The ability to differentiate induced-pluripotent stem cells into cardiomyocytes (iPSC-CMs) has opened up novel avenues for potential cardiac therapies. However, iPSC-CMs exhibit a range of somewhat immature functional properties. This study explored the development of the beta-adrenergic receptor (βAR) pathway, which is crucial in regulating contraction and signifying the health and maturity of myocytes. We explored the compartmentation of β₂AR-signalling and phosphodiesterases (PDEs) in caveolae, as functional nanodomains supporting the mature phenotype. Förster Resonance Energy Transfer (FRET) microscopy was used to study the cyclic adenosine monophosphate (cAMP) levels in iPSC-CMs at day 30, 60, and 90 following βAR subtype-specific stimulation. Subsequently, the PDE2, PDE3, and PDE4 activity was investigated using specific inhibitors. Cells were treated with methyl-β-cyclodextrin (MβCD) to remove cholesterol as a method of decompartmentalising β₂AR. As iPSC-CMs mature with a prolonged culture time, the caveolae density is increased, leading to a reduction in the overall cytoplasmic cAMP signal stimulated through β₂AR (but not β₁AR). Pan-phosphodiesterase inhibition or caveolae depletion leads to an increase in the β₂AR-stimulated cytoplasmic cAMP. Moreover, with time in culture, the increase in the βAR-dependent cytoplasmic cAMP becomes more sensitive to cholesterol removal. The regulation of the β₂AR response by PDE2 and 4 is similarly increased with the time in culture. We conclude that both the β₂AR and PDEs are restricted to the caveolae nanodomains, and thereby exhibit a tighter spatial restriction over the cAMP signal in late-stage compared to early iPSC-CMs.

中文翻译：

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iPSC-CM 的年龄依赖性成熟导致 β2AR-cAMP 信号传导的分隔增强

将诱导多能干细胞分化为心肌细胞 (iPSC-CM) 的能力为潜在的心脏治疗开辟了新途径。然而，iPSC-CM 表现出一系列有些不成熟的功能特性。本研究探索了 β-肾上腺素能受体 (βAR) 通路的发展，该通路在调节收缩和表明肌细胞的健康和成熟方面至关重要。我们探索了 β _2的划分小窝中的 AR 信号和磷酸二酯酶 (PDE)，作为支持成熟表型的功能性纳米结构域。Förster 共振能量转移 (FRET) 显微镜用于研究 βAR 亚型特异性刺激后第 30、60 和 90 天 iPSC-CM 中的环磷酸腺苷 (cAMP) 水平。随后，使用特定抑制剂研究了 PDE2、PDE3 和 PDE4 活性。用甲基-β-环糊精 (MβCD) 处理细胞以去除胆固醇，作为分解 β ₂ AR 的方法。随着 iPSC-CM 随着培养时间的延长而成熟，小窝密度增加，导致通过 β ₂ AR（但不是 β ₁应）。泛磷酸二酯酶抑制或caveolae 耗竭导致β2 _AR刺激的细胞质cAMP 增加。此外，随着培养时间的延长，βAR 依赖性细胞质 cAMP 的增加对胆固醇的去除变得更加敏感。PDE2 和 4对 β ₂ AR 反应的调节同样随着培养时间的增加而增加。我们得出结论，β ₂ AR 和 PDE 都被限制在小窝纳米域中，因此与早期的 iPSC-CM 相比，在后期对 cAMP 信号表现出更严格的空间限制。