Yolanda Benito

Diet-Induced Aortic Valve Disease in Mice Haploinsufficient for the Notch Pathway Effector RBPJK/CSL

Objective—Calcific aortic valve disease is similar to atherosclerosis in that both diseases result from chronic inflammation and endothelial dysfunction. Heterozygous NOTCH1 mutations have been associated to calcific aortic disease and a bicuspid aortic valve. We investigated whether mice with genetic inactivation of the Notch signaling pathway are prone to develop valve disease when exposed to a predisposing diet. Methods and Results—Using Doppler echocardiography, histology, immunohistochemistry, quantitative gene expression analysis, and cell culture assays, we examined the effect of a hypercholesterolemic diet supplemented with vitamin D on mice heterozygous for null mutations in the Notch1 receptor or the effector transcription factor gene RBPJk. After 16 weeks on the hyperlipidemic diet, calcific aortic disease was detected in heterozygous RBPJk mice. Analysis of valve leaflets revealed macrophage infiltration, enhanced collagen deposition, proosteogenic protein expression, and calcification. Heterozygous null Notch1 mice displayed milder histopathologic changes and did not develop any significant hemodynamic disturbance. Valvular disease correlated with reduced expression of the Notch target gene Hey1 in valves of RBPJk heterozygous mice fed the hyperlipidemic diet. Consistent with the in vivo data, Notch signaling inhibition in porcine valve interstitial cells led to downregulation of HEY1 transcription, activation of osteogenic markers, and increased calcified nodule formation. Conclusion—We show that Notch signaling disruption via RBPJk heterozygous inactivation results in aortic valve disease. Notch1 heterozygous mice do not show functional impairment, suggesting that additional Notch receptors may be involved in aortic valve homeostasis and disease. Our data establish a genetic mouse model of calcific aortic valve disease and may help to identify a patient population with reduced valvular NOTCH signaling at risk for developing this disease.

Topology of Blood Transport in the Human Left Ventricle by Novel Processing of Doppler Echocardiography

Novel processing of Doppler-echocardiography data was used to study blood transport in the left ventricle (LV) of six patients with dilated cardiomyopathy and six healthy volunteers. Bi-directional velocity field maps in the apical long axis of the LV were reconstructed from color-Doppler echocardiography. Resulting velocity field data were used to perform trajectory-based computation of Lagrangian coherent structures (LCS). LCS were shown to reveal the boundaries of blood injected and ejected from the heart over multiple beats. This enabled qualitative and quantitative assessments of blood transport patterns and residence times in the LV. Quantitative assessments of stasis in the LV are reported, as well as characterization of LV vortex formations from E-wave and A-wave filling.

Validation of Noninvasive Indices of Global Systolic Function in Patients With Normal and Abnormal Loading Conditions A Simultaneous Echocardiography Pressure-Volume Catheterization Study

Background—Noninvasive indices based on Doppler echocardiography are increasingly used in clinical cardiovascular research to evaluate left ventricular global systolic chamber function. Our objectives were to clinically validate ultrasound-based methods of global systolic chamber function to account for differences between patients in conditions of abnormal load, and to assess their sensitivity to load confounders. Methods and Results—Twenty-seven patients (8 dilated cardiomyopathy, 10 normal ejection fraction, and 9 end-stage liver disease) underwent simultaneous echocardiography and left heart catheterization with pressure-conductance instrumentation. The reference index, maximal elastance (Emax), was calculated from pressure–volume loop data obtained during acute inferior vena cava occlusion. A wide range of values were observed for left ventricular systolic chamber function (Emax: 2.8±1.0 mm Hg/mL), preload, and afterload. Among the noninvasive indices tested, the peak ejection intraventricular pressure difference showed the best correlation with Emax (R=0.75). A significant but weaker correlation with Emax was observed for ejection fraction (R=0.41), midwall fractional shortening (R=0.51), global circumferential strain (R=−0.53), and strain rate (R=−0.46). Longitudinal strain and strain rate failed to correlate with Emax, as did noninvasive single-beat estimations of this index. Principal component and multiple regression analyses demonstrated that peak ejection intraventricular pressure difference was less sensitive to load, whereas ejection fraction and longitudinal strain and strain rate were heavily influenced by afterload. Conclusions—Current ultrasound methods have limited accuracy to characterize global left ventricular systolic chamber function in a given patient. The Doppler-derived peak ejection intraventricular pressure difference should be preferred for this purpose because it best correlates with the reference index and is more robust in conditions of abnormal load.

Noninvasive Estimation of the Rate of Relaxation by the Analysis of Intraventricular Pressure Gradients

Background—During late ejection, myocardial relaxation causes systolic flow to decelerate and stop, and this phenomenon is coupled with the generation of a pressure gradient inside the left ventricle (LV). We hypothesized that the peak reverse ejection intraventricular pressure difference (REIVPD) between the LV apex and the outflow tract could be a useful method to improve the assessment of LV relaxation using Doppler echocardiography. Methods and Results—Three sets of animal experiments and 1 clinical study were designed. In 6 pigs, a close relationship between REIVPD and the intensity of the relaxation wave (Rrm=0.89) was demonstrated using wave intensity analysis of high-fidelity pressure-volume-velocity data. In 19 animals, REIVPD sensitively detected modifications of the lusotropic state and closely correlated with the time constant of LV relaxation (&tgr;) within animals (Rrm=−0.93). Load-dependence analysis in 5 pigs showed that REIVPD remained stable up to values of 35% to 40% acute preload reduction. Clinical validation was tested in 50 patients (23 with normal systolic function) undergoing simultaneous Doppler echocardiography and high-fidelity LV pressure measurements on the same beat. REIVPD and tissue Doppler mitral annulus velocity (e′) were independently related to &tgr;, but the REIVPD · e′ product correlated better with &tgr; than either variable separately (bootstrap-corrected correlation coefficients: R=−0.84 versus −0.71, and −0.70, respectively, P<0.05). Area under the receiver operating characteristic curve to predict impaired relaxation (&tgr;>50 ms) for e′ · REIVPD was 0.96 (95% confidence interval, 0.85 to 0.99). Conclusions—The Doppler-derived REIVPD provides a sensitive, reliable, reproducible, and relatively load-independent index of the rate of LV relaxation. Combined with tissue Doppler measurements of longitudinal function, this method improves noninvasive assessment of LV relaxation in the clinical setting.

A clinical method for mapping and quantifying blood stasis in the left ventricle

In patients at risk of intraventrcular thrombosis, the benefits of chronic anticoagulation therapy need to be balanced with the pro-hemorrhagic effects of therapy. Blood stasis in the cardiac chambers is a recognized risk factor for intracardiac thrombosis and potential cardiogenic embolic events. In this work, we present a novel flow image-based method to assess the location and extent of intraventricular stasis regions inside the left ventricle (LV) by digital processing flow-velocity images obtained either by phase-contrast magnetic resonance (PCMR) or 2D color-Doppler velocimetry (echo-CDV). This approach is based on quantifying the distribution of the blood Residence Time (TR) from time-resolved blood velocity fields in the LV. We tested the new method in illustrative examples of normal hearts, patients with dilated cardiomyopathy and one patient before and after the implantation of a left ventricular assist device (LVAD). The method allowed us to assess in-vivo the location and extent of the stasis regions in the LV. Original metrics were developed to integrate flow properties into simple scalars suitable for a robust and personalized assessment of the risk of thrombosis. From a clinical perspective, this work introduces the new paradigm that quantitative flow dynamics can provide the basis to obtain subclinical markers of intraventricular thrombosis risk. The early prediction of LV blood stasis may result in decrease strokes by appropriate use of anticoagulant therapy for the purpose of primary and secondary prevention. It may also have a significant impact on LVAD device design and operation set-up.

Diastolic chamber properties of the left ventricle assessed by global fitting of pressure-volume data: improving the gold standard of diastolic function

In cardiovascular research, relaxation and stiffness are calculated from pressure-volume (PV) curves by separately fitting the data during the isovolumic and end-diastolic phases (end-diastolic PV relationship), respectively. This method is limited because it assumes uncoupled active and passive properties during these phases, it penalizes statistical power, and it cannot account for elastic restoring forces. We aimed to improve this analysis by implementing a method based on global optimization of all PV diastolic data. In 1,000 Monte Carlo experiments, the optimization algorithm recovered entered parameters of diastolic properties below and above the equilibrium volume (intraclass correlation coefficients = 0.99). Inotropic modulation experiments in 26 pigs modified passive pressure generated by restoring forces due to changes in the operative and/or equilibrium volumes. Volume overload and coronary microembolization caused incomplete relaxation at end diastole (active pressure > 0.5 mmHg), rendering the end-diastolic PV relationship method ill-posed. In 28 patients undergoing PV cardiac catheterization, the new algorithm reduced the confidence intervals of stiffness parameters by one-fifth. The Jacobian matrix allowed visualizing the contribution of each property to instantaneous diastolic pressure on a per-patient basis. The algorithm allowed estimating stiffness from single-beat PV data (derivative of left ventricular pressure with respect to volume at end-diastolic volume intraclass correlation coefficient = 0.65, error = 0.07 ± 0.24 mmHg/ml). Thus, in clinical and preclinical research, global optimization algorithms provide the most complete, accurate, and reproducible assessment of global left ventricular diastolic chamber properties from PV data. Using global optimization, we were able to fully uncouple relaxation and passive PV curves for the first time in the intact heart.

A labdane diterpene exerts ex vivo and in vivo cardioprotection against post-ischemic injury: Involvement of AKT-dependent mechanisms

Therapeutic approaches to protect the heart from ischemia/reperfusion (I/R) injury are an area of intense research, as myocardial infarction is a major cause of mortality and morbidity. Diterpenes are bioactive natural products with great therapeutic potential. In the present study, we have investigated the in vivo cardioprotective effects of a labdane diterpene (DT1) against cardiac I/R injury and the molecular mechanisms involved. DT1 attenuates post-ischemic injury via an AKT-dependent activation of HIF-1α, survival pathways and inhibition of NF-κB signaling. Myocardial infarction (MI) was induced in Wistar rats occluding the left coronary artery (LCA) for 30min followed by 72h reperfusion. DT1 (5mg/kg) was intravenously administered at reperfusion. In addition, we investigated the mechanisms of cardioprotection in the Langendorff-perfused model. Cardioprotection was observed when DT1 was administered after myocardial injury. The molecular mechanisms involved the activation of the survival pathway PDK-1, AKT and AMPK, a reduced phosphorylation of PKD1/2 and sustained HIF-1α activity, leading to increased expression of anti-apoptotic proteins and decreased caspase-3 activation. Pharmacological inhibition of AKT following MI and prior to DT1 challenge significantly decreased the cardioprotection afforded by DT1 therapy at reperfusion. Cardiac function after MI was significantly improved after DT1-treatment, as evidenced by hemodynamic recovery and decreased myocardial infarct size. These findings demonstrate an efficient in vivo cardioprotection by diterpene DT1 against I/R when administered at reperfusion, opening new therapeutic strategies as adjunctive therapy for the pharmacological management of I/R injury.

Stasis Mapping Using Ultrasound: A Prospective Study in Acute Myocardial Infarction

During the subacute phase of acute myocardial infarction (AMI), the incidence of left ventricular thrombosis (LVT) can be as high as 15% to 20%. A method for assessing the risk of LVT would be of particular value in the setting of AMI, because prophylactic anticoagulation must be balanced against

ABNORMAL EXERCISE HEMODYNAMICS IN PATIENTS WITH LOW GRADIENT AORTIC STENOSIS AND PRESERVED EJECTION FRACTION: A SIMULTANEOUS STRESS ECHO-RIGHT HEART CATHETERIZATION STUDY

The systemic arterial load modulates the clinical impact of aortic stenosis (AS). The hemodynamic response to exercise has never been measured invasively in patients with low-gradient (LG) AS and normal ejection fraction. Twenty patients (77 ± 6 years old; 17 female) with LGAS (mean pressure

In Vivo Measurements of Blood Transport Patterns and Stasis in the Human Left Ventricle

Because the left ventricle (LV) is not completely emptied during systole, oxygenated blood from the left atrium interacts with residual blood from preceding cycles. It is hypothesized that LV flow is optimal for transporting blood under normal conditions; yet proving this remains a challenge. Furthermore, clinical evaluation of LV hemodynamics has tremendous diagnostic importance for patients with cardiomyopathy. We have performed Doppler-echocardiography on 6 patients with dilated cardiomyopathy and 6 healthy volunteers. Using novel processing of the color-Doppler data, bi-directional velocity field maps in the apical long axis plane were derived. Resulting flow data was used to perform Lagrangian coherent structure (LCS) computation, which enabled novel characterization of the transport topology in the LV during filling and ejection. This framework was used to quantify stasis in the LV, which can be used as a surrogate for diagnosing pumping deficiencies and thrombosis risk. This framework also enables characterization of LV vortices, which have previously received much attention using Eulerian characterizations. The framework presented here uncovers the well-defined boundaries to both E-wave and A-wave filling vortices, which has not been previously reported.© 2013 ASME