Speckle variance optical coherence tomography of blood flow in the beating mouse embryonic heart

Efficient separation of blood and cardiac wall in the beating embryonic heart is essential and critical for experiment‐based computational modelling and analysis of early‐stage cardiac biomechanics. Although speckle variance optical coherence tomography (SV‐OCT) relying on calculation of intensity variance over consecutively acquired frames is a powerful approach for segmentation of fluid flow from static tissue, application of this method in the beating embryonic heart remains challenging because moving structures generate SV signal indistinguishable from the blood. Here, we demonstrate a modified four‐dimensional SV‐OCT approach that effectively separates the blood flow from the dynamic heart wall in the beating mouse embryonic heart. The method takes advantage of the periodic motion of the cardiac wall and is based on calculation of the SV signal over the frames corresponding to the same phase of the heartbeat cycle. Through comparison with Doppler OCT imaging, we validate this speckle‐based approach and show advantages in its insensitiveness to the flow direction and velocity as well as reduced influence from the heart wall movement. This approach has a potential in variety of applications relying on visualization and segmentation of blood flow in periodically moving structures, such as mechanical simulation studies and finite element modelling. Picture : Four‐dimensional speckle variance OCT imaging shows the blood flow inside the beating heart of an E8.5 mouse embryo.     

A Novel Atlas-Based Strategy for Understanding Cardiac Dysfunction in Patients with Congenital Heart Disease

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease. Infants diagnosed with TOF require surgical interventions to survive into adulthood. However, as a result of postoperative structural malformations and long-term ventricular remodeling, further interventions are often required later in life. To help identify those at risk of disease progression, serial cardiac magnetic resonance (CMR) imaging is used to monitor these patients. However, most of the detailed information on cardiac shape and biomechanics contained in these large four-dimensional (4D) data sets goes unused in clinical practice for lack of efficient and comprehensive quantitative analysis tools. While current global metrics of cardiac size and function, such as indexed ventricular mass and volumes, can identify patients at risk of further complications, they are not adequate to explain the underlying mechanisms causing the postoperative malfunctions, and help cardiologists plan optimal personalized treatments. We are proposing a novel approach that uses 4D ventricular shape models derived from CMR imaging exams to generate statistical atlases of ventricular shape and finite-element models of ventricular biomechanics to identify specific features of cardiac shape and biomechanical properties that explain variations in ventricular function. This study has the potential to discover novel biomarkers that precede adverse ventricular remodeling and dysfunction.     

Structure and function of the hearts of lizards and snakes

With approximately 7000 species, snakes and lizards, collectively known as squamates, are by far the most species‐rich group of reptiles. It was from reptile‐like ancestors that mammals and birds evolved and squamates can be viewed as phylogenetically positioned between them and fishes. Hence, their hearts have been studied for more than a century yielding insights into the group itself and into the independent evolution of the fully divided four‐chambered hearts of mammals and birds. Structurally the heart is complex and debates persist on rudimentary issues such as identifying structures critical to understanding ventricle function. In seeking to resolve these controversies we have generated three‐dimensional (3D) models in portable digital format (pdf) of the anaconda and anole lizard hearts (‘typical’ squamate hearts) and the uniquely specialized python heart with comprehensive annotations of structures and cavities. We review the anatomy and physiology of squamate hearts in general and emphasize the unique features of pythonid and varanid lizard hearts that endow them with mammal‐like blood pressures. Excluding pythons and varanid lizards it is concluded that the squamate heart has a highly consistent design including a disproportionately large right side (systemic venous) probably due to prevailing pulmonary bypass (intraventricular shunting). Unfortunately, investigations on rudimentary features are sparse. We therefore point out gaps in our knowledge, such as the size and functional importance of the coronary vasculature and of the first cardiac chamber, the sinus venosus, and highlight areas with implications for vertebrate cardiac evolution.     

The role of basement membranes in cardiac biology and disease

Basement membranes (BMs) are highly specialised extracellular matrix (ECM) structures that within the heart underlie endothelial cells (ECs) and surround cardiomyocytes and vascular smooth muscle cells. They generate a dynamic and structurally supportive environment throughout cardiac development and maturation by providing physical anchorage to the underlying interstitium, structural support to the tissue, and by influencing cell behaviour and signalling. While this provides a strong link between BM dysfunction and cardiac disease, the role of the BM in cardiac biology remains under-researched and our understanding regarding the mechanistic interplay between BM defects and their morphological and functional consequences remain important knowledge-gaps. In this review, we bring together emerging understanding of BM defects within the heart including in common cardiovascular pathologies such as contractile dysfunction and highlight some key questions that are now ready to be addressed.     

绿茶浸出液对蟾蜍离体心脏活动的影响

利用斯氏蛙心灌流方法,以心率和心搏幅度为评价指标,研究了5%、10%、20%、25%、30%、40%6个浓度梯度的绿茶浸出液对蟾蜍离体心脏活动的影响。结果表明,25%～30%的绿茶浸出液对蟾蜍的心率和心肌收缩力都有显著增强作用(P<0.05),低于25%和为40%时,虽有影响,但不显著(P>0.05)。因此25%～30%的绿茶浸出液具有一定的强心效应。     

Conditional Gene Targeting of Connexin43: Exploring the Consequences of Gap Junction Remodeling in the Heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

Simultaneous orientation and cellular force measurements in adult cardiac myocytes using three-dimensional polymeric microstructures

A number of techniques have been developed to monitor contractile function in isolated cardiac myocytes. While invaluable observations have been gained from these methodologies in understanding the contractile processes of the heart, they are invariably limited by their in vitro conditions. The present challenge is to develop innovative assays to mimic the in vivo milieu so as to allow a more physiological assessment of cardiac myocyte contractile forces. Here we demonstrate the use of a silicone elastomer, poly(dimethylsiloxane) (PDMS), to simultaneously orient adult cardiac myocytes in primary culture and measure the cellular forces in a three-dimensional substrate. The realignment of adult cardiac myocytes in long-term culture (7 days) was achieved due to directional reassembly of the myofibrils along the parallel polymeric sidewalls. The cellular mechanical forces were recorded in situ by observing the deformation of the micropillars embedded in the substrate. By coupling the cellular mechanical force measurements with on-chip cell orientation, this novel assay is expected to provide a means of a more physiological assessment of single cardiac myocyte contractile function and may facilitate the future development of in vitro assembled functional cardiac tissue.     

Soft tissue impact characterisation kit (STICK) for ex situ investigation of heart rhythm responses to acute mechanical stimulation

Both mechanical induction and mechanical termination of arrhythmias have been reported in man. Examples include pre-cordial impacts by sports implements (baseballs, pucks) that can trigger arrhythmias, including ventricular fibrillation, or via the so-called pre-cordial thump, used as an emergency resuscitation measure to convert arrhythmias to normal sinus node rhythm. These interventions have been partially reproduced in experimental studies on whole animals. Relating observations at the system's level to underlying mechanisms has been difficult, however, largely because of: (i) a deficit in efficient and affordable pharmacological agents to selectively target (sub-)cellular responses in whole animal studies, and (ii) the lack of suitable experimental models to study the above responses at intermediate levels of functional and structural integration, such as the isolated heart or cardiac tissue. This paper presents a soft tissue impact characterisation kit (STICK), suitable for quantitative investigations into the effects of acute mechanical stimulation on cardiac electro-mechanical function in rodent isolated heart or tissue preparations. The STICK offers independent control over a range of relevant biophysical parameters, such as impact location and energy, pre-impact projectile speed and contact area, as well as over the timing of a mechanical stimulus relative to the cardiac cycle (monitored via electrocardiogram, ECG, here recorded directly from the cardiac surface). Projectile deceleration upon interaction with the tissue is monitored, contact-free, with a resolution of 175 μm, providing information on tissue deformation dynamics, force, pressure and work of the mechanical intervention. In order to study functional effects of cardiac mechanical stimulation in the absence of tissue damage, impacts must be limited (for juvenile Guinea pig heart) to 2–2.5 mJ in the slack left ventricle (diastolic impact) and 5–10 mJ in contracture (systolic impact), as confirmed by enzyme assay and histological investigation. Impacts, timed to coincide with the early T-wave of the ECG, are capable of triggering short runs of ventricular fibrillation. Thus, the STICK is a suitable tool for the study of acute cardiac mechano-electric feedback effects, caused by short impulse-like mechanical stimulation, at the level of the isolated organ or tissue.     

分子动力学模拟与分子生物力学

生物大分子的微观结构动力学决定其生物学功能,其力学-化学耦合规律是分子生物力学的重点关注方向。分子动力学模拟是耦合生物大分子力学-化学性质微观结构动力学基础的有效手段,其结果可用于预测结构-功能关系、指导实验设计和诠释实验结果。本文简要介绍了分子动力学模拟的方法学特点、基本工作原理及其在分子生物力学中的应用,并展望了未来可能的发展方向和应用前景。     

Quantification of left ventricular mechanical dyssynchrony by conductance catheter in heart failure patients

Mechanical dyssynchrony is an important codeterminant of cardiac dysfunction in heart failure. Treatment, either medical, surgical, or by pacing, may improve cardiac function partly by improving mechanical synchrony. Consequently, the quantification of ventricular mechanical (dys)synchrony may have important diagnostic and prognostic value and may help to determine optimal therapy. Therefore, we introduced new indexes to quantify temporal and spatial aspects of mechanical dyssynchrony derived from online segmental conductance catheter signals obtained during diagnostic cardiac catheterization. To test the feasibility and usefulness of our approach, we determined cardiac function and left ventricular mechanical dyssynchrony by the conductance catheter in heart failure patients with intraventricular conduction delay (n = 12) and in patients with coronary artery disease (n = 6) and relatively preserved left ventricular function. The heart failure patients showed depressed systolic and diastolic function. However, the most marked hemodynamic differences between the groups were found for mechanical dyssynchrony, indicating a high sensitivity and specificity of the new indexes. Comparison of conductance catheter-derived indexes with septal-to-lateral dyssynchrony derived by tissue-Doppler velocity imaging showed highly significant correlations. The proposed indexes provide additional, new, and quantitative information on temporal and spatial aspects of mechanical dyssynchrony. They may refine diagnosis of cardiac dysfunction and evaluation of interventions, and ultimately help to select optimal therapy.     

A framework for biomechanics simulations using four-chamber cardiac models

Computational cardiac models have been extensively used to study different cardiac biomechanics; specifically, finite-element analysis has been one of the tools used to study the internal stresses and strains in the cardiac wall during the cardiac cycle. Cubic-Hermite finite element meshes have been used for simulating cardiac biomechanics due to their convergence characteristics and their ability to capture smooth geometries compactly–fewer elements are needed to build the cardiac geometry–compared to linear tetrahedral meshes. Such meshes have previously been used only with simple ventricular geometries with non-physiological boundary conditions due to challenges associated with creating cubic-Hermite meshes of the complex heart geometry. However, it is critical to accurately capture the different geometric characteristics of the heart and apply physiologically equivalent boundary conditions to replicate the in vivo heart motion. In this work, we created a four-chamber cardiac model utilizing cubic-Hermite elements and simulated a full cardiac cycle by coupling the 3D finite element model with a lumped circulation model. The myocardial fiber-orientations were interpolated within the mesh using the Log-Euclidean method to overcome the singularity associated with interpolation of orthogonal matrices. Physiologically equivalent rigid body constraints were applied to the nodes along the valve plane and the accuracy of the resulting simulations were validated using open source clinical data. We then simulated a complete cardiac cycle of a healthy heart and a heart with acute myocardial infarction. We compared the pumping functionality of the heart for both cases by calculating the ventricular work. We observed a 20% reduction in acute work done by the heart immediately after myocardial infarction. The myocardial wall displacements obtained from the four-chamber model are comparable to actual patient data, without requiring complicated non-physiological boundary conditions usually required in truncated ventricular heart models.     

Functional hypervariability and gene diversity of cardioactive neuropeptides

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.     

Isolation,Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca²⁺ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.     

The representation of heart development in the gene ontology

An understanding of heart development is critical in any systems biology approach to cardiovascular disease. The interpretation of data generated from high-throughput technologies (such as microarray and proteomics) is also essential to this approach. However, characterizing the role of genes in the processes underlying heart development and cardiovascular disease involves the non-trivial task of data analysis and integration of previous knowledge. The Gene Ontology (GO) Consortium provides structured controlled biological vocabularies that are used to summarize previous functional knowledge for gene products across all species. One aspect of GO describes biological processes, such as development and signaling.In order to support high-throughput cardiovascular research, we have initiated an effort to fully describe heart development in GO; expanding the number of GO terms describing heart development from 12 to over 280. This new ontology describes heart morphogenesis, the differentiation of specific cardiac cell types, and the involvement of signaling pathways in heart development. This work also aligns GO with the current views of the heart development research community and its representation in the literature. This extension of GO allows gene product annotators to comprehensively capture the genetic program leading to the developmental progression of the heart. This will enable users to integrate heart development data across species, resulting in the comprehensive retrieval of information about this subject.The revised GO structure, combined with gene product annotations, should improve the interpretation of data from high-throughput methods in a variety of cardiovascular research areas, including heart development, congenital cardiac disease, and cardiac stem cell research. Additionally, we invite the heart development community to contribute to the expansion of this important dataset for the benefit of future research in this area.     

Picometer-scale conformational heterogeneity separates functional from nonfunctional states of a photoreceptor protein

Protein structural fluctuations occur over a wide spatial scale, ranging from minute, picometer-scale displacements, to large, interdomain motions and partial unfolding. While large-scale protein structural changes and their effects on protein function have been the focus of much recent attention, small-scale fluctuations have been less well studied, and are generally assumed to have proportionally smaller effects. Here we use the bacterial photoreceptor photoactive yellow protein (PYP) to test if subtle structural changes do, indeed, imply equally subtle functional effects. We flash froze crystals of PYP to trap the protein's conformational ensemble, and probed the molecules in this ensemble for their ability to facilitate PYP's biological function (i.e., light-driven isomerization of its chromophore). Our results indicate that the apparently homogeneous structural state observed in a 0.82 A crystal structure in fact comprises an ensemble of conformational states, in which subpopulations with nearly identical structures display dramatically different functional properties.     

MicroRNAs in a hypertrophic heart: from foetal life to adulthood

The heart is the first organ to form and undergoes adaptive remodelling with age. Ventricular hypertrophy is one such adaptation, which allows the heart to cope with an increase in cardiac demand. This adaptation is necessary as part of natural growth from foetal life to adulthood. It may also occur in response to resistance in blood flow due to various insults on the heart and vessels that accumulate with age. The heart can only compensate to this increase in workload to a certain extent without losing its functional architecture, ultimately resulting in heart failure. Many genes have been implicated in cardiac hypertrophy, however none have been shown conclusively to be responsible for pathological cardiac hypertrophy. MicroRNAs offer an alternative mechanism for cellular regulation by altering gene expression. Since 1993 when the function of a non‐coding DNA sequence was first discovered in the model organism Caenorhabditis elegans, many microRNAs have been implicated in having a central role in numerous physiological and pathological cellular processes. The level of control these antisense oligonucleotides offer can often be exploited to manipulate the expression of target genes. Moreover, altered levels of microRNAs can serve as diagnostic biomarkers, with the prospect of diagnosing a disease process as early as during foetal life. Therefore, it is vital to ascertain and investigate the function of microRNAs that are involved in heart development and subsequent ventricular remodelling. Here we present an overview of the complicated network of microRNAs and their target genes that have previously been implicated in cardiogenesis and hypertrophy. It is interesting to note that microRNAs in both of these growth processes can be of possible remedial value to counter a similar disease pathophysiology.     

Biomechanical properties of native and tissue engineered heart valve constructs

Due to the increasing number of heart valve diseases, there is an urgent clinical need for off-the-shelf tissue engineered heart valves. While significant progress has been made toward improving the design and performance of both mechanical and tissue engineered heart valves (TEHVs), a human implantable, functional, and viable TEHV has remained elusive. In animal studies so far, the implanted TEHVs have failed to survive more than a few months after transplantation due to insufficient mechanical properties. Therefore, the success of future heart valve tissue engineering approaches depends on the ability of the TEHV to mimic and maintain the functional and mechanical properties of the native heart valves. However, aside from some tensile quasistatic data and flexural or bending properties, detailed mechanical properties such as dynamic fatigue, creep behavior, and viscoelastic properties of heart valves are still poorly understood. The need for better understanding and more detailed characterization of mechanical properties of tissue engineered, as well as native heart valve constructs is thus evident. In the current review we aim to present an overview of the current understanding of the mechanical properties of human and common animal model heart valves. The relevant data on both native and tissue engineered heart valve constructs have been compiled and analyzed to help in defining the target ranges for mechanical properties of TEHV constructs, particularly for the aortic and the pulmonary valves. We conclude with a summary of perspectives on the future work on better understanding of the mechanical properties of TEHV constructs.