Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs
Open accessAbstract
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 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. 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 Ca 2+ -ATPase, Ad-PLB transduction significantly attenuated electrically evoked Ca 2+ 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 I Ca,L , implicating a PLB-specific effect. Conclusions—: 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.
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Engineered cardiac thin films, micro-tissues, and strips permit measurement of twitch force.
“engineered cardiac thin films, micro-tissues and strips allow the assessment of twitch force [9,12–19]”
From Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells (2018) · cites this paper as [18]
The Methods section on microtissue force quantification describes measuring forces from engineered cardiac microtissues, and the Results report their measured twitch tension. The Results also describe twitch-force measurements in engineered cardiac tissue strips. Our copy does not address cardiac thin films, so it supports only two of the three formats listed. (LLM, conf=0.85)
Experimental — accuracy not yet measuredTranscript analysis of hvCOC and other engineered constructs showed changes consistent with prior maturation studies.
“Transcript analysis of 2D-cultured clusters [24] and alternative engineered constructs, including human ventricular cardiac micro-tissues (hvCMT; ~1000 cells) [18], tissue strips (hvCTS; ~106 cells) [14] and hvCOC made of identical constituent hPSC-vCMs, revealed changes consistent with our previous maturation studies [18,24–28].”
From Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells (2018) · cites this paper as [18]
The Results identify hvCMTs as engineered microtissues of approximately 1,000 ventricular cardiomyocytes, so the source supports that part of the citing sentence. The Transcriptomic Analysis methods describe RNA analysis of hES2-vCMs, not of hvCMTs; the Discussion says their global transcriptome was unchanged after PLB overexpression. This source therefore does not establish transcript changes in the engineered constructs consistent with maturation studies. No page numbers are available in our copy. (LLM, conf=0.86)
Experimental — accuracy not yet measuredVerapamil decreased stroke volume, cardiac output, and developed pressure, with P-V loops showing decreases in end-systolic pressure and stroke work.
“Consistent with a negative inotropic effect resulting from reduced Ca2+ entry that we reported in our previous studies with hvCTS and hvCMT [14,18], the stroke volume, cardiac output, and developed pressure decreased (shown in Fig. 4a) with the P-V loops likewise exhibiting decreases in end-systolic pressure and stroke work (Fig. 4b).”
From Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells (2018) · cites this paper as [18]
The Results section reports reduced twitch tension in hvCMTs after PLB transduction, supporting a negative inotropic effect. But the Results on Ca²⁺ transients say PLB transduction did not alter L-type Ca²⁺ current; the paper does not report verapamil treatment or the cited pressure–volume measures. In the citing sentence, those measures are presented as the citing authors’ own Fig. 4 results, while the citation is attached to the prior-study comparison. (LLM, conf=0.82)
Experimental — accuracy not yet measuredhvCOC showed augmented sensitivity to adrenergic stimulation and both chronotropic and inotropic effects compared with hvCTS and hvCMT systems constructed with the same hPSC-vCMs.
“Functionally, we observed an augmented sensitivity of the hvCOC to adrenergic stimulation and the presence of both chronotropic and inotropic effects when compared with the hvCTS and hvCMT systems constructed with the same hPSC-vCMs [14,18].”
From Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells (2018) · cites this paper as [18]
The Results section reports that β-adrenergic stimulation increased beating frequency but not twitch tension in hvCMTs, and says cardiac tissue strips likewise showed a positive chronotropic but negligible inotropic response. The paper therefore supports the comparator response described in the citing sentence, but it does not assess hvCOC or establish its greater sensitivity; the citing authors present that as their own observation. No page number is available in our copy. (LLM, conf=0.84)
Experimental — accuracy not yet measuredThose constructs do not permit pressure- and volume-based measures that directly reflect pump performance.
“but not pressure- and volume-based metrics directly representing pump performance of the heart”
From Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells (2018) · cites this paper as [18]
Experimental — accuracy not yet measured
2D hPSC-vCMs, 3D hvCMTs and hvCTSs have been reported to show positive chronotropic but weak or null inotropic responses to β-adrenergic stimulation.
“Pharmacologically, 2D-cultured hPSC-vCMs [18], as well as 3D hvCMTs [21] and hvCTSs [14] created from such cells, have been reported to display positive chronotropic but weak or even null inotropic responses to b-adrenergic stimulation (Supplemental Fig. 5).”
From Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells (2018) · cites this paper as [18]
Developed pressure exhibited a negative pressure-frequency relationship in hvCOC.
“Developed pressure exhibited a negative pressure-frequency relationship (Fig. 3f), similar to the negative Ca2+ transient-frequency data in hvCMs [24] and negative force-frequency relationships observed for hvCMT [18], hvCTS [14] and newborn human heart muscle [30], as commonly seen with engineered heart tissues.”
From Bioengineering an electro-mechanically functional miniature ventricular heart chamber from human pluripotent stem cells (2018) · cites this paper as [18]
The citation is attached to the comparison with a negative force–frequency relationship in hvCMT, not to the hvCOC pressure result, which the citing sentence assigns to its own Figure 3f. The source’s Results section on hvCMT responses reports beating frequency and twitch tension under isoproterenol and notes that frequency can affect force, but it does not establish a negative force–frequency relationship. Searches for that relationship and for pacing-rate effects found no decisive passage; page numbers were unavailable. (LLM, conf=0.72)
Experimental — accuracy not yet measured
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- Authors
- Chen, G., Li, S., Karakikes, I., Ren, L., Zi-Ying Chow, M., Chopra, A., Keung, W., Yan, B., Chan, C. W. Y., Costa, K. D., Kong, C.-W., Hajjar, R. J., Chen, C. S., & Li, R. A.
- Affiliations
- Boston University (US)
- From the Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, Manhattan, NY (G.C., I.K., K.D.C., R.J.H., R.A.L.); Department of Physiology (G.C., S.L., L.R., M.Z.-Y.C., W.K., C.-W.K., R.A.L.), Stem Cell and Regenerative Medicine Consortium (G.C., S.L., L.R., M.Z.-Y.C., W.K., B.Y., C.W.Y.C., C.-W.K., R.A.L.), Department of Anatomy (C.W.Y.C.), LKS Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong; Department of Bioengineering, Boston University, MA (A.C., C.S.C.);...
- ORCID
- Venue
- Circulation: Arrhythmia and Electrophysiology
- Published
- 2015-02
- Volume
- 8
- Issue
- 1
- Pages
- 193-202
- ISSN
- 1941-3084 (ISSN-L)
- 1941-3149
- PMC
- PMC5884688
- OpenAlex
- W2171560520
- Cited by
- 38
- References
- 34
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 3 · 2023: 6 · 2022: 1 · 2021: 2 · 2020: 4 · 2019: 8 · 2018: 4 · 2017: 2 · 2016: 2 · 2015: 3
- FWCI
- 0.96
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Circulation Arrhythmia and Electrophysiology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- The HKU Scholars Hub (University of Hong Kong) (submittedVersion)
- Funders
- National Heart, Lung, and Blood Institute (R01HL119046, R01HL128072, R01HL093183, R01HL117505)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiac electrophysiology and arrhythmias
- Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
- Keywords
- phospholamban, human pluripotent stem cell-derived cardiomyocytes, ventricular myocytes, iPSC-derived cardiomyocytes, embryonic stem cells, inotropic effect, beta-adrenergic stimulation, isoproterenol, sarco/endoplasmic reticulum Ca2+-ATPase, calcium transients, cardiomyocyte maturation, adenoviral gene transfer
- MeSH terms
- Phospholamban, Adrenergic beta-Agonists, Calcium-Binding Proteins, Cell Differentiation, Cells, Cultured, Humans, Isoproterenol, Mutation, Myocardial Contraction, Phenotype, Sarcoplasmic Reticulum, Transduction, Genetic, Transfection, Signal Transduction, Up-Regulation, Coculture Techniques, Calcium Signaling, Tissue Engineering, Myocytes, Cardiac, Sarcoplasmic Reticulum Calcium-Transporting ATPases, Embryonic Stem Cells, Induced Pluripotent Stem Cells
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