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

Abstract

Human (h) pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), serve as potentially unlimited ex vivo sources of cardiomyocytes (CMs) for disease modelling, drug discovery, cardiotoxicity screening and future clinical applications, including cardiac repair. Despite these advantages, a well-accepted obstacle to the widespread application of human PSC-derived CMs (hPSC-CMs) is their developmentally immature phenotype in terms of 〖Ca〗^(2+) handling and β-adrenergic signalling. To study these phenotypes, I used a cardiac ventricular specification protocol to differentiate multiple lines of hESCs and hiPSCs into CMs characterised by ventricular-like electrophysiological profiles. The hPSC-derived ventricular CMs (hPSC-VCMs) and engineered cardiac microtissues (hCMT) created from these cells displayed positive chronotropic (i.e. spontaneous rate) but null inotropic responses (i.e. contractile force) to β-adrenergic stimulation. These findings led me to hypothesize that phospholamban (PLB), an inhibitor of sarco/endoplasmic reticulum 〖Ca〗^(2+)-ATPase (SERCA) robustly present in adult CMs but poorly expressed in hPSC-VCMs, might account for the phenotypic differences observed between hPSC-VCMs and adult cardiomyocytes.

Since PLB inhibition of SERCA is relieved by β-adrenergic stimulation in adult CMs, I investigated the functional consequences of altered PLB expression in hPSC-VCMs to obtain insights into developmental maturation. To induce the over-expression of wild-type PLB or a pseudophosphorylated (S16E) PLB, hPSC-VCMs were transduced by recombinant adenoviruses (Ad), Ad-PLB or Ad-S16E-PLB. As anticipated from the inhibitory effect of unphosphorylated PLB on SERCA, Ad-PLB transduction significantly (p<0.01) attenuated electrically evoked 〖Ca〗^(2+) transient amplitudes and prolonged 50% decay times. An important finding is that Ad-PLB-transduced hPSC-VCMs, rather than other groups, uniquely responded to isoproterenol, a β-adrenergic agonist. Consistent with relief from inhibition after phosphorylation of PLB, the Ad-S16E-PLB-transduced hPSC-VCMs exhibited an intermediate phenotype. Similar trend of functional changes were observed in Ad-PLB- and Ad-S16E-PLB-transduced hCMTs. Mechanistically, PLB overexpression altered neither the global transcriptomic profile nor the L-type calcium current (ICa,L). Taken together, these results demonstrate that forced expression of PLB directly restores the positive inotropic response of hPSC-VCMs to β-adrenergic stimulation.

The results reported in this dissertation provide a better mechanistic understanding of the immaturity of hESC-VCMs and are expected to improve the construction of disease models and transplantable prototypes with adult-like physiological responses.