3 Results
3.1 IPO is not effective in diabetic myocardial I/R injury rats
To investigate whether IPO has cardioprotective effects against myocardial I/R injury in diabetic rats, myocardial infarction size and biochemical markers of myocardial injury after I/R injury were examined. As shown in Figure 1(A), I/R infarction sizes in diabetic rats were larger than those in non-diabetic control rats. IPO significantly decreased infarct sizes in non-diabetic rats, but not in diabetic rats. As shown in Figure 1(B) and (C), CK-MB and LDH levels were significantly increased in diabetic rats compared with non-diabetic rats. IPO noticeably reduced CK-MB and LDH release in non-diabetic rats, but not in diabetic rats. These results showed that IPO improved cardiac functional recovery in non-diabetic rats subjected to I/R, but it had no effect on diabetic rats.
We next measured the changes in DJ-1, Bcl-xL and apoptosis related protein Cleaved Caspase-3 induced by the above processes. As shown in Figure 1(D) (E), DJ-1 and Bcl-xL expressions levels were significantly down-regulated in diabetic rats compared with non-diabetic rats, I/R increased DJ-1 expression levels from non-diabetic rats, and IPO induced further increases in DJ-1 expression levels. However, these changes were not observed in diabetic groups. In addition, we investigated the myocardial I/R induced cardiomyocytes apoptosis and found that was alleviated by IPO treatment in non-diabetic rats but does not work in diabetic rats, Figure1 (F). These results suggest that IPO was able to increased DJ-1 and Bcl-xL expression in non-diabetic rats, as well as decreased Cleaved Caspase-3 expression, but this effect is lost in diabetes.
3.2 Myocardial overexpression of DJ-1 during IPO induced cardioprotection
To futher determine whether hyperglycaemia-induced DJ-1 inhibition compromises IPO induced cardioprotection in diabetic rats, we overexpressed the DJ-1 protein in myocardial tissue from diabetic rats via AAV9-CMV-DJ-1 injections. At 3 weeks after AAV9-CMV-DJ-1 infection, we determined that DJ-1 protein expression levels in treated rats were nearly 2.2-fold higher than those in control group. As shown in Figure 2 (A-C), DJ-1 overexpression alone slightly but not significantly reduced infarct sizes, CK-MB and LDH releases, whereas the combination of DJ-1 overexpression and IPO markedly decreased infarct sizes, CK-MB and LDH levels, indicating that DJ-1 overexpression restores IPO-induced cardioprotection in diabetic rats. We subsequently investigated the effects of the above treatments in cardiac mitochondrial DJ-1, Bcl-xL and cardiac cleaved caspase-3 levels. As shown in Figure 2 (D-E), the combination of cardiac AAV9-CMV-DJ-1 infection and IPO significantly activated cardiac mitochondrial DJ-1, Bcl-xL content and inhibited cardiac Cleaved Caspase-3 level. As shown in Table 1, DJ-1 overexpression alone slightly increased heart rate, LVSP, +dp/dt or −dp/dt, but those did not reach statistical differences. However, in the presence of DJ-1 overexpression, IPO significantly improved heart rate, LVSP, +dp/dt and −dp/dt in diabetic rats.
3.4 IPO induces DJ-1 transfer into mitochondria in the diabetic rats
To test whether hyperglycemia and IPO affect simultaneously the subcellular location of DJ-1 in cardiomyocytes, cytoplasmic and mitochondrial fractions were prepared, and mitochondrial and cytoplasmic DJ-1 levels were examined by Western blot. As presented in Figure 3(A), IPO treatment increased the levels of mitochondrial DJ-1protein relative to the IR treated. Importantly, our results demonstrated that IPO treatment increased the mitochondrial/cytoplasmic ratio of DJ-1 in the diabetic rats subjected to IR, suggesting that IPO promotes DJ-1 translocation from the cytosol to the mitochondria, show in Figure 3(B). Overall, these results suggest a possible involvement of IPO modulation DJ-1 translocation from the cytosol to the mitochondria.
3.5 HPO preserved the protective effects of overexpress DJ-I
Additional investigations were performed using embryonic rat cardiomyocyte-derived H9c2 cells. As shown in Figure 4(A-B), HG stimulation noticeably decreased cell viability and increased LDH release compared with the LG group. These effects were amplified by hypoxia-reoxygenation(H/R). HPO significantly reversed these effects under LG conditions, but not under HG conditions. However, DJ-1 significantly restored the protective effects of HPO, as demonstrated by increased cell viability and decreased LDH release. We subsequently investigated the effects of the above treatments on mitochondrial DJ-1, Bcl-xL and cellular Cleaved Caspase-3 levels. As shown in Figure 4(C-E), AAV9-CMV-DJ-1 mediated DJ-1 overexpression alone did not significantly affect the expression of DJ-1, Bcl-xL in mitochondria, nor did it affect Cleaved Caspase-3 expression in H9c2 cells exposed to HG. However, the combination of AAV9-CMV-DJ-1 infection and IPO significantly increased the mitochondrial DJ-1, Bcl-xL levels, reduced cardiomyocytes apoptosis.
3.6 HPO induced DJ-1 translocate in mitochondria and promoted binding to Bcl-xL
To further confirm the effects of HPO combine with DJ-1 can alleviate high glucose-induced H9c2 H/R injury, we overexpressed DJ-1 using pEX-2-EGFP-DJ-1 transfected and examined its effects on Bcl-xL. The overexpress efficiency of pEX-2-EGFP-DJ-1 is shown in Figure 5 (A). As show in Figure 5 (B-E), overexpress of DJ-1 did not significantly change mitochontrail Bcl-xL protein levels. However, with HPO, Bcl-xL protein levels were significantly higher in the mitochondria. Therefore, we presume that DJ-1 may play a major role as molecular chaperone in affecting some important protein during HPO. We next examined the binding of mitochontrial DJ-1 to Bcl-xL in H9c2 cell subjected to HPO. The binding of DJ-1 to Bcl-xL was assessed by co-immunoprecipitation. Mitochondrial lysates were immunoprecipitated with anti-DJ-1 antibody. Precipitates were immunoblotted for DJ-1, and Bcl-xL respectively. After co-immunoprecipitation analysis, a novel and intriguing interaction between DJ-1 and Bcl-xL was discovered (Figure 5F). Furthermore, members of other apoptosis families, such as Bax, Bcl-2, and Mcl-1, coincidently have no detected interaction with DJ-1(Figure 5G).
3.7 Cys-106 of DJ-1 is required for its binding to Bcl-xL
Although our data implicate a functional relationship between DJ-1 and Bcl-xL, the mechanistic basis underlying this relationship is not well understood. The ability of DJ-1 to protect against oxidative stress is dependent on C106 oxidation and oxidation-driven mitochondrial localization. To examine if the interaction between DJ-1 and Bcl-xL is mediated by the oxidative state of DJ-1, we created a C106A mutant to examine whether C106 is required for DJ-1 to bind to Bcl-xL. DJ-1(C106A), which cannot be oxidized, bound to Bcl-xL much less than DJ-1 did in co-immunoprecipitation assays and could not bind to Bcl-xL without HPO treatment in cells. We performed co-immunoprecipitation experiments. As expected, C106A interacted with Bcl-xL but not C106A mutant as shown in Figure 6 (A), and lower bind to Flag-Bcl-xL without HPO in H9c2 cells, Figure 6 (B). These results suggest that the interactions between DJ-1 and Bcl-xL are oxidation-dependent.
3 Discussion
In the present study, we demonstrate that IPO can alleviate diabetic myocardial ischemic reperfusion injury by promoting DJ-1 transfer to mitochondria. (1) AAV9-mediated DJ-1 overexpression, restored the IPO induced cardioprotection in diabetic heart, and (2) DJ-1 translocates to mitochondria under IPO treatment. The precise localization of DJ-1 in mitochondria has been confirmed by several groups to be in the outer mitochondrial membrane, although it has also been reported to be inserted into the inter-membrane space or in the mitochondrial matrix. In our observations, in diabetic heart under IPO treatment, DJ-1 binds to Bcl-xL (a typical Bcl-2 family protein that primarily localizes in the outer mitochondrial membrane) in the mitochondria. (3) DJ-1 directly bound to Bcl-xL in a C106-dependent manner, the interactions between DJ-1 and Bcl-xL are oxidation-dependent.
Recent studies have shown that IPO significantly protects cardiomyocytes against I/R injury in diabetes[2, 3]. IPO maintains mitochondrial homoeostasis, attenuates oxidative stress, regulates autophagy and alleviates apoptosis[21-23]. Our present study demonstrated that IPO confers protection against myocardial I/R injury in non-diabetic rats, but not in diabetic rats, a finding consistent with those of previous studies[2, 3], demonstrating that hyperglycaemia-induced DJ-1 inhibition may be responsible for the loss of IPO-induced cardioprotection in diabetes.
Under basal conditions, endogenous DJ-1 is present in the cytosol and nucleus. Upon oxidation, DJ-1 translocates from the cytosol to the outer mitochondrial membrane, a process which is required for its cardioprotective effect. It has been suggested that DJ-1 binds to electron transport chain complexes and is required for the latter's normal function such that knockdown of DJ-1 inhibits complex I activity[24]. Experimental studies have demonstrated that the activation of DJ-1 in response to myocardial I/R injury protects the heart by regulating the SUMOylation status of Drp1 and attenuating excessive mitochondrial fission[12]. A recent study has also shown that mouse embryonic lacking DJ-1 had impaired mitochondrial respiration due to complex I inhibition, increased mitochondrial oxidative stress, reduced mitochondrial membrane potential, more fragmented mitochondria, and impaired mitophagy, findings which confirmed that DJ-1 is required for normal mitochondrial function[25, 26]. Furthermore, we observed that IPO was able to increased DJ-1 expression and modulation DJ-1 translocate from the cytosol to the mitochondria in non-diabetic rats, but not in diabetic rats. We, therefore, overexpressed the DJ-1 protein in myocardial tissue from diabetic rats via AAV9-CMV-DJ-1 injections, found that DJ-1 translocates to mitochondria restored the sensitivity of IPO induced cardioprotection in diabetic heart. Importantly, overexpress of DJ-1 activation the protection associated with preconditioning, suggesting that ischemic preconditioning mediates its protective effect through the IPO-dependent activation of DJ-1. More recent studies have suggested that DJ-1 may inhibit oxidative stress-induced apoptotic cell death by interacting with ASK1 directly and preventing its dissociation with Thioredoxin 1, a factor which inhibits ASK1 activity under basal conditions and reducing JNK activity[27]. Furthermore, DJ-1 has been reported to prevent apoptotic cell death by activating and phosphorylating the anti-apoptotic protein kinase, Akt, by suppressing PTEN activity[28]. A previous study showed that the protective effect of DJ-1 in the mitochondria and the interaction between DJ-1 and Bcl-xL are dependent on the oxidation state of Cys106[29].
Bcl-2 family proteins play important roles in the control of mitochondrial cell death. Bcl-2 family proteins include both anti-apoptotic and pro-apoptotic molecules[9]. The anti-apoptotic protein Bcl-xL (primarily localized to the outer mitochondrial membrane) plays a role in keeping mitochondria intact to inhibit cytochrome C release[30]. In our present study, we found that DJ-1 binds to Bcl-xL in mitochondia by IPO treatment in diabetic heart. Upon IPO treatment, the interaction between DJ-1 and Bcl-xL is enhanced. It has been reported that DJ-1 inhibits the ubiquitination of NF-E2-related factor 2 by preventing its association with its E3 ligase Keap1[31]. It is therefore possible that the interaction of DJ-1 with Bcl-xL blocks the relevant E3 ligases from interacting with and ubiquitinating Bcl-xL. Interestingly, DJ-1's translocation to mitochondria under oxidative stress is dependent on oxidation of its Cys-106 site. However, DJ-1(C106A), a loss of oxidized form of DJ-1, does not binds to Bcl-xL in the mitochondria and fails to perform its protective function under IPO treatment in diabetic heart. We found that IPO promotes the translocation of DJ-1 to the mitochondria and its binding to Bcl-xL, and the binding of DJ-1 and Bcl-xL is dependent on the oxidative state of DJ-1. However, DJ-1(Cys-106A) exhibits very low binding affinity to Bcl-xL in co-immunoprecipitation assays. This means oxidation of DJ-1 at Cys-106 is important for its protective effects. For instance, the oxidation of Cys-106 is required for DJ-1 protection against mitochondrial fragmentation and cell death, and DJ-1 fails to protect cells when Cys-106 is in reduced state. Taken together with our findings, we propose that the protective effects of IPO in diabetic I/R injury heart are mediated by the enhanced interactions between DJ-1 and Bcl-xL in mitochontrial.