Long-term effects of empagliflozin on excitation-contraction-coupling in human induced pluripotent stem cell cardiomyocytes

Abstract

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 μmol/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 Ca2+ transient amplitude, diastolic Ca2+ levels, Ca2+ transient kinetics, or sarcoplasmic Ca2+ load after 2 weeks or 8 weeks of treatment. Confocal microscopy determining possible effects on proarrhythmogenic diastolic Ca2+ release events showed that in iPSC-CM, Ca2+ 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.

Direct cardiac effects of SGLT2 inhibitors

“…Various studies have demonstrated that SGLT2is reduce cytosolic Ca 2+ in cardiomyocytes under different conditions: EMPA (1 μM) for 2 h reduced lipopolysaccharide (LPS)-increased cytosolic Ca 2+ in a human cardiomyocyte derived cell line in the absence of albumin [ 15 ]; 10–500 nM EMPA reduced doxorubicin (DOXO)-increased cytosolic Ca 2+ in cultured HL-1 adult mouse cardiomyocytes in the presence of albumin [ 16 ]; However, not all studies reported effects on ion homeostasis: no decrease was observed in cytosolic Ca 2+ or Na + following short treatment (> 5 min) with DAPA (1 μM) in isolated cardiomyocytes in the absence of albumin from high-salt treated Dahl-sensitive rats [ 17 ], and no effects of EMPA (0.5 μM) on Ca 2+ transient, sarcoplasmic Ca 2+ load and diastolic sarcoplasmic Ca 2+ leak in hiPSC-CMs in the absence of albumin were observed [ 18 ].…”

Potential Mechanisms of SGLT2 Inhibitors for the Treatment of Heart Failure With Preserved Ejection Fraction

“…Likewise, we performed a blinded experimental long-term study of human induced pluripotent stem cell cardiomyocytes from healthy subjects as clinical effects occur after weeks or months which is in contrast to many experimental study designs where acute exposure to these drugs have been performed. In our respective study 2 months of treatment with empagliflozin showed no impact on Ca 2+ homeostasis and EC-coupling proteins (Pabel et al, 2020b). In a study based on Dahl saltsensitive rats with high-salt diet serving as a HFpEF model, the authors reported that dapagliflozin beneficially affects Ca 2+ and Na + overload after in vivo treatment but not after direct treatment of cardiomyocytes (Cappetta et al, 2020).…”

Effects of Atrial Fibrillation on the Human Ventricle

“…For assessing diastolic sarcoplasmic reticulum Ca 2+ sparks, iPSC-CMs were loaded with Fluo-4 am (10 µmol/L, Invitrogen) in the presence of Pluronic F-127 0.05 % w/v (Life Technologies) for 15 minutes and then incubated with Tyrode’s solution for 15 minutes. 25 , 26 Ca 2+ spark measurements were performed at room temperature by using a laser scanning confocal microscope (LSM 7 Pascal, Zeiss). Fluo-4 was excited by an argon ion laser (488 nm), and emitted fluorescence was collected through a 505 nm long-pass emission filter.…”