Electro-metabolic coupling in multi-chambered vascularized human cardiac organoids

The study of cardiac physiology and disease is hindered by physiological differences between humans and small-animal models. Here, we report the generation of multi-chambered vascularized human cardiac organoids under anisotropic stress, and their applicability to study electro-metabolic coupling in cardiac tissue. The organoids are derived from human induced pluripotent stem cells, and integrate sensors for the simultaneous measurement of oxygen uptake, extracellular field potentials and cardiac contraction at resolutions higher than 10 Hz. The microphysiological system allowed us to find that 1-Hz cardiac respiratory cycles are coupled with electrical activity rather than with mechanical activity, that calcium oscillations drive a mitochondrial respiration cycle, that the pharmaceutical or genetic inhibition of electro-mitochondrial coupling results in arrhythmogenic behaviour, and that the induction of arrythmia by the chemotherapeutic mitoxantrone can be partially reversed by the co-administration of metformin. Microphysiological cardiac systems may further facilitate the study of the mitochondrial dynamics of cardiac rhythms and advance the understanding of cardiac physiology. Overall design: Recent work demonstrated that geometric confinement induces a central cavity to form in cardiac organoids . Other developmental studies showed that complex partitioning only occurs following the development of the cardiac vasculature. To mimic this developmental step, we seeded a mixture of hiPSC-derived cardiomyocytes (hiPSC-CMs) and rat primary cardiac microvascular endothelial cells (CECs) in geometrically-confining microwells. The tissue contracted into a single mass in 4 days and started beating after 10 days. Cardiac organoids were cultured over the next weeks, acquiring smooth exterior and synchronized behaviour following 25 days of culture. GFP-expressing endothelial cells reveal that vascular networks begin forming on day 10, developing into complex circumferentially-aligned networks by day 25. To validate these structural findings we carried out RNA sequencing of the cardiac organoids, comparing their expression to multiple human cardiac tissues, and hiPSC-CMs from two-dimensional cultures. Cardiac organoids showed expression signatures associated with pacemaker (sinoatrial and atrioventricular nodes), endocardium, and epicardium cells, as well as cardiac fibroblasts.

心脏生理学与疾病的研究常因人类与小型动物模型间的生理学差异而受阻。本研究报道了在各向异性应力（anisotropic stress）条件下构建多腔室血管化人类心脏类器官（multi-chambered vascularized human cardiac organoids）的方法，以及其在研究心脏组织电代谢耦合（electro-metabolic coupling）中的应用价值。
该类器官源自人类诱导多能干细胞（human induced pluripotent stem cells），集成了可同时测量摄氧量、细胞外场电位与心脏收缩活动的传感器，测量分辨率高于10 Hz。本微生理系统的研究结果显示：1 Hz的心脏呼吸周期与电活动而非机械活动存在耦合关系；钙振荡可驱动线粒体呼吸周期；电-线粒体耦合的药物或遗传抑制会引发致心律失常行为；化疗药物米托蒽醌（mitoxantrone）诱导的心律失常可通过联合施用二甲双胍（metformin）得到部分逆转。心脏微生理系统可进一步助力心脏节律线粒体动力学研究，深化对心脏生理学的认知。
总体实验设计：既往研究表明，几何约束可诱导心脏类器官形成中央空腔；另有发育学研究证实，复杂的分区结构仅在心脏血管系统发育后才会形成。为模拟这一发育步骤，我们将人类诱导多能干细胞分化的心肌细胞（hiPSC-derived cardiomyocytes）与大鼠原代心脏微血管内皮细胞（cardiac microvascular endothelial cells, CECs）的混合细胞接种于几何约束的微孔中。该组织在培养第4天时收缩为单一实体，第10天开始自主搏动。后续数周内持续培养心脏类器官，至第25天时其外观趋于光滑，并表现出同步收缩行为。表达绿色荧光蛋白（GFP）的内皮细胞观测显示，血管网络于第10天开始形成，至第25天发育为复杂的环向对齐网络。为验证上述结构发现，我们对心脏类器官进行了RNA测序，并将其基因表达谱与多个人类心脏组织样本及二维培养的hiPSC-CMs进行比对。结果显示，心脏类器官呈现出与起搏细胞（窦房结与房室结细胞）、心内膜细胞、心外膜细胞及心脏成纤维细胞相关的表达特征。