Refractory cardiogenic shock (CS) often requires veno-arterial extracorporeal membrane oxygenation (VA-ECMO) to sustain end-organ perfusion. Current animal models result in heterogenous cardiac injury and frequent episodes of refractory ventricular fibrillation. Thus, we aimed to develop an innovative, clinically relevant, and titratable model of severe cardiopulmonary failure. Six sheep (60 ± 6 kg) were anaesthetized and mechanically ventilated. VA-ECMO was commenced and CS was induced through intramyocardial injections of ethanol. Then, hypoxemic/hypercapnic pulmonary failure was achieved, through substantial decrease in ventilatory support. Echocardiography was used to compute left ventricular fractional area change (LVFAC) and cardiac Troponin I (cTnI) was quantified. After 5 h, the animals were euthanised and the heart was retrieved for histological evaluations. Ethanol (58 ± 23 mL) successfully induced CS in all animals. cTnI levels increased near 5000-fold. CS was confirmed by a drop in systolic blood pressure to 67 ± 14 mmHg, while lactate increased to 4.7 ± 0.9 mmol/L and LVFAC decreased to 16 ± 7%. Myocardial samples corroborated extensive cellular necrosis and inflammatory infiltrates. In conclusion, we present an innovative ovine model of severe cardiopulmonary failure in animals on VA-ECMO. This model could be essential to further characterize CS and develop future treatments. Up to 80% of patients with CS develop concomitant acute respiratory failure and require ventilatory support4. CS can be caused by multiple aetiologies, with acute myocardial infarction (AMI) being the primary cause. Despite improvement in management and advances in therapeutic options, CS is still associated with a mortality of approximately 50% and poor outcomes5. Management of CS patients includes treatment of the cause (e.g. coronary reperfusion in AMI) and restoration of adequate end-organ perfusion. The latter can be achieved with medications, ie vasopressors and inotropes, mechanical circulatory support (MCS), or both. MCS use has increased exponentially over the last decade as indications have broadened and early implementation has been shown to improve survival8. Several MCS systems are used to treat CS, ranging from percutaneous mini-invasive devices—intra-aortic balloon pump (IABP) or micro-axial pumps—to more invasive techniques such as veno-arterial extracorporeal membrane oxygenation (VA-ECMO) and TandemHeart9. Among these devices, ECMO use has increased dramatically due to improved management, reduced complications, easier percutaneous access, and improvements in regional planning10.Cardiogenic shock (CS) is defined as a critical reduction in cardiac output (CO) resulting in end-organ hypoperfusion12. For instance, the impact on left ventricular (LV) distension and overload, the consequences of differential hypoxia on the brain and coronary arteries and the effect of lower body hyperoxia14. Considerable progress in improving ECMO has been made, due to the use of large mammalian models of CS and comprehensive experimentation aimed at clinical translation. Indeed, due to the high level of similarities to human in major determinants of myocardial work and energy consumption, e.g. heart rate and vascular wall to-lumen-ratios, large animals remain the method of choice for clinical translation in this field15. Nevertheless, the development of a reliable and reproducible large animal model of CS supported by VA-ECMO with significant clinical translatability has been challenging. In particular, reproducing a stable cardiogenic shock model by ligating major coronary arteries often resulted in refractory and life-threatening ventricular arrhythmias19. Other methods, such as carbon monoxide poisoning or drug-induced CS (beta-blockers), have resulted in heterogeneous outcomes and cause a global decrease in ventricular contractility, different to regional wall motion abnormalities usually seen in myocardial infarction21.The rapid surge in the use of ECMO over the last decade has also highlighted potential adverse effects of ECMO on the heart and other organs Thus, the primary aim of this study was to characterise a novel ovine model of CS and pulmonary failure supported by VA-ECMO. In particular, we aimed to develop a model with high level of reproducibility, and reduced risk of refractory arrhythmias that could be reliably applied to assess novel therapeutic strategies and devices that could improve patients outcomes.23.We present our study methods and results in accordance with the ARRIVE Guidelines for reporting  in vivo research and according to recommendations provided by the European Extracorporeal Life Support Organisation Innovations Workgroup24. Sheep were managed according to protocols approved by the UAEC.This study was approved by the University Animal Ethics Committee (UAEC) of Queensland University of Technology (QUT)  and adhered to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes of the National Health and Medical Research Council (NHMRC)Animal experiments were carried out at the QUT Medical Engineering Research Facility (MERF). Sheep were sourced from the Warwick Saleyards, transported to MERF, and agisted for 2 weeks prior to the beginning of each experiment. During the acclimatization period, sheep had access to food/water ad libitum until the night prior to study commencement, when they fasted. They were regularly examined by MERF veterinarians, and blood tests were performed to confirm the health of the animal (data not provided) prior to experimental procedures. In addition, during the acclimatization period, an echocardiography was performed to assess for normal heart and valvular functions.Six female Merino Cross Dorset non-pregnant sheep (baseline body temperature 39.0 ± 0.5 °C) between 1 and 3 years-old weighing 60 ± 6 kg were used for the study. On the day of the experiment, sheep were transferred to the operative theatre through a custom-made restraining trolley  of 15 per minute and inspiratory fraction of oxygen (FiO2) of 40%. Settings were adjusted to maintain an arterial partial pressure of oxygen (PaO2) between 60 and 100 mmHg and an arterial partial pressure of carbon dioxide (PaCO2) between 35 and 45 mmHg. Maintenance of anaesthesia was provided by continuous infusion of midazolam (0.2 to 0.7 mg/kg/h), fentanyl (0.2 to 0.7 mcg/kg/h) and ketamine (0.3 to 0.6 mg/kg/h). Depth of anaesthesia and analgesia was regularly assessed, and medications were adjusted accordingly. A 14-Fr nasogastric tube was gently advanced through the nose and gastric residuals were continuously suctioned throughout the experiment. A 7.5-Fr Swan-Ganz catheter  was inserted into the left vascular sheath. Finally, 3-Fr arterial cannulas  were inserted under ultrasound guidance using Seldinger technique via a soft-tip guidewire  into the left and right radial arteries  of 8 mL/kg, positive end-expiratory pressure (PEEP) of 5 cmHThe ECMO circuit comprised a Rotaflow console, centrifugal pump, tubing and a low resistance oxygenator . The circuit was primed using 0.9% saline and heated at 37 °C via a heater-cooler machine . Animal was placed in the supine position for cannulation  free wall, parallel and superficial to the LV surface. To avoid intravascular and/or intracavity injections, ethanol was administered only during needle pullback with a 1 mL syringe and a hypodermic 29-G needle or 32-mm 23-G needles for the septum. Additional Movie File shows this in more detail  below 30%;Reduction of arterial systolic blood pressure below 90 mmHg for more than 10 min;Arterial blood lactate > 4 mmol/L;Urinary output < 0.5 mL/kg/h.Amiodarone (6 mg/kg) and lidocaine (8 mg/kg) were administered prophylactically to avoid ventricular arrhythmias during the procedure. A left lateral mini-thoracotomy was performed at the 52O, RR was set to 5/min and FiO2 was set at 21%. Thus, after 1 h from ventilatory adjustment, ARF was confirmed through the withdrawal of blood from the right radial artery , indicating PaO2 below 60 mmHg.After the development of CS, we allowed a 1 h of stabilization, ventilatory support was then substantially decreased to develop acute respiratory failure (ARF): VT was reduced to 4 mL/kg, PEEP was reduced to 0 cmHshock), ARF development (Thypoxia) and hourly during the 5-h follow-up period  using standard procedures. Slides were examined by an independent, blinded, specialist veterinary pathologist and scored for features listed in Table Data are presented as mean ± standard error of the mean (SEM) or median with interquartile range (IQR) when appropriate. A dependent t-test was fitted to determine the effect of cardiorespiratory failure induction in the animals. A repeated measures one-way ANOVA was used to analyse dynamic changes in cTnI levels and echocardiographic parameters. To assess the impact of number of injections on key parameters of cardiac failure we carried out a restricted maximum likelihood analysis, based on repeated measures approach, including number of injection and times of assessment upon and after development of cardiac failure . A (co)variance structure was used to model the within-subject errors and the Kenward-Roger approximations to estimate denominator degrees of freedom. For each continuous variable, the overall F test was first assessed for significance (p ≤ 0.05). If it was significant, all two-sided comparisons among groups were performed. Each pair-wise comparison was corrected using Bonferroni test, to control for the experiment-wise error rate. Normality of the residuals of the mixed models were assessed to confirm reliability of the model. Statistical analyses were performed using R statistical software package version 4.0.2 (2020-06-22) (R Core Team 2020).All six animals survived (100%) until the end of the study and were included in the final analysis. In addition, baseline body temperature and haemoglobin were 39.0 ± 0.5 °C and 10.9 ± 1.4 g/dL, respectively.2 from 98.3 ± 36.2 mmHg to 45.5 ± 12.6 mmHg (p = 0.01) and pH , but no significant change in PaCO2 .A median number of 53 injections  of 96% ethanol was administered to the animals to achieve CS. An associative trend between number of injections and global circumferential strain was found (p = 0.051) on linear mixed model analysis, while number of injections did not impact lactate (p = 0.612), global radial strain (p = 0.249) or FAC (p = 0.139). Table Figure After development of CS, epicardial echocardiography revealed a statistically significant increase in both left ventricular end-diastolic Fig. A and endCardiac tissue histological assessment of the left ventricle showed substantial injury Fig. , charact2 of the LV endocardial surface was scarred by electrogram criteria, corresponding to 19.7% of the chamber surface area. Conversely, electroanatomic mapping of the RV was unremarkable. Epicardial voltages were relatively preserved compared to the endocardium and showed only patchy scar zones. Left ventricular electrogram morphology showed marked peak-to-peak signal attenuation but no isolated, split, or fractionated electrograms were seen , and administration of further amiodarone (10 mg/kg) and lidocaine (8.3 mg/kg), while VA-ECMO blood flow rate was temporarily and appropriately increased to 4 L/min.1. These were further corroborated by pathognomonic biochemical, macroscopic, and histopathological features. Acute respiratory failure was induced by lowering mechanical ventilatory support and confirmed by pre-defined hypoxic criteria in ABG analysis.We present a reproducible ovine model of severe cardiopulmonary failure supported by VA-ECMO. CS was induced by ethanol injection and confirmed by significant changes in pre-defined hemodynamic (SAP < 90 mmHg), metabolic (lactate > 4 mmol/L) and echocardiographic criteria (LVEF < 30%), consistent with CS definitions from contemporary clinical trials and guidelines22. Methodological limitations of large animal trials can risk the clinical translation of their findings, hence particular attention was paid to confirm the extent of shock in all animals26.Heart failure models should be induced to reflect various types of clinical situations, especially for mechanical circulatory support devices (such as ECMO) for profound cardiogenic shock. A systematic review by our group found that 7 out of the 18 studies investigating CS were unsuccessful at meeting a wide range of CS diagnostic criteria and would rather be described as acute heart failure models, rather than cardiogenic shock models27. To reduce the loss of animals, antiarrhythmics were administered before ethanol injection, and this resulted in ventricular fibrillation in only one animal. Complete heart block developed in one animal, likely due to severe ethanol-induced injury to the atrio-ventricular conduction system. These serious arrhythmogenic events were limited and did not affect the mortality rate of our animal cohort. To further characterize this model electrophysiologically, both endo- and epicardial electroanatomic substrate mapping were performed in one animal. This showed that almost 20% of the endocardial LV surface had electrograms attenuated to an extent consistent with scar. This attenuation was not confluent but was seen in discrete zones of regional endocardial and epicardial LV myocardium corresponding to injection sites. The electrograms in these zones were attenuated but did not exhibit fractionation or isolated delayed components. This is consistent with the histological demonstration of dense confluent cellular necrosis in the injected regions without intervening surviving myofiber bundles. Epicardial voltages were relatively preserved, consistent with a zone of subepicardial myocyte preservation related to the depth of ethanol injection. Thus, formation of myocardial necrosis is directly related to injury site. Increased number of sites injected relates to expansion of scar area, allowing for titration of left-ventricular failure.Ventricular fibrillation is the most frequent side-effect of previous experimental cardiogenic shock models, leading to death in up to 50% of animalsInjury can be created regionally, and wall motion abnormality can be localized directly,The extent of injury is potentially titratable – researchers can study decreasing levels of heart failure,.) with high precision in multiple animals.The pattern of injury can replicate a desired clinical scenario , experimental acute heart failure models have predominantly been created through coronary artery occlusions . However, the latter can frequently lead to critical and unpredictable adverse events, such as refractory hemodynamic instability caused by ventricular arrhythmias or sudden cardiac arrest34. Other models, such as global hypoxia, esmolol-induced LV failure or carbon monoxide poisoning have limited translatability, as they induce global LV failure which is rarely seen in AMI patients. Therefore, in comparison with previous models, our model overcomes limitations of achieving a myocardial injury which is either excessively severe or marginal, without possibility to titrate the degree of injury. Furthermore, we conducted ECMO cannulation prior to shock induction to ensure high survival despite the considerable degree of heart failure.To date, pre-clinical heart failure models have employed a range of different animals  and methods 35. Secondly, only six animals were evaluated, yet consistent results were found amongst all the evaluated animal subjects. Thirdly, to achieve ethanol administration, the thorax was opened, which is known to modify the heart–lung interactions in mechanically ventilated patients36. Indeed, opening the thorax will cancel the effect of MV on stressed vascular volume, in particular the increase in RV afterload, decrease in LV afterload and venous return. Nevertheless, an open chest model was essential to titrate and evaluate injury in these animals. In addition, we carried out cardiac mapping in only one animal, due to the extensive costs of these assessments. Although we obtained critical findings, further corroboration of electrophysiological impairments associated with ethanol injections should be obtained in future studies. Finally, although ethanol was injected into the myocardium while slowly pulling out the needle to avoid inoculation into the bloodstream, ultrasound-guided injections could further improve safety of the intervention.Several limitations of our model should be highlighted. Firstly, whilst on peripheral VA-ECMO, ejection fraction decreases as afterload increases. Thus, during ECMO, left ventricular outflow tract velocity time integral could be a reliable estimate of heart function, independently of the afterload. Due to dissimilarities between the human and sheep thorax anatomy, measurement of outflow tract velocity time integral was not obtained. Yet, to minimize the effect of increased afterload, we obtained our echocardiographic assessments at baseline and at CS, using only 1 L/min of VA-ECMO support to minimize afterload, as is often applied in clinical practice to assess the possibility of weaning patients from VA-ECMO37. This so-called cardiac overload is clinically relevant, since increased filling pressures can promote pulmonary oedema and formation of intraventricular thrombi with embolic events, which can drastically worsen patient outcomes irrespective of the use of VA-ECMO39. The effect of increasing VA-ECMO flow on left-ventricular dimensions has been comprehensively assessed by Ostadal et al. in a porcine model of cardiogenic shock18. The authors reported an increase of left ventricular volumes as ECMO flow was incrementally increased. Similar findings were present in our model: EDA and ESA increased significantly after full-flow ECMO support was initiated. Importantly, use of unloading devices, i.e. Impella is associated with decreased mortality at the cost of increased bleeding rates40. Although other unloading methods, such as pharmacological therapies or LV venting exist, increased LV afterload is a crucial clinical complication. Thus, in conjunction with continuous measurement of intracardiac pressures and volumes, our model could be crucial for future studies investigating innovative unloading technologies for VA-ECMO during CS. Furthermore, whilst we recognize this model as a novel tool to investigate device therapies in CS, we must acknowledge its limitations for pharmacological experimentation. CS was achieved through direct toxic myocardial necrosis, which doesn’t reproduce ischemia and reperfusion phases with a penumbra myocardial area and the complex phenomenon of re-entry which triggers frequently lethal ventricular arrhythmias.As previously outlined, although ECMO can provide essential restoration of end-organ perfusion, this technology is not devoid of potential hazards. Of these, it’s increasingly recognized that peripherally instituted VA-ECMO can hamper cardiac recovery as it exerts stress and strain on the left ventricular myocardiumWe herein described and comprehensively assessed a novel model of severe cardiogenic shock and ARF achieved by direct intramural injection of ethanol to the left ventricle and marginal support by the mechanical ventilator. Our methods produced a clinically relevant decline in left ventricular performance with a hemodynamic profile consistent with CS. Our model is stable, and injury can be induced in a site-specific manner, making it highly suitable for future studies investigating device therapies in CS which aim to reproduce a specific clinical scenario; and takes critical care  closer to personalised medicine.Supplementary Information 1.Supplementary Video S1.


Background and objectives: Current guidelines criteria do not satisfactorily discriminate responders to cardiac resynchronization therapy (CRT). QRS amplitude is an established index to recognize the severity of myocardial disturbance and might be a key to optimal patient selection for CRT. Materials and Methods: (1) Initial R-wave amplitude, (2) S-wave amplitude, and (3) a summation of maximal R- or R′-wave amplitude and S-wave amplitude were measured at baseline. These parameters were averaged according to right (V1 to V3) or left (V4 to V6) precordial leads. The impact of these parameters on response to CRT, which was defined as a decrease in left ventricular end-systolic volume ≥15% at six-month follow-up, was investigated. Results: Among 47 patients  who received guideline-indicated CRT implantation, 25 (53%) achieved the definition of CRT responder. Among baseline electrocardiogram parameters, only the higher S-wave amplitude in right precordial leads was an independent predictor of CRT responders  at a cutoff of 1.44 mV. The cutoff was independently associated with cumulative incidence of heart failure readmission and appropriate electrical defibrillation following CRT implantation . Conclusions: Prominent S-wave in right precordial leads might be a promising index to predict left ventricular reverse remodeling and greater clinical outcomes following CRT implantation. Guidelines ,2,3 statQRS amplitude is an established index to recognize myocardial damage or replacement of fibrosis in various cardiac disorders ,10,11,12A total of 69 heart failure patients who received CRT implantation between March 2010 and December 2020 at our institute were included in this retrospective study. All patients met the following guideline-directed criteria: (1) New York Heart Association functional class II–IV; (2) left ventricular ejection fraction ≤35% or <50% if dependent on right ventricular pacing rhythm; (3) QRS duration ≥120 ms. The study was conducted in compliance with the Declaration of Helsinki and was approved by the institutional review board at the University of Toyama. Informed consent was obtained from all patients.Baseline clinical characteristics such as demographic, laboratory, and echocardiographic parameters were retrieved from the electronic medical records.Electrocardiograms (ECGs) were recorded with an amplification of 1 cm/mV. Parameters such as QRS axis, QRS duration in lead II, QRS morphology classified into right bundle branch block, LBBB, and intraventricular conduction disturbance were measured. In precordial leads, initial R-wave amplitude, S-wave amplitude, and QRS amplitude consisting of maximal R- or R′-wave amplitude plus S-wave amplitude were measured and were averaged according to right (V1 to V3) or left (V4 to V6) precordial leads , and left ventricular ejection fraction (LVEF)) were collected within one week before CRT implantation (baseline) and six months later. A CRT responder was defined as a subject who achieved a reduction of LVESV ≥ 15% at six months following CRT implantation as a primary endpoint . PatientClinical events including a composite endpoint , worsening of heart failure requiring unplanned hospitalization, and appropriate electrical defibrillation for ventricular tachyarrhythmias (only for those with implanted defibrillators) were counted.p-value < 0.05 was considered as statistically significant. Data analysis was performed using SPSS v16.0 . Data were expressed as the mean and standard deviation for normally distributed variables and as the median with the interquartile range for non-normally distributed data. Continuous data were compared using t-test or the Mann–Whitney test, as appropriate. Categorical data were expressed as numbers and percentages and compared using chi-squared test.Two-sided p < 0.05 in the univariate analyses. Receiver-operating characteristics analysis was performed to calculate a cutoff of continuous variables to predict CRT responders.Logistic regression analyses were performed to investigate the predictors of CRT responders among baseline variables, including ECG parameters. Multivariate analyses were performed for those with Cumulative incidence of clinical events was stratified by the cutoff of independent variables and compared between the two groups using a log-rank test. Univariate and multivariate Cox proportional hazard ratio regression analyses were performed to investigate the impact of ECG parameters on clinical outcomes, which were adjusted for other considerable variables, including age, male sex, ischemic etiology, left ventricular ejection fraction, the serum levels of B-type natriuretic peptide, and LBBB ,17,18.Among 69 patients included in this study, 22 without follow-up echocardiograms were excluded from the primary analyses and only included in the secondary analyses.A total of 47 patients who received CRT implantation and completed paired echocardiograms tests were included in the primary analyses . Twenty-Averaged QRS amplitude in right precordial leads, averaged S-wave amplitude in right precordial leads, and averaged S-wave amplitude in left precordial leads were analyzed by multivariate logistic regression analysis adjusted for beta-blockers, systolic blood pressure, and QRS axis, which were significant in the univariate analyses . FinallyROC analysis showed a cutoff of 1.44 mV for the S-wave amplitude in right precordial leads to best predict CRT responder, with an area under the curve of 0.787, sensitivity of 84.0%, and specificity of 68.2% . RepreseAmong 69 patients who had at least baseline ECGs, baseline characteristics between patients with averaged S-wave in right precordial leads < 1.44 mV and those with S-wave ≥ 1.44 mV were compared . Patientp < 0.05 for both).Among 69 patients (median 515 (261–1583) days follow-up) who had at least baseline ECGs, cumulative incidences of the composite endpoint, heart failure readmission, and appropriate electrical defibrillation (applicable for 53 patients with implanted defibrillators) were lower in the patients with higher S-wave amplitude in V1–3 A–C. Cox When we excluded those with LBBB, similar trends remained, although some of them did not reach statistical significance except for heart failure readmission A–C.We investigated the association between baseline QRS amplitude in precordial leads and echocardiographic response to CRT. Although we implanted CRT according to the guideline-recommended criteria, only 53% were CRT responders. The higher S-wave amplitude in right precordial leads was associated with greater cardiac reverse remodeling and clinical outcomes following CRT implantation.CRT improves left ventricular electrophysiological desynchrony using additional left ventricular lead, particularly in those with LBBB. However, not all candidates enjoy satisfactory cardiac reverse remodeling following guideline-indicated CRT implantation. Many recent studies define CRT responder as achieving a reduction of LVESV ≥15% in echocardiographic assessment following a certain period after CRT implantation . Wide QRThe results of this study are supported by the fact that prominent S-waves in right precordial leads are observed in a typical LBBB. However, in this study, some patients were CRT responders and had good clinical outcomes irrespective of the existence of LBBB. Other studies consistently demonstrated that some patients with right bundle branch block or nonspecific ventricular conduction delay also showed favorable response to CRT despite the lack of LBBB ,22. InteWe speculate that the presence of S-wave in right precordial leads might indicate conduction disturbance in the left ventricle irrespective of the type of bundle branch block. The existence of prominent S-wave, a novel and simple index to predict CRT responder that we propose here, might include most of LBBB and some part of non-LBBB.Moreover, the existence of prominent S-wave was associated with small left atrium in this study. Previous studies described that the left atrial area and function are associated with CRT response ,24. The The left axis deviation, which reflects a left anterior fascicular block, is an independent predictor of clinical outcomes following CRT implantation ,25,26. ABy adding the prominent S-wave in right precordial leads to the current guidelines criteria, we might be able to further discriminate CRT responders who can enjoy greater reverse remodeling and clinical outcomes, especially in a non-LBBB pattern such as in intraventricular conduction disturbance. Of note, given a high sensitivity, a prominent S-wave would be more useful to predict non-responders to CRT.First, this was a retrospective observational study including small sample size from a single center, and further prospective randomized larger and multicenter trials are needed. Second, the number of patients with ischemic heart disease was small compared with the previous large cohort, indicating selection bias might exist in this study. Of course, QRS morphology such as right bundle branch block or LBBB, which was not included in adjusted parameters for CRT response, should affect the S-wave amplitude impact. Third, although we used the use of beta-blocker for the adjustment, we cannot completely exclude the impact of beta-blocker use. Fourth, several factors such as pericardial effusion, obesity, and pulmonary emphysema might affect QRS amplitude. Finally, the development of new devices and techniques for the implantation might affect CRT response.The existence of prominent S-wave in right precordial leads would be a key to further discriminate CRT responders and non-responders in addition to the current guideline criteria.


Resistant hypertension (RH) is a major contributor to cardiovascular diseases and is associated with increased all-cause and cardiovascular mortality. Cardiac changes such as impaired left ventricular (LV) function, left ventricular hypertrophy (LVH), myocardial fibrosis, and enlarged left atrium (LA) are consequences of chronic exposure to an elevated blood pressure. The purpose of this review article is to demonstrate the potential benefits of using STE as a non-invasive imaging technique in the assessment of cardiac remodeling in patients with hypertension and specifically in uncontrolled and RH population.It is well-recognized that conventional transthoracic echocardiography is a useful analytic imaging modality to evaluate hypertension-mediated organ damage (HMOD) and in a resistant hypertensive population. More recently two-dimensional speckle tracking echocardiography (STE) has been utilized to provide further risk assessment to this population.Recent data has shown that STE is a new promising echocardiographic marker to evaluate early stage LV dysfunction and myocardial fibrosis over conventional 2D parameters in patients with cardiovascular diseases. Hypertension (HTN) remains the leader of cardiovascular mortality among several risk factors . It has Impaired left ventricular (LV) function, left ventricular hypertrophy (LVH), and myocardial fibrosis are recognized markers of target organ damage, compromised in patients with long standing HTN –11. HoweMyocardial strain refers to the percentage deformation of the myocardium during the cardiac cycle. It represents the extent of regional myocardial deformation in a specified period of time in three orthogonal directions . All were determined by length, thickness, and shortening, using the formula ε = (L-Lο) / Lo, where ε indicates strain (has a unit of %), L indicates length after deformation, and Lo indicates baseline length. Strain rate (SR) refers to the speed at which the myocardium deforms (velocity changes/distance) •.Initially, two techniques were introduced to assess myocardial strain: (i) cardiac magnetic resonance (CMR) in the late 1980s • and  with and without LVH, suggesting that LV mechanical abnormalities precede the development of LVH –29.Normal myocardium consists of cardiac myocytes 30–40%) and non-myocyte components (60–70%) [–40% and Recent studies , 31–33 hFurthermore, fibrosis may have a possible direct effect on the rearrangement of myocardial sheets in subendocardial layers , 37 wherLongitudinal, circumferential, and radial dysfunction do not occur in tandem with longitudinal subendocardial fibers being prone to being compromised first in several pathologies , 41. GLSp < 0.001 per 1% decrease). This association persisted after multivariable adjustment for the following parameters: . [p < 0.01).Studies have shown that GLS might be beneficial as an independent predictor of cardiovascular outcomes in general population –59 and ind E/e).  Similarlnd E/e).  retrospep = 0.003). This difference in the responses between the two genders could be due to the differences in GLS baseline values, where females had higher GLS compared to males. In addition, the association between female gender and improvement in GLS is unclear and has yet to be examined in the general population to confirm sex differences associated with LV function. Another observation found an improvement in GLS by -0.46% for every 5 kg/m2 reduction in body mass index (BMI) (p = 0.015).Cheng et al.  examinedSimilar findings have been reported by other studies , 65 whicNotably, the mid-myocardial layer may remain unchanged or even increased compared to the longitudinal function, which probably explain the well-preserved function reflected by EF , 51, 66 However, longitudinal function is not always the earliest predictor in all circumstances. Previous studies have reported that all three planes of function  may decline in HF, signifying a decompensation mechanism of the LV and impaired myocardial layers as a response to increase myocardial wall stress and disease progress , 69, 70.Rotation, twist, and torsion are several terms to describe additional deformation of the LV caused by the helical arrangement of myocardial fibers. LV rotation is defined as an apical counterclockwise movement and basal clockwise movement in systole. During systole, the LV stores potential energy, which is subsequently released in early diastole. Twist and untwist play an important role by storing and releasing this energy which leads to LV diastolic relaxation and early diastolic filling. Twist/untwist (°) and rate (°/s) are calculated as the net difference between basal clockwise and apical anticlockwise rotation and rotation rate . TorsionMyocardial fiber orientation is a fundamental feature of the myocardium, and it has a substantial role in systolic function. STE imaging is a new non-invasive cardiovascular imaging modality that can be used in clinical practice to understand the mechanism of cardiac deformation, particularly in patients with early compensation of myocardial function and in patients with RH. Using STE also offers comprehensive evaluation to detect the underlying impaired systolic function in several pathologies, including HTN, to deliver optimal management plan. Furthermore, this powerful and valuable technique provides accurate and objective measures on global/regional contractile function.


The cardiovascular mechanisms of injury are poorly understood and speculative. We aim to use multimodality cardiac imaging including cardiac magnetic resonance (CMR) imaging, computed tomography coronary angiography (CTCA) and positron emission tomography with 2-deoxy-2-[fluorine-18]fluoro-This is a single-centre exploratory observational study aiming to recruit 50 patients with COVID-19 infection who will undergo cardiac biomarker sampling. Of these, 30 patients will undergo combined CTCA and 18F-FDG-PET/CT, followed by CMR. Prevalence of obstructive and non-obstructive atherosclerotic coronary disease will be assessed using CTCA. CMR will be used to identify and characterise myocardial disease including presence of cardiac dysfunction, myocardial fibrosis, myocardial oedema and myocardial infarction. 18F-FDG-PET/CT will identify vascular and cardiac inflammation. Primary endpoint will be the presence of cardiovascular pathology and the association with troponin levels.The results of the study will identify the presence and modality of cardiac injury associated COVID-19 infection, and the utility of multi-modality imaging in diagnosing such injury. This will further inform clinical decision making during the pandemic.Trial Registration: This study has been retrospectively registered at the ISRCTN registry (ID ISRCTN12154994) on 14th August 2020. Accessible at https://www.isrctn.com/ISRCTN12154994 The number of people diagnosed with the coronavirus disease 2019 (COVID-19) , 4. ThesAt present, the cardiovascular pathology of injury in COVID-19 patients remain poorly understood, with the underlying mechanism being speculative , 7. To dUsing cardiac magnetic resonance (CMR) imaging –13, compOur principal aims are to identify the cardiac pathophysiological mechanisms related to COVID-19 infection that would influence care and clinical outcomes and provide insight into potential therapeutic targets.This is a single-centre exploratory observational study involving participants diagnosed with COVID-19 receiving care at the Aga Khan University Hospital in Nairobi, Kenya. We aim to recruit around 50 patients with COVID-19 infection with 20 to 30 patients undergoing cardiac imaging. If feasible we will aim to image 15–20 with plasma troponin levels, using a high-sensitivity assay, above the 99th centile upper reference limit (URL) and 5–10 patients with troponin levels within the normal reference range. We will additionally identify a control population of individuals where COVID-19 is suspected but excluded following testing. We will attempt to match controls by age and sex to the COVID-19 positive population. All patients not requiring non-invasive and invasive ventilation will be eligible for recruitment.Inclusion criteria for patients diagnosed with COVID-19 include those over the age of 18 years, testing positive on PCR testing and ideally within 2 weeks of a positive test. Patients will be excluded based on the following conditions: (1) prior diagnosis of myocardial infarction, coronary revascularisation or cardiac surgery; (2) requiring invasive or non-invasive ventilation; (3) inability to undergo CT or CMR scanning; (4) severe renal failure (estimated glomerular filtration rate < 30 mL/min); (5) Major allergy to iodinated contrast media/gadolinium; (6) pregnancy or breast feeding; (7) inability to give informed consent; (8) contraindication to imaging example metal fragments in the eye. Similarly exclusion criteria will apply to patients over the age of 18 years chosen as controls and testing negative for COVID-19.We will also explore stratification by clinical parameters of COVID-19 severity. As such we will also provide baseline characteritics and imaging parameters by clinical requirement of oxygen supplementation.All staff involved in scanning patients will wear personal protective equipment (PPE) in line with hospital policy, and follow protocols created by the study team including physicians from the infectious diseases team. Patients will be scanned last on the list followed by a deep clean to the scanning area to prevent nosocomial cross infection. Routine clinical scans will be prioritised over research scans. No aerosol producing procedures will be performed.At initial recruitment, patients will undergo blood sampling for high-sensitivity cardiac troponin, N-terminal pro B-type natriuretic peptide and high-sensitivity C Reactive protein. A subset of patients will be selected for multimodality cardiovascular imaging. These participants will undergo two visits for a combined CTCA and 18F-FDG-PET/CT as soon as possible, followed by a CMR on another visit. All CTCA and 18F-FDG-PET/CT will be performed within 2 weeks of presentation to maximise sensitivity of the scan. All efforts will be made to ensure CMR scans are also performed within 2 week, however scanning beyond this point will be performed if it is logistically impossible to perform the scan within this window.CTCA scanning will be ECG-gated and performed in diastole during a single breath hold with prospective electrocardiographic gating, detector collimation 64 × 0.625 mm, tube voltage 120 kV and window of acquisition 70–90% (or wider if necessary due to heart rate). Tube current will vary depending on BMI using a pre-specified manufacturer protocol. After the acquisition of scout images, CTCA will be performed with iodinated contrast (Ultravist 370 mg/mL) in a biphasic injection protocol. Image acquisition will be triggered by contrast enhancement of 100 HU in the ascending aorta. In order to optimise the quality of the CTCA scan images, heart rate control will be pharmacologically achieved if appropriate, and in addition sublingual glyceryl trinitrate will be administered.Participants will undergo 18F-FDG-PET/CT imaging after high-fat, low-carbohydrate meal for 24 h with 18 h fast to reduce physiologic myocardial FDG uptake , 16. TheStandard CMR images will be acquired as previous –19. In bGadolinium-based contrast agent will be intravenously administered. Myocardial infarction and fibrosis will be identified by the presence of hyperenhancement in the myocardium in late enhancement images, acquired using a T1-inversion sequence with inversion time optimised on a patient-by-patient basis . MultiplAll scans will be anonymised, with analysts blinded to all blood results and other scanning modalities. The cardiac MRIs will be analysed in Manchester (UK), PET scans in Bristol (UK) and CTCAs in Edinburgh (UK). There will be no discussion regarding scans between the centres. When a second opinion is needed, it will be sought from an independent radiologist in Nairobi (Kenya). All analysis will be performed by expert cardiologists trained in the relevant cardiac imaging modalities. The second opinion, when sought, will be performed by expert cardiothoracic radiologists.The presence of coronary artery disease in each major coronary artery, and the main side branches will be documented based on degree of stenosis. In addition presence of coronary calcification, other cardiac or vascular pathology, pericardial pathology and aortic/main pulmonary artery diameter will be recorded.Arterial inflammation: Aortic inflammation will be assessed as previous , 22. In Myocardial inflammation: Myocardial uptake will be assessed as previous –27. In bVisual assessment of myocarditis:A visual assessment of FDG uptake (on diagnostic scans) that signifies myocarditis will be categorised: None—1, focal on diffuse—2, Focal—3, Diffuse—4.Objective assessment of myocarditis:Each segment of the 17 segment model, will be assessed for myocardial uptake visually. In addition, regions of interest within each myocardial segment of the 17-segment model will be drawn avoiding the blood pool and liver.A mean cardiac SUV will be derived from the average of the maximum SUV values in serial short axis measurements (Sum of each segment /17).Raw SUV uptake compared to blood pool uptake will be calculated for each segment, and categorised.TBR (target-to-background-ratio) will be calculated by dividing each cardiac segment SUV by the mean blood pool SUVEach segment indexed to the mean blood pool will also be categorised (0—uptake < mean blood pool uptake),1—uptake = mean blood pool uptake,2—uptake > mean blood pool uptake but < liver uptake, 3—(uptake > liver uptake)Assessment of each segment of the 17 segment model will be performed in 3 different ways as follows.Ejection fraction, regional wall motion abnormalities, myocardial fibrosis, oedema and presence of infarction will be determined as previously described , 13, 29.Identify the proportion of patients with biochemical evidence of myocardial injury or strain with the presence of elevated cardiac troponin.Identify significant atherosclerotic disease potentially causing cardiac injury with the presence of obstructive coronary artery disease on CTCA (> 50%).Identify myocardial disease using CMR by determining ejection fraction, presence of myocardial infarction (sub-endocarditis gadolinium enhancement), myocarditis  or myocardial oedema Identify vascular inflammation by 18F-FDG-PET/CT by quantifying increased FDG uptake in aorta. Determining the presence of different patterns of FDG uptake in myocardium will be a secondary exploratory outcome.We have four primary objectives:Based on published literature we based our sample size calculation on the assumption that 1 in 4 of hospitalised COVID-19 patients will have elevated cardiac troponin . In sampP value of less than 0.05 will indicate statistical significance.Baseline clinical and imaging data will will be expressed as the mean ± standard deviation (SD) for parametric data and median (interquatile range) for non-parametric data. Categorical data will be presented as proportions. We will conduct hypothesis testing based on imaging parameters comparing (a) controls and patients and (b) within patients, those stratified by troponin concentrations. For within patient analysis we will attempt to compare individuals stratified above and below the 99th centile for cardiac troponin concentration. If the number of patients above the 99th centile for cardiac troponin are small, we will group patients by equal strata of cardiac troponin value. We will also explore stratification by clinical parameters of COVID-19 severity. As such we will provide also provide baseline characteritics and imaging parameters by clinical requirement of oxygen supplementation. In order to understand cardiac complications secondary to COVID-19, this study aims to utilise multimodality cardiac imaging to identify any morphological and functional cardiovascular changes associated with infection. To date, whilst data has shown that myocardial injury is prevalent in patients with COVID-19, cardiac imaging has either been limited to case reports and small case series, performed after recovery using single modalities or using study designs highly susceptible to selection/reporting –10.To our knowledge, our study represents the first multi-modality cardiac imaging study, with each technique attempting to elucidate specific pathophysiological processes. CTCA will examine the presence of coronary disease associated directly or indirectly with elevated cardiac troponin concentrations, and its absence would imply Type 2 myocardial infarction in the correct context. CMR will evaluate cardiac function and dysfunction including structural evidence of heart failure resulting from either ischaemic or non-ischaemic cardiomyopathy (e.g. stress cardiomyopathy). It will also identify myocardial infarction or fibrosis caused by myocarditis. 18F-FDG-PET/CT can assess for myocardial and vascular inflammation that would delineate if myocardial oedema (seen on CMR) is caused by direct cardiac injury or as part of a generalised inflammatory process.Identification of each pathophysiological process will inform future studies in evaluating optimal choices of therapeutic agents; (1) anti-platelet and anti-coagulation agents for atherosclerotic disease or thrombotic occlusions; (2) Blood pressure and oxygen support in the presence of type 2 myocardial infarction; (3) anti-viral or anti-inflammatory strategies for a primary myocarditic processes; (4) early and long term cardioprotective therapy for myocardial dysfunction.There are several limitations and considerations that need to be taken into account with regards to our study design. First, given the imaging strategy undertaken, patients must be fit enough for example to breathhold. As such our study primarily aims to recruit COVID-19 cases who are not requiring invasive or non-invasive ventilation. Our findings may therefore not be generalisable to severe cases of COVID-19. Second, recent evidence has shown the role of anti inflammatories in managing patients with COVID-19. These drugs are likely to modulate the degree of inflammatory signal seen in the myocardium and arterial tree and may underestimate the true pathophysiological effect of the disease on the heart. Thirdly, manual selection or regions of interest during scan analysis is liable to error. This may be compounded by diminished scan quality due to infected patients being short of breath or tachycardic. Finally, the observational trial aims to recruit a relatively small number of patients, and clinical outcomes will not be evaluated. However there is a lack of data and well designed studies investigating COVID-19 cardiovascular injury, and the study should provide valuable insights.


Arrhythmogenic cardiomyopathy is a myocardial disorder characterized by ventricular arrhythmias, right and/or left ventricular involvement, and fibrofatty infiltrations in the myocardium. We report a family diagnosed with arrhythmogenic left ventricular cardiomyopathy (ALVC) and depict their echocardiographic characteristics.Fifteen family members were divided into three groups based on whether they carried the TMEM43 mutation and had been diagnosed with ALVC. Eight of them had TMEM43 mutations, and four were diagnosed with ALVC according to the Padua criteria. Only the proband experienced sudden cardiac death and had a dilated left ventricle. Left ventricular ejection fraction was reduced in two patients; however, left ventricular global longitudinal strain was depressed in three patients. Low QRS voltages in limb leads were evident in three patients, and five patients had frequent ventricular premature contractions. Late gadolinium enhancement was evident in three patients. Left ventricular layer-specific strain showed that the transmural strain gradient ratio was increased in patients diagnosed with ALVC, and it was elevated in the genotype-positive and phenotype-negative groups compared with healthy individuals.Global left ventricular longitudinal strain better evaluated left ventricular function than left ventricular ejection fraction. The transmural strain gradient ratio was elevated in patients diagnosed with ALVC, suggesting that it was useful for the evaluation of ALVC. An expert panel of the Heart Rhythm Society (HRS) defined arrhythmogenic cardiomyopathy (ACM) as an arrhythmogenic disorder of the myocardium that is not explained by ischemia, hypertensive, or valvular disease . ArrhythThe study was approved by the local research ethics committee, and all individuals provided written informed consent. Fifteen pedigree members were included in this study, and four of them were diagnosed with ALVC at the First Affiliated Hospital of Soochow University. Clinical evaluation included 12-lead ECG, 24-h Holter, echocardiography and contrast-enhanced cardiac magnetic resonance (CE-CMR). Clinical manifestations, disease history, and family history of the pedigree members were also collected.Diagnosis was based on the Padua criteria for ALVC. These criteria were developed by a multidisciplinary team of basic researchers and clinical cardiologists at the Medical School of the University of Padua by taking into account the tissue characterization findings by contrast-enhanced cardiac magnetic resonance, depolarization/repolarization ECG abnormalities, and ventricular arrhythmia features for the diagnosis of the left ventricular phenotype, which are grouped into six categories . LV lateEchocardiographic measurements were performed by a board-certified echocardiographer. Two-dimensional biographic imaging of all subjects was performed using GE Vivid E95 equipment (Norway) with a 2.5 MHz transducer. All images were stored in a digital format for offline analysis . Dynamic 2D images of the apical four- and two-chamber views were used to analyze the auto-EF, and three anchor points were set within the LV cavity, including two at the level of the mitral valve annulus and one at the LV apex. Endocardial borders were then detected and traced automatically by the software during the whole heart cycle to calculate left ventricular end-diastolic volume, left ventricular end-systolic volume, and left ventricular ejection fraction (LVEF). When needed, corrections were performed manually.Left ventricular longitudinal strain was assessed using two-dimensional speckle tracking echocardiography in three standard apical views. The region of interest was adjusted to ensure optimal analysis of most of the LV walls based on the American Society of Echocardiography /European Association of Echocardiography consensus statement . Each apAll participants underwent CMR at 1.5 T or 3 T . All images were analyzed using CVI42 software . Measurements were performed by two experienced cardiologists blinded to both clinical and genetic data (E.N. and S.M.F.). LV late gadolinium enhancement (scar) quantification was performed in the short-axis slices using manually drawn endocardial and epicardial borders and a semiautomated 5 SD approach with minimal manual adjustment and was expressed in grams and as a percentage of the total LV mass.The chip capture sequencing technique was used to capture all exons and shear sites related to ACM, and high-throughput sequencing was used to test the genetic mutations. Sanger sequencing was performed to verify suspected mutation sites responsible for the disease, which were detected by high-throughput sequencing. All gene assay were performed using Mygenostics.Statistical analysis was performed using SPSS 25.0 and GraphPad Prism 8.0.1. The family members were divided into three groups based on whether they carried the TMEM43 mutation and had been diagnosed with ALVC. Differences in continuous variables between the two groups were analyzed using Student’s t test.Figure TMEM43 mutations were detected in eight family members, of which 4 were diagnosed with ALVC according to the Padua criteria Table , which wPadua criteria: Major criteria include left ventricular late gadolinium enhancement (LGE) of ≥ 1 bull’s eye segment (s) of CE-CMR and family history/genetics. Minor criteria include global LV systolic dysfunction with or without LV dilatation, regional LV hypokinesia or akinesia of the LV free wall, septum, or both, inverted T waves in the left precordial leads (V4-V6), low QRS voltages in limb leads, frequent ventricular premature contractions (> 500 per 24 h), nonsustained or sustained ventricular tachycardia with RBBB morphology; and family history and/or genetics. Diagnosis is fulfilled by 2 major or 1 major and 2 minor criteria from different categories.Global longitudinal strain and left ventricular ejection fraction were reduced, while the number of ventricular premature contractions in 24 h and LVEDVI were increased in patients diagnosed with ALVC. Global longitudinal strain and LVEF in genotype-positive and phenotype-negative patients were not significantly reduced compared to those in healthy individuals Figs.  and 4.FiThe left ventricular layer-specific strain showed that the transmural strain gradient ratio was increased in patients diagnosed with ALVC, and it was elevated in the genotype-positive and phenotype-negative groups compared with that in healthy individuals Figs.  and 6.FiArrhythmogenic cardiomyopathy is an inherited cardiac disease characterized by fibrofatty replacement of the myocardium, in both the right and left ventricles . We repoMutant TMEM43 was first described in families from Newfoundland (Canada) in 2008, as the cause of a fully penetrant aggressive disease with a high incidence of malignant ventricular arrhythmias, and was defined as ACM/ARVC type 5 . HoweverInverted T waves in left precordial leads, low QRS voltages in limb leads, frequent ventricular premature contractions, and nonsustained or sustained ventricular tachycardia were ECG depolarization, repolarization abnormalities, and ventricular arrhythmias in the Padua criteria . In the Five family members underwent CMR, and LGE was evident in three patients, suggesting cardiac scarring. Fibrofatty myocardial replacement was confirmed by endomyocardial biopsy in prior studies , 8. PrevLeft ventricular ejection fraction was reduced in two patients, while GLS was decreased in all 4 patients who were diagnosed with ALVC according to the Padua criteria, suggesting that left ventricular strain better evaluates left ventricular function than left ventricular ejection fraction. Region left ventricular akinesia or dyskinesia was evident in five patients, and it was more apparent in the posterior and lateral LV walls. The reduction of the segmental left ventricular longitudinal strain in bull’s eye map was similar to that of two-dimensional echocardiography, which showed that left ventricular strain values were more frequently reduced in the posterior and lateral left ventricular walls. Regional left ventricular hypokinesia or akinesia was one of the diagnostic criteria for ALVC; however, the reduction in left ventricular strain in the bull’s eye segments was more sensitive and accurate than two-dimensional echocardiography, which should be considered in the future diagnostic criteria for ALVC.Left ventricular layer-specific strain showed that the transmural strain gradient ratio was increased in patients diagnosed with ALVC, and it was elevated in the group with genotype-positive and phenotype-negative patients compared with that in the normal group. A previous study showed that fibrofatty myocardial replacement affects the subepicardial left ventricle more than other layers , which mIn this family diagnosed with ALVC, global left ventricular longitudinal strain better evaluated left ventricular function than left ventricular ejection fraction, and the segmental left ventricular strain was more accurate and consistent with 2D echocardiography. The transmural strain gradient ratio was elevated in patients diagnosed with ALVC, suggesting that layer-specific strain is useful for evaluating patients with ALVC.


Pseudomonas aeruginosa (Pa) provokes NEU1 sialidase-mediated MUC1 ectodomain (MUC1-ED) desialylation and MUC1-ED shedding from murine lungs in vivo. Here, we asked whether Pa in the lungs of patients with ventilator-associated pneumonia might also increase MUC1-ED shedding. The levels of MUC1-ED and Pa-expressed flagellin were dramatically elevated in bronchoalveolar lavage fluid (BALF) harvested from Pa-infected patients, and each flagellin level, in turn, predicted MUC1-ED shedding in the same patient. Desialylated MUC1-ED was only detected in BALF of Pa-infected patients. Clinical Pa strains increased MUC1-ED shedding from cultured human alveolar epithelia, and FlaA and FlaB flagellin-expressing strains provoked comparable levels of MUC1-ED shedding. A flagellin-deficient isogenic mutant generated dramatically reduced MUC1-ED shedding compared with the flagellin-expressing wild-type strain, and purified FlaA and FlaB recapitulated the effect of intact bacteria. Pa:MUC1-ED complexes were detected in the supernatants of alveolar epithelia exposed to wild-type Pa, but not to the flagellin-deficient Pa strain. Finally, human recombinant MUC1-ED dose-dependently disrupted multiple flagellin-driven processes, including Pa motility, Pa biofilm formation, and Pa adhesion to human alveolar epithelia, while enhancing human neutrophil-mediated Pa phagocytosis. Therefore, shed desialylated MUC1-ED functions as a novel flagellin-targeting, Pa-responsive decoy receptor that participates in the host response to Pa at the airway epithelial surface.We previously reported that flagellin-expressing  This common nosocomial infection occurs in approximately 10% of ventilated patients2. Although the reported incidences per 1000 ventilator days has decreased from 3.1 to 0.9 in medical intensive care units (ICUs), and from 5.2 to 2.0 in surgical ICUs, attributable morbidity and mortality remain relatively high4. All-cause mortality associated with VAP is reportedly 20% to 50%, while attributable mortality is estimated to be 13%5. VAP is associated with prolonged hospitalization and increased cost. In risk-matched patients either with or without VAP, the presence of pneumonia added an average of US $40,000 to hospital costs and 10 days to total time on mechanical ventilation, ICU stay, and/or overall hospitalization6. Prolonged intubation is a primary driver of infection, with risk of VAP increasing by 2% per day, starting in the second week post-intubation7. Other modifiable risk factors include the use of antacids, supine position, and receipt of enteral nutrition. Non-modifiable risk factors include coma, age greater than 60 years, head trauma, chronic obstructive pulmonary disease, and male gender. Intervention-related factors, such as neurologic or thoracic surgery and reintubation, contribute to VAP as well7.Ventilator-associated pneumonia (VAP) is defined by the appearance of a new pulmonary infiltrate of infectious origin occurring more than 48 h after endotracheal (ET) intubation1. This translates to positive and negative likelihood ratios of 2.5 and 0.06, respectively8. However, no known combination of signs and symptoms provides a definitive diagnosis. Addition of noninvasive, semiquantitative sputum cultures by ET aspirate, can help with organism- and susceptibility-directed therapy1. In these critically-ill patients, the absence of a reliable diagnostic approach can lead to over-diagnosis and overuse of broad-spectrum antibiotics, encouraging the development of multidrug-resistant (MDR) pathogens9.For the diagnosis of VAP, the findings of a new pulmonary infiltrate in the setting of fever, purulent sputum, leukocytosis, and hypoxemia provide a reported sensitivity of 69% and specificity of 75%10. Whether pneumonia ensues is dictated by organism virulence factors, inoculum size, and the host immune response11. In the ventilated patient, the presence of an ET tube impairs the cough reflex, decreasing the ability to expel pathogens. Over time, the ET tube luminal surface serves as a nidus for bacterial colonization and biofilm formation that can be impregnable to antibiotics. These bacteria can then be introduced into the lower respiratory tract by positive ventilatory pressure10.The predominant mechanisms underlying VAP pathogenesis include microaspiration of oropharyngeal secretions and bacterial biofilm formation within the ET tubePseudomonas aeruginosa (Pa), a flagellated, Gram-negative bacillus, is a leading pathogen implicated in VAP, with a reported frequency approaching 25%12. Nonventilated patients who are debilitated with burns, neutropenia, or cystic fibrosis are also at increased risk of Pa acquisition13. Pa commonly colonizes healthcare workers, and in one study, was recovered from 22% of hospital rooms8. Hospital reservoirs include showers, cleaning equipment, bronchoscopes, endoscopes and respiratory therapy equipment13. Antibacterial-susceptible strains that colonize patients can become MDR through ubiquitous antibiotic use and selective pressure.15. This mucus blanket made up of two distinct yet interacting layers, a highly viscous gel layer overlying a periciliary liquid layer. Members of the mucin family comprise the major protein component of mucus and are divided into secreted and cell-associated mucins. Of the 5 major mucins that have been detected in the lung, two are secreted, MUC5AC and MUC5B, and three are cell-tethered, MUC1, MUC4, and MUC16. MUC1 is comprised of an NH2-terminal MUC1 ectodomain (MUC1-ED) coupled to a COOH-terminal MUC1 cytoplasmic domain (MUC1-CD)15. The MUC1-ED contains a variable number of highly sialylated, 20-amino acid (aa) tandem repeats distal to a juxtamembranous glycine-serine protease recognition site. The proline-rich MUC1-ED exhibits an extended, rod-like conformation that protrudes higher above the EC surface than other membrane-associated proteins16. Here, the MUC1-ED is strategically positioned to interact with flagellin-expressing Pa in the airway lumen. The MUC1-ED is up to 90% carbohydrate, almost entirely through more than 500 predicted O-linked glycosylation sites within its tandem repeats17. The relatively short, 72-aa MUC1-CD contains multiple binding sites for cytosolic signaling molecules20.The first host barrier with which Pa interacts in the airways is the mucus blanket which covers the epithelium21 293 cells enhanced their adhesiveness for wild-type Pa, NEU1 overexpression in Toll-like receptor (TLR)5-expressing HEK293 cells did not21. Although NEUs are well established virulence factors for multiple bacterial pathogens24, this is the only case, to our knowledge, where a prokaryote hijacks a host sialidase to influence its pathogenicity. At the same time, we also found that NEU1-mediated MUC1-ED desialylation uncovers a juxtramembranous protease recognition site, permitting MUC1-ED cleavage by endogenous sheddases and release from the EC surface27  production, and neutrophil-mediated Pa phagocytosis.We had previously established that Pa-expressed flagellin engages the MUC1-ED expressed on the surface of airway epithelial cells (ECs)22. We asked whether MUC1-ED levels also might be increased in BALF from patients with Pa airway infection. Between 12 June 2013 and 20 February 2020, BALF was collected from 61 patients undergoing mechanical ventilation and clinically diagnosed with VAP in three separate units at the University of Maryland Medical Center . Of these 61 patients, the BALF of 13 (21.3%) were culture-positive for Pa, 32 (52.5%) culture-positive for other microorganisms, 6 of which were polymicrobial, and 16 (26.2%) culture-negative (Table vs. Pa-negative (n = 48) patients. Pa-infected VAP patients were more likely to have been admitted to the long-term Comprehensive Pulmonary Rehabilitation Unit (CPRU) (61.5% vs. 12.5%), and experienced fewer ventilator-free days (8.2 vs. 18.7 days), compared with Pa-negative patients (Table vs. 81.2%). None of the other clinical/demographic parameters measured were different between Pa-infected patients and Pa-negative patients.We previously reported that Pa increases MUC1-ED shedding using an in vivo mouse model of Pa pneumoniaMUC1-ED levels in these 61 BALF samples were quantified by enzyme-linked immunosorbent assay (ELISA) and normalized to total BALF protein. Mean ± S.E. normalized MUC1-ED levels were 4.01 ± 0.19 µg/mg BALF protein  in the BALF from the 13 Pa-infected VAP patients compared with 0.71 ± 0.05 µg/mg  in the BALF from the 32 patients infected with other microorganisms and 0.52 ± 0.08 µg/mg  in the BALF from the 16 culture-negative patients , which binds to subterminal galactose residues only after removal of terminal sialic acid, was used to address this question. First, we validated the specificity of the PNA lectin. As anticipated, PNA recognized asialofetuin, but not sialylated fetuin s and BALFs simultaneously collected from a subset of VAP patients (n = 15), including 2 patients infected with Pa, 6 patients infected with other microorganisms, and 7 culture-negative patients. No correlation was observed between MUC1-ED levels in the paired BALF and TA samples 4 as a bacterial lipoprotein mimetic, and CpG oligodeoxynucleotide (ODN) 1826 as a bacterial DNA mimetic, each failed to increase MUC1-ED shedding FlaA and rFlaB, cloned from PAK and PA01, respectively, stimulated MUC1-ED shedding -derived flagellin30. STm-derived flagellin provoked far less MUC1-ED shedding than did Pa-derived flagellin, excluding participation of TLR5  dose-dependently increased MUC1-ED shedding compared with an identical inoculum of PAK/fliC¯  broth, a mean lung burden of 5.52 × 108 CFU/mg  was estimated for the 13 Pa-infected VAP patients  was estimated for the Pa-infected VAP patients  was estimated for the Pa-infected VAP patients  to MUC1-ED levels in BALFs that cultured positive for FlaB-expressing Pa (n = 6) . However, as with MUC1-ED levels  to establish its binding to the intact Pa flagellum. Gold-labeled anti-mouse IgG secondary antibody immunolocalized to the Pa flagellar filament following incubation of the bacteria with human rMUC1-ED and mouse anti-MUC1-ED antibody -8 production, and pulmonary leukostasis, predominantly with polymorphonuclear leukocytes (PMNs)35. In a prospective study of mechanically ventilated ICU patients across 56 sites in 11 countries, the incidence of Pa pneumonia ranged from 13.5% to 19.4%36. In a review of 24 studies, Pa was the most frequent isolate from ICU VAP patients, accounting for 24.4% of all microorganisms isolated12. Of the 13 Pa-positive patients in our study, 8 (61.5%) were identified in a chronic ventilator Comprehensive Pulmonary Rehabilitation Unit and 5 (38.5%) in an acute tertiary care medical ICU  compared with Pa-negative patients (18.7 ± 10.0 days).In the current study, 21.3% of mechanically ventilated patients were infected with Pa. Pa is the second most common microorganism isolated in U.S. hospital-acquired lung infections with 90,000 cases per year22. More specifically, purified Pa-derived flagellin dose-dependently increased MUC1-ED shedding from the murine airway epithelium into the brochoalveolar compartment. In the current study, BALF levels of the two Pa flagellin types, FlaA and FlaB, each could be tightly correlated with BALF levels of shed MUC1-ED in the same patient. Pa flagellin levels predicted MUC1-ED levels in a concentration-dependent manner. In these same patients, we found a greater than 6.0-fold increase in the mean ± S.E. MUC1-ED level in the BALF of Pa-infected VAP patients (4.01 ± 0.19 µg/mg BALF protein) compared with that observed in Pa-negative patients (0.66 ± 0.05 µg/mg) , whereas all patients who were infected with other microorganisms or culture-negative had MUC1-ED levels ≤ 1.55 µg/mg . Based on these data, 2.0 µg MUC1-ED/mg BALF protein provides a reliable diagnostic cut-off value to discriminate between Pa-infected and non-Pa-infected patients. Elevated BALF MUC1-ED levels may offer a predictive biomarker for Pa lung infection in VAP patients. The MUC1-ED ELISA is more rapid, less labor intensive, and less expensive than conventional bacterial cultures. However, the MUC1-ED ELISA identifies only Pa, and unlike bacterial cultures, does not provide antibiotic sensitivities. Further studies are required to establish whether elevated BALF MUC1-ED levels might also result from S. maltophilia lung infections.The current study supports Pa-derived flagellin as a ligand for the MUC1-ED, and suggests that flagellin engagement of the cell-associated MUC1-ED provokes MUC1-ED shedding in the airways of VAP patients. Using an in vivo experimental murine model to explore lung responsiveness to Pa, we have established flagellin-driven MUC1-ED shedding from the airway EC surface as a novel, Pa-selective host response to lung infectiontro Fig. C, whethe22. While NEU1 has historically been studied in context of its indispensable role in glycan catabolism37, far less is known about its role in the host response to septic processes, including Pa lung infection. We previously established NEU1 as the predominant sialidase expressed in human airway epithelial cells23. Silencing of NEU1 expression reduces total human lung airway EC sialidase activity by more than 70%. We detected NEU1 immunostaining in the superficial epithelium along the entire human airway, including the brush border23. This NEU1 expression pattern closely correlates with that known for MUC1 in these same tissues39. The MUC1-ED isolated from BALF of Pa-infected patients was desialylated  disrupting one or more flagellin-driven processes would be predicted to protect against Pa pathogenesis. At MUC1-ED concentrations similar to those found in the BALF of Pa-infected patients, the Pa flagellin-targeting, E. coli-expressed human rMUC1-ED decoy receptor reduced Pa motility, adhesion to alveolar ECs and biofilm formation, as well as IL-8 production, and enhanced neutrophil-mediated Pa phagocytosis. Whether MUC1-ED bound to Pa flagellin functions as an authentic opsonin is unclear. Human rMUC1-ED exerted no measurable bacteriostatic or bactericidal activity. Through its ability to impair flagellin-driven Pa motility, MUC1-ED may interfere with Pa penetration of the mucus blanket40, invasion of the epithelium23, movement through the paracellular pathway into subepithelial tissues41, and/or Pa resistance to mucociliary clearance42. Inhibition of flagellin-dependent Pa adhesion to host airway epithelia likely decreases Pa colonization and invasion of subepithelial tissues. Disruption of the MUC1-ED-Pa flagellin receptor-ligand interaction blocks MUC1-driven downstream intracellular signaling43, biosynthesis of proinflammatory cytokines/chemokines45, pulmonary leukostasis22, and neutrophil recruitment to the bronchoalveolar compartment46. On the other hand, NEU1-mediated release of sialic acid residues from the highly sialylated MUC1-ED substrate may facilitate their incorporation into sialic acid-rich Pa biofilms33 and/or dampen complement activation47, thereby contributing to Pa pathogenesis.Pa flagellin constitutes an important virulence factor through bacterial motility, adhesion to and invasion of host epithelia, and inflammatory gene expression via engagement of host TLR5S. maltophilia, but not other recognized airway pathogens, increased MUC1-ED levels in cell culture supernatants and purified rFlaA and rFlaB flagellins recapitulated the effect of intact Pa bacteria. E. coli-expressed, human rMUC1-ED dose-dependently inhibited Pa motility, adhesion to alveolar epithelia, biofilm formation, and IL-8 production, while at the same time, enhanced neutrophil-mediated Pa phagocytosis, without influencing bacterial growth. The current studies expand our previous observation of a flagellin-provoked, NEU1-mediated generation of a MUC1-ED decoy receptor in a mouse model of Pa pneumonia22 to human in vivo pathophysiology. Measurement of MUC1-ED and flagellin BALF levels offer a rapid, reliable means to identify ventilated patients with Pa pneumonia that might serve as a guide for empiric antibiotic therapy. Of note, the mechanism of action of most conventional antibiotics requires bacterial replication and cell wall remodeling13. It is conceivable that human rMUC1-ED, which itself does not alter bacterial replication, might be combined with antibiotics for synergistic therapy against MDR Pa.In conclusion, MUC1-ED and Pa-derived flagellin were detected in the BALF of Pa-infected VAP patients at dramatically elevated levels compared with those found in Pa-negative patient BALF. Extending the ex vivo studies to an in vitro model of Pa infection, stimulation of human alveolar ECs with either Pa or This study was conducted in accordance with the Declaration of Helsinki and other local statutes or regulations protecting subjects in biomedical research, and was approved by the Institutional Review Boards of the University of Maryland Baltimore (protocol numbers HP-00059183 and HP-00083805). Informed consent was obtained from all participants.48. Clinical diagnosis of VAP was based on findings of a new pulmonary infiltrate by chest x-ray in the setting of fever, purulent sputum, leukocytosis, and diminished oxygenation1.This study was conducted using BALF collected from VAP patients at the University of Maryland Medical Center . VAP patients were admitted either to the 29-bed Medical Intensive Care Unit (MICU) or the 9-bed Critical Care Resuscitation Unit (CCRU) of the University Teaching Hospital, or received prolonged mechanical ventilation in the 14-bed Comprehensive Pulmonary Rehabilitation Unit (CPRU) of the long-term acute care University Specialty Hospital. Patient data were collected as part of routine hospital standard of care, including age, race, gender, body mass index (BMI), ICU location, ventilator-free days, antibiotics administered, chest x-ray, APACHE II score, and microbiology culture results. Bacterial identification of surveillance cultures was done at the University of Maryland Medical Center Clinical Microbiology Laboratory following standard laboratory procedures using the Vitek MS for bacterial identification and Vitek-2 system for antimicrobial susceptibilities  and interpreted according to Clinical and Laboratory Standards Institute (CLSI) criteria49.BAL was performed on VAP patients undergoing standard of care diagnostic bronchoscopy. Briefly, after conscious sedation with fentanyl and midazolam, and local anesthesia with 2% lidocaine, the bronchoscope was wedged in a 3rd or 4th order bronchus, after which 125 ml of sterile pyrogen-free 0.9% NaCl was injected in 25-ml aliquots. The BALF was retrieved, pooled, filtered through sterile gauze, centrifuged at 450 xg to remove cells, and the supernatant concentrated 25-fold by ultrafiltration and stored until use at − 80 °C. Tracheal aspiration was performed using a 12 French siliconized polyvinyl chloride tracheal aspiration probe  as describedL. pneumophila, S. pneumoniae, H. influenzae, S. aureus, and K. pneumoniae were previously described52. S. maltophilia strain 810–2 was obtained from the American Type Culture Collection . All bacteria were cultured at 37 °C in LB medium  , and quantified spectrophotometrically at A600. In selected experiments, Pa strains PA01 and PAK were cultured at 37 °C in DMEM containing 10% FBS  in the presence or absence of confluent monolayers of A549 human alveolar ECs (American Type Culture Collection).Pa strains PA01, GFP-PA01, PAK, and PAK/fliC¯, Pa clinical strains isolated from pneumonia patients, 54. Pa and STm flagellins were incubated with polymyxin B-agarose (Thermo Fisher Scientific) to remove LPS, after which less than 0.1 endotoxin units/µg of protein was detected by the Limulus amebocyte lysate assay.An overnight culture of PA01 was centrifuged at 5000×g for 30 min, resuspended in Krebs–Ringer buffer, and incubated for 1 h at 37 °C. The bacteria were collected by centrifugation and the supernatant was filtered through a 0.22 μm pore membrane and the filtrate boiled for 20 min. The filtrate was concentrated by centrifugal ultrafiltration, adjusted to pH 6.0, and flagellin purified by sequential ion exchange chromatography using Macro-Prep High S and Macro-Prep High Q resins (Bio-Rad). Flagellin was purified from STm strain CVD1925 as describedFlaA (GenBank accession no. CP020659.1) and FlaB (GenBank accession no. AE004091.2) genes from PAK and PA01, respectively, were codon optimized for expression in Escherichia coli , cloned into pUC57 and pJET1.2, respectively, and the plasmids transformed into E. coli NEB 5-alpha competent cells . The FlaA and FlaB genes were amplified by PCR, cloned into pTrcHis-TOPO (Thermo Fisher Scientific), and expressed in E. coli BL21 competent cells (New England Biolabs) for production and purification of 6XHis-tagged rFlaA and rFlaB proteins54. The 6X-His-FlaA and FlaB proteins were purified on a nickel-nitrilotriacetic acid affinity column (Thermo Fisher Scientific), their identities verified by immunoblotting with anti-FlaA and anti-FlaB antibodies, and purities confirmed by a single Coomassie blue-stained protein band following SDS-PAGE.The DNA sequences of the 5 cells/well) in 24-well plates were incubated for increasing times at 37 °C with 1.0 × 107 CFUs/well of WT-PAK or the PAK/fliC¯ flagellin-deficient strain, or were incubated for 24 h with increasing inocula WT-PAK or PAK/fliC¯. In other experiments, A549 cells were incubated for 24 h with 1.0 × 107 CFUs/well of Pa strains PA01, WT-PAK, or PAK/fliC¯, Pa clinical strains isolated from pneumonia patients, S. maltophilia, L. pneumophila, S. pneumoniae, H. influenzae, S. aureus, or K. pneumoniae. In still other experiments, A549 cells were incubated with 100 ng/well of Pa LPS , 10 µg/well of Pam3Cys-Ser-(Lys)4 (Sigma), 10 µg/well of CpG ODN 1826 , 10 ng/well of Pa or STm flagellins, 10 ng/well of Pa rFlaA or rFla flagellins, or the PBS vehicle control. MUC1-ED levels in cell culture supernatants were measured by ELISA and normalized to total cell protein.A549 cells . The wells were incubated for 2 h at 22 °C with 200 µg/ml of HRP-conjugated goat anti-mouse IgG secondary antibody , and bound antibodies detected with tetramethylbenzidine substrate (KPL) at A22.Equal protein aliquots of BALFs were resolved by SDS-PAGE. In order to adequately visualize the > 250 kDa MUC1-ED protein, discontinuous gel electrophoresis was performed under reducing conditions in 3% stacking/5% separating acrylamide gels containing 25 mM Tris–HCl, 190 mM glycine, 0.1% SDS, pH 8.3 until the 150 kDa prestained protein molecular weight marker reached the bottom of the gel. The resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes  and the membranes probed with rabbit anti-mouse MUC1-ED antibody, followed by HRP-conjugated goat anti-rabbit IgG secondary antibody and enhanced chemiluminescence reagents (Thermo Fisher Scientific), as describedEqual protein aliquots of BALF were incubated with PNA immobilized on agarose beads  to selectively enrich for PNA-binding proteins and the PNA-bound proteins resolved by SDS-PAGE as described above. The resolved proteins were transferred to PVDF membranes, and the membranes processed for MUC1-ED immunoblotting as above. Asialofetuin (1.0 µg) was used as a positive control for PNA-binding proteins whereas fetuin (1.0 µg) was used as a negative control.600 = 0.5), were resuspended in LB medium, stab-inoculated into 0.3% LB agar plates, incubated overnight, and colony diameters measured as an indicator of bacterial motility, as described22. In other experiments, PAK were preincubated with increasing concentrations rMUC1-ED, washed, resuspended in LB medium, and analyzed for motility.Bacteria in mid-log phase (A5 cells/well) were cultured in DMEM containing 10% FBS in 24-well plates. The cells were incubated for 24 h at 37 °C with 1.0 × 108 CFUs/well of WT-PAK or the PAK/fliC¯ flagellin-deficient strain. The bacteria were collected by centrifugation of culture supernatants at 5,000 xg for 10 min, washed 3 times with PBS, pH 7.4, and lysed. Equal protein aliquots (10 µg) of the lysates were processed for MUC1-ED immunoblotting as above.A549 cells (2.0 × 10NcoI and EcoRI restriction sites was amplified by PCR from a full-length MUC1-pcDNA3 plasmid55 and the amplicons subcloned into the pBAD/6X-His plasmid (Thermo Fisher Scientific). The plasmid was transformed into E. coli NEB 5-alpha competent cells (New England Biolabs), the cells were cultured to mid-log phase (A600 = 0.5), induced for 3 h with 0.01% arabinose, and lysed by sonication. The 6X-His epitope-tagged human rMUC1-ED was purified on a nickel-nitrilotriacetic acid affinity column, its identity verified by immunoblotting with anti-human MUC1-ED antibody, and purity confirmed by a single Coomassie blue-stained protein band following SDS-PAGE.Human MUC1-ED incorporating flanking 56.Immunogold electron microscopy using a Tecnai T12 transmission electron microscope (Thermo Fisher Scientific) to detect rMUC1-ED binding to Pa flagellin was performed as previously described600 = 0.1 in M9 medium, incubated for 48 h at 37 °C in 96-well plates with increasing concentrations rMUC1-ED, washed with deionized water, stained with 0.1% crystal violet for 10 min at 22 °C, and washed with deionized water. The crystal violet was solubilized with 30% acetic acid for 10 min at 22 °C and A595 measured.Overnight cultures of PAK in LB medium were adjusted to A23, incubated for 40 min at 37 °C with PA01 (2.0 × 107 CFUs/well) in the presence of increasing concentrations of rMUC1-ED or the PBS vehicle control, and washed 3 times with PBS, pH 7.4. Bound bacteria were released with 0.05% trypsin, and CFUs quantified on LB agar plates.A549 cells were cultured in DMEM containing 10% FBS. The cells were infected with adenovirus encoding NEU1 at a multiplicity of infection of 100 in 24-well plates as described600 = 0.5) in LB medium. The bacteria were centrifuged, washed, and diluted in PBS to obtain the desired number of bacteria. GFP-PA01 were incubated for 10 min at 22 °C with increasing concentrations of rMUC1-ED or the PBS vehicle control as described57. Approximately 1.0 × 106 human PMNs for each reaction were added to the bacteria in a 96 well microtiter plate, incubated for 2 h at 37 °C, aliquots added to LB agar, and the viable colonies quantified after overnight incubation.An overnight culture of GFP-PA01 was inoculated into 10 ml of fresh LB medium and grown to mid-log phase (A59 using our prior pilot study of BALF MUC1-ED levels in Pa-positive (n = 4) vs. Pa-negative (n = 11) patients21. The null hypothesis was defined as the mean BALF MUC1-ED level of Pa-positive patients being equal to the mean BALF MUC1-ED level of Pa-negative patients. Sample sizes for the Pa-positive group (n = 13) and Pa-negative group (n = 48) in the current study achieve 100% power to detect a difference of 3.33 µg MUC1-ED/mg BALF protein between the null hypothesis and the alternative hypothesis that the mean BALF MUC1-ED level of Pa-positive patients in the pilot study is 3.87 µg/mg BALF protein and the mean BALF MUC1-ED level of Pa-negative patients in the pilot study is 0.54 µg/mg BALF protein, with group standard deviations of 0.50 and 0.31, respectively, and with a significance level  of 0.05 using a two-sided two-sample t-test.For comparison of patient clinical and demographic parameters, values were expressed as mean ± S.D. All other values were expressed as mean ± S.E. The two-tailed Student’s t test, analysis of variance followed by Tukey’s post hoc analysis, and linear regression were performed using the Microsoft Excel Analysis ToolPak add-in. Statistical significance was set at p < 0.05. Power and sample size analyses were performed as describedSupplementary Information.