Introduction
Diabetic cardiomyopathy, a prominent cardiovascular complication, has been recognised as a microvascular disease and a primary cause of morbidity and mortality in diabetic patients. The pathogenesis of diabetic cardiomyopathy involves coronary endothelial cell (EC) dysfunction, fibrosis, cardiac myocyte dysfunction and cardiac myocyte necrosis. We recently reported that coronary ECs from diabetic mice are dysfunctional in regulation of vascular tone and angiogenesis [
1]. In addition, we and other investigators [
2,
3] demonstrated that EC apoptosis is significantly increased in the diabetic heart; however, the molecular mechanism is not clear.
Recent work has highlighted the importance of mitochondrial dynamics in cells and animal physiology. Because mitochondria constantly fuse and divide, an imbalance of these two processes dramatically alters overall mitochondrial morphology [
4]. It is now clear that mitochondrial dynamics play important roles in mitochondrial functions, including development, apoptosis and functional complementation of mitochondrial DNA (mtDNA) mutations by content mixing [
5‐
11].
There are at least five proteins which regulate mitochondrial fusion and fission: optic atrophy 1 (OPA1), mitofusins 1 and 2 (MFN1, MFN2), dynamin-related protein 1 (DRP1) and fission 1 (FIS1). MFN1, MFN2 and OPA1 are essential for mitochondrial fusion and FIS1 and DRP1 are required for mitochondrial fission in mammals [
7,
12]. MFN1 and MFN2 reside on the outer membrane with the N-terminal GTPase, where it is predicted to have a coil protruding into the cytosol [
13], while OPA1 is an intermembrane-space protein [
14]. These proteins work together to promote mitochondrial fusion [
15]. DRP1 exists largely in a cytosolic pool, but a fraction localises to puncta on mitochondria [
16]. FIS1 has a single transmembrane domain at the C-terminal end such that the bulk of the molecule is exposed to the cytosol [
17]. It has been suggested that FIS1 recruits DRP1 from the cytosol to mitochondria for the fission reaction [
17,
18]. Those proteins are associated with different kinds of disease, most of which are related to neuropathy [
19‐
25]. Leinninger et al. previously reported that a change in the DRP1 protein level is caused by hyperglycaemia [
26]. Other studies indicate a close relationship between MFN2 and type 2 diabetes and obesity [
27,
28].
Mitochondria are a primary source of reactive oxygen species (ROS) during production of ATP by the complexes of the respiratory chain. While ROS production is occurring in mitochondria throughout life, mtDNA is more sensitive than genomic DNA to ROS-induced damage, as it is not protected by histones and its repair capabilities are limited [
29]. There is increasing evidence showing the involvement of the superoxide anion (O
2
−
) in the pathogenesis of diabetes-associated vascular complications [
30,
31]. High-glucose treatment of cultured endothelial cells induces overproduction of mitochondrial O
2
−
[
32]. In vivo studies show overproduction of cytosolic O
2
−
in aortic endothelial cells in type 1 diabetes [
33‐
35]. There is, however, no direct evidence demonstrating the change of mitochondrial O
2
−
concentration in vivo or the relationship between O
2
−
concentration and mitochondrial fission in ECs in diabetes.
In this study, we show increased mitochondrial fragmentation, decreased OPA1 and increased DRP1 protein production in mouse coronary endothelial cells (MCECs) isolated from diabetic mice. In addition, we found that morphological changes in mitochondria that occurred as a result of diabetes were restored by O2
−
scavenger treatment without changing OPA1 and DRP1 protein levels.
Discussion
For the first time, a quantification of mitochondrial morphological changes in living cells, comparing control and diabetic cells, was performed. Mitochondria in MCECs isolated from control mice appear as elongated tubular structures with many branches. In contrast, MCECs from diabetic mice have more fragmented mitochondria with significantly smaller volume than mitochondria in control MCECs (Fig.
1). The physiological significance of the continual fusion and fission of mitochondria is still under debate. A possible function for fusion could be a rescue mechanism for damaged mitochondria involving exchange of mtDNA and/or mitochondrial protein [
11]. The predominant gene responsible for autosomal-dominant optic atrophy has been identified as
OPA1 [
19,
20] and those patients exhibit significantly lower copy number of mitochondrial DNA molecules, which may result from decreased mitochondrial fusion [
21]. A recent report demonstrated that
Fis1 transfection in clone 9 cells led to increased cell apoptosis. A targeted null mutation of either
Mfn1 or
Mfn2 results in mid-gestational lethality [
23]. Another study shows a naturally occurring neuropathy is associated with
Mfn2 mutation [
24]. In addition, MFN2 was identified as a suppressor of obesity [
27,
28] and hypertension [
25]. Taken together, these data suggest that increased mitochondrial fission disrupts the normal function of different kinds of cells and may cause organ failure and systemic disease. Figure
2 demonstrates that the fusion-related protein OPA1 level is significantly decreased and fission-related protein DRP1 is significantly increased in MCECs from diabetic mice compared with control mice, but other fusion- or fission-related protein levels do not change (MFN2 and FIS1) or change in a way which is contrary to our expectations (MFN1). The decrease in OPA1 and the increase in DRP1 protein level might be, at least in part, one of the causes of increased mitochondrial fragmentation in diabetic MCECs. As the increase in MFN1 protein is supposed to increase mitochondrial fusion, we hypothesise that in this cell type MFN1 is not a major player in the determination of mitochondrial morphology and further study is required to test this hypothesis.
It has been documented that hyperglycaemia induces excess ROS production, including O
2
−
, in many cell types [
31‐
35,
40‐
43] and local ROS formation is considered to be a major contributing factor to endothelial dysfunction, including endothelial cell apoptosis [
42‐
44], abnormalities in cell cycling [
44] and delayed replication [
41]. Our data demonstrate that MCECs isolated from diabetic mice exhibit significantly higher production of cytosolic and mitochondrial O
2
−
(Fig.
3) and, interestingly, that chronic reduction of O
2
−
by TEMPOL administration increased mitochondrial volume to a level even above that of control MCECs (Fig.
5a,b) without changing OPA1 and DRP1 protein levels (Fig.
5c–e). This suggests that O
2
−
concentration might serve as a direct regulator of mitochondrial morphology. Our ex vivo data also support this hypothesis. As seen in Fig.
6, HCECs exposed to high glucose for 24 h show significantly decreased mitochondrial volume, which is reversed by treatment with the O
2
−
scavenger TEMPOL. This high-glucose-treatment-induced mitochondrial fragmentation was also decreased by
Drp1–shRNA transfection (Fig.
8), implying that these morphological changes by 24 h-high-glucose exposure are not mediated by the fusion-related protein OPA1, but by DRP1 protein upregulation and/or by excess O
2
−
production in the cytosol and mitochondria. In addition, exogenous O
2
−
overproduction by DETA or menadione directly changed the mitochondrial morphology and increased fragmentation (Fig.
7). These data suggest that increased O
2
−
in ECs may potentially cause mitochondrial fragmentation in the diabetic heart.
There is increasing evidence to show that ROS can directly initiate mitochondrial fragmentation in different cell types [
45‐
48]; the detailed mechanisms are, however, still unclear. It is known that O
2
−
can activate protein kinase C (PKC) in ECs [
49,
50], although PKC activation can also stimulate O
2
−
generation. It is thus possible that morphological dynamics in mitochondria could be controlled by PKC activation initiated by O
2
−
. Our data show that treatment with 100 nmol/l phorbol 12-myristate 13-acetate (PMA, a PKC activator) significantly decreased mitochondrial volume (vehicle 0.01% (vol./vol.) DMSO, 0.24 ± 0.01 μm
3; PMA, 0.15 ± 0.02 μm
3;
p < 0.05,
n = 5 each group). Although these data imply that PKC directly regulates mitochondrial morphology, it doesn’t define the relationship between PKC and O
2
−
. It would be necessary to use a PKC inhibitor to identify whether O
2
−
-mediated mitochondrial fission is controlled by PKC activation in diabetic MCECs.
These data suggest that O2
−
overproduction, a decrease in the fusion-related protein OPA1 and an increase in the fission-related protein DRP1 in MCECs in diabetic mice lead to mitochondrial fragmentation, and that treatment with an O2
−
scavenger may help improve mitochondrial function by decreasing mitochondrial fragmentation.