Rationale:: Cardiomyocytes (CMs) 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 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 extracellular matrix in combination with growth factors known to promote cardiogenesis. Methods and Results:: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. Conclusions:: Dynamic extracellular matrix application promoted epithelial–mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.

Heart failure (HF) commonly results in atrial fibrillation (AF) and fibrosis, but how the distribution of fibrosis impacts AF dynamics has not been studied. HF was induced in sheep by ventricular tachypacing (220 bpm, 6 to 7 weeks). Optical mapping (Di-4-ANEPPS, 300 frames/sec) of the posterior left atrial (PLA) endocardium was performed during sustained AF (burst pacing) in Langendorff-perfused HF (n=7, 4 μmol/L acetylcholine; n=3, no acetylcholine) and control (n=6) hearts. PLA breakthroughs were the most frequent activation pattern in both groups (72.0±4.6 and 90.2±2.7%, HF and control, respectively). However, unlike control, HF breakthroughs preferentially occurred at the PLAs periphery near the pulmonary vein ostia, and their beat-to-beat variability was greater than control (1.93±0.14 versus 1.47±0.07 changes/[beats/sec], respectively, P <0.05). On histological analysis (picrosirius red), the area of diffuse fibrosis was larger in HF (23.4±0.4%) than control (14.1±0.6%; P <0.001, n=4). Also the number and size of fibrous patches were significantly larger and their location was more peripheral in HF than control. Computer simulations using 2-dimensional human atrial models with structural and ionic remodeling as in HF demonstrated that changes in AF activation frequency and dynamics were controlled by the interaction of electrical waves with clusters of fibrotic patches of various sizes and individual pulmonary vein ostia. During AF in failing hearts, heterogeneous spatial distribution of fibrosis at the PLA governs AF dynamics and fractionation.

Background—: Human pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. Methods and Results—: Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal ( SCN5A , Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Conclusions—: Maturation of human stem cell–derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers.

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. New methods of generating and optically mapping impulse propagation of large human iPSC-CM cardiac monolayers are needed. Objective:: Our first aim was to develop an imaging platform with versatility for multiparameter 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 illumination, a multiband 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 (≥1 cm), 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 multiparametric 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 to now 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.

Histone H3 lysine 4 (H3K4me) methyltransferases and their cofactors are essential for embryonic development and the establishment of gene expression patterns in a cell-specific and heritable manner. However, the importance of such epigenetic marks in maintaining gene expression in adults and in initiating human disease is unclear. Here, we addressed this question using a mouse model in which we could inducibly ablate PAX interacting (with transcription-activation domain) protein 1 (PTIP), a key component of the H3K4me complex, in cardiac cells. Reducing H3K4me3 marks in differentiated cardiomyocytes was sufficient to alter gene expression profiles. One gene regulated by H3K4me3 was Kv channel-interacting protein 2 (Kcnip2), which regulates a cardiac repolarization current that is downregulated in heart failure and functions in arrhythmogenesis. This regulation led to a decreased sodium current and action potential upstroke velocity and significantly prolonged action potential duration (APD). The prolonged APD augmented intracellular calcium and in vivo systolic heart function. Treatment with isoproterenol and caffeine in this mouse model resulted in the generation of premature ventricular beats, a harbinger of lethal ventricular arrhythmias. These results suggest that the maintenance of H3K4me3 marks is necessary for the stability of a transcriptional program in differentiated cells and point to an essential function for H3K4me3 epigenetic marks in cellular homeostasis.

Background— The posterior left atrium (PLA) and pulmonary veins (PVs) have been shown to be critical for atrial fibrillation (AF) initiation. However, the detailed mechanisms of reentry and AF initiation by PV impulses are poorly understood. We hypothesized that PV impulses trigger reentry and AF by undergoing wavebreaks as a result of sink-to-source mismatch at specific PV-PLA transitions along the septopulmonary bundle, where there are changes in thickness and fiber direction. Methods and Results— In 7 Langendorff-perfused sheep hearts AF was initiated by a burst of 6 pulses (CL 80 to 150ms) delivered to the left inferior or right superior PV ostium 100 to 150 ms after the sinus impulse in the presence of 0.5 μmol/L acetylcholine. The exposed septal-PLA endocardial area was mapped with high spatio-temporal resolution (DI-4-ANEPPS, 1000-fr/s) during AF initiation. Isochronal maps for each paced beat preceding AF onset were constructed to localize areas of conduction delay and block. Phase movies allowed the determination of the wavebreak sites at the onset of AF. Thereafter, the PLA myocardial wall thickness was quantified by echocardiography, and the fiber direction in the optical field of view was determined after peeling off the endocardium. Finally, isochrone, phase and conduction velocity maps were superimposed on the corresponding anatomic pictures for each of the 28 episodes of AF initiation. The longest delays of the paced PV impulses, as well as the first wavebreak, occurred at those boundaries along the septopulmonary bundle that showed sharp changes in fiber direction and the largest and most abrupt increase in myocardial thickness. Conclusion— Waves propagating from the PVs into the PLA originating from a simulated PV tachycardia triggered reentry and vagally mediated AF by breaking at boundaries along the septopulmonary bundle where abrupt changes in thickness and fiber direction resulted in sink-to-source mismatch and low safety for propagation.