The pathophysiological mechanisms underlying the remarkable cardiac effects of empagliflozin in the EMPA-REG OUTCOME trial are still unresolved. The SGLTs are a family of proteins able to translocate glucose in different tissues [
32]. Both SGLT2 and SGLT1 co-transport Na+ together with glucose. While SGLT1 is expressed in small intestine, lungs, kidneys, liver and cardiac myocytes, SGLT2 has been primarily found in the kidney, but it is also expressed by pancreatic alpha cells. We demonstrated that SGLT2 is expressed in murine cardiac myocytes of C57BL/6 mice. Notwithstanding the previous report by Yoshii et al., that did not find transcription of the SGLT2 gene in mice hearts [
33], we were able to demonstrate SGLT2 expression in C57BL/6 mice hearts using 3 different methodologies: transcription as shown by RT-PCR (Fig.
5b); protein expression, using western blotting (Fig.
5a); and immunohistochemistry (Additional file
1: Figure S5). This apparent discrepancy of results might be related to some differences between the work by Yoshii and colleagues and ours [
33]. First, they only performed PCR experiments (n = 3), whereas we found consistent results using two different techniques. It should be noted that protein expression levels we found were higher than one might have expected from SGLT2 transcription levels, suggesting that post-transcriptional regulation might be particularly relevant to SGLT2 expression in this model. In this regard, it should be mentioned that SGLT2 is target of multiple microRNAs, a class of post-transcriptional regulators of gene expression (Additional file
1: Table S1). In particular, the highly conserved miR-296-5p, recently found to be involved in the modulation of cardiac hypertrophy [
34] and myocardial fibrosis [
35] was able to promote healing of diabetic wounds, directly targeting SGLT2 [
36]. In addition, SGLT2 translation is also influenced by glucose level [
37]. Our findings of SGLT2 expression in mice are in line with the recent demonstration of expression SGLT2 in both mouse [
38] and human heart tissue [
39]. The same authors also report that high glucose levels were associated with a significant increase in expression levels of both of SGLT1 (7.1 folds; p < 0.01) and SGLT2 (7.5-folds; p < 0.01) and that treatment with empagliflozin was able to restore physiological SGLT1 and SGLT2 expression levels, opening the way for potential relevance of our finding in humans [
39].
The expression of functional SGLT1 in human hearts was already demonstrated by von Lewinsky et al. [
40], that reported that SGLT1 contributes to the positive inotropic effect exerted by insulin in patients with end-stage HF. Hence, our direct targeting of cardiac SGLT2 receptors is a further potential mechanism.
Therefore, our hypothesis is that SGLT2 inhibition may affect the tissue and cellular Na+ homeostasis that is responsible for the excitation–contraction coupling and the mitochondrial redox regulation in cardiomyocytes. In the 2000s Despa et al. [
41] observed that [Na+]i is increased in failing cardiac myocytes, as a consequence of increased Na+ influx via the late Na+ current, increased activity of the sarcolemmal Na+/H+ exchanger (NHE), and a reduction in Na+/K+ ATPase activity. Moreover, SGLT1 expression in the heart was found to be upregulated both in animal models of type 2 diabetes and in patients with diabetic cardiomyopathy, and its activity contributes to the increase in [Na+]i.
Moreover, Baartscheer et al. recently reported that EMPA reduced [Na+]i and [Ca2+]c in isolated ventricular myocytes independently of the presence of glucose [
42]. In line with this pathophysiological hypothesis, the beneficial effect exerted by EMPA in our experimental model was independent of any significant impact on glycemia, glycosuria or diuresis ad demonstrated by the experiments performed using metabolic cages.
EMPA is reported to reduce glycemia by increasing glycosuria and natriuresis in diabetic humans [
43]. However, in line with human findings we did not observe significantly lower glycemia in EMPA-treated non-diabetic mice. Furthermore, in line with the latter findings we did not observe an increase in glycosuria or diuresis compared to mice not receiving EMPA, thus excluding that the effect of EMPA on LV structure and function could have been mediated by changes in blood volume or loading conditions. Hence, cardiac functional and structural improvements in EMPA-treated non-diabetic mice occurred independently of glycemia, glycosuria or diuresis.
In light of these considerations, it is tempting to speculate that our positive results on LV function in non-diabetic mice may be—at least in part—related to direct effects of EMPA on cardiac ion homeostasis. By decreasing [Na+]i and restoring mitochondrial Ca2+ handling, EMPA may ameliorate mitochondrial energetic mismatch and production of ROS, thus interrupting the vicious circle which underlies Na+ overload and oxidative stress in cardiomyocytes.