Long-term effects of empagliflozin on excitation-contraction-coupling in human induced pluripotent stem cell cardiomyocytes
Creators: name: Sossalla, Samuel
name: Pabel, Steffen
name: Reetz, Florian
name: Dybkova, Nataliya
name: Shomroni, Orr
name: Salinas, Gabriela
name: Mustroph, Julian
name: Hammer, Karin P.
name: Hasenfuss, Gerd
name: Hamdani, Nazha
name: Maier, Lars S.
name: Streckfuss-Bömeke, Katrin
Publisher: name: Universität Regensburg
Year: 2020
Subjects: subject: PROTEIN-KINASE-II; NA+/H+-EXCHANGER; INHIBITION; HEART; DANTROLENE; MOUSE; LEAK; Empagliflozin; EC-coupling; Electrophysiology; iPSC-CM
subject: 610 Medizin

description: The SGLT2 inhibitor empagliflozin improved cardiovascular outcomes in patients with diabetes. As the cardiac mechanisms remain elusive, we investigated the long-term effects (up to 2 months) of empagliflozin on excitation-contraction (EC)-coupling in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) in a blinded manner. IPSC from 3 donors, differentiated into pure iPSC-CM (4 differentiations), were treated with a clinically relevant concentration of empagliflozin (0.5 mu mol/l) or vehicle control. Treatment, data acquisition, and analysis were conducted externally blinded. Epifluorescence microscopy measurements in iPSC-CM showed that empagliflozin has neutral effects on Ca(2+)transient amplitude, diastolic Ca(2+)levels, Ca(2+)transient kinetics, or sarcoplasmic Ca(2+)load after 2 weeks or 8 weeks of treatment. Confocal microscopy determining possible effects on proarrhythmogenic diastolic Ca(2+)release events showed that in iPSC-CM, Ca(2+)spark frequency and leak was not altered after chronic treatment with empagliflozin. Finally, in patch-clamp experiments, empagliflozin did not change action potential duration, amplitude, or resting membrane potential compared with vehicle control after long-term treatment. Next-generation RNA sequencing (NGS) and mapped transcriptome profiles of iPSC-CMs untreated and treated with empagliflozin for 8 weeks showed no differentially expressed EC-coupling genes. In line with NGS data, Western blots indicate that empagliflozin has negligible effects on key EC-coupling proteins. In this blinded study, direct treatment of iPSC-CM with empagliflozin for a clinically relevant duration of 2 months did not influence cardiomyocyte EC-coupling and electrophysiology. Therefore, it is likely that other mechanisms independent of cardiomyocyte EC-coupling are responsible for the beneficial treatment effect of empagliflozin. Key messages This blinded study investigated the clinically relevant long-term effects (up to 2 months) of empagliflozin on cardiomyocyte excitation-contraction (EC)-coupling. Human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) were used to study a human model including a high repetition number of experiments. Empagliflozin has neutral effects on cardiomyocyte Ca2+ transients, sarcoplasmic Ca2+ load, and diastolic sarcoplasmic Ca2+ leak. In patch-clamp experiments, empagliflozin did not change the action potential. Next-generation RNA sequencing, mapped transcriptome profiles, and Western blots of iPSC-CM untreated and treated with empagliflozin showed no differentially expressed EC-coupling candidates.