Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Feb 28;11(3):234.
doi: 10.3390/bioengineering11030234.

Contractile and Genetic Characterization of Cardiac Constructs Engineered from Human Induced Pluripotent Stem Cells: Modeling of Tuberous Sclerosis Complex and the Effects of Rapamycin

Veniamin Y Sidorov  1   2 , Tatiana N Sidorova  3 , Philip C Samson  1   4 , Ronald S Reiserer  1   4 , Clayton M Britt  1   4 , M Diana Neely  5 , Kevin C Ess  5 , John P Wikswo  1   2   4   6
Affiliations

Contractile and Genetic Characterization of Cardiac Constructs Engineered from Human Induced Pluripotent Stem Cells: Modeling of Tuberous Sclerosis Complex and the Effects of Rapamycin

Veniamin Y Sidorov et al. Bioengineering (Basel). .

Abstract

The implementation of three-dimensional tissue engineering concurrently with stem cell technology holds great promise for in vitro research in pharmacology and toxicology and modeling cardiac diseases, particularly for rare genetic and pediatric diseases for which animal models, immortal cell lines, and biopsy samples are unavailable. It also allows for a rapid assessment of phenotype-genotype relationships and tissue response to pharmacological manipulation. Mutations in the TSC1 and TSC2 genes lead to dysfunctional mTOR signaling and cause tuberous sclerosis complex (TSC), a genetic disorder that affects multiple organ systems, principally the brain, heart, skin, and kidneys. Here we differentiated healthy (CC3) and tuberous sclerosis (TSP8-15) human induced pluripotent stem cells (hiPSCs) into cardiomyocytes to create engineered cardiac tissue constructs (ECTCs). We investigated and compared their mechano-elastic properties and gene expression and assessed the effects of rapamycin, a potent inhibitor of the mechanistic target of rapamycin (mTOR). The TSP8-15 ECTCs had increased chronotropy compared to healthy ECTCs. Rapamycin induced positive inotropic and chronotropic effects (i.e., increased contractility and beating frequency, respectively) in the CC3 ECTCs but did not cause significant changes in the TSP8-15 ECTCs. A differential gene expression analysis revealed 926 up- and 439 down-regulated genes in the TSP8-15 ECTCs compared to their healthy counterparts. The application of rapamycin initiated the differential expression of 101 and 31 genes in the CC3 and TSP8-15 ECTCs, respectively. A gene ontology analysis showed that in the CC3 ECTCs, the positive inotropic and chronotropic effects of rapamycin correlated with positively regulated biological processes, which were primarily related to the metabolism of lipids and fatty and amino acids, and with negatively regulated processes, which were predominantly associated with cell proliferation and muscle and tissue development. In conclusion, this study describes for the first time an in vitro TSC cardiac tissue model, illustrates the response of normal and TSC ECTCs to rapamycin, and provides new insights into the mechanisms of TSC.

Keywords: I-Wire cardiac construct; heart-on-a-chip; hiPSC; mTOR; rapamycin; tuberous sclerosis complex.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
Procedure for creating engineered cardiac tissue constructs (ECTCs). (A) Photograph of the mold. (B) Mold filled with fibrinogen-based matrix and iPSCs. (C) Differentiated cardiomyocytes self-organized into an ECTC suspended between two stationary wires. (D) Schematic representation of cardiomyocyte differentiation protocol.
Figure 2
Figure 2
Histological imaging of ECTC on 45th day of differentiation. (A) ECTC in PDMS device located in the middle of the channel. The black dot on the right side of ECTC represents the position of the flexible probe. (B) H&E staining of longitudinal section with insert of cross-section. (C–F) Immunofluorescence of ECTC cross-section stained against cardiac Troponin T (cTnT, red), connexin-43 (C × 43, green), and DAPI (blue). (G–I) Images of the staining against collagen I. Scale bar in (B,C,G–I) is 100 µm, and in (D–F) is 20 µm. (J–L) Higher-magnification immunofluorescence images of the longitudinal sections of ECTC for cTnT and connexin-43. Scale bar is 20 µm. Yellow arrow points in the longitudinal direction of the construct.
Figure 3
Figure 3
β-adrenergic stimulation in CC3 ECTC. (A) Representative uncalibrated contraction traces in control (upper) and at 1 µM isoproterenol. (B) Chronotropic effect of isoproterenol. (C) Developed force during control and isoproterenol application. (D) Effect of isoproterenol on Frank–Starling force–tension relationship, * p < 0.05, N = 8 different engineered cardiac tissue constructs (ECTCs).
Figure 4
Figure 4
Inotropic response of the ECTC to treatment with 10 nM rapamycin. (A,C) Representative superimposed developed force traces as a function of applied transverse force in control (left) and after rapamycin application (right) are shown for CC3 (A) and TSP8-15 (C) cardiac constructs. (B,D) Frank–Starling force–tension relationship for CC3 (B), N = 9 ECTCs and TSP8-15 (D), N = 8 ECTCs. Values are means ± SD. * p < 0.05.
Figure 5
Figure 5
Chronotropic response of the CC3 and TSP8-15 ECTCs to application of 10 nM rapamycin. Difference in frequency of spontaneous beating rate before and after treatment with rapamycin for CC3 ECTC (A) and TSP8-15 (B). (C) Faster beating rate in control TSP8-15. (D) Increase in contraction frequency by rapamycin in CC3 cardiac constructs (p < 0.01, N = 36 recordings), and (E) no significant alteration in TSP8-15 constructs (N = 32 recordings). Values are means ± SD. In total, 9 CC3 and 8 TSP8-15 ECTCs were analyzed. Different ECTCs are marked with gray and black in (A,B).
Figure 6
Figure 6
Scattered (left) and volcano (right) plots of RNA sequencing data. Transcriptomic comparison in CC3 vs. TSP8-15 ECTCs (A–C), CC3 vs. CC3 and rapamycin (D–F), and TSP8-15 vs. TSP8-15 and rapamycin (G–I). Red dots indicate up-regulated and blue dots down-regulated genes. Black dots indicate not DEGs. CPM, count per million. FC, fold change. False discovery rate (FDR) < 0.05.
Figure 7
Figure 7
Fold change (FC) of cardiac-specific TSP8-15 genes that are differentially expressed relative to those in CC3.
Figure 8
Figure 8
Gene ontology enrichment analysis of CC3 vs. TSP8-15 ECTCs, up- (A) and down-regulated (B) DEG sets. The top 15 GO terms for biological processes (red), cellular components (blue), and molecular function (green) are shown. The numbers in the column are the counts of genes in overlap.
Figure 9
Figure 9
Gene ontology enrichment analysis of up- (A) and down-regulated (B) gene sets. Genes were differentially expressed in response to rapamycin treatment of CC3 ECTCs. Bar charts show the top 15 GO annotations and terms for biological processes (red), cellular components (blue), and molecular function (green). The numbers in the column are the counts of genes in overlap.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Watson D.E., Hunziker R., Wikswo J.P. Fitting tissue chips and microphysiological systems into the grand scheme of medicine, biology, pharmacology, and toxicology. Exp. Biol. Med. 2017;242:1559–1572. doi: 10.1177/1535370217732765. - DOI - PMC - PubMed
    1. Seki T., Fukuda K. Methods of induced pluripotent stem cells for clinical application. World J. Stem Cells. 2015;7:116–125. doi: 10.4252/wjsc.v7.i1.116. - DOI - PMC - PubMed
    1. Tohyama S., Fujita J., Fujita C., Yamaguchi M., Kanaami S., Ohno R., Sakamoto K., Kodama M., Kurokawa J., Kanazawa H., et al. Efficient Large-Scale 2D Culture System for Human Induced Pluripotent Stem Cells and Differentiated Cardiomyocytes. Stem Cell Rep. 2017;9:1406–1414. doi: 10.1016/j.stemcr.2017.08.025. - DOI - PMC - PubMed
    1. Orlova K.A., Crino P.B. The tuberous sclerosis complex. Ann. N. Y. Acad. Sci. 2010;1184:87–105. doi: 10.1111/j.1749-6632.2009.05117.x. - DOI - PMC - PubMed
    1. Mühler E.G., Turniski-Harder V., Engelhardt W., von Bernuth G. Cardiac involvement in tuberous sclerosis. Heart. 1994;72:584–590. doi: 10.1136/hrt.72.6.584. - DOI - PMC - PubMed

LinkOut - more resources