Elaine Wan

Enhanced Efferocytosis of Apoptotic Cardiomyocytes Through Myeloid-Epithelial-Reproductive Tyrosine Kinase Links Acute Inflammation Resolution to Cardiac Repair After Infarction

Rationale: Efficient clearance of apoptotic cells (efferocytosis) is a prerequisite for inflammation resolution and tissue repair. After myocardial infarction, phagocytes are recruited to the heart and promote clearance of dying cardiomyocytes. The molecular mechanisms of efferocytosis of cardiomyocytes and in the myocardium are unknown. The injured heart provides a unique model to examine relationships between efferocytosis and subsequent inflammation resolution, tissue remodeling, and organ function. Objective: We set out to identify mechanisms of dying cardiomyocyte engulfment by phagocytes and, for the first time, to assess the causal significance of disrupting efferocytosis during myocardial infarction. Methods and Results: In contrast to other apoptotic cell receptors, macrophage myeloid-epithelial-reproductive tyrosine kinase was necessary and sufficient for efferocytosis of cardiomyocytes ex vivo. In mice, Mertk was specifically induced in Ly6cLO myocardial phagocytes after experimental coronary occlusion. Mertk deficiency led to an accumulation of apoptotic cardiomyocytes, independently of changes in noncardiomyocytes, and a reduced index of in vivo efferocytosis. Importantly, suppressed efferocytosis preceded increases in myocardial infarct size and led to delayed inflammation resolution and reduced systolic performance. Reduced cardiac function was reproduced in chimeric mice deficient in bone marrow Mertk; reciprocal transplantation of Mertk+/+ marrow into Mertk−/− mice corrected systolic dysfunction. Interestingly, an inactivated form of myeloid-epithelial-reproductive tyrosine kinase, known as solMER, was identified in infarcted myocardium, implicating a natural mechanism of myeloid-epithelial-reproductive tyrosine kinase inactivation after myocardial infarction. Conclusions: These data collectively and directly link efferocytosis to wound healing in the heart and identify Mertk as a significant link between acute inflammation resolution and organ function.

Enhanced Efferocytosis of Apoptotic Cardiomyocytes Through MER Tyrosine Kinase Links Acute Inflammation Resolution to Cardiac Repair After Infarction

Rationale: Efficient clearance of apoptotic cells (efferocytosis) is a prerequisite for inflammation resolution and tissue repair. After myocardial infarction, phagocytes are recruited to the heart and promote clearance of dying cardiomyocytes. The molecular mechanisms of efferocytosis of cardiomyocytes and in the myocardium are unknown. The injured heart provides a unique model to examine relationships between efferocytosis and subsequent inflammation resolution, tissue remodeling, and organ function. Objective: We set out to identify mechanisms of dying cardiomyocyte engulfment by phagocytes and, for the first time, to assess the causal significance of disrupting efferocytosis during myocardial infarction. Methods and Results: In contrast to other apoptotic cell receptors, macrophage myeloid-epithelial-reproductive tyrosine kinase was necessary and sufficient for efferocytosis of cardiomyocytes ex vivo. In mice, Mertk was specifically induced in Ly6cLO myocardial phagocytes after experimental coronary occlusion. Mertk deficiency led to an accumulation of apoptotic cardiomyocytes, independently of changes in noncardiomyocytes, and a reduced index of in vivo efferocytosis. Importantly, suppressed efferocytosis preceded increases in myocardial infarct size and led to delayed inflammation resolution and reduced systolic performance. Reduced cardiac function was reproduced in chimeric mice deficient in bone marrow Mertk; reciprocal transplantation of Mertk+/+ marrow into Mertk−/− mice corrected systolic dysfunction. Interestingly, an inactivated form of myeloid-epithelial-reproductive tyrosine kinase, known as solMER, was identified in infarcted myocardium, implicating a natural mechanism of myeloid-epithelial-reproductive tyrosine kinase inactivation after myocardial infarction. Conclusions: These data collectively and directly link efferocytosis to wound healing in the heart and identify Mertk as a significant link between acute inflammation resolution and organ function.

Diet-induced obesity causes long QT and reduces transcription of voltage-gated potassium channels

In humans, obesity is associated with long QT, increased frequency of premature ventricular complexes, and sudden cardiac death. The mechanisms of the pro-arrhythmic electrophysiologic remodeling of obesity are poorly understood. We tested the hypothesis that there is decreased expression of voltage-gated potassium channels in the obese heart, leading to long QT. Using implanted telemeters, we found that diet-induced obese (DIO) wild-type mice have impaired cardiac repolarization, demonstrated by long QT, as well as more frequent ventricular ectopy, similar to obese humans. DIO mice have reduced protein and mRNA levels of the potassium channel Kv1.5 caused by a reduction of the transcription factor cyclic AMP response element binding protein (CREB) in DIO hearts. We found that CREB knock-down by siRNA reduces Kv1.5, CREB binds to the Kv1.5 promoter in the heart, and CREB increases transcription of mouse and human Kv1.5 promoters. The reduction in CREB protein during lipotoxicity can be rescued by inhibiting protein kinase D (PKD). Our results identify a mechanism for obesity-induced electrophysiologic remodeling in the heart, namely PKD-induced reduction of CREB, which in turn decreases expression of the potassium channel Kv1.5.

Aberrant sodium influx causes cardiomyopathy and atrial fibrillation in mice

Increased sodium influx via incomplete inactivation of the major cardiac sodium channel Na(V)1.5 is correlated with an increased incidence of atrial fibrillation (AF) in humans. Here, we sought to determine whether increased sodium entry is sufficient to cause the structural and electrophysiological perturbations that are required to initiate and sustain AF. We used mice expressing a human Na(V)1.5 variant with a mutation in the anesthetic-binding site (F1759A-Na(V)1.5) and demonstrated that incomplete Na+ channel inactivation is sufficient to drive structural alterations, including atrial and ventricular enlargement, myofibril disarray, fibrosis and mitochondrial injury, and electrophysiological dysfunctions that together lead to spontaneous and prolonged episodes of AF in these mice. Using this model, we determined that the increase in a persistent sodium current causes heterogeneously prolonged action potential duration and rotors, as well as wave and wavelets in the atria, and thereby mimics mechanistic theories that have been proposed for AF in humans. Acute inhibition of the sodium-calcium exchanger, which targets the downstream effects of enhanced sodium entry, markedly reduced the burden of AF and ventricular arrhythmias in this model, suggesting a potential therapeutic approach for AF. Together, our results indicate that these mice will be important for assessing the cellular mechanisms and potential effectiveness of antiarrhythmic therapies.

Secondhand Smoking Is Associated With Vascular Inflammation

BACKGROUND The relative risk for cardiovascular diseases in passive smokers is similar to that of active smokers despite almost a 100-fold lower dose of inhaled cigarette smoke. However, the mechanisms underlying the surprising susceptibility of the vascular tissue to the toxins in secondhand smoke (SHS) have not been directly investigated. The aim of this study was to investigate directly vascular endothelial cell function in passive smokers. METHODS Using a minimally invasive method of endothelial biopsy, we investigated directly the vascular endothelium in 23 healthy passive smokers, 25 healthy active smokers, and 23 healthy control subjects who had never smoked and had no regular exposure to SHS. Endothelial nitric oxide synthase (eNOS) function (expression of basal eNOS and activated eNOS [phosphorylated eNOS at serine1177 (P-eNOS)]) and expression of markers of inflammation (nuclear factor-κB [NF-κB]) and oxidative stress (nitrotyrosine) were assessed in freshly harvested venous endothelial cells by quantitative immunofluorescence. RESULTS Expression of eNOS and P-eNOS was similarly reduced and expression of NF-κB was similarly increased in passive and active smokers compared with control subjects. Expression of nitrotyrosine was greater in active smokers than control subjects and similar in passive and active smokers. Brachial artery flow-mediated dilation was similarly reduced in passive and active smokers compared with control subjects, consistent with reduced endothelial NO bioavailability. CONCLUSIONS Secondhand smoking increases vascular endothelial inflammation and reduces active eNOS to a similar extent as active cigarette smoking, indicating direct toxic effects of SHS on the vasculature.

Intracardiac myocardial elastography in canines and humans in vivo

Intracardiac echocardiography (ICE) is a useful imaging modality which is used during RF ablation procedures to identify anatomical structures. Utilizing ICE in conjunction with myocardial elastography (ME) can provide additional information on the mechanical properties of cardiac tissue and provide information on mechanical changes caused by ablation. The objective of this study was to demonstrate that ICE can be used at high frame rate using a diverging beam transmit sequence to image myocardial strain and differentiate myocardial tissue properties before, during, and after ablation for a clinical ablation procedure. In this feasibility study, three normal canines and eight patients with atrial fibrillation (AF) were studied in vivo. A 5.8-MHz ICE transducer was used to image the heart with a diverging beam transmit method achieving 1200 frames per second (fps). Cumulative axial displacement estimation was performed using 1-D cross-correlation with a window size of 2.7 mm and 95% overlap. Axial cumulative strains were estimated in the left atrium (LA) and right atrium (RA) using a least-squares estimator with a kernel of 2 mm on the axial displacements. In the canine case, radial thickening was detected in the lateral wall and in the interatrial septum during LA emptying. For AF patients, the mean absolute strain in the ablated region was lower (6.7 ± 3.1%) than before the ablation (17.4 ± 9.3%) in LA at the end of the LA emptying phase. In the cavotricuspid isthmus (CTI) region, mean absolute strain magnitude at the end of the RA emptying phase was found to be higher during ablation (43.0 ± 18.1%) compared with after ablation (33.7 ± 15.8%). Myocardial strains in the LA of an AF patient were approximately 2.6 times lower in the ablated region than before ablation. This initial feasibility indicates that ME can be used as a new imaging modality in conjunction with ICE in RF ablation guidance and lesion monitoring.

Electromechanical wave imaging (EWI) validation in all four cardiac chambers with 3D electroanatomic mapping in canines in vivo

Characterization and mapping of arrhythmias is currently performed through invasive insertion and manipulation of cardiac catheters. Electromechanical wave imaging (EWI) is a non-invasive ultrasound-based imaging technique, which tracks the electromechanical activation that immediately follows electrical activation. Electrical and electromechanical activations were previously found to be linearly correlated in the left ventricle, but the relationship has not yet been investigated in the three other chambers of the heart. The objective of this study was to investigate the relationship between electrical and electromechanical activations and validate EWI in all four chambers of the heart with conventional 3D electroanatomical mapping. Six (n  =  6) normal adult canines were used in this study. The electrical activation sequence was mapped in all four chambers of the heart, both endocardially and epicardially using the St Judes EnSite 3D mapping system (St. Jude Medical, Secaucus, NJ). EWI acquisitions were performed in all four chambers during normal sinus rhythm, and during pacing in the left ventricle. Isochrones of the electromechanical activation were generated from standard echocardiographic imaging views. Electrical and electromechanical activation maps were co-registered and compared, and electrical and electromechanical activation times were plotted against each other and linear regression was performed for each pair of activation maps. Electromechanical and electrical activations were found to be directly correlated with slopes of the correlation ranging from 0.77 to 1.83, electromechanical delays between 9 and 58 ms and R 2 values from 0.71 to 0.92. The linear correlation between electrical and electromechanical activations and the agreement between the activation maps indicate that the electromechanical activation follows the pattern of propagation of the electrical activation. This suggests that EWI may be used as a novel non-invasive method to accurately characterize and localize sources of arrhythmias.

Validation of electromechanical wave imaging in a canine model during pacing and sinus rhythm

BACKGROUND Accurate determination of regional areas of arrhythmic triggers is of key interest to diagnose arrhythmias and optimize their treatment. Electromechanical wave imaging (EWI) is an ultrasound technique that can image the transient deformation in the myocardium after electrical activation and therefore has the potential to detect and characterize location of triggers of arrhythmias. OBJECTIVES The objectives of this study were to investigate the relationship between the electromechanical and the electrical activation of the left ventricular (LV) endocardial surface during epicardial and endocardial pacing and during sinus rhythm as well as to map the distribution of electromechanical delays. METHODS In this study, 6 canines were investigated. Two external electrodes were sutured onto the epicardial surface of the LV. A 64-electrode basket catheter was inserted through the apex of the LV. Ultrasound channel data were acquired at 2000 frames/s during epicardial and endocardial pacing and during sinus rhythm. Electromechanical and electrical activation maps were synchronously obtained from the ultrasound data and the basket catheter, respectively. RESULTS The mean correlation coefficient between electromechanical and electrical activation was 0.81 for epicardial anterior pacing, 0.79 for epicardial lateral pacing, 0.69 for endocardial pacing, and 0.56 for sinus rhythm. CONCLUSION The electromechanical activation sequence determined by EWI follows the electrical activation sequence and more specifically in the case of pacing. This finding is of key interest in the role that EWI can play in the detection of the anatomical source of arrhythmias and the planning of pacing therapies such as cardiovascular resynchronization therapy.

Intracardiac myocardial elastography for lesion quantification in cardiac radiofrequency ablation

Radiofrequency ablation of the myocardium is used to treat various cardiac arrhythmias. The size, spacing, and transmurality of lesions have been shown to affect the success of the ablation procedure; however, there is currently no method to directly image the size and formation of ablation lesions in real time. Intracardiac myocardial elastrography has been used previously to image the reduction in end-systolic strain in the ablated region as a result of the lesion formation. However, the relationship between end-systolic strain change and lesion size has not been investigated. In this study, a large animal model is used to establish a relationship between the area affected by the strain reduction and lesion volume. Ablation lesions (n=10) were created in the left ventricular epicardium in five anesthetized canines. A clinical intracardiac echocardiography machine was programmed to emit a custom diverging beam sequence at 600 Hz and used to image the ablation site before and after the induction of a lesion. Cumulative strains were estimated over systole using a normalized cross-correlation displacement algorithm and a Savitzky-Golay strain kernel. The reduction in strain as a result of the ablation was computed by comparing cumulative end-systolic strains before and after ablation. Lesion volume was also measured ex vivo and compared to the area of significant strain change (>8% reduction) for each lesion. A good correlation was found between the area of significant strain change and lesion volume (r2 = 0.86). These results indicate that end-systolic strain measured using ME can be used to estimate the size of lesions induced during an RF ablation procedure, potentially assisting clinicians in lesion formation assessment during the procedure.

Technical Note: A 3‐D rendering algorithm for electromechanical wave imaging of a beating heart

Purpose Arrhythmias can be treated by ablating the heart tissue in the regions of abnormal contraction. The current clinical standard provides electroanatomic 3‐D maps to visualize the electrical activation and locate the arrhythmogenic sources. However, the procedure is time‐consuming and invasive. Electromechanical wave imaging is an ultrasound‐based noninvasive technique that can provide 2‐D maps of the electromechanical activation of the heart. In order to fully visualize the complex 3‐D pattern of activation, several 2‐D views are acquired and processed separately. They are then manually registered with a 3‐D rendering software to generate a pseudo‐3‐D map. However, this last step is operator‐dependent and time‐consuming. Methods This paper presents a method to generate a full 3‐D map of the electromechanical activation using multiple 2‐D images. Two canine models were considered to illustrate the method: one in normal sinus rhythm and one paced from the lateral region of the heart. Four standard echographic views of each canine heart were acquired. Electromechanical wave imaging was applied to generate four 2‐D activation maps of the left ventricle. The radial positions and activation timings of the walls were automatically extracted from those maps. In each slice, from apex to base, these values were interpolated around the circumference to generate a full 3‐D map. Results In both cases, a 3‐D activation map and a cine‐loop of the propagation of the electromechanical wave were automatically generated. The 3‐D map showing the electromechanical activation timings overlaid on realistic anatomy assists with the visualization of the sources of earlier activation (which are potential arrhythmogenic sources). The earliest sources of activation corresponded to the expected ones: septum for the normal rhythm and lateral for the pacing case. Conclusions The proposed technique provides, automatically, a 3‐D electromechanical activation map with a realistic anatomy. This represents a step towards a noninvasive tool to efficiently localize arrhythmias in 3‐D.