Background
Diabetes mellitus (DM) has been reported to be an independent predictor of cardiovascular morbidity and mortality in patients with ischemic heart disease [
1], as well as heart failure [
2], while direct deleterious effects of DM on hearts also have been reported [
3]. However, few investigations of the cardiac consequences of type 2 diabetes, particularly in regard to sensitivity to ischemia, have been performed [
4‐
6]. It is recognized that cytoplasmic Ca
2+ overload is an important mechanism in myocardial ischemic injury [
7‐
10]. Cytoplasmic Ca
2+ overload occurs via a combination of activities of Na
+/H
+ exchanger (NHE) and Na
+/Ca
2+ exchanger (NCX), with the latter functioning in the reverse mode [
9‐
13]. Changes in cytoplasmic Ca
2+ concentration ([Ca
2+]
i) during ischemia have not been evaluated in type 2 diabetic hearts. We previously reported that a highly increased intracellular Na
+ concentration ([Na
+]
i), mainly via NHE, caused enhanced sensitivity to ischemia in type 2 diabetic
db/db mouse hearts [
5]. Therefore, we speculated that NHE plays a key role in cytoplasmic Ca
2+ overload in ischemic cardiomyocytes of type 2 diabetic hearts.
The purpose of the present study was to determine changes in diastolic [Ca
2+]
i and Ca
2+ transient amplitude during ischemia and reperfusion in isolated type 2 diabetic
db/db mouse hearts loaded with fura-2, a fluorescent dye [
8,
14]. The correlation between [Ca
2+]
i and cardiac function was investigated, as well as the importance and role of NHE in [Ca
2+]
i changes.
Discussion
The main finding obtained in the present study was a marked decrease in cytoplasmic Ca2+ overload during ischemia-reperfusion in hearts from diabetic db/db mice in the presence of the NHE inhibitor cariporide. This was accompanied by significantly improved recovery of ventricular function on reperfusion, as assessed from our findings of a lower increase in diastolic pressure and increased recovery of developed pressure.
In agreement with previous reports that used mice under similar experimental conditions (i.e., same age and similar control perfusion conditions) [
4‐
6], we found no difference in ventricular function between control and diabetic hearts (Table
2). Basal alterations in [Ca
2+]
i in hearts from diabetic
db/db mice have been reported by several investigators. Belke et al. noted increased Ca
2+ leakage from the sarcoplasmic reticulum (SR) [
19], while impaired [Ca
2+]
i cycling due to reductions in expressions of both sarcolemmal Ca
2+ channels and SR Ca
2+ release channels were demonstrated by Pereira et al. [
23]. In the present study, during the control perfusion period, time to peak and decay time of Ca
2+ transient were significantly prolonged in
db/db hearts (Figure
1 and Table
2). Our observations are in agreement with the above reports and represent a consistent feature of ventricular myocytes in diabetic hearts [
20]. It has been shown that increased saturated fatty acid levels impair Ca
2+ handling and contraction in a reactive oxygen species (ROS)-dependent manner in normal cardiomyocytes [
15]. However, it remains to be determined whether increased fatty acid utilization interferes with Ca
2+ handling in diabetic
db/db mouse hearts. Basal alterations in [Ca
2+]
i might precipitate cytoplasmic Ca
2+ overload during ischemia-reperfusion, as we observed in the present
db/db hearts. On the other hand, cytoplasmic Ca
2+ overload in these hearts was largely prevented by the presence of the NHE inhibitor cariporide (Figure
6). This finding indicates a major role for the ionic exchanger NHE in Ca
2+ overload. It is known that increases in [Na
+]
i in ischemic cardiomyocytes [
5] generate Ca
2+ loading via reverse Na
+/Ca
2+ exchanger, which in turn mediates much of the damage incurred upon reperfusion [
24]. In this context azelnidipine [
25] and ranolazine [
26] have also been shown to be efficient at reducing intracellular calcium accumulation.
NHE is a key element in the physiological response of [Ca
2+]
i and Ca
2+ signaling in cardiomyocytes [
27‐
29], and its activation has been reported to be correlated with cardiac hypertrophy via this system [
17,
27,
29]. The present results are in line with our previous report that showed higher [Na
+]
i increase during ischemia and severely impaired post-ischemic cardiac function in hearts from diabetic
db/db mice. In that study, NHE inhibitor cariporide reduced ischemia-induced [Na
+]
i increase and improved post-ischemic cardiac function in
db/db hearts [
5]. Likewise, elevation of diastolic fura-2 ratio in the present study was attenuated during ischemia and reperfusion and functional alterations on reperfusion were markedly reduced by cariporide in diabetic
db/db hearts. Taken together, our previous [
5] and present results suggest that the role of NHE is more important for ischemia-induced cytoplasmic Ca
2+ overload and myocardial damage in diabetic
db/db hearts than in control
db/+ hearts. Moreover, enhanced NHE activity during ischemia in
db/db hearts can be inferred from these results. Indeed, enhanced NHE activity in ventricular myocytes has been reported in another genetic model of type 2 diabetes, the Goto-Kakizaki rat [
17]. In addition, the use of HCO
3- free, HEPES buffered perfusion solution might have enhanced the contribution of NHE1 in the present study [
30]. Among known factors stimulating cardiac NHE1 activity are [Ca
2+]
i[
31,
32] and angiotensin II [
33‐
35]. In ventricular myocytes of diabetic
db/db mice, NHE1 activity might be stimulated by basal alterations in [Ca
2+]
i, due to increased Ca
2+ leakage [
18]. Future work will have to examine NHE1 activity in cardiac myocytes of diabetic
db/db mice.
Inhibition of an NHE isoform located in the mitochondrial membrane and reduction of mitochondrial Ca
2+ ([Ca
2+]
m) overload by one of the NHE blockers during ischemia-reperfusion have been reported [
36]. We cannot exclude the possibility that cariporide may have exerted some effects on mitochondrial NHE and [Ca
2+]
m in the present study. In addition, though these effects of cariporide might be different between diabetic and non-diabetic hearts, [Ca
2+]
m cannot be selectively measured with the methods employed in this study. Finally, NHE inhibitors have recently been shown to affect mitochondria by blunting MPTP formation and ROS release [
37]. An overproduction of mitochondrial ROS has indeed been shown in the heart in obesity related diabetes [
38]. Further studies are still needed to investigate the role of ROS formation in ischemia-induced calcium overload in hearts of diabetic
db/db mice.
Concerning clinical applications, blockade of NHE may provide salutary effects for diabetic patients with ischemic heart disease through an interaction with PKB/Akt [
39,
40] and other mechanisms [
29,
31‐
35]. However, it is unfortunate that the majority of trials conducted to test the effects of NHE inhibitors in ischemic heart disease cases have failed [
41]. Intrinsically, valuable actions of NHE might be inhibited excessively in those trials. Hence, the methods or timings to inhibit NHE activity should be studied. For example, partial inhibition of aldosterone-induced genomic synthesis of NHE by eplerenone [
42] may be an important choice.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RA conceived of the study, carried out the experiments, and drafted the manuscript. SS and TN participated in the fluorescence measurement study. IT, DF, and MY conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final version of the manuscript.