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    • The experimental design of this

    2018-10-24

    The experimental design of this model provides several differences to standard 2D culture: EHTs are anchored between two flexible silicone posts, which generate a preload, and EHTs perform auxotonic contractile work (the physiological form of cardiac contraction) against elastic posts. This results in macroscopically distinguishable muscle bundles, a high degree of CM alignment and orientation, good sarcomeric organization, and cross-striated CM approaching the classical rod shape of adult ventricular myocytes. These results compare favorably with human embryonic stem cell- and hiPSC-CM cultured in classical monolayers that are polymorphic and small, isotropically oriented, and have disarrayed sarcomeres and very immature ultrastructural organization (reviewed in Yang et al., 2014). Furthermore, TEM, confocal immunofluorescence microscopy, and western blot analysis revealed that CM in EHTs have a relatively high mitochondrial density and express classical calcium-handling proteins. TEM and staining for caveolin and junctophilin are compatible with nascent t tubules, but the data clearly do not qualify as proof of an established t-tubular system in hiPSC-CM in EHTs. Others have shown that culture time is important for the structural and functional development of hiPSC-CM in 2D (Lundy et al., 2013) and that 1-year-old CM cultured at very low density exhibits increased size, longer and better organized sarcomeres, greater transcript levels of cardiac marker proteins, longer action potentials, and signs of metabolic switch that could be mimicked by overexpressing the microRNA let-7 (Kuppusamy et al., 2015). Since EHTs are cultured for only 2–3 weeks before the first measurements, other factors such as the 3D TAPI-1 muscle-like environment and load likely participate in this development process. The impact of load on cardiomyocyte development is also supported by a recent study in neonatal rat EHTs demonstrating that increasing the load by inserting metal braces induced a phenotype of pathological cardiac hypertrophy in rat EHTs (Hirt et al., 2012), indicating that culture under isometric conditions (stiff posts or 2D cultures on plastic surfaces) may in fact induce a pathological phenotype. Somewhat surprisingly, EHTs could readily be generated not only from hiPSC-CM preparations with 60%–90% purity, but also from two commercially available hiPSC-CM (CDI, Axiogenesis), which, by genetic selection, are essentially free of non-myocytes, suggesting that previous findings of a mandatory requirement of 25% non-cardiomyocytes in collagen-based EHTs (Naito et al., 2006) do not apply for human tissues generated with fibrin. Contractile force in CM is the result of a fine-tuned interplay between electrical activation, calcium handling, and myofilament activation. To obtain a comprehensive picture of inotropic regulation in hiPSC-EHT, we analyzed the effects of eight compounds (calcium, ouabain, Bay K-8644, EMD-57033, isoprenaline, rolipram, ryanodine, and verapamil) that modulate contractile function by different modes of action. The data suggest that the hiPSC-EHT model replicates characteristic effects on inotropy and kinetics of contraction. The level of precision in replicating inotropic effects in hiPSC-EHTs is, to the best of our knowledge, unmatched by other approaches. For example, edge detection in 2D hiPSC-CM demonstrated high sensitivity and specificity in detecting modulators of inotropy, but failed to discriminate positive from negative inotropic agents (Pointon et al., 2015). The positive inotropic effects described in our study are smaller than in human non-failing heart tissue (Figure S5F). These differences are likely related to a lack of full maturation of CM in hiPSC-EHTs: Mitochondrial density and substructure, sarcomeric organization, intercellular connections, and t tubules are still clearly less developed in hiPSC-CM in EHTs than in native heart tissue. EHTs beat spontaneously, regulation of contractile function by physiological interventions was smaller, and a force-frequency relationship was absent. A key prerequisite for inotropic responses of CM is the precise spatial configuration of t tubules and sarcoplasmic reticulum. Even though PRP indicated functional sarcoplasmic reticulum and TEM analysis/caveolin-3/junctophilin-2 immunofluorescence suggests tubular structures in close proximity to Z lines, the irregularity and the small size indicate a very immature t-tubule/SR system. In native myocytes, the t-tubule microarchitecture is highly organized with distances to the SR of 12–15 nm. Small deviations of the spatial distance have been associated with altered calcium-induced calcium-release mechanism (Gómez et al., 1997). The much greater abnormalities of the t-tubule/SR organization in hiPSC-CM would also be compatible with the lack of a force-frequency relationship. Along the same lines, detubulation of rat myocytes by formamide-induced osmotic shock was associated with prolonged contraction peaks and calcium transients, and lack of a positive force-frequency relationship, but a preserved FDAR, positive force-calcium relationship, and positive PRP (Ferrantini et al., 2014). A study comparing human ventricular heart tissue of newborns (<2 weeks) and infants (3–15 months) observed t tubules, positive force-frequency relationship, and FDAR in infants, but only FDAR in newborns (Wiegerinck et al., 2009). PRP is directly related to the capacity of the SR to store and release calcium. The small PRP in hiPSC-EHTs suggest that the SR is incompletely developed, but functional. The link between small PRP and immaturity is also supported by a previous study demonstrating an increase in PRP with rabbit cardiomyocyte maturation between postnatal days 1–4, 14–21, and >180 (Boucek et al., 1987). On the other hand, while non-failing human heart tissue has been mainly evaluated under isometric conditions, force in hiPSC-EHTs is measured under physiological auxotonic conditions. The impact of this difference is not well characterized, but one study demonstrated 50% lower forces for auxotonic versus isometric conditions in adult rat ventricular CM (Nishimura et al., 2004).