Modeling inherited cardiac diseases with iPS-derived engineered heart tissue
Zusammenfassung der Projektergebnisse
The concept of patient-specific human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) test models is highly attractive, but still in its infancy due to heterogeneity and immaturity of hiPSC-CM and lack of models to analyze physiological parameter like contractile force. The objective of this proposal was to further develop and apply a model for hiPSC-CM disease modeling with high predictivity based on contractile force. To this end, we have (i) established an automated contractility/calcium transient test system which allows the sequential measurement of calcium transients (FURA-2) and force (video-optical) under perfusion and electrical stimulation and demonstrated species-specific differences in contraction and calcium transient kinetics for rat, mouse and human EHTs. (il) In a proof-of-principle study we showed that the engineered heart tissue (EHT) model is suitable to study functional consequences of gene mutations by characterizing EHT from a Mybpc3-targeted knock-out (KO) and knock-in (Kl) mouse model. We demonstrated that KO and Kl EHTs developed higher forces and have shorter contraction and relaxation times than control. They exhibited (1) lower sensitivity to the negative inotropic effect of verapamil, (2) smaller positive inotropic effects of isoprenaline and (3) attenuated positive inotropic responses to EMD 57033 (Ca^2+ sensitizer). This data suggests that the consequence of gene mutations can be detected by contractile analysis of EHTs. (iii) We optimized a protocol for cost effective, large scale production of cardiomyocytes based on small molecule/growth factor based sequential mesodermal and cardiac differentiation from hiPSC with an efficiency of 84±6%. (iv) We characterized the proteome of hiPSC-CMs in 2D versus EHT format and identified a pattern of protein expression similar to fetal versus adult human non failing heart, suggesting that aspects of developmental hypertrophy are replicated in EHT development. (v) We created a total of 33 disease-specific hiPSC lines with identified mutation in 27 cases and are currently setting up genome editing protocols. (vi) We extensively characterized EHTs from a control hiPSC line based on contractile force and found that hiPSC-EHTs behave essentially very similar to hESC-EHTs and show a great degree of similarity to human non-failing heart tissue in a variety of pharmacological and physiological assays. Overall, in this project, we showed that the EHT model is suitable to study functional consequences of gene mutations and that contractile force regulation in hIPSC-EHT replicates many aspects of human heart tissue. This study paves the way to now perform disease modeling of cardiac diseases with cardiac disease specific hiPSC lines and isogenic controls in EHT format.
Projektbezogene Publikationen (Auswahl)
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The beat goes on: human heart muscle from pluripotent stem cells. Circ. Res. 109, 2-4 (2011)
Eschenhagen, T.
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Increased afterload induces pathological cardiac hypertrophy: a new in vitro model. Basic Res. Cardiol. 107, 307 (2012)
Hirt MN, Sörensen NA, Bartholdt LM, Boeddinghaus J, Schaaf S, Eder A, Vollert I, Stöhr A, Schulze T, Witten A, Stoll M, Hansen A, Eschenhagen T
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Physiological aspects of cardiac tissue engineering. Am. J. Physiol. Heart Circ. Physiol. 303, H133-43 (2012)
Eschenhagen, T., Eder, A., Vollert, l. & Hansen, A.
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Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice. J. Mol. Cell. Cardiol. 63C, 189-198(2013)
Stöhr A, Friedrich FW, Flenner F, Geertz B, Eder A, Schaaf S, Hirt MN, Uebeler J, Schlossarek S, Carrier L, Hansen A, Eschenhagen T
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Automated analysis of contractile force and Ca^2+ transients in engineered heart tissue. Am. J. Physiol. Heart Circ. Physiol. 306, H1353-63 (2014)
Stoehr A, Neuber C, Baldauf C, Vollert I, Friedrich FW, Flenner F, Carrier L, Eder A, Schaaf S, Hirt MN, Aksehirlioglu B, Tong CW, Moretti A, Eschenhagen T, Hansen A
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Cardiac tissue engineering: state of the art. Circ. Res. 114, 354- 67 (2014)
Hirt, M. N., Hansen, A. & Eschenhagen, T.
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Effects of proarrhythmic drugs on relaxation time and beating pattern in rat engineered heart tissue. Basic Res. Cardiol. 109, 436 (2014)
Eder A, Hansen A, Uebeler J, Schulze T, Neuber C, Schaaf S, Yuan L, Christ T, Vos MA, Eschenhagen T
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Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation. J. Mol. Cell. Cardiol. 74,151-61 (2014)
Hirt MN, Boeddinghaus J, Mitchell A, Schaaf S, Börnchen C, Müller C, Schulz H, Hubner N, Stenzig J, Stoehr A, Neuber C, Eder A, Luther PK, Hansen A, Eschenhagen T
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Modelling sarcomeric cardiomyopathies in the dish: from human heart samples to iPSC cardiomyocytes. Cardiovasc. Res. 105, 424-38 (2015)
Eschenhagen, T., Mummery, C. & Knollmann, B. C.
