An integrated platform for simultaneous multi-well field potential recording and Fura-2-based calcium transient ratiometry in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes.
BACKGROUND
Human induced pluripotent stem cell-derived cardiomyocytes are available from various sources and they are being evaluated for safety testing. Several platforms are available offering different assay principles and read-out parameters: patch-clamp and field potential recording, imaging or photometry, impedance measurement, and recording of contractile force. Routine use will establish which assay principle and which parameters best serve the intended purpose.


METHODS
We introduce a combination of field potential recording and calcium ratiometry from spontaneously beating cardiomyocytes as a novel assay providing a complementary read-out parameter set. Field potential recording is performed using a commercial multi-well multi-electrode array platform. Calcium ratiometry is performed using a fiber optic illumination and silicon avalanche photodetectors. Data condensation and statistical analysis are designed to enable statistical inference of differences and equivalence with regard to a solvent control.


RESULTS
Simultaneous recording of field potentials and calcium transients from spontaneously beating monolayers was done in a nine-well format. Calcium channel blockers (e.g. nifedipine) and a blocker of calcium store release (ryanodine) can be recognized and discriminated based on the calcium transient signal. An agonist of L-type calcium channels, FPL 64176, increased and prolonged the calcium transient, whereas BAY K 8644, another L-type calcium channel agonist, had no effect. Both FPL 64176 and various calcium channel antagonists have chronotropic effects, which can be discriminated from typical "chronotropic" compounds, like (±)isoprenaline (positive) and arecaidine propargyl ester (negative), based on their effects on the calcium transient.


CONCLUSIONS
Despite technical limitations in temporal resolution and exact matching of composite calcium transient with the field potential of a subset of cells, the combined recording platform enables a refined interpretation of the field potential recording and a more reliable identification of drug effects on calcium handling.
Drug Discovery Support, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65, 88397 Biberach, Germany. Electronic address: Georg.Rast@boehringer-ingelheim.com.
Georg
G
Rast
Infrastructure, Safety & Environment, Engineering, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65, 88397 Biberach, Germany.
Jürgen
J
Weber
Konitel GmbH, Hauptstrasse 2, 88437 Äpfingen, Germany.
Christoph
C
Disch
Infrastructure, Safety & Environment, Engineering, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65, 88397 Biberach, Germany.
Elmar
E
Schuck
Global Biometrics and Clinical Applications, Medicine, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65, 88397 Biberach, Germany.
Carina
C
Ittrich
Drug Discovery Support, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65, 88397 Biberach, Germany.
Brian D
BD
Guth
D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry; D016257:Fura-2 / Q000737:chemistry; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D032383:Myocytes, Cardiac / Q000187:drug effects*; D009543:Nifedipine / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D011874:Radiometry / Q000379:methods*; D012433:Ryanodine / Q000494:pharmacology
D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D011758:Pyrroles; C071658:FPL 64176; D012433:Ryanodine; D009543:Nifedipine; D002118:Calcium; D016257:Fura-2
Calcium transient; Cardiomyocyte; Field potential; Fura-2; Human induced pluripotent stem cell (hiPSC); Methods; Multi-electrode array; Ratiometry