350 Chapter 13 proteasome activity (PKA/HSP90 signaling), metabolic homeostasis (fatty acid oxidation), physiology (calcium handling, increased contractility, and β-adrenergic response).8–10 Next, we and others showed that alterations in energy sources are a simple, yet effective method to metabolically switch hiPSC-CMs from glucose to fatty acid oxidation by providing oxidative substrates adapted to the metabolic needs of hiPSC-CMs.11–13 According to a recent article in Nature, a fatty acid called γ-linolenic acid (GLA) found in the milk of nursing mice has been found to trigger a transformation in the metabolic pathways by binding retinoid X receptors (RXRs), enabling the cells to rapidly mature after birth.14 This unexpected finding reveals a role for GLA in promoting the early murine heart maturation, which could be applied to improve hiPSC-CM maturation. In Chapter 6, we demonstrate that prolonged cultured and metabolically stimulated cardiomyocytes increase hypoxia susceptibility, which is more reminiscent of adult CMs. The metabolic maturation of hiPSC-CMs represents an improved in vitro model to study human ischemic heart disease and genetic cardiomyopathies.11,15 In this thesis, we combined biochemical strategies such as prolonged culturing of a monolayer with metabolic and hormonal supplements to induce maturation in vitro. Here, sarcomeric organization, CM length, and sarcomere spacing were improved, resulting in a morphology similar to human adult CMs (Figure 1A). Although many of the measurable maturation parameters such as; surface area, bi-nuclearity, gene expression, M-banding, and sarcomere spacing are improved in these hiPSC-CMs to mimic the human adult CM level, the upstroke velocity, resting membrane potential, and T-tubuli are almost reaching the parameter levels (Figure 1B). Nevertheless, if the maturation status of hiPSC-CMs could even further improve or accelerate in the described parameters, a significant increase in disease modeling can be expected. For example, increased gene expression levels of ventricular ion channels and calcium-handling genes are considered to be hallmarks of maturation.16 Still, complex mRNAbased mechanisms driven by alternative splicing and RNA-binding proteins may need to be manipulated to improve CM maturation further.17 Abolishing depolarization-associated genes HCN4, CACNA1H, and SLC8A1, along with overexpressing the ion channel KCNJ2 changes extensively as hiPSC-CM grafts mature in vivo, thereby reducing arrhythmic events after cell graft transplantation.18 However, the one major challenge remaining is to localize the fully functional gap junction protein Connexin 43 (Cx43) in the intercalated discs. In metabolically matured hiPSC-CMs, a rather low Cx43 expression and localization are found (Figure 1C), causing a significant reduction of intercellular coupling and leading to slow electrical signal propagation.19 The formation of functional gap junctions is essential for modulating cardiac electric activities and the absence of Cx43 leads to the incidence of ventricular tachyarrhythmias.20 Interestingly, even during the development and maturation of human cardiomyocytes, spatiotemporal localization of Cx43 takes 7 years to move from the lateral membranes to the intercalated disks.21 In summary, after 70 years of many cell culture improvements, we now have create
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