Different Responses to Drug Safety Screening Targets between Human Neonatal and Infantile Heart Tissue and Cardiac Bodies Derived from Human-Induced Pluripotent Stem Cells.

AIMS: Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) have become a promising tool in cardiovascular safety pharmacology. Immaturity of iPS-CMs remains an ongoing concern. We compared electrophysiological and contractile features of cardiac bodies (hiPS-CBs) derived from human-induced pluripotent stem cells and human neonatal and infantile myocardial slices relevant for drug screening. METHODS AND RESULTS: Myocardial tissue slices were prepared from biopsies obtained from patients undergoing surgery for hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot (TOF). Electrophysiological features and response to Ik,r blockade as well as contractile properties were investigated using microelectrodes and isometric force measurements and were compared to hiPS-CBs. Both native myocardial tissue slices as well as hiPS-CBs showed action potential prolongation after Ik,r blockade, but early afterdepolarisations could be observed in native myocardial tissue slices only. The force-frequency relationship (FFR) varied at lower frequencies and was negative throughout at higher frequencies in hiPS-CBs. In contrast, native myocardial tissue slices exhibited positive, negative, and biphasic FFRs. In contrast to native myocardial tissue slices, hiPS-CBs failed to show an inotropic response to ß-adrenergic stimulation. Although all groups showed ß-adrenergic induced positive lusitropy, the effect was more pronounced in myocardial tissue slices. CONCLUSION: hiPS-CBs were able to reproduce AP prolongation after Ik,r blockade, but to a lesser extent compared to human neonatal and infantile myocardial tissue slices. Early afterdepolarisations could not be induced in hiPS-CBs. Contractile force was differently regulated by ß-adrenergic stimulation in hiPS-CBs and the native myocardium. If used for cardiotoxicity screening, caution is warranted as hiPS-CBs might be less sensitive to pharmacologic targets compared to the native myocardium of neonates and infants.

The Interaction between Adult Cardiac Fibroblasts and Embryonic Stem Cell-Derived Cardiomyocytes Leads to Proarrhythmic Changes in In Vitro Cocultures.

Transplantation of stem cell-derived cardiomyocytes is one of the most promising therapeutic approaches after myocardial infarction, as loss of cardiomyocytes is virtually irreversible by endogenous repair mechanisms. In myocardial scars, transplanted cardiomyocytes will be in immediate contact with cardiac fibroblasts. While it is well documented how the electrophysiology of neonatal cardiomyocytes is modulated by cardiac fibroblasts of the same developmental stage, it is unknown how adult cardiac fibroblasts (aCFs) affect the function of embryonic stem cell-derived cardiomyocytes (ESC-CMs). To investigate the effects of aCFs on ESC-CM electrophysiology, we performed extra- and intracellular recordings of murine aCF-ESC-CM cocultures. We observed that spontaneous beating behaviour was highly irregular in aCF-ESC-CM cocultures compared to cocultures with mesenchymal stem cells (coefficient of variation of the interspike interval: 40.5 ± 15.2% versus 9.3 ± 2.0%, p = 0.008) and that action potential amplitude and maximal upstroke velocity (V max) were reduced (amplitude: 52.3 ± 1.7 mV versus 65.1 ± 1.5 mV, V max: 7.0 ± 1.0 V/s versus 36.5 ± 5.3 V/s), while action potential duration (APD) was prolonged (APD50: 25.6 ± 1.0 ms versus 16.8 ± 1.9 ms, p < 0.001; APD90: 52.2 ± 1.5 ms versus 43.3 ± 3.3 ms, p < 0.01) compared to controls. Similar changes could be induced by aCF-conditioned medium. We conclude that the presence of aCFs changes automaticity and induces potentially proarrhythmic changes of ESC-CM electrophysiology.

Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and ß-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 µM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05). Ni(2+) was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca(2+)-release decreased force development to 54 ± 3% in ventricular (n=13, p<0.05) and to 52 ± 8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca(2+)-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169 ± 7% force compared to baseline, n=8, p<0.05) and 10 s (198 ± 9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after ß-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

Electrophysiological characteristics of embryonic stem cell-derived cardiomyocytes are cell line-dependent.

BACKGROUND: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs) requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs) of different murine ESC lines. METHODS: Two wild-type (D3 and R1) and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7) were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP) and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC) promoter. Action potentials (APs) were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. RESULTS: Spontaneous AP frequency and AP duration (APD) as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. CONCLUSION: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

Mesenchymal stem cells and their conditioned medium improve integration of purified induced pluripotent stem cell-derived cardiomyocyte clusters into myocardial tissue.

Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) might become therapeutically relevant to regenerate myocardial damage. Purified iPS-CMs exhibit poor functional integration into myocardial tissue. The aim of this study was to investigate whether murine mesenchymal stem cells (MSCs) or their conditioned medium (MScond) improves the integration of murine iPS-CMs into myocardial tissue. Vital or nonvital embryonic murine ventricular tissue slices were cocultured with purified clusters of iPS-CMs in combination with murine embryonic fibroblasts (MEFs), MSCs, or MScond. Morphological integration was assessed by visual scoring and functional integration by isometric force and field potential measurements. We observed a moderate morphological integration of iPS-CM clusters into vital, but a poor integration into nonvital, slices. MEFs and MSCs but not MScond improved morphological integration of CMs into nonvital slices and enabled purified iPS-CMs to confer force. Coculture of vital slices with iPS-CMs and MEFs or MSCs resulted in an improved electrical integration. A comparable improvement of electrical coupling was achieved with the cell-free MScond, indicating that soluble factors secreted by MSCs were involved in electrical coupling. We conclude that cells such as MSCs support the engraftment and adhesion of CMs, and confer force to noncontractile tissue. Furthermore, soluble factors secreted by MSCs mediate electrical coupling of purified iPS-CM clusters to myocardial tissue. These data suggest that MSCs may increase the functional engraftment and therapeutic efficacy of transplanted iPS-CMs into infarcted myocardium.

Baicalin maintains late-stage functional cardiomyocytes in embryoid bodies derived from murine embryonic stem cells.

BACKGROUND/AIMS: Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells. METHODS: The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence. RESULTS: Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to ß-adrenergic and muscarinic stimulations. CONCLUSION: Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells.

Contractile properties of early human embryonic stem cell-derived cardiomyocytes: beta-adrenergic stimulation induces positive chronotropy and lusitropy but not inotropy.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide the unique opportunity to study the very early development of the human heart. The aim of this study was to investigate the effect of calcium and beta-adrenergic stimulation on the contractile properties of early hESC-CMs. Beating clusters containing hESC-CMs were co-cultured in vitro with noncontractile slices of neonatal murine ventricles. After 5-7 days, when beating clusters had integrated morphologically into the damaged tissue, isometric force measurements were performed during spontaneous beating as well as during electrical field stimulation. Spontaneous beating stopped when extracellular calcium ([Ca²âº](ec)) was removed or after administration of the Ca²âº channel blocker nifedipine. During field stimulation at a constant rate, the developed force increased with incremental concentrations of [Ca²âº](ec). During spontaneous beating, rising [Ca²âº](ec) increased beating rate and developed force up to a [Ca²âº](ec) of 2.5 mM. When [Ca²âº](ec) was increased further, spontaneous beating rate decreased, whereas the developed force continued to increase. The beta-adrenergic agonist isoproterenol induced a dose-dependent increase of the frequency of spontaneous beating; however, it did not significantly change the developed force during spontaneous contractions or during electrical stimulation at a constant rate. Force developed by early hESC-CMs depends on [Ca²âº](ec) and on the L-type Ca²âº channel. The lack of an inotropic reaction despite a pronounced chronotropic response after beta-adrenergic stimulation most likely indicates immaturity of the sarcoplasmic reticulum. For cell-replacement strategies, further maturation of cardiac cells has to be achieved either in vitro before or in vivo after transplantation.

Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells.

Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC-CMs) and iPS cells (iPS-CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5-7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force-frequency relationship (FFR), effects of Ca(2+), Ni(2+), nifedipine, ryanodine, beta-adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC-CMs and iPS-CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca(2+) and decreased with nifedipine. Force was reduced by Ni(2+). Isoproterenol (1 microM) increased the force most pronounced in embryonic tissue (207+/-31%, n=7; ESC-CMs: 123+/-5%, n=4; iPS-CMs: 120+/-4%, n=8). EC(50) values were similar. Contractile properties of iPS-CMs and ESC-CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the beta-adrenergic response of iPS-CMs and ESC-CMs.

Generation of functional murine cardiac myocytes from induced pluripotent stem cells.

BACKGROUND: The recent breakthrough in the generation of induced pluripotent stem (iPS) cells, which are almost indistinguishable from embryonic stem (ES) cells, facilitates the generation of murine disease- and human patient-specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well-established murine ES cell line. METHODS AND RESULTS: With the use of a standard embryoid body-based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell-derived cardiomyocytes show typical features of ES cell-derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 (Mesp1), friend of GATA2 (FOG-2), GATA-binding protein 4 (GATA4), NK2 transcription factor related, locus 5 (Nkx2.5), T-box 5 (Tbx5), T-box 20 (Tbx20), atrial natriuretic factor (ANF), myosin light chain 2 atrial transcripts (MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), alpha-myosin heavy chain (alpha-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric alpha-actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca(2+) fluctuations with amplitudes of Ca(2+) transients comparable to ES cell cardiomyocytes. Simultaneous Ca(2+) release within clusters of iPS cell-derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the beta-adrenergic and muscarinic signaling cascade in these cells. CONCLUSIONS: iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.