Beta-adrenergic and Muscarinic Modulation of Human Embryonic Stem Cell-derived Cardio-myocytes

Abstract

Background: Embryonic stem cells provide the most promising tool for cell replacement therapy including transplantation of human embryonic stem (hES) cell- derived cardiomyocytes in the infarcted area of the heart. Here we provide data for differentiation of cardiomyocytes from hES cells and firstly describe their hormonal modulation. Methods: Using Micro-Electrode Arrays as a novel electrical mapping technique of beating cardiomyocyte clusters within whole hES cell aggregates, we were able to measure the field potential generation and morphology changes during hormonal modulation. Results: We found that isoproterenol provokes, similar to the mouse ES cell system, a strong positive chronotropic effect with an EC₅₀ of around 10^-8 M. Moreover, isoproterenol stimulated with a higher EC₅₀ value the slow field potential amplitude, FP_slow, indicating a stimulation of Ca²⁺ channels in ventricular-like ES cell-derived cardiomyocytes which is shown to be clearly independent from frequence modulation. In contrast, carbachol (10 µM) produced a transient negative chronotropic effect but had no effect on FP_slow. Conclusion: The Micro-Electrode system allows measurement of ionic channel modulation and chronotropic responsiveness in a pharmacological screening setup. Moreover, all our data indicate that cardiomyocytes derived from human embryonic stem cells exhibit a physiological response to the major hormones of the vegetative nervous system and might therefore serve as an ideal candidate for the use in cell replacement strategies.

Gene-trapped mouse embryonic stem cell-derived cardiac myocytes and human genetics implicate AKAP10 in heart rhythm regulation

“…This finding could suggest that beat-to-beat variability is intrinsic to individual cardiac myocytes in culture, or it could reflect the complex paracrine signaling among cells in an EB. The biology of cardiac myocytes in EBs is still just beginning to be explored, but early findings suggest that adrenergic and cholinergic systems develop within EBs (30). Although we are just beginning to understand this system, our results suggest that studies in hESC-derived myocytes will be relevant to cardiovascular genetics and pharmacology.…”

“…Also, it would be impossible to adequately evaluate the 646V SNP in mice because mice are WT for this allele. Fortunately, recent advances in human ESC culture may soon make genetic manipulations common with mouse ESCs in human ESC-derived myocytes (30). Then, for the first time, we will have a genetically tractable system to directly examine human cardiac myocytes.…”

Generation and Characterization of Functional Cardiomyocytes from Rhesus Monkey Embryonic Stem Cells

“…Contaminating DNA was digested by DNAse I (Stratagene, La Jolla, CA, http://www.stratagene.com) for 15 minutes at 37°C followed by phenol/chloroform extraction. After precipitation, 750 ng of RNA was used for Oligo(dT) [12][13][14][15][16][17][18] -primed cDNA synthesis with Superscript II, a modified moloney murine leukemia virus reverse transcriptase (Invitrogen Corporation). One microliter of cDNA was amplified by polymerase chain reaction (PCR) with 1.25 U of REDTaq DNA-Polymerase (Sigma-Aldrich) in REDTaq 10ϫ reaction buffer, using 0.4 M of each primer and 0.4 M dNTPs in a 25-l reaction.…”

“…Data were analyzed offline with a customized toolbox programmed with MATLAB (The MathWorks, Natick, MA, http://www. mathworks.com) to detect and characterize FPs as has been described previously [12,25]. The frequency of contractions was calculated by measuring the distance between respective FP minima (FP MIN ) and denoted inter-spike interval (ISI).…”

Embryonic stem cells and cardiomyocyte differentiation: phenotypic and molecular analyses

“…Cardiomyocytes derived from hES cells typically have a beating rate of approximately 30-130 beats per minute, and respond to a number of pharmacological agents involving adrenoreceptors or muscarinic receptors. For example, the beating rate and amplitude of contraction of hES-CMs increase following the addition of isoproterenol, a β1 adrenergic receptor (β1-AR) agonist, while negative chronotropic responses have also been observed with carbachol, a muscarinic agonist [35]. In mES cells this responsiveness to adrenergic and muscarinic agonists depends on the cell type and degree of differentiation, which ultimately may also prove true for the human derivatives.…”