Class effects of SGLT2 inhibitors in mouse cardiomyocytes and hearts: inhibition of Na⁺/H⁺ exchanger, lowering of cytosolic Na⁺ and vasodilation

We investigated whether SGLT2i exhibited similar effects on NHE by studying intracellular Na⁺ and pH. Figure 1a shows reduced recovery of intracellular pH after NH₄ ⁺ pulse with all three SGLT2i (EMPA 6.69 ± 0.09, DAPA 6.77 ± 0.12 and CANA 6.80 ± 0.18 vs vehicle 7.09 ± 0.09; p < 0.001 for all three comparisons). Furthermore, [Na⁺]_c was reduced with all three SGLT2i (Fig. 1b) after 10 min (in mmol/l: EMPA 10.0 ± 0.5, DAPA 10.7 ± 0.7 and CANA 11.0 ± 0.9 vs vehicle 12.7 ± 0.7; p < 0.001 for all three comparisons).

Interactions between SGLT2i and NHE were explored using a homology model of NHE. The extracellular part of the protein model was probed to gain insight into the possible binding mode of SGLT2i to NHE. All three SGLT2i displayed high binding affinity to the extracellular Na⁺-binding site of NHE (in kJ/mol: EMPA −34.3, DAPA −32.2, CANA −37.2) and adopted similar binding conformation with the glucoside moiety oriented towards the Na⁺-binding site and the aglycone part of the inhibitors lining the side of the extracellular aperture (Fig. 1c). Binding of a glucose molecule to the NHE homology model showed that glucose bound in an identical orientation to the glucoside part of the SGLT2i, but with a reduced affinity of −24.3 kJ/mol, illustrating the importance of the hydrophobic part of the SGLT2i to ensure efficient binding.

Combined, these data show that EMPA, DAPA and CANA inhibit NHE activity through binding to the Na⁺-binding site of NHE-1. Secondary to this, they reduce [Na⁺]_c in isolated cardiomyocytes.

Perfusion pressure at t = 0 min was not significantly different between groups (in mmHg: vehicle 79.4 ± 2.7, EMPA 79.9 ± 2.4, DAPA 79.3 ± 4.1, CANA 79.3 ± 2.5). After 30 min following drug administration, EMPA and CANA resulted in vasodilation, compared with vehicle, in isolated constant-flow perfused mouse hearts (vehicle vs EMPA p < 0.05, CANA p < 0.05, DAPA p = 0.410) (Fig. 2a). Furthermore, cardiac functional performance (rate pressure product), cardiac energetic status (PCr/ATP) and O₂ consumption were unchanged after administration of the SGLT2i (Fig. 2b–d, respectively).

Our experiments examining NHE activity and [Na⁺]_c in cardiomyocytes showed that the inhibitory effect on cardiac NHE is not specific to EMPA but extends to other SGLT2i. In our previous work [4], we showed that EMPA treatment not only lowers [Na⁺]_c but reduces [Ca²⁺]_c and increases mitochondrial Ca²⁺. Since DAPA and CANA also inhibit NHE activity and reduce [Na⁺]_c, we postulate that these SGLT2i reduce [Ca²⁺]_c and increase mitochondrial Ca²⁺ similar to EMPA, and ultimately optimise cardiac mitochondrial function and energetics. Because both [Na⁺]_c and NHE activity are increased in diabetic and failing hearts, SGLT2i should have an even stronger effect on these in diseased cardiomyocytes. Furthermore, preclinical studies with known NHE inhibitors have clearly shown reductions in the development of hypertrophy and heart failure [13, 14], which support NHE inhibition and its consequential [Na⁺]_c-lowering as a potential class effect of SGLT2i to combat heart failure.

SGLT2i are targeted to SGLT2 by the glucosyl part of the molecule, while binding affinity is determined by the attached hydrophobic moiety. Our docking studies indicate that the hydrophilic glucosyl part of the SGLT2i orients towards the hydrophilic Na⁺-binding site. The composition of the hydrophobic aglycone part of the SGLT2i appears to be an important determinant in their binding to NHE. The NHE molecule exists in a low- and high-affinity form for intracellular protons, regulated by pH and mitogens [15]. This conformational heterogeneity may be essential in SGLT2i binding, especially in disease states where NHE activity threshold is shifted towards its high-affinity form.

Our data on isolated mice hearts revealed a direct vasodilation effect of EMPA and CANA, but not DAPA, at constant glucose concentration. Oelze et al [16] have previously shown that EMPA normalised endothelial function in aortic rings from streptozotocin-induced rat models of diabetes, an effect that was also detected with ipragliflozin in a similar mouse model [17]. However, because EMPA treatment in these studies also caused a large reduction in plasma glucose levels, it is impossible to interpret these data towards EMPA exerting direct vascular effects. Wang et al [18] reported that NHE activation in hyperglycaemic endothelial cells led to increased intracellular Ca²⁺ and reduced endothelial nitric oxide synthase levels and impaired relaxation of aortic rings from streptozotocin-induced rat models of diabetes, while NHE inhibition abolished these effects. Assuming that NHE inhibition by SGLT2i also occurred in other cells than cardiomyocytes in our intact heart experiments, vasodilation by SGLT2i may therefore be related to lowering of [Ca²⁺]_c in endothelial cells or vascular smooth muscle cells after NHE inhibition. Finally, no changes were observed for cardiac workload, energetic status and metabolic function in healthy hearts. The functional and energetic status of healthy hearts was already optimal and could not be improved by treatment with SGLT2i. Interestingly, a preliminary study in db/db mice found that EMPA administration acutely improved PCr/ATP [19]. In our experiments, we did notice a non-significant trend of increased O₂ consumption in EMPA-treated hearts (p = 0.054), which may possibly indicate increased activation of mitochondrial energy metabolism.

We cannot explain why DAPA did not significantly induce vasodilation in healthy hearts. The non-significant results for DAPA in relation to vasodilation could in part be explained by the relatively low sample size. Here, we only studied the direct effects of SGLT2i for 30 min in isolated hearts. Chronic cardiac effects of SGLT2i may be studied in the future in in vivo models to translate and understand drug effects in individuals who use SGLT2i daily. Another limitation of this study is the lack of a diabetic model to investigate direct cardiac effects of SGLT2i. Nonetheless, the results in healthy cells and hearts suggest that these direct effects of SGLT2i may happen regardless of diabetes, opening the possibility to explore SGLT2i in other cardiac diseases where increased NHE activity is a driver of the disease, such as heart failure and hypertrophy. Thus, future research should also examine the effects of SGLT2i on cardiac physiology and metabolism in diabetic and failing hearts.

In conclusion, EMPA, DAPA and CANA all exhibit direct cardiac effects through NHE inhibition and [Na⁺]_c reduction. EMPA and CANA, but not DAPA, induce coronary dilation of the intact heart.