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Background: SGLT2i directly inhibit the cardiac sodium-hydrogen exchanger-1 (NHE1) in isolated ventricular cardiomyocytes (CMs). However, other studies with SGLT2i have yielded conflicting results. This may be explained by methodological factors including cell isolation techniques, cell types and ambient pH. In this study, we tested whether the use of protease XIV (PXIV) may abrogate inhibition of SGLT2i on cardiac NHE1 activity in isolated rabbit CMs or rat cardiomyoblast cells (H9c2), in a pH dependent manner. Methods: Rabbit ventricular CMs were enzymatically isolated from Langendorff-perfused hearts during a 30-min perfusion period followed by a 25-min after-dissociation period, using a collagenase mixture without or with a low dose PXIV (0.009 mg/mL) present for different periods. Empagliflozin (EMPA) inhibition on NHE activity was then assessed at pH of 7.0, 7.2 and 7.4. In addition, effects of 10 min PXIV treatment were also evaluated in H9c2 cells for EMPA and cariporide NHE inhibition. Results: EMPA reduced NHE activity in rabbit CMs that were not exposed to PXIV treatment or undergoing a 35-min PXIV treatment, independent of pH levels. However, when exposure time to PXIV was extended to 55 min, NHE inhibition by Empa was completely abolished at all three pH levels. In H9c2 cells, NHE inhibition by EMPA was evident in non-treated cells but lost after 10-min incubation with PXIV. NHE inhibition by cariporide was unaffected by PXIV. Conclusion: The use of protease XIV in cardiac cell isolation procedures obliterates the inhibitory effects of SGLT2i on NHE1 activity in isolated cardiac cells, independent of pH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Schumacher, Van Amersfoorth, Fiolet, Baartscheer, Veldkamp, Coronel and Zuurbier.)

Sodium glucose cotransporter 2 inhibitors (SGLT2i) are the first antidiabetic compounds that effectively reduce heart failure hospitalization and cardiovascular death in type 2 diabetics. Being explicitly designed to inhibit SGLT2 in the kidney, SGLT2i have lately been investigated for their off-target cardiac actions. Here, we review the direct effects of SGLT2i Empagliflozin (Empa), Dapagliflozin (Dapa), and Canagliflozin (Cana) on various cardiac cell types and cardiac function, and how these may contribute to the cardiovascular benefits observed in large clinical trials. SGLT2i impaired the Na + /H + exchanger 1 (NHE-1), reduced cytosolic [Ca 2+ ] and [Na + ] and increased mitochondrial [Ca 2+ ] in healthy cardiomyocytes. Empa, one of the best studied SGLT2i, maintained cell viability and ATP content following hypoxia/reoxygenation in cardiomyocytes and endothelial cells. SGLT2i recovered vasoreactivity of hyperglycemic and TNF-α-stimulated aortic rings and of hyperglycemic endothelial cells. Anti-inflammatory actions of Cana in IL-1β-treated HUVEC and of Dapa in LPS-treated cardiofibroblast were mediated by AMPK activation. In isolated mouse hearts, Empa and Cana, but not Dapa, induced vasodilation. In ischemia-reperfusion studies of the isolated heart, Empa delayed contracture development during ischemia and increased mitochondrial respiration post-ischemia. Direct cardiac effects of SGLT2i target well-known drivers of diabetes and heart failure (elevated cardiac cytosolic [Ca 2+ ] and [Na + ], activated NHE-1, elevated inflammation, impaired vasorelaxation, and reduced AMPK activity). These cardiac effects may contribute to the large beneficial clinical effects of these antidiabetic drugs.

Objective: Metabolic inhibition causes a decline in mechanical performance and, if prolonged, myocardial contracture and cell death. The decline in mechanical performance is mainly due to altered intracellular calcium handling, which is under control of the Na(+)/Ca(2+)-exchanger (NCX) The driving force of the NCX (ΔG(ncx)) determines the activity of NCX. The aim of this study was to describe the relation between ΔG(ncx) and calcium homeostasis during metabolic inhibition., Methods: In left ventricular rabbit myocytes, during metabolic inhibition (2 mmol/L sodium cyanide), sodium ([Na(+)](i)), calcium ([Ca(2+);](i)), and action potentials were determined with SBFI, indo-1, and the patch clamp technique. Changes of ΔG(ncx) were calculated., Results: During metabolic inhibition: The first 8 min [Na(+)](i) remained constant, systolic calcium decreased from 532 ± 28 to 82 ± 13 nM, diastolic calcium decreased from 121 ± 12 to 36 ± 10 nM and the sarcoplasmic reticulum (SR) calcium content was depleted for 85 ± 3%. After 8 min [Na(+);](i) and diastolic calcium started to increase to 30 ± 1.3 mmol/L and 500 ± 31 nM after 30 min respectively. The action potential duration shortened biphasically. In the first 5 min it shortened from 225 ± 12 to 153 ± 11 ms and remained almost constant until it shortened again after 10 min. After 14 min action potential and calcium transients disappeared due to unexcitability of the myocytes. This resulted in an increased of the time average of ΔG(ncx) from 6.2 ± 0.2 to 7.7 ± 0.3 kJ/mol during the first 3 min, where after it decreased and became negative after about 15 min., Conclusion: Metabolic inhibition caused an early increase of ΔG(ncx) caused by shortening of the action potential. The increase of ΔG(ncx) contributed to decrease of diastolic calcium, calcium transient amplitude, SR calcium content, and contractility. The increase of diastolic calcium started after ΔG(ncx) became lower than under aerobic conditions.