Phospholamban as a Crucial Determinant of the Inotropic Response of Human Pluripotent Stem Cell–Derived Ventricular Cardiomyocytes and Engineered 3-Dimensional Tissue Constructs

Abstract

BACKGROUND: 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 beta-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in beta-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. METHODS AND RESULTS: 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. CONCLUSIONS: Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to beta-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.