Local tissue mechanics control cardiac pacemaker cell embryonic patterning.

Cardiac pacemaker cells (CPCs) initiate the electric impulses that drive the rhythmic beating of the heart. CPCs reside in a heterogeneous, ECM-rich microenvironment termed the sinoatrial node (SAN). Surprisingly, little is known regarding the biochemical composition or mechanical properties of the SAN, and how the unique structural characteristics present in this region of the heart influence CPC function remains poorly understood. Here, we have identified that SAN development involves the construction of a "soft" macromolecular ECM that specifically encapsulates CPCs. In addition, we demonstrate that subjecting embryonic CPCs to substrate stiffnesses higher than those measured in vivo results in loss of coherent electrical oscillation and dysregulation of the HCN4 and NCX1 ion channels required for CPC automaticity. Collectively, these data indicate that local mechanics play a critical role in maintaining the embryonic CPC function while also quantitatively defining the range of material properties that are optimal for embryonic CPC maturation.

Adherens junction engagement regulates functional patterning of the cardiac pacemaker cell lineage.

Cardiac pacemaker cells (CPCs) rhythmically initiate the electrical impulses that drive heart contraction. CPCs display the highest rate of spontaneous depolarization in the heart despite being subjected to inhibitory electrochemical conditions that should theoretically suppress their activity. While several models have been proposed to explain this apparent paradox, the actual molecular mechanisms that allow CPCs to overcome electrogenic barriers to their function remain poorly understood. Here, we have traced CPC development at single-cell resolution and uncovered a series of cytoarchitectural patterning events that are critical for proper pacemaking. Specifically, our data reveal that CPCs dynamically modulate adherens junction (AJ) engagement to control characteristics including surface area, volume, and gap junctional coupling. This allows CPCs to adopt a structural configuration that supports their overall excitability. Thus, our data have identified a direct role for local cellular mechanics in patterning critical morphological features that are necessary for CPC electrical activity.

Simplified platform for mosaic in vivo analysis of cellular maturation in the developing heart.

Cardiac cells develop within an elaborate electro-mechanical syncytium that continuously generates and reacts to biophysical force. The complexity of the cellular interactions, hemodynamic stresses, and electrical circuitry within the forming heart present significant challenges for mechanistic research into the cellular dynamics of cardiomyocyte maturation. Simply stated, it is prohibitively difficult to replicate the native electro-mechanical cardiac microenvironment in tissue culture systems favorable to high-resolution cellular/subcellular analysis, and current transgenic models of higher vertebrate heart development are limited in their ability to manipulate and assay the behavior of individual cells. As such, cardiac research currently lacks a simple experimental platform for real-time evaluation of cellular function under conditions that replicate native development. Here we report the design and validation of a rapid, low-cost system for stable in vivo somatic transgenesis that allows for individual cells to be genetically manipulated, tracked, and examined at subcellular resolution within the forming four-chambered heart. This experimental platform has several advantages over current technologies, chief among these being that mosaic cellular perturbations can be conducted without globally altering cardiac function. Consequently, direct analysis of cellular behavior can be interrogated in the absence of the organ level adaptions that often confound data interpretation in germline transgenic model organisms.

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo.

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

Dynamic Cellular Integration Drives Functional Assembly of the Heart's Pacemaker Complex.

Impulses generated by a multicellular, bioelectric signaling center termed the sinoatrial node (SAN) stimulate the rhythmic contraction of the heart. The SAN consists of a network of electrochemically oscillating pacemaker cells encased in a heterogeneous connective tissue microenvironment. Although the cellular composition of the SAN has been a point of interest for more than a century, the biological processes that drive the tissue-level assembly of the cells within the SAN are unknown. Here, we demonstrate that the SAN's structural features result from a developmental process during which mesenchymal cells derived from a multipotent progenitor structure, the proepicardium, integrate with and surround pacemaker myocardium. This process actively remodels the forming SAN and is necessary for sustained electrogenic signal generation and propagation. Collectively, these findings provide experimental evidence for how the microenvironmental architecture of the SAN is patterned and demonstrate that proper cellular arrangement is critical for cardiac pacemaker biorhythmicity.

Optical Electrophysiology in the Developing Heart.

The heart is the first organ system to form in the embryo. Over the course of development, cardiomyocytes with differing morphogenetic, molecular, and physiological characteristics are specified and differentiate and integrate with one another to assemble a coordinated electromechanical pumping system that can function independently of any external stimulus. As congenital malformation of the heart presents the leading class of birth defects seen in humans, the molecular genetics of heart development have garnered much attention over the last half century. However, understanding how genetic perturbations manifest at the level of the individual cell function remains challenging to investigate. Some of the barriers that have limited our capacity to construct high-resolution, comprehensive models of cardiac physiological maturation are rapidly being removed by advancements in the reagents and instrumentation available for high-speed live imaging. In this review, we briefly introduce the history of imaging approaches for assessing cardiac development, describe some of the reagents and tools required to perform live imaging in the developing heart, and discuss how the combination of modern imaging modalities and physiological probes can be used to scale from subcellular to whole-organ analysis. Through these types of imaging approaches, critical insights into the processes of cardiac physiological development can be directly examined in real-time. Moving forward, the synthesis of modern molecular biology and imaging approaches will open novel avenues to investigate the mechanisms of cardiomyocyte maturation, providing insight into the etiology of congenital heart defects, as well as serving to direct approaches for designing stem-cell or regenerative medicine protocols for clinical application.