Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs.
Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs.
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DOI：
10.1161/circep.114.002049
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发表时间：
2015-03
期刊：
影响因子：
--
通讯作者：
Li RA
中科院分区：
文献类型：
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作者：
Chen G;Li S;Karakikes I;Ren L;Chow MZ;Chopra A;Keung W;Yan B;Chan CW;Costa KD;Kong CW;Hajjar RJ;Chen CS;Li RA
人(H)胚胎干细胞(ESCs)和诱导多能干细胞(IPSCs)是一种潜在的无限体外培养心肌细胞(CMS)来源。然而，他们不成熟的表型一直是一个被广泛接受的障碍。HESC/IPSC来源的心室(V)CMS及其工程心脏微组织(HvCMTs)对β-肾上腺素能刺激表现出正性变时性反应，但无变力性反应。鉴于磷蛋白(Plb)在成体中的强势表达，但在hESC/IPSC-VCM中表达很少，以及它在β-肾上腺素能信号转导中的生物学作用，我们研究了PLB在hESC/IPSC-VCM和hvCMTs中表达的功能后果。首先，我们证实了PLB蛋白在hESC(HES2，H9)和IPSC来源的VCM和成人VCM中存在差异表达。用重组腺病毒(Ad)Ad-PLB或Ad-S16E-PLB转导hES2-VCM，分别过表达野生型PLB和伪磷酸化突变株。从未磷酸化的PLB对肌细胞/内质网Ca~(2+)-ATPase的抑制作用来看，Ad-PLB转导显著减弱电诱发的Ca~(2+)瞬变幅度，延长50%的衰减时间。重要的是，Ad-PLB转导的hES2-VCM对异丙肾上腺素有独特的反应。Ad-S16E-PLB转导的hES2-VCms表现为中间表型。在H9和IPSC-VCM中也观察到了同样的趋势。在方向上，Ad-PLB转导和Ad-S16E转导的hvCMTs也有类似的结果。然而，Ad-PLB既不改变全局转录组，也不改变ICA，L，暗示了PLB特有的效应。基因工程上调hESC/IPSC-VCM中PLB的表达可恢复对β-肾上腺素能刺激的正性变力反应。这些结果不仅对hESC/IPSC-VCMS的不成熟提供了更好的机制理解，而且还将导致改进的疾病模型和具有成体相似生理反应的可移植原型。
Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However, a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to β-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in β-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. First, we confirmed that PLB protein was differentially expressed in hESC (HES2, H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point–mutated variant, respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase, Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly, Ad-PLB–transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB–transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally, similar results were also seen with Ad-PLB–transduced and Ad-S16E–transduced hvCMTs. However, Ad-PLB altered neither the global transcriptome nor ICa,L, implicating a PLB-specific effect. Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to β-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses.