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Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs.

ABSTRACT: 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 ?-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in ?-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 ?-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.

SUBMITTER: Chen G

PROVIDER: S-EPMC5884688 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs.

Chen Gaopeng G Li Sen S Karakikes Ioannis I Ren Lihuan L Chow Maggie Zi-Ying MZ Chopra Anant A Keung Wendy W Yan Bin B Chan Camie W Y CW Costa Kevin D KD Kong Chi-Wing CW Hajjar Roger J RJ Chen Christopher S CS Li Ronald A RA

Circulation. Arrhythmia and electrophysiology 20141210 1

<h4>Background</h4>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 ...[more]

PMID: 25504561

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Project description:Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) grown in engineered heart tissue (EHT) can be used for drug screening, disease modeling, and heart repair. However, the immaturity of hiPSC-CMs currently limits their use. Because mechanical loading increases during development and facilitates cardiac maturation, we hypothesized that afterload would promote maturation of EHTs. To test this we developed a system in which EHTs are suspended between a rigid post and a flexible one, whose resistance to contraction can be modulated by applying braces of varying length. These braces allow us to adjust afterload conditions over two orders of magnitude by increasing the flexible post resistance from 0.09 up to 9.2??N/?m. After three weeks in culture, optical tracking of post deflections revealed that auxotonic twitch forces increased in correlation with the degree of afterload, whereas twitch velocities decreased with afterload. Consequently, the power and work of the EHTs were maximal under intermediate afterloads. When studied isometrically, the inotropy of EHTs increased with afterload up to an intermediate resistance (0.45??N/?m) and then plateaued. Applied afterload increased sarcomere length, cardiomyocyte area and elongation, which are hallmarks of maturation. Furthermore, progressively increasing the level of afterload led to improved calcium handling, increased expression of several key markers of cardiac maturation, including a shift from fetal to adult ventricular myosin heavy chain isoforms. However, at the highest afterload condition, markers of pathological hypertrophy and fibrosis were also upregulated, although the bulk tissue stiffness remained the same for all levels of applied afterload tested. Together, our results indicate that application of moderate afterloads can substantially improve the maturation of hiPSC-CMs in EHTs, while high afterload conditions may mimic certain aspects of human cardiac pathology resulting from elevated mechanical overload.

Dataset Information

Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs.

Publications

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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