Rationale Cardiomyocytes differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research including disease modeling and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix (ECM) is also fundamentally involved in cardiac development from the earliest morphogenetic events such as gastrulation. Objective We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using ECM in combination with growth factors known to promote cardiogenesis. Methods and Results PSCs were cultured as monolayers on Matrigel, an ECM preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin+ mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, BMP4, and bFGF) generated cardiomyocytes with high purity (up to 98%) and yield (up to 11 cardiomyocytes/input PSC) from multiple PSC lines. The resulting cardiomyocytes progressively mature over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial and ventricular cardiomyocytes were observed, and monolayers of electrically coupled cardiomyocytes modeled cardiac tissue and basic arrhythmia mechanisms. Conclusions Dynamic ECM application promoted EMT of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I K1 ), which is important for maintenance of the cell resting membrane potential, and the sodium current (I Na ), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I K1 modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I K1 -I Na interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I K1 -I Na interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na V 1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na v 1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na v 1.5-Kir2.1 interactions. The reciprocal modulation between Na v 1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.reentry | scaffolding proteins | conduction velocity | protein trafficking I n the heart, the inward rectifying potassium current (I K1 ) is the major current responsible for the maintenance of the resting membrane potential (RMP), whereas the sodium current (I Na ) provides the largest fraction of the inward depolarizing current that flows during an action potential (1). It is well-known that a relationship exists between these two ionic currents that is crucial for proper cardiac electrical function; disruption of this balance results in changes in sodium channel availability, cell excitability, action potential duration, and conduction velocity (2). Accordingly, I K1 -I Na interactions are important in stabilizing and controlling the frequency of the electrical rotors that are responsible for the most dangerous cardiac arrhythmias, including ventricular tachycardia and fibrillation (3).Post synaptic density, discs large, and zonula occludens-1 (PDZ) domain proteins link different and in many cases, multiple proteins to macromolecular complexes through interactions with their various domains. More than 70 PDZ d...

Rationale Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) offer a powerful in-vitro tool to investigate disease mechanisms and to perform patient-specific drug screening. To date electrophysiological analysis of iPSC-CMs has been limited to single cell recordings or low resolution microelectrode array mapping of small cardiomyocyte aggregates. A new method of generating and optically mapping impulse propagation of large human iPSC-CM cardiac monolayers is needed. Objective Our first aim was to develop an imaging platform with versatility for multi-parameter electrophysiological mapping of cardiac preparations, including human iPSC-CM monolayers. Our second aim was to create large electrically coupled human iPSC-CM monolayers for simultaneous action potential and calcium wave propagation measurements. Methods and Results A fluorescence imaging platform based on electronically-controlled light-emitting-diode (LED) illumination, a multi-band emission filter and single camera sensor was developed and utilized to monitor simultaneously action potential and intracellular calcium wave propagation in cardiac preparations. Multiple large diameter (≥1cm) electrically coupled human cardiac monolayers were then generated that propagated action potentials and calcium waves at velocities similar to those commonly observed in rodent cardiac monolayers. Conclusions The multi-parametric imaging system presented here offers a scalable enabling technology to measure simultaneously action potential and intracellular calcium wave amplitude and dynamics of cardiac monolayers. The advent of large-scale production of human iPSC-CMs makes it possible now to generate sufficient numbers of uniform cardiac monolayers that can be utilized for the study of arrhythmia mechanisms and offers advantages over commonly used rodent models.