The present study demonstrates for the first time that the kidney-targeted therapeutic agent, EMPA, directly reduces myocardial intracellular Na2+ through an interaction with the NHE, independent of cardiac SGLT inhibition. The observed decreased [Ca2+]c and increased [Ca2+]m are likely to have occurred secondary to the decrease in [Na+]c via the sarcolemmal and mitochondrial Na+/Ca2+ exchanger (NCX), respectively [6, 7]. Dapagliflozin, another SGLT2 inhibitor with a slightly different chemical composition, was recently shown to reduce cell shortening after 5 min but not after 3 h of treatment. It was also found to reduce systolic but not diastolic Ca2+ in cardiomyocytes from models of type 1 diabetes (but not in control cardiomyocytes) . In comparison with dapagliflozin, the current study indicates a stronger and longer lasting effect of EMPA on cardiomyocytes. It is suggested that further research is required to compare the effects of different classes of SGLT2 inhibitors on cardiomyocytes characteristics.
EMPA: cardiac NHE and SGLT2
Our observations that the effects of EMPA were independent of glucose presence is in agreement with a previous observation that SGLT2 is absent from cardiac tissue . However, SGLT1 is present in the heart [9, 10], potentially explaining the increase in [Na+]c with increased circulating glucose. It was recently reported that SGLT1 within the diseased human heart is mainly localised in capillaries and not in cardiomyocytes . This is in contrast with other observations that SGLT1 is localised in the T tubules of isolated cardiomyocytes . In the current study, the effects of glucose on [Na+]c were detected in isolated cardiomyocytes; therefore, it is unlikely that glucose release from the capillaries plays a role in this effect. However, further research is needed to study the role of SGLT1 within the intact heart. We used EMPA concentrations in the range of clinically measured plasma concentrations (≤1 μmol/l) [1, 12], well below the IC50 of SGLT1 (8.3 μmol/l) for EMPA . Therefore, EMPA does not exert its cardiac effects through SGLT1inhibition.
EMPA raises cardiac [Ca2+]m
Previous studies have demonstrated that increasing [Na+]c results in decreased [Ca2+]m through increased mitochondrial efflux via the mitochondrial NCX . This is very likely to be the mechanism underlying the EMPA-induced elevation in [Ca2+]m, observed in the present study. Mitochondrial Ca2+ is considered to be an important activator of ATP synthesis and of the antioxidant enzymatic network [5, 14]. Knowing that in vivo mitochondrial impairment, energy deficiency and increased oxidative stress [5, 14, 15] are hallmarks of failing hearts, restoring [Ca2+]m (e.g. increasing [Ca2+]m that is otherwise reduced due to Na+ loading) is predicted to be beneficial in this condition. Indeed, in a recent study, it was demonstrated that increasing mitochondrial Ca2+ during heart failure development was associated with the prevention of sudden death and overt heart failure . This is in contrast to conditions where [Ca2+]m is already elevated, such as during reperfusion injury phenomena, in which further increases in [Ca2+]m can be detrimental.
EMPA and heart failure
The propensities for arrhythmias, oxidative stress and heart failure are all associated with, and at least partly driven by, intracellular cardiomyocyte Na+ and Ca2+ loading [2, 3, 5, 6, 14]. Hyperglycaemia (as reported in this study) and diabetes  also result in intracellular Na+ and Ca2+ loading, possibly contributing to the reported interaction between hyperglycaemia/diabetes and cardiovascular diseases.
Previous studies have demonstrated that chronic inhibition of NHE prevents or mitigates heart failure in animal models [16, 17]. Although clinical studies of NHE inhibition have been performed in the setting of acute coronary syndromes (and largely show a neutral effect) no clinical studies have been performed using NHE inhibition in the chronic setting of heart failure and diabetes. Therefore, these types of studies are awaited. We surmise that the beneficial cardiovascular effects of EMPA are, at least in part, attributed to NHE inhibition. However, the current results do not allow for the generation of conclusive statements about a positive or negative role on cardiac mitochondrial function following EMPA treatment. The next area that needs to be evaluated is whether EMPA does indeed result in beneficial functional cardiac effects, such as increased ATP production, oxygen consumption and/or antioxidant capacity. Whether the reductions in both cardiac [Na+]c and [Ca2+]c with EMPA treatment, and the downstream effect of elevated [Ca2+]m contribute to the primary mechanisms underlying the cardiovascular benefits observed in the EMPA-REG OUTCOME trial needs to be examined in future research.
In conclusion, our data demonstrate that the kidney-targeted therapeutic agent EMPA has direct cardiac effects, decreases cardiac [Na+]c and [Ca2+]c and increases cardiac [Ca2+]m via inhibition of the cardiac NHE.