Angela deAlmeida

Hemodynamics Is a Key Epigenetic Factor in Development of the Cardiac Conduction System

Abstract— The His-Purkinje system (HPS) is a network of conduction cells responsible for coordinating the contraction of the ventricles. Earlier studies using bipolar electrodes indicated that the functional maturation of the HPS in the chick embryo is marked by a topological shift in the sequence of activation of the ventricle. Namely, at around the completion of septation, an immature base-to-apex sequence of ventricular activation was reported to convert to the apex-to-base pattern characteristic of the mature heart. Previously, we have proposed that hemodynamics and/or mechanical conditioning may be key epigenetic factors in development of the HPS. We thus hypothesized that the timing of the topological shift marking maturation of the conduction system is sensitive to variation in hemodynamic load. Spatiotemporal patterns of ventricular activation (as revealed by high-speed imaging of fluorescent voltage-sensitive dye) were mapped in chick hearts over normal development, and following procedures previously characterized as causing increased (conotruncal banding, CTB) or reduced (left atrial ligation, LAL) hemodynamic loading of the embryonic heart. The results revealed that the timing of the shift to mature activation displays striking plasticity. CTB led to precocious emergence of mature HPS function relative to controls whereas LAL was associated with delayed conversion to apical initiation. The results from our study indicate a critical role for biophysical factors in differentiation of specialized cardiac tissues and provide the basis of a new model for studies of the molecular mechanisms involved in induction and patterning of the HPS in vivo.

Role of RyR2 Phosphorylation at S2814 during Heart Failure Progression

Rationale: Increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure (HF) progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in HF development and associated diastolic sarcoplasmic reticulum Ca2+ leak is unclear. Objective: Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with nonischemic and ischemic forms of HF. Methods and Results: Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with nonischemic, but not with ischemic, HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development after transverse aortic constriction compared with wild-type littermates. After transverse aortic constriction, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor. Cardiomyocytes from S2814A mice exhibited significantly lower sarcoplasmic reticulum Ca2+ leak and improved sarcoplasmic reticulum Ca2+ loading compared with wild-type mice after transverse aortic constriction. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice after experimental myocardial infarction. Conclusions: Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological sarcoplasmic reticulum Ca2+ leak and HF development in nonischemic forms of HF such as transverse aortic constriction in mice.

Increased Ventricular Preload Is Compensated by Myocyte Proliferation in Normal and Hypoplastic Fetal Chick Left Ventricle

Hemodynamics influence cardiac development, and alterations in blood flow may lead to impaired cardiac growth and malformations. The developing myocardium adapts to augmented workload by increasing cell number (hyperplasia). The aim of this study was to determine the influence of alterations in ventricular preload on fetal myocyte proliferation by manipulation of intracardiac shunting at the atrial level. We hypothesized that partial clipping of the right atrial appendage would increase the blood flow to the left ventricle and, in turn, lead to an increase in chamber volume and myocardial mass based on myocyte proliferation. Using an ex ovo culture setup, we performed partial right atrial clipping on embryonic day 8 chick embryos. Ultrasound imaging was performed before and after the surgery to assess the changes in left ventricular volume. Sampling after 24 hours was preceded by 2 hour of pulse-labeling with 5-bromodeoxyuridine. Ultrasound imaging showed that partial right atrial clipping led to a significant increase in left ventricular end-diastolic volume, demonstrating increased blood flow and preload. Anti–5-bromodeoxyuridine immunolabeling revealed a significant increase in myocyte proliferation in the left ventricle and atrium. No significant changes were found in the right heart structures. Increased left ventricular myocyte proliferation and myocardial mass after right atrial clipping was also observed in embryos with experimental left ventricular hypoplasia. These results demonstrate the ability of fetal myocardium to respond to increased preload by myocyte hyperplasia and support the rationale for prenatal surgical interventions in certain cases of congenital heart disease such as hypoplastic left heart syndrome.

High‐frequency ultrasonographic imaging of avian cardiovascular development

The chick embryo has long been a favorite model system for morphologic and physiologic studies of the developing heart, largely because of its easy visualization and amenability to experimental manipulations. However, this advantage is diminished after 5 days of incubation, when rapidly growing chorioallantoic membranes reduce visibility of the embryo. Using high‐frequency ultrasound, we show that chick embryonic cardiovascular structures can be readily visualized throughout the period of Stages 9–39. At most stages of development, a simple ex ovo culture technique provided the best imaging opportunities. We have measured cardiac and vascular structures, blood flow velocities, and calculated ventricular volumes as early as Stage 11 with values comparable to those previously obtained using video microscopy. The endocardial and myocardial layers of the pre‐septated heart are readily seen as well as the acellular layer of the cardiac jelly. Ventricular inflow in the pre‐septated heart is biphasic, just as in the mature heart, and is converted to a monophasic (outflow) wave by ventricular contraction. Although blood has soft‐tissue density at the ultrasound resolutions and developmental stages examined, its movement allowed easy discrimination of perfused vascular structures throughout the embryo. The utility of such imaging was demonstrated by documenting changes in blood flow patterns after experimental conotruncal banding. Developmental Dynamics 236:3503–3513, 2007.

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure. TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure. The murine TAC model was first validated by Rockman et al., and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries. With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Fibroblast Growth Factor-2 regulates proliferation of cardiac myocytes in normal and hypoplastic left ventricles in the developing chick

The developing heart increases its mass predominantly by increasing the number of contained cells through proliferation. We hypothesized that addition of fibroblast growth factor-2, a factor previously shown to stimulate division of the embryonic myocytes, to the left ventricular myocardium in an experimental model of left heart hypoplasia created in the chicken would attenuate phenotypic severity by increasing cellular proliferation. We have established an effective mode of delivery of fibroblast growth factor-2 to the chick embryonic left ventricular myocardium by using adenovirus vectors, which was more efficient and better tolerated than direct injection of recombinant fibroblast growth factor-2 protein. Injection of control adenovirus expressing green fluorescent protein did not result in significant alterations in myocytic proliferation or cell death compared with intact, uninjected, controls. Co-injection of adenoviruses expressing green fluorescent protein and fibroblast growth factor-2 was used for verification of positive injection, and induction of proliferation, respectively. Treatment of both normal and hypoplastic left ventricles with fibroblast growth factor-2 expressing adenovirus resulted in to 2 to 3-fold overexpression of fibroblast growth factor-2, as verified by immunostaining. An increase by 45% in myocytic proliferation was observed following injection of normal hearts, and an increase of 39% was observed in hypoplastic hearts. There was a significant increase in anti-myosin immunostaining in the hypoplastic, but not the normal hearts. We have shown, therefore, that expression of exogenous fibroblast growth factor-2 in the late embryonic heart can exert direct effects on cardiac myocytes, inducing both their proliferation and differentiation. These data suggest potential for a novel therapeutic option in selected cases of congenital cardiac disease, such as hypoplastic left heart syndrome.