Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions. Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR). Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

Abstract-To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43 Ϫ/Ϫ mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, g j , was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye. Compared with wild-type (Cx43 ϩ/ϩ ) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43 ϩ/Ϫ strands and was absent in Cx43 Ϫ/Ϫ strands. Cx45 signal was reduced by 46% in Cx43 ϩ/Ϫ strands and to the limit of detection in Cx43 Ϫ/Ϫ strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in Ϸ 2% of myocytes. Intercellular conductance, g j , was reduced by 32% in Cx43 ϩ/Ϫ cell pairs and by 96% in Cx43 Ϫ/Ϫ cell pairs. The symmetrical dependence of g j on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43Ϫ/Ϫ cells. Propagation in Cx43 Ϫ/Ϫ strands was very slow (2.1 cm/s versus 52 cm/s in Cx43 ϩ/ϩ ) and highly discontinuous, with simultaneous excitation within and long conduction delays (2 to 3 ms) between individual cells. Propagation was abolished by 1 mmol/L heptanol, indicating residual junctional coupling. In summary, knockout of Cx43 in ventricular myocytes leads to very slow conduction dependent on the presence of Cx45. Electrical field effect transmission does not contribute to propagation in synthetic strands. Key Words: Cx43 Ⅲ Cx45 Ⅲ very slow electrical propagation Ⅲ discontinuous propagation Ⅲ heptanol C onnexin (Cx) proteins form intercellular channels enabling the intercellular exchange of ions and small molecules. 1 In the heart, they facilitate rapid, coordinated electrical excitation, a prerequisite for normal rhythmic contraction. Three different connexins, Cx43, Cx40, and Cx45, are expressed in heart. 2,3 Cx43, the most abundant connexin, is found in ventricular and atrial myocardium. Cx40 is expressed in atrial tissue and the cardiac conduction system. Although Cx45 expression in working ventricular myocardium is modest compared with Cx43, Cx45 is vital for early embryogenesis and cardiac development. 4,5 Cx45 is also expressed in the sinoatrial and atrioventricular nodes. It colocalizes with Cx40 in the conduction system, 6 and with Cx43 in ventricular myocardium. 7 The three cardiac connexins form channels with unique properties. 8 Although multiple connexins are coexpressed in cardiac tissues, their interactions and contributions to electrical or metabolic function are not understood completely. Cx43 and Cx45 likely form heteromeric/heterotypic channels, 9 -12 which may fulfill distinct functions. Cardiac diseases that lead to arrhythmias are associated with gap junction remodeling. [13][14][15] Therefore, know...

Abstract-Atrial tissue expresses both connexin 40 (Cx40) and 43 (Cx43) proteins. To assess the relative roles of Cx40 and Cx43 in atrial electrical propagation, we synthesized cultured strands of atrial myocytes derived from mice with genetic deficiency in Cx40 or Cx43 expression and measured propagation velocity (PV) by high-resolution optical mapping of voltage-sensitive dye fluorescence. The amount of Cx40 and/or Cx43 in gap junctions was measured by immunohistochemistry and total or sarcolemmal Cx43 or Cx40 protein by immunoblotting. Progressive genetic reduction in Cx43 expression decreased PV from 34Ϯ6 cm/sec in Cx43 ϩ/ϩ to 30Ϯ8 cm/sec in Cx43 ϩ/Ϫ and 19Ϯ11 cm/sec in Cx43 Ϫ/Ϫ cultures. Concomitantly, the cell area occupied by Cx40 immunosignal in gap junctions decreased from 2.0Ϯ1.6% in Cx43 ϩ/ϩ to 1.7Ϯ0.5% in Cx43 ϩ/Ϫ and 1.0Ϯ0.2% in Cx43 Ϫ/Ϫ strands. In contrast, progressive genetic reduction in Cx40 expression increased PV from 30Ϯ2 cm/sec in Cx40 ϩ/ϩ to 40Ϯ7 cm/sec in Cx40 ϩ/Ϫ and 45Ϯ10 cm/sec in Cx40 Ϫ/Ϫ cultures. Concomitantly, the cell area occupied by Cx43 immunosignal in gap junctions increased from 1.2Ϯ0.9% in Cx40 ϩ/ϩ to 2.8Ϯ1.4% in Cx40 ϩ/Ϫ and 3.1Ϯ0.6% in Cx40 Ϫ/Ϫ cultures. In accordance with the immunostaining results, immunoblots of the Triton X-100 -insoluble fraction revealed an increase of Cx43 in gap junctions in extracts from Cx40-ablated atria, whereas total cellular Cx43 remained unchanged. Our results suggest that the relative abundance of Cx43 and Cx40 is an important determinant of atrial impulse propagation in neonatal hearts, whereby dominance of Cx40 decreases and dominance of Cx43 increases local propagation velocity. (Circ Res. 2006;99:1216-1224.)Key Words: atrial myocyte Ⅲ basic science Ⅲ cardiac gap junction connexins Ⅲ cardiovascular genomics Ⅲ cell culture Ⅲ conduction velocity Ⅲ connexin 40 Ⅲ connexin 43 Ⅲ mapping Ⅲ neonatal mouse cardiomyocytes Ⅲ optical mapping C onnexin (Cx) proteins enable the intercellular exchange of ions and small regulatory molecules and are important determinants of cardiac electrical propagation. 1 Three major connexins, Cx43, Cx40, and Cx45, are expressed in heart. 2,3 Cx43 is abundant in ventricular and atrial myocardium; Cx40 is expressed in atrial tissue and in the Purkinje system; Cx45 is present in the sinoatrial (SA) and atrioventricular (AV) nodes and colocalizes with Cx43 in ventricular myocardium. 4,5 Cx43 and Cx40 each form channels with relatively large pores, whereas Cx45 forms narrow channels (unitary channel conductances of 75, 130, and 30 pS, respectively). 6 Ventricular myocytes express abundant Cx43 and small amounts of Cx45. In contrast, atrial myocytes express large amounts of Cx43 and Cx40. In ventricular myocytes and transfected cells, Cx43 and Cx45 colocalize in gap junctions and may form heteromeric/heterotypic channels. 5,7-10 Heterotypic Cx43/Cx40 gap junction channels have been described in HeLa cell pairs, whereas the role of heteromeric Cx43/ Cx40 channels has not been fully clarified. 7,11 Changes in cell-to-cell...