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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. [ABSTRACT FROM AUTHOR]

The advent of induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) has revolutionized the cardiovascular research field. Now it is possible to generate a virtually unlimited supply of patient specific pluripotent stem cells and cardiomyocytes that can be used for research purposes, drug toxicity testing and/or regenerative medicine therapies. The most immediate application for this technology is in vitro disease modeling and in vitro drug toxicity testing. To date the majority of disease modeling and drug toxicity testing has been performed on single hiPSC-CMs in culture. However, the study of complex cardiac arrhythmia mechanisms requires a more physiological model system of electrically and mechanically connected hiPSC-CMs that function as a syncytium-like the cardiomyocytes of the adult heart. This review focuses on the work that has been performed recently using hiPSC-CM 2D monolayers for the study of cardiac electrical impulse propagation. [ABSTRACT FROM AUTHOR]

Abstract: Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers. [Copyright &y& Elsevier]

Background: Persistent atrial fibrillation (PAF) results in electromechanical and structural remodeling by mechanisms that are poorly understood. Myofibroblast proliferation and fibrosis are major sources of structural remodeling in PAF. Myofibroblasts also interact with atrial myocytes via direct physical contact and release of signaling molecules, which may contribute to remodeling. Objective: To determine whether myofibroblasts contribute to atrial myocyte electromechanical remodeling via direct physical contact and platelet-derived growth factor (PDGF) signaling. Methods: Myofibroblasts and myocytes from adult sheep atria were co-cultured for 24 hours. Alternatively adult sheep atrial myocytes were exposed to 1ng/mL recombitant PDGF AB peptide for 24 hours. Results: Myocytes making contact with myofibroblasts demonstrated significant reduction (P ≤ .05) in peak L-type calcium current density, shortening of action potential duration (APD), and reduction in calcium transients. These effects were blocked by pretreatment with a PDGF-AB neutralizing anti-body. Heterocellular contact also severely disturbed the localization of the L-type calcium channel. Myocytes exposed to recombinant PDGF-AB peptide for 24 hours demonstrated reduced APD50, APD80 and Peak L-type calcium current. Pretreatment with a PDGF-AB neutralizing antibody prevented these effects. Finally, while control atrial myocytes did not respond in a 1:1 manner to pacing frequencies of 3Hz or higher, atrial myocytes from hearts that were tachypaced for 2 months and normal myocytes treated with PDGF-AB for 24 hours could be paced up to 10Hz. Conclusions: In addition to leading to fibrosis, atrial myofibroblasts contribute to electromechanical remodeling of myocytes via direct physical contact and release of PDGF-AB, which may be a factor in PAF-induced remodeling. [Copyright &y& Elsevier]

Background: Collecting electrophysiological and molecular data from the murine conduction system presents technical challenges. Thus, only little advantage has been taken of numerous genetically engineered murine models to study excitation through the cardiac conduction system of the mouse. Objective : To develop an approach for isolating murine cardiac Purkinje cells (PCs), to characterize major ionic currents and to use the data to simulate action potentials (APs) recorded from PCs. Methods : Light microscopy was used to isolate and identify PCs from apical and septal cells. Current and voltage clamp techniques were used to record APs and whole cell currents. We then simulated a PC AP on the basis of our experimental data. Results : APs recorded from PCs were significantly longer than those recorded from ventricular cells. The prominent plateau phase of the PC AP was very negative (∼−40 mV). Spontaneous activity was observed only in PCs. The inward rectifier current demonstrated no significant differences compared to ventricular myocytes (VMs). However, sodium current density was larger, and the voltage-gated potassium current density was significantly less in PCs compared with myocytes. T-type Ca2+ currents (ICa,T) were present in PCs but not VMs. Computer simulations suggest that ICa,T and cytosolic calcium diffusion significantly modulate AP profile recorded in PCs, as compared to VMs. Conclusions: Our study provides the first comprehensive ionic profile of murine PCs. The data show unique features of PC ionic mechanisms that govern its excitation process. Experimental data and numerical modeling results suggest that a smaller voltage-gated potassium current and the presence of ICa,T are important determinants of the longer and relatively negative plateau phase of the APs. [Copyright &y& Elsevier]

Background: Inherited arrhythmias can be caused by mutations in the cardiac ryanodine receptor (RyR2). The cellular source of these arrhythmias is unknown. Isolated RyR2R4496C mouse ventricular myocytes display arrhythmogenic activity related to spontaneous Ca2+ release during diastole. On the other hand, recent whole-heart epicardial and endocardial optical mapping data demonstrate that ventricular arrhythmias in the RyR2R4496C mouse model of catecholaminergic polymorphic ventricular tachycardia (CPVT) originate in the His–Purkinje system, suggesting that Purkinje cells, and not ventricular myocytes, may be the cellular source of arrhythmogenic activity. The relative effect of the RyR2R4496C mutation on calcium homeostasis in ventricular myocytes versus Purkinje cells is unknown. Objective: This study sought to determine which cardiac cell type is more severely affected, in terms of calcium handling, by expression of the RyR2R4496C mutant channel: the ventricular myocytes or the Purkinje cells. Methods and Results: To discriminate Purkinje cells from ventricular myocytes, we crossed the RyR2R4496C mouse model of CPVT with the Cx40EGFP/+ transgenic mouse. This genetic cross yields Purkinje cells that express eGFP, and therefore fluoresce green when excited by the appropriate wavelength; ventricular myocytes, which do not express connexin 40, are not green. Intracellular calcium was measured in each cell type using calcium-sensitive probes. Purkinje cells of the RyR2R4496C mouse model of CPVT show an approximately 2× greater rate (P < .05) and approximately 2× to 3× greater amplitude (P < .000001) of spontaneous calcium release events than ventricular myocytes isolated from the same heart. Conclusion: These results demonstrate that focally activated arrhythmias originate in the specialized electrical conducting cells of the His–Purkinje system in the RyR2R4496C mouse model of CPVT. [Copyright &y& Elsevier]