related alterations in cardiovascular structure and function are commonly associated with chronic inflammation mediated by various factors. ProinflThe natriuretic peptides (NPs) represent a family of structurally related but genetically distinct peptide hormones that include atrial NP (ANP), brain/B-type NP (BNP) and C-type NP (CNP). ANP andCNP is released in response to pro-inflammatory cytokines, possibly involving interactions between macrophageal cytokines and vascular endothelium,17. ThisNTproCNP has a longer half-life and is present in the blood at higher concentrations than biologically active CNP. As such, NTproCNP may in fact demonstrate even more consistent data than CNP in terms of clinical application . FurtherIn this study, we hypothesise that NTproCNP, being closely related to the inflammatory processes present in ageing, may show correlation with impairments in myocardial relaxation, in an elderly community-based population, prior to development of cardiovascular disease.The subjects were recruited from the Cardiac Ageing Study (CAS), a prospThe study sample consisted of men and women who were recruited between 2014 and 2017 and who had no self-reported history of physician-diagnosed cardiovascular disease  or cancer. Written informed consent was obtained from participants upon enrolment. The SingHealth Centralised Institutional Review Board (2014/628/C) had approved the study protocol. All methods were performed in accordance with the relevant guidelines and regulations.As previously described ,22, all We performed standard echocardiography for each subject using ALOKA α10 with a 3.5 MHz probe. Standard echocardiography that involved 2-D, M-mode, pulse Doppler and tissue Doppler imaging, was performed in the standard parasternal and apical  views, and three cardiac cycles were recorded. We measured left ventricular ejection fraction (LVEF), left atrial (LA) volume and LA volume index. Using Doppler echocardiography, trans-mitral flow E and A wave with the sample volume position at the tip of the mitral valve leaflets from the apical 4-chamber view were recorded. E/A ratio was computed as a ratio of peak velocity flow in early diastole E (m/s) to peak velocity flow in late diastole by atrial contraction A (m/s). From the apical 4-chamber view, pulsed wave tissue Doppler imaging was performed with the sample volume at the septal and lateral annulus. The frame rate was between 80 and 100 frames per second. All measurements were measured by the same operator and the measurements were averaged over three cardiac cycles and adjusted by the RR interval.Plasma levels of NTproCNP were measured using the NT-proCNP ELISA kit (DRG Instruments GmbH Germany), according to the manufacturer’s assay procedures. Briefly, 50 μL of assay buffer was added into each well, followed by 20 μL of plasma samples and 50 μL of conjugate. The plate was sealed and incubated at room temperature for three hours. Subsequently, the plate was washed five times before 100 μL of substrate was added to each well, then incubated for another thirty minutes at room temperature in the dark. 50 μL of stop solution was added and read at 450 nm immediately. Plasma levels of NTproBNP were measured with NTproBNP assay  using monoclonal electrochemiluminescence immunoassay (ECLIA) method.We first examined bivariable association of subject clinical characteristics, cardiac function and biomarkers with impaired myocardial relaxation. Impaired myocardial relaxation was defined as ratio of peak velocity flow in early diastole E (m/s) to peak velocity flow in late diastole by atrial contraction A (m/s) less than 0.84. (mean E/A ratio was 0.84 in our study sample).The t test and chi-square test were used for continuous variables and categorical variables respectively. Continuous variables are reported as a mean with standard deviation (SD).Multivariable logistic regression models were constructed to assess the association of NTproCNP with impaired myocardial relaxation. The initial univariate logistic regression model examined the individual association with demographic variables and clinical covariates. Those variables associated in the univariate analysis with a p < 0.05 were candidate NTproCNP effect modifiers. The potential effects of these candidates were then assessed individually via multivariable logistic regression.P value of <0.05 was considered significant.All statistical analyses were performed using STATA 13 . For all analysis, a two-tailed 2). Notably, NTproBNP levels were low in the entire cohort (median 80 pg/ml interquartile range 45–150), with no differences in NTproBNP levels between those with impaired myocardial relaxation and those with preserved myocardial relaxation (median 74 vs 84 pg/ml)  were included in the analysis. Of these 93 participants, 55 (59%) had impaired myocardial relaxation (ratio of peak velocity flow in early diastole E (m/s) to peak velocity flow in late diastole by atrial contraction A (m/s) <0.84). Participants with impaired myocardial relaxation did not have other features of diastolic dysfunction: mean ratio of peak velocity flow in early diastole to peak early diastolic septal mitral annular velocity was 9.92 ± 2.3, mean peak early diastolic septal mitral annular velocity 0.06 ± 0.01, mean pulmonary artery systolic pressure 27.1 ± 7.4 mmHg and mean left atrial volume index 23.0 ± 7.0 (ml/m4 pg/ml) .We studied the sensitivity and specificity of NTproCNP for determining impaired myocardial relaxation. NTproCNP value of ≥19 pmol/L was associated with impaired myocardial relaxation .The baseline characteristics of the study sample are shown in In a multivariable regression model adjusting for age, body mass index and NTproBNP, NTproCNP was associated with female gender .At the univariate level, female gender  and NTproCNP levels ≥ 19  were associated with impaired myocardial relaxation as denoted by the E/A ratio. Participants with impaired myocardial function (E/A ratio < 0.84) were significantly more likely to be female and less likely to have a high NTproCNP. After multivariable adjustments, NTproCNP remained independently associated with impaired myocardial relaxation . These results are presented in In this analysis, we evaluated the utility of NTproCNP as a biomarker of myocardial function in aged community adults. Among community adults, NTproCNP was independently associated with impaired myocardial relaxation, a common manifestation of myocardial ageing in ageing adults. With agThis community cohort of elderly adults did not have clinical cardiovascular disease, such as heart failure, as suggested by relatively low NTproBNP levels. These low levels of NTproBNP are below suggested cut points for excluding asymptomatic heart failure in elderly adults, of particular relevance to our study. TherefoAge-related diastolic dysfunction has been attributed to an increased passive stiffness, which is associated with increases in left ventricular fibrosis. Lower NCurrently, there are limited studies on NTproCNP in cardiovascular disease in pre-specified cohorts. Our study advances current understanding on NTproCNP as a potential biomarker for cardiovascular states. To the best of our knowledge, we are the first to show a direct association between NTproCNP and measures of myocardial function in community elderly adults. Existing studies exploring NTproCNP as a novel biomarker have largely focused their attention on cohorts with clinical cardiovascular disease –29, withNotably, despite the well-established role of NTproBNP as a diagnostic and prognostic biomarker of heart disease such as heart failure, we did Our hypothesis-generating observations are interesting and deserve clarification from future mechanistic studies. However, we postulate that our observations may be explained by differences in functional characteristics between NTproBNP and NTproCNP. NTproBNP is primarily secreted by cardiomyocytes in response to increased ventricular stretching and serves a natriuretic function, particularly observed in heart failure states, where natriuretic function is critical in restoring fluid balance in heart failure. On the other hand, in non-heart failure states, NTproCNP may represent a ‘protective’ function, such as in cardioprotection, to maintain cardiovascular function in health,32. CNP Further, in the evaluation of NTproCNP as a potential biomarker of myocardial function in ageing, our study attempted to determine a cut-off value of NTproCNP that could identify impaired myocardial relaxation with reasonable specificity. Larger studies in the future are necessary to confirm our observations about appropriate cut-offs for NTproCNP, adjusted to specificity or sensitivity thresholds. However, our data provides a glimpse of how NTproCNP levels may be handled in similar investigations.We acknowledge limitations of our study. Firstly, NTproCNP levels are assumed to reflect levels of CNP. It is possible that NTproCNP and CNP levels do not correlate perfectly in milieu. However, prior reports have suggested significant correlations observed between NTproCNP and CNP such that NTproCNP could be used as a suitable substitute for assessment of CNP status in the circulation. SecondlIn summary, our findings suggest that NTproCNP could be a suitable candidate biomarker for cardiac ageing, advancing our understanding of mechanisms involved in cardiac ageing.This study demonstrates the utility of NTproCNP as a potential biomarker of cardiac ageing. NTproCNP may be a promising circulating biomarker that can provide insights into myocardial ageing states, useful for future mechanistic and interventional studies.


Thousands of articles have discussed the concept of successful aging, yet no existed scale to measure its components among old-old (age 75+) in urban China. This study used a mixed method to develop a valid scale in Shanghai from July 2016 to Nov 2018. We first conducted 97 semi-structured, in-depth interviews on community-dwelling old-old and listed 70 items based on the findings and previous studies . Ten multidisciplinary professionals and eight old-old had focus groups to evaluate the content validity of the scale. We used the final 22 items to survey 208 community-dwelling old-old in twelve neighborhoods. The mean age is 81, with 40% females. The principal component analysis showed a 13-item scale representing four domains , with 0.67 total reliability (Cronbach's Alpha). This study provided a set of quality indicators to evaluate old-old’s life in urban China.