The present study is the first to identify beneficial effects of the SGLT-2 inhibitor, empagliflozin, on AF in a diabetic rat model of HFD + STZ. The major findings of this study are as follows: (1) empagliflozin attenuated DM-induced atrial structural remodeling such as LA interstitial fibrosis, atrial myocyte hypertrophy and effectively suppressed serum MDA, hs-CRP levels in diabetic rats; (2) it effectively prevented DM-induced atrial electrical remodeling (indicated by IACT) and AF inducibility; (3) it ameliorated atrial mitochondrial respiratory dysfunction, preserved mitochondrial membrane potential, improved the mitochondrial biogenesis, and may regulated mitochondrial fusion and division functions of atrial myocytes in diabetic rats.
Atrial remodeling and diabetes mellitus
AF and DM are intertwined disorders that are linked through oxidative stress and inflammation. DM exacerbates atrial electrical and structural remodeling, thereby facilitating AF initiation and maintenance [
13,
14]. Conversely, hence the presence of AF indicates an increased risk in cardiovascular event and mortality amongst DM patients [
4,
15,
16]. The initial stage of DM-induced myocardial changes is characterized by increased fibrosis and stiffness, which is reflected by reduced early diastolic filling, increased atrial filling and enlargement, and elevated LV end-diastolic pressure [
17,
18]. We further demonstrated that LAD, IVST, LVPWT and LVEDP were significantly increased in the DM rats compared with controls, which was consistent with results of the previous studies.
Although the precise pathophysiological mechanisms of DM-induced AF have not been fully elucidated, structural and electrical remodeling are two major contributors to the AF substrate, which refers to the factors that predispose to arrhythmogenesis [
19]. Atrial interstitial fibrosis and replacement fibrosis are hallmarks of arrhythmogenic structural remodeling, producing electrical conduction heterogeneity and disturbance and eventually AF [
16,
20].
Previously, our team has employed different animal models for evaluating the pathophysiological mechanisms that underlie adverse cardiac remodeling in diabetes. For example, alloxan-induced diabetic rabbits exhibit atrial interstitial fibrosis and increased AF inducibility, which are associated with prolonged AERP dispersion and IACT [
14,
15]. In this study, we used the HFD + STZ injection to induce diabetes. Some animals did not meet the criteria of developing diabetes after a single injection of STZ. In these animals, a second dose was injected. Whilst this may increase phenotypic variability between the experimental animals, this double injection approach has been used by other research groups. A previous study used high-fat diet combined with 30 mg/kg STZ twice injection presented a typical characteristic of T2DM as insulin resistance, hyperglycemia, and represented a stable disease model of T2DM [
16].
Data from the present study further support the notion that AF is associated with atrial interstitial fibrosis and electrical conduction heterogeneity. Moreover, indirect evidence supporting the involvement of increased inflammation and oxidative stress in DM-induced atrial remodeling is presented by our data on the serum biochemical examination, where the content of lipid oxidation products, MDA and hs-CRP, are sharply raised while SOD is reduced in the circulation of diabetic rats. Thus, evidence from this study supports the notion that DM contributes to the induction of AF through the promotion of atrial remodeling.
Mitochondrial dysfunction, atrial remodeling and diabetes mellitus
Recently, several studies in DM models have reported mitochondrial function dysfunction in the myocardium as an important pathological change [
11,
18,
19]. Cardiomyocytes contain a relatively large number of mitochondria and mitochondrial oxidative phosphorylation provides 90% of intracellular ATP production in cardiomyocytes. An emerging body of evidence shows that mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase are the predominant mechanisms by which ROS is generated in the diabetic heart [
21]. Hyperpolarization of the mitochondrial membrane and impaired mitochondrial function promote mitochondrial ROS production. ROS can damage mitochondrial membrane structure and oxidize lipids to yield reactive lipid peroxidation products [
22]. Moreover, ROS can activate mitochondrial uncoupling, leading to reduce cardiac efficiency. Altered mitochondrial Ca
2+ handling further promotes mitochondrial respiratory dysfunction, which ultimately leads to cell death under increased oxidative stress. Our previous work has found that increased mitochondrial reactive oxygen species production rate, depolarized mitochondrial membrane potential, and mitochondrial swelling in diabetic rabbits [
23]. In the present study, we extend these findings by reporting abnormal mitochondrial membrane potential and mitochondrial respiratory control ratio, increased ROS production, as well as abnormal expression levels mitochondrial proteins in the DM rats.
Mitochondrial abnormalities may play an important role for the increased propensity for AF in DM. Previous studies have demonstrated that abnormal mitochondrial structure and function prompted atrial structural and electrical remodeling [
24,
25]. Damage to mitochondrial function leads to the increasing extracellular matrix, causing further contractile dysfunction, hypertrophy and cardiac fibrosis [
26]. In addition, mitochondrial dysfunction was associated with altered cardiac electrical properties, giving rise to action potential heterogeneities in conduction or repolarization in both the ventricles and the atria [
23,
27]. In this present study, we observed higher AF inducibility in the DM group, and markedly reduced by EMPA. These findings complement previous studies focusing on alterations in ventricular structure and electrical function in diabetic animals and the effects of anti-diabetic treatment upon these. For example, empagliflozin was shown to improve left ventricular diastolic function a genetic mouse model of T2DM [
20]. Furthermore, the SGLT-2 inhibitor dapagliflozin was shown to suppress prolonged ventricular-repolarization through augmentation of mitochondrial function and protection against the generation of reactive oxygen and nitrogen species in insulin-resistant metabolic syndrome rats [
28].
PGC-1a is a crucial promoter of mitochondrial biogenesis, regulated by adenosine monophosphate kinase (AMPK) and induced by NRF-1 and Tfam [
29,
30]. There is evidence that the SGLT-2 inhibitor attenuates the up-regulation of the cardiac Na
+/H
+ exchanger (NHE) in vitro in mouse cardiac fibroblasts stimulated with lipopolysaccharides (LPS) via AMPK activation [
31]. In our study, we found that the protein level of PGC-1a, NRF-1, and Tfam expressed in LA tissue were reduced in the DM group, which corresponds to the impaired mitochondrial biogenesis in DM described by recent studies. Moreover, the protein expression level of DRP-1 and Mfn-1, mitochondrial division and fusion proteins respectively, in addition to OPA-1, are also lower in DM group, suggesting that both the quality and function of atrial myocardial mitochondria is reduced in DM, resulting in suppressed mitochondrial fusion and division.
SGLT-2 inhibitor, empagliflozin and potential cardiac beneficial effects
SGLT-2 is a sodium–glucose cotransporter exclusively expressed in the kidney. They are mostly localized in the brush border membrane of the proximal tubule epithelial cells in the S1 segment of the proximal convoluted tubule [
32,
33]. SGLT-2 expression is significantly increased in diabetic humans, rats, and db/db mice [
34‐
36]. It is correlated with glomerular hyperfiltration, increased glucose reabsorption, and elevated plasma glucose [
37]. Conversely, SGLT-2 inhibition leads to natriuresis, osmotic diuresis, plasma volume contraction. Recognizing the physiological effects of SGLT-2, SGLT-2 inhibitors have emerged as a new class of plasma glucose-lowering medication. Besides from beneficial effects on parameters such as glucose concentration, weight, blood pressure and albuminuria, SGLT-2 inhibitors potentially reduce the risk of cardiovascular mortality and HF [
38]. Recently, several large-scale randomized controlled trials, the EMPA-REG OUTCOME, DECLARE-TIMI and CANVAS, provided evidence that SGLT-2 inhibitors reduce the risk of cardiovascular events significantly when compared to the use of placebo [
4,
5,
39]. Other teams have contributed to the understanding of cardiac remodeling in diabetic rat models using SGLT-2 inhibitors [
40‐
45]. The cardiac protection effect of SGLT-2 inhibitor was not only in DM, still in cardiorenal syndrome (CRS) animals. Yang et al. [
46] found that compared with CRS animals, LVEF was remarkably preserved and LV remodeling was substantially suppressed in CRS animals treated by EMPA. The finding may support the results of a previous clinical trial [
4]. In addition, EMPA also ameliorates type 2 diabetes-induced ultrastructural remodeling of the neurovascular unit and neuroglia in the female db/db mouse [
47].
Several hypotheses about the potential mechanisms of SGLT‑2 inhibitors responsible for cardioprotection have been identified [
48,
49]. These include prevention of (i) cardiac inflammation and oxidative stress, (ii) apoptosis, (iii) ionic homeostasis, and mitochondrial dysfunction. Indeed, Durak et al. [
28] suggested a new insight into another SGLT-2 inhibitor DAPA-associated cardioprotection, including suppression of prolonged ventricular-repolarization through augmentation of mitochondrial function and oxidative stress. Furthermore, Lee et al. [
50] have found that EMPA could change Ca
2+ regulation, late Na
+ and Na
+/H
+-exchanger current, and electrophysiological characteristics in DM cardiomyopathy.
To the best of our knowledge, this study is the first to examine the protective effects SGLT-2 inhibitor on the diabetic atria and explore the roles of mitochondrial dysfunction as an underlying pathogenic mechanism driving this atrial remodelling. Our study demonstrated that the SGLT-2 inhibitor, EMPA, have favorable effects in ameliorating arrhythmic substrate, improving electrophysiological abnormalities, and reducing AF inducibility. Meanwhile, we also found that SGLT-2 inhibitors increased LA tissue mitochondrial respiration function and the protein expression levels of PGC-1a, NRF-1, Tfam, DRP-1, Mfn-1 and OPA-1, indicating that EMPA can improve the impaired mitochondrial function in DM. With reduced mitochondrial impairment and atrial remodeling in DM, the present study provides evidence for potential clinical use of EMPA upon the prevention of DM-induced AF.
However, it is not clear the relative contributions of direct cardiac versus systemic effects towards cardioprotection mediated by SGLT-2 inhibition. For example, DM is associated with heart failure, and SGLT-2 inhibitors could reduce the risk of cardiovascular mortality by preventing HF [
38,
39]. Indeed, there are close and complex links between heart failure with preserved ejection fraction (HFpEF) and AF, with shared risk factors and the higher AF incidence in HFpEF may independently contribute to poor clinical outcomes [
51]. In this study, we discovered that EMPA improved diastolic dysfunction on diabetic rats. It is likely that the improvement in HF partly contributed to reduced risk of AF inducibility by high-dose EMPA. In our animal model, both glucose and lipid profiles were reversed by EMPA, and thus the cardiac protective effects must have been partially mediated by alterations in lipid and glucose levels. To dissect the relative contributions of glucose-dependent and glucose-independent effects, further studies need to examine the effects of empagliflozin on cardiac function in non-diabetic models such as heart failure or hypertension models. Moreover, we found direct amelioration of atrial electrical and structural substrate and prevention of mitochondrial dysfunction by empagliflozin, and thus direct cardiac effects may operate in this setting.
In addition, Na
+ and Ca
2+ handling abnormalities contribute to cardiac contractile dysfunction, as well to the perpetuation and progression of AF. Baartscheer et al. [
52] demonstrated the direct myocardial effects of empagliflozin on Ca
2+ and Na
+ concentrations independently of SGLT-2 activity. Thus, in isolated ventricular myocytes from rabbits and rats incubated with empagliflozin for 3 h, empagliflozin decreased cytoplasmic Na
+ ([Na
+]
c) and Ca
2+ ([Ca
2+]
c) and also increased mitochondrial Ca
2+ concentration ([Ca
2+]
m). Empagliflozin can exert effects by decreasing myocardial [Na
+]
c and [Ca
2+]
c and increasing [Ca
2+]
m through the inhibition of the Na
+/H
+ exchanger (NHE) directly [
46]. In addition, it has been shown that empagliflozin improved LV diastolic function by increasing sarcoplasmic endoplasmic reticulum Ca
2+-ATPase (SERCA2a) activity in diabetic mice [
53]. Hence, further in-depth study and important knowledge gaps need to be addressed by a multidisciplinary experimental and computational approach to investigate the specific roles of NHE, Na
+ and Ca
2+ ionic concentrations in atrial muscle, as has been performed for skeletal muscle [
54].