Diazoxide Preconditioning Research Articles

Diazoxide Preconditioning of Nonhuman Primate Pancreas Improves Islet Isolation Outcomes by Mitochondrial Protection.

Previously, we showed that diazoxide (DZ), an effective ischemic preconditioning agent, protected rodent pancreas against ischemia-reperfusion injury. Here, we further investigate whether DZ supplementation to University of Wisconsin (UW) solution during pancreas procurement and islet isolation has similar cytoprotection in a preclinical nonhuman primate model. Cynomolgus monkey pancreata were flushed with UW or UW + 150 μM DZ during procurement and preserved for 8 hours before islet isolation. First, a significantly higher islet yield was observed in UW + DZ than in UW (57,887 vs 23,574 IEq/pancreas and 5396 vs 1646 IEq/g). Second, the DZ treated islets had significantly lower apoptotic cells per islet (1.64% vs 9.85%). Third, DZ significantly inhibited ROS surge during reperfusion with a dose-response manner. Fourth, DZ improved in vitro function of isolated islets determined by mitochondrial potentials and calcium influx in responses to glucose and KCI. Fifth, the DZ treated islets had much higher cure rate and better glycemia control in diabetic mice transplant model. This study showed a strong mitochondrial protection of DZ on nonhuman primate islets against ischemia-reperfusion injury that provides strong evidence for its clinical application in islet and pancreas transplantation.

Diazoxide preconditioning of endothelial progenitor cells from streptozotocin-induced type 1 diabetic rats improves their ability to repair diabetic cardiomyopathy.

Type 1 diabetes mellitus (DM) is a strong risk factor for the development of diabetic cardiomyopathy (DCM) which is the leading cause of morbidity and mortality in the type 1 diabetic patients. Stem cells may act as a therapeutic agent for the repair of DCM. However, deteriorated functional abilities and survival of stem cells derived from type 1 diabetic subjects need to be overcome for obtaining potential outcome of the stem cell therapy. Diazoxide (DZ) a highly selective mitochondrial ATP-sensitive K(+) channel opener has been previously shown to improve the ability of mesenchymal stem cells for the repair of heart failure. In the present study, we evaluated the effects of DZ preconditioning in improving the ability of streptozotocin-induced type 1 diabetes affected bone marrow-derived endothelial progenitor cells (DM-EPCs) for the repair of DCM in the type 1 diabetic rats. DM-EPCs were characterized by immunocytochemistry, flow cytometry, and reverse transcriptase PCR for endothelial cell-specific markers like vWF, VE cadherin, VEGFR2, PECAM, CD34, and eNOS. In vitro studies included preconditioning of DM-EPCs with 200 μM DZ for 30 min followed by exposure to either 200 μM H2O2 for 2 h (for oxidative stress induction) or 30 mM glucose media (for induction of hyperglycemic stress) for 48 h. Non-preconditioned EPCs with and without exposure to H2O2 and 30 mM high glucose served as controls. These cells were then evaluated for survival (by MTT and XTT cell viability assays), senescence, paracrine potential (by ELISA for VEGF), and alteration in gene expression [VEGF, stromal derived factor-1α (SDF-1α), HGF, bFGF, Bcl2, and Caspase-3]. DZ preconditioned DM-EPCs demonstrated significantly increased survival and VEGF release while reduced cell injury and senescence. Furthermore, DZ preconditioned DM-EPCs exhibited up-regulated expression of prosurvival genes (VEGF, SDF-1α, HGF, bFGF, and Bcl2) on exposure to H2O2, and VEGF and Bcl2 on exposure to hyperglycemia while down regulation of Caspase-3 gene. Eight weeks after type 1 diabetes induction, DZ preconditioned, and non-preconditioned DM-EPCs were transplanted into left ventricle of diabetic rats (at a dose of 2 × 10(6) DM-EPCs/70 μl serum free medium). After 4 weeks, DZ preconditioned DM-EPCs transplantation improved cardiac function as assessed by Millar's apparatus. There was decrease in collagen content estimated by Masson's trichrome and sirius red staining. Furthermore, reduced cell injury was observed as evidenced by decreased expression of Caspase-3 and increased expression of prosurvival genes Bcl2, VEGF, and bFGF by semi-quantitative real-time PCR. In conclusion, the present study demonstrated that DZ preconditioning enhanced EPCs survival under oxidative and hyperglycemic stress and their ability to treat DCM.

Diazoxide preconditioning of endothelial progenitor cells improves their ability to repair the infarcted myocardium.

Reduced survival and homing of the transplanted cells in the oxidative stressed ischemic environment limits the potential outcome of cell therapies for myocardial ischemia. Diazoxide (DZ), a highly selective mitochondrial ATP sensitive K(+) channel opener, is known to improve the survival and therapeutic ability of mesenchymal stem cells and skeletal myoblasts for the repair of heart failure. The current study explored the effect of DZ preconditioning in improving the ability of endothelial progenitor cells (EPCs) to counteract, in vitro oxidative stress, and to repair the infarcted myocardium. The EPCs were preconditioned by 30 min incubation with 200 μM DZ followed by exposure to 200 μM hydrogen peroxide (H2 O2 ) for 2 h. Non-preconditioned EPCs with and without exposure to H2 O2 were used as control. DZ preconditioning of EPCs resulted in significantly reduced cell injury as shown by reduced lactate dehydrogenase release and expression of annexin V-PE in comparison to untreated EPCs. Furthermore, DZ preconditioned EPCs exhibited upregulated expression of prosurvival genes (VEGF, SDF-1α, PCNA, and Bcl2 ), improved chemokines release (VEGF, IGF, and SDF-1α), viability, Akt phosphorylation and tube formation. In vivo experiments involved transplantation of DZ preconditioned and untreated EPCs in the left ventricle after permanent ligation of left anterior descending coronary artery in rats. The results demonstrated that DZ EPCs transplanted group showed significant reduction in infarct size along with robust cell proliferation, angiogenesis and improvement in cardiac function. The current study demonstrates that DZ preconditioning enhances EPCs survival under oxidative stress in vitro and their ability to treat myocardial infarction.

Diazoxide preconditioning is mediated by mechanistic target of rapamycin (mTOR) and S6 Kinase (S6K)

Diazoxide (DZ), a mitochondrial ATP-sensitive potassium channel opener, is protective in models of brain ischemia. The cellular signaling pathways activated by DZ preconditioning are not clear. We examined the role of the mTOR pathway in delayed DZ-induced preconditioning of cultured rat primary cortical neurons. Neurons were treated for three days with 500 µM DZ or feeding medium and then exposed to three hours of continuous normoxia in DMEM with glucose or with oxygen glucose deprivation (OGD) followed by reoxygenation and feeding medium. OGD decreased viability by 50 percent, depolarized mitochondria and reduced mitochondrial respiration, whereas DZ treatment improved viability and mitochondrial respiration, and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Diazoxide induced neuroprotection was associated with increased phosphorylation of protein kinase B (Akt), mTOR, and the major mTOR downstream substrate, S6 Kinase (S6K). Previously, we have reported that wortmannin and rapamycin abolished the protective effects of DZ. We expanded our study using torin-1, a structurally distinct mTOR inhibitor, as well as S6K targeted siRNA. Both approaches abolished the protective effects of DZ. Diazoxide effects on mitochondrial membrane potential or reactive oxygen species production were not affected by rapamycin. We conclude that DZ protects against OGD by activation of the Akt-mTOR-S6K pathway rather than by reduced ROS production and mitochondrial membrane depolarization. This work was supported by NIH grants HL-077731, HL-030260, HL-065380, and HL-093554.

Diazoxide Preconditioning of Non-Human Primate Pancreas During Procurement and Preservation Improves Islet Isolation Outcomes and Function.

Objective: Diazoxide (DZ) has been shown as an effective pharmacological ischemic preconditioning agent. In a previous report, we showed that DZ had a strong protection of rodent islets against ischemia-reperfusion injury. In this follow-up study, we are investigating whether DZ supplementation to University of Wisconsin (UW) solution during pancreas harvesting and preservation has similar cytoprotection in non-human primate. Research Design and Methods: Cynomolgus monkey pancreata were flushed with UW (n = 3) or UW + 150 μM DZ (n = 3) during pancreas harvesting and preserved for 8 hrs prior to islet isolation. Islet yields, in vitro, and in vivo function were evaluated. Results: (i) Significantly higher islet yields were observed in the UW+DZ group than in the UW group (5,7887 vs. 23,574 IEq/pancreas; p = 0.02. and 5,396 vs. 1,646 IEq/gram; p = 0.019). (ii) The DZ-treated islets had higher insulin content (59.22 vs. 40.35 ng/islet; p = 0.006). The islets from the UW + DZ group post culture have significantly higher insulin positive cells per islet (96.15% vs. 82.74%; p = 0.004). (iv)The DZ-treated islets had a significant lower apoptotic cells per islet post culture (1.64% vs. 9.85%, p = 0.0015). (v) The DZ-treated islets had improved in vitro calcium influx in response to glucose challenge and in vivo function reflected in a higher cure rate and shorter days needed to reverse diabetes in a nude mouse transplant model. Conclusion: This study in NHP confirmed our previous observation in rodent model, showing similar DZ cellular protection on islet cells against ischemia-reperfusion injury that provides a strong evidence for future DZ application in human islet and pancreas transplantation.

Effect of Diazoxide Preconditioning on Cultured Rat Myocardium Microvascular Endothelial Cells against Apoptosis and Relation of PI3K/Akt Pathway.

Anti-apoptotic mechanism for cell protection on reperfusion may provide a new method to reduce reperfusion injury. The aim of the present study is to explore the effect of mitochondrial ATP sensitive potassium channel (Mito-KATP) opener diazoxide (DZ) preconditioning on hypoxia/reoxygen (H/R) injury of rat myocardium microvascular endothelial cells (MMECs) against apoptosis and relation of PI3K/Akt pathway. Animal experimentation. The rat MMECs were cultivated, and H/R model was made to imitate ischemia-reperfusion injury. The cells were seeds in 96-wellplates (100μL/hole) or in 6cm diameter dishes (2 mL/dish) with the density of 1×106/mL and randomly divided into 4 groups (n=6 each): control group (Group N), hypoxia-regoxygen group (Group H/R), Diazoxide preconditioning+H/R group (Group DZ) and Diazoxide preconditioning +mitochondrial KATP blocker 5-hydroxydecanoate (5-HD) + H/R group (Group DZ+5-HD). The cells were exposed to 2h hypoxia followed by 2h reoxygenation. Diazoxide 100μmol/L and diazoxide 100μmol/L+ 5-HD100μmol/L were added to the culture medium 2h before hypoxia in DZ and DZ+5-HD groups respectively. Each group was observed the proliferation in MTT, apoptotic rate in Annexin V-FITC/PI double standard, cell structure of Hoechst staining, and the levels of PI3K, Akt and p53 mRNA by RT-qPCR. Compared with Group N, apoptotic rate of Group H/R increased (p<0.01) and the vitality decreased significantly (p<0.05), and the expression of PI3K, Akt and p53 mRNA elevated in Group H/R (p<0.05). Compared with Group H/R, apoptotic rate and p53 mRNA level of Group DZ depressed significantly (p<0.01, p<0.05), while the vitality, PI3K and Akt mRNA levels increased (p<0.05). Compared with Group DZ, apoptotic rate and p53 mRNA level of Group DZ+5-HD increased significantly (p<0.01, p<0.05), but the vitality, PI3K and Akt mRNA levels decreased (p<0.05). Under the condition of H/R, mito-KATP opened by DZ may depend on PI3K/Akt pathway to regulate expression level of the downstream p53 mRNA to inhibit apoptosis and improve viability of MMECs at the same time.

Diazoxide preconditioning antagonizes cytotoxicity induced by epileptic seizures.

Diazoxide, an activator of mitochondrial ATP-sensitive potassium channels, can protect neurons and astrocytes against oxidative stress and apoptosis. In this study, we established a cellular model of epilepsy by culturing hippocampal neurons in magnesium-free medium, and used this to investigate effects of diazoxide preconditioning on the expression of inwardly rectifying potassium channel (Kir) subunits of the ATP-sensitive potassium. We found that neuronal viability was significantly reduced in the epileptic cells, whereas it was enhanced by diazoxide preconditioning. Double immunofluorescence and western blot showed a significant increase in the expression of Kir6.1 and Kir6.2 in epileptic cells, especially at 72 hours after seizures. Diazoxide pretreatment completely reversed this effect at 24 hours after seizures. In addition, Kir6.1 expression was significantly upregulated compared with Kir6.2 in hippocampal neurons after seizures. These findings indicate that diazoxide pretreatment may counteract epileptiform discharge-induced cytotoxicity by suppressing the expression of Kir subunits.

MTOR Pathway Mediates Diazoxide Preconditioning in Cultured Neurons

Diazoxide (DZ), a mitochondrial ATP‐sensitive potassium channel opener, is protective in models of brain ischemia but the signaling pathway is not clear. The mammalian target of Rapamycin (mTOR) pathway is a major regulator of protein synthesis and has been shown to be involved in protection during cerebral ischemia. We tested the hypothesis that DZ protects neurons via mechanisms involving the mTOR pathway. Cultured cortical neurons were treated with DZ, and Rapamycin (R) or Wortmannin (W) and exposed to 3 hours of oxygen‐glucose deprivation (OGD) followed by reoxygenation. Viability, mitochondrial membrane potential (Ψm), of neurons and western blots of cell homogenates were performed. OGD reduced viability (100% to 46.7% ± 0.9, p&lt;0.05) and DZ partially rescued the viability (64.9% ± 4.5, p&lt;0.05). W, R, DZ + R, DZ + W, DZ + R + W, however, did not alter viability after OGD. Ψm decreased after OGD (to 49.9% ± 9.5, p&lt;0.05 vs. Ctrl) compared to untreated controls (Ctrl). DZ reduced Ψm under normoxic conditions (65.3% ± 6.2, p&lt;0.05 vs. Ctrl) and further decreased it after OGD (26.2% ± 2.9, p&lt;0.05 vs. Ctrl). R or W did not affect Ψm at normoxia but decreased it with OGD (50.9% ± 8.4, p&lt;0.05 and 53.9% ± 8.9, p&lt;0.05; respectively; both vs. Ctrl). Immuno‐blot analysis showed decreased phosphorylated S6‐ Kinase (phospho‐S6K) expression (29.9% ± 6.9, p&lt;0.05) following OGD that was rescued by DZ (206.5% ± 52.8, p&lt;0.05 vs. OGD). DZ + R, DZ+W, DZ + R + W decreased phospho‐S6K when compared to Ctrl. Thus, DZ preconditioning is mediated by mTOR pathway by phosphorylation of S6K. Supported by HL‐030260, HL‐065380, HL077731, &amp; HL093554.

Diazoxide preconditioning protects cerebrum against ischemia/reperfusion injury via mitochondrial calcium uniporter

Objective To investigate whether diazoxide preconditioning protects cerebrum against i(s)chemia reperfusion injury via mitochondrial calcium uniporter(MCU)or not.Methods The ischemia reperfusion model was performed by thread occlusion of middle cerebral artery for 2 h,and then 24 h reperfusion.Fifty-two rats were randomly divided into four groups:group A (sham-operated group),group B(ischemia/reperfusion group),group C(diazoxide group),group D(diazozide-spermine group).After 24 h reperfusion,neuropsychological score,the mitochondrial calcium content([Ca2+]m),,the activity of superoxide dismutase (SOD)and glutathione peroxidase(GSH-Px),the content of malondialdehyde(MDA)and nitric oxide(NO)in ischemic hemisphere were observed in above groups respectively.Results Compared with group B,diazoxide preconditioning in group C improved neuropsychological score(13.1 ±0.8,P＜0.01),improved the activity of SOD(143 ±-18)U/mgprot and GSH-Px(84 ±8)U/mgprot(P＜0.05),and decreased the content of MDA(0.799 ±0.092)nmol/mgprot and NO(0.216±0.138)nmol/mgprot(P＜0.05)and[Ca2+]m (293±7)nmol/mgprot(P＜0.01).Spermine in group D attenuated the beneficial effects of diazoxide preconditioning in group C.Compared with group C,spermine attenuated neuropsychological score(8.0±0.7,P＜0.01)and the activity of SOD(128±7)U/mgprot and GSH-Px(73 ±6)U/mgprot(P＜0.05),and improved[Ca2+]m(322±10)nmol/mgprot(P＜0.01)and the content of MDA (0.955±0.141)nmol/mgprot and NO(0.575±0.292)nmol/mgprot(P＜0.05).Conclusions The effects of diazoxide preconditioning against cerebral ischemia reperfusion injury by attenuating[Ca2+]m and peroxidation injury in rats may involve in the suppression of MCU during reperfusion. Key words: Diazoxide; Spermine; Mitochondria; Brain ischemia; Reperfusion

Activation of IL-11/STAT3 Pathway in Preconditioned Human Skeletal Myoblasts Blocks Apoptotic Cascade Under Oxidant Stress

To determine whether our novel approach of diazoxide-induced stem cell preconditioning might be extrapolated to human skeletal myoblasts to support their survival under lethal oxidant stress. Using an in vitro model of H(2)O(2) treatment of human skeletal myoblasts, we report the ability of diazoxide-preconditioned human skeletal myoblasts to express cytokines and growth factors, which act in an autocrine and paracrine fashion to promote their own survival. Preconditioning of skeletal myoblasts was cytoprotective and significantly reduced their apoptotic index (p < 0.05). IL-11 gene and protein expression was significantly increased in preconditioned skeletal myoblasts. Transfection of skeletal myoblasts with IL-11-specific siRNA incurred their death under oxidant stress. The cytoprotective effect of diazoxide preconditioning was blocked by Erk1/2 inhibitor PD98059 (20-100 µM), which abrogated STAT-3 phosphorylation, thus confirming a possible involvement of Erk1/2/STAT3 signaling downstream of IL-11 in cell survival. We also investigated the time course of subcellular changes and signaling pathway of skeletal myoblasts apoptosis under oxidant stress before and after preconditioning. Apoptosis was induced in skeletal myoblasts with 100-500 µM H(2)O(2) for time points ranging from 1 to 24 h. Release of lactate dehydrogenase, disruption of the mitochondrial membrane potential and cytochrome-c translocation into cytoplasm were the earliest signs of apoptosis. Total Akt protein remained unchanged whereas marked reduction in pAkt was observed in the native skeletal myoblasts. Terminal dUTP nick end-labeling and annexin-V positivity were significantly increased after 4 h. Ultra-structure studies showed condensed chromatin, shriveled nuclei and swollen mitochondria. These data suggest that skeletal myoblasts undergo apoptosis under oxidant stress in a time-dependent manner and preconditioning of skeletal myoblasts significantly prevented their apoptosis via IL-11/STAT3 signaling.

Long-term diazoxide preconditioning attenuates the cardioprotective effect against isoproterenol induced myocardial injury in a rat model

Short-term diazoxide (DZ) preconditioning exerts efficient protective effect on myocardial ischemia injury. This study aimed to investigate the effects of long term DZ preconditioning on isoproterenol (ISO) induced myocardial injury. DZ (20 mg/kg/d) was intraperitoneally injected into rats for different durations (1, 2, 4, 6 and 8 w) and myocardial injury was then introduced through intraperitoneal injection of ISO. The mitochondrial respiratory function and the cardiac troponin(cTnI) level in the blood were measured. In addition, the mRNA and protein expressions of SUR2A were detected. After DZ preconditioning for 1~4 w, the myocardial protective effect was observed in this ISO induced myocardial injury model and was similar to that after 3 days of DZ preconditioning. After DZ preconditioning for 6 to 8 w, the protective effect was markedly attenuated, characterized by impaired mitochondrial respiratory function and increased blood cTnI level. Furthermore, qRT-PCR and western blot assay revealed no significant difference in the mRNA and protein expressions of SUR2A among groups. Long term DZ preconditioning (≥6 w) may attenuate the protective effect against the ISO-induced myocardial injury, which might be associated with the persistent opening of mitochondrial ATP-sensitive potassium channel. Key words: Diazoxide preconditioning, isoproterenol-induced myocardial injury, mitochondrial adenosine triphosphate -sensitive potassium channel.

Myocardial ischaemic and diazoxide preconditioning both increase PGC-1α and reduce mitochondrial damage

Objectives — Pretreatment with diazoxide, a mitochondrial ATP-sensitive potassium channel (mito KATP) opener, was found to protect the rat heart against ischaemia-reperfusion (I/R) injury by mimicking ischaemic preconditioning (IPC). However, the protection mechanisms have not been fully clarified yet. We hypothesize that molecular regulation of mitochondrial energetics is integral to this cardioprotective programme. We explored the involvement of peroxisome proliferator-activated receptor g coactivator-1-1α (PGC-1α) in the effect of IPC and diazoxide preconditioning (DPC) with regard to its role in protection against I/R injury.Methods — 30 Wistar rats were used to establish the Langendorff isolated perfused heart model. Rats were randomly divided into 5 groups, 6 in each group: (1) the I/R group: after 30 min of equilibration perfusion, the heart was subjected to 30 min of ischaemia and 1h of reperfusion; (2) the IPC group: after 10 min of equilibration perfusion, the heart was subjected to two times 5 min ischaemia and 5 min of reperfusion, followed by 30 min of ischaemia and 1h of reperfusion; (3) the DPC group: after 10 min of equilibration perfusion, the heart was given two times a K-H perfusion solution containing diazoxide (100 mmol/l) for 5 min then a non-diazoxide K-H perfusion solution for 5 min, followed by 30 min of ischaemia and 1h of reperfusion; (4) a blank control group: an equal amount of saline was used instead of diazoxide. The perfusion procedure was the same as in the DPC group; (5) the dimethyl sulfoxide (DMSO) group: DMSO was applied instead of diazoxide, and the perfusion procedure was the same as in the DPC group. Cardiac apex muscle was cut for frozen section. Immunohistochemistry staining of PGC-1α was performed and average absorbance was calculated. An electron microscope was used for Flameng scoring of the myocardial mitochondria.Results — The average absorbance values of PGC-1α were: I/R group (3.88 ± 1.72), IPC group (10.94 ± 5.23), DPC group (8.40 ± 3.64), blank control group (3.55 ± 1.56) and DMSO group (4.16 ± 0.52), respectively. The expression of PGC-1α was significantly increased in the IPC and DPC groups and the differences were statistically significant compared to the I/R, blank control and DMSO groups, i.e., P < 0.01 for IPC group and P < 0.05 for DPC group. However, there was no significant difference between the IPC and DPC groups (P > 0.05). Flameng score: IPC group (0.44 ± 0.13), DPC group (0.47 ± 0.10), I/R group (1.78 ± 0.14), blank control group (1.70 ± 0.03) and DMSO group (1.68 ± 0.06). The Flameng score of the IPC and DPC groups was statistically significantly different as compared to the I/R group, blank control group and DMSO group (P < 0.01), but no significant difference was detected between the IPC and DPC groups (P > 0.05).Conclusion — IPC and DPC have a protective effect on myocardial mitochondria, and their mechanism of action may be related to activation and over-expression of PGC-1α.

E0014 The role of peroxisome proliferatoractivated receptor coactivator 1 in myocardial ischaemic preconditioning and diazoxide preconditioning

ObjectiveIschemic preconditioning (IPC) is an important endogenous protective mechanism for ischaemia-reperfusion (I/R) injury. Currently, it is believed that opening of the mitochondrial ATP-sensitive potassium channel (mitoKATP channel) plays an important...

Transplantation of Mesenchymal Stem Cells Preconditioned with Diazoxide, a Mitochondrial ATP-Sensitive Potassium Channel Opener, Promotes Repair of Myocardial Infarction in Rats

Myocardial infarction (MI) causes myocardium injury and scar formation, and the transmural infarction is associated with ventricular hypofunction. Stem cell transplantation therapy has improved cardiac function in animal models of MI. However, the poor survival of the donor cells in the host myocardium hampers the therapeutic efficacy of stem cell transplantation. Diazoxide, a mitochondrial ATP-sensitive potassium channel opener, has been applied to suppress cell apoptosis and promote cell survival. We therefore assessed the effects of diazoxide on the selected mesenchymal stem cells (SMSCs). Pretreatment of SMSCs with diazoxide (200 micromol/L) for 30 min protected cells from oxidative stress injury by upregulating the expression of basic fibroblast growth factor and hepatocyte growth factor mRNAs and phospho-Akt and by preventing mitochondral cytochrome c translocation into the cytoplasm. Expression of mRNAs and proteins was detected by RT-PCR and western blot analyses. Thirty min after establishment of MI (the ligation of the left anterior descending of coronary artery) in female rats, the male rat SMSCs preconditioned with diazoxide were injected at four sites on the edge of the infarcted area. At 4 weeks after cell tranplantation, the donor cells in the recipient myocardium were tracked with Y chromosome. Preconditioning with diazoxide improved the survival rate of the transplanted SMSCs, compared to the untreated SMSCs. Moreover, transplantation of the diazoxide-pretreated SMSCs reduced the infarct size and increased left ventricular function, as judged by transthoracic echocardiography. In conclusion, diazoxide preconditioning is effective to promote SMSCs survival under oxidative stress and attenuates cardiac injury in MI.

Diabetes counteracts the protective effect of the diazoxide preconditioning on ischemic reperfused rat heart

To explore the impact of diabetic condition on the protective effect of diazoxide preconditioning (DPC) on ischemic-reperfused (I/R) myocardium in rats. Thirty normal male Sprague-Dawley rats were divided into 3 groups, including non-diabetic control group, non-diabetic I/R group, and non-diabetic I/R DPC group. Thirty diabetic male rats were also divided into the same 3 groups. The Langendorff isolated heart perfusion models were established. The control groups had a 90 min perfusion without any intervention. The I/R groups had a 30 min equilibration period, a 30 min ischemia, and a 30 min reperfusion. The I/R DPC groups had a 10 min equilibration, two cycles of 100 micromol/L diazoxide perfusion, 5 min each, followed by a 5 min diazoxide-free period before the 30 min ischemia and a 30 min reperfusion. The recovery rate of the left ventricular function, including cardiac output, left ventricular developed pressure (LVDP), and the maximum change rate of left ventricular pressure rise and fall (+/- dp/dt(max)) were recorded. The activity of creatine kinase in coronary outflow and activities of malonyldialdehyde, and superoxide dismutase in myocardium were detected. Myocardial water content was also assessed. In non-diabetic rats, the content of creatine kinase, malonyldialdehyde and water content were significantly decreased in I/R DPC group compared with those in I/R group. Furthermore, in I/R DPC group, the activity of superoxide dismutase and the recovery rate of the left ventricular function, including cardiac output, LVDP and +/- dp/dt(max), were significantly increased compared with those in I/R group (P < 0.05). By contrast, there were no significant changes between I/R DPC group and I/R group in diabetic rats (P > 0.05). Diabetes counteracts the protective effect of the diazoxide preconditioning on ischemic reperfused rat heart, which may be related with acute insulin resistance in cardiomyocytes.

Protective effect of ischemia and diazoxide preconditioning on postischemic reperfused myocardium and possible mechanism thereof

To study the protective mechanism of ischemic preconditioning (IPC) and diazoxide preconditioning (DPC) against myocardium ischemia-reperfusion (I/R) injury. The hearts were taken out from 30 male Wistar rats and were divided randomly into 3 equal groups: I/R group undergoing 30-min equilibration perfusion and 30-min ischemia and then 60-min reperfusion, IPC group undergoing 10-min equilibration perfusion and then two cycles of 5 min ischemia interspersed with 5 min reperfusion prior to 30 min ischemia and a 60-min reperfusion, and DPC group undergoing 10-min equilibration perfusion and 2 cycles of 5 min of 100 microM diazoxide perfusion followed by 5-min drug-free period before the 30 min ischemia and 60-min reperfusion. Frozen sections of myocardium at the cardiac apex were made and immunohistochemical staining was used to detect the expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha). Ultrathin sections 70 nm thick were made and transmission electron microscopy was used to detect the structure of the mitochondria with the Flameng scoring system. The PGC-1alpha expression of the IPC and DPC groups were significantly higher than that of the I/R group (P<0.01 and P<0.05), however, there was no significant difference in PGC-1alpha is expression between the IPC and DPC groups (P >0.05). The Flameng scores of the IPC and DPC groups were 0.44 +/- 0.13 and 0.47 +/- 0.10 respectively, both significantly higher than that of the I/R group (1.78 +/- 0.14, both P <0.01), however, there was no significant difference between IPC and DPC groups (P>0.05). IPC and DPC can protect myocyte mitochondria from the injury of ischemia/ reperfusion. The cardioprotective effects of IPC and DPC may be concerned with the activation and high expression of PGC-1alpha.

Preconditioning Enters the Era of “Physiological Proteomics”

See related article, pages 706–714 Cardiac ischemic preconditioning was first described in 1986 by Murry et al,1 who reported that brief periods of ischemia dramatically reduced myocardial infarct size caused by a subsequent, more prolonged period of ischemia. Since the initial report, the phenomenon has been shown to occur in many cell types, including human cardiomyocytes,2 raising hopes that ischemic injury to human myocardium might be prevented. The discovery that pretreatment with several pharmacological agents including adenosine, diazoxide, bradykinin, and insulin could also precondition the heart and prevent subsequent ischemic damage, provided an as yet unfulfilled promise that drugs could be developed which limit cardiac cell death caused by myocardial infarction in patients.3 As is always true of complex physiological problems, it is likely that understanding the fundamental molecular mechanism of preconditioning will be necessary to develop effective medical therapies. In recent years the mitochondrion has become the subject of intense scrutiny for the key to preconditioning. The formation of the mitochondrial permeability transition pore (mPTP) is an essential step in ischemia-induced cardiomyocyte death because its formation accounts for collapse of the mitochondrial membrane potential and failure of oxidative phosphorylation to produce ATP which leads to cardiomyocyte death. Furthermore, inhibitors and activators of the mPTP inhibit or favor preconditioning, respectively, suggesting both the mPTP and the mitochondrion are key participants.3,4 Further evidence that the mitochondrion is key to the initial physiological response to preconditioning is provided by Arrell et al in this issue of Circulation Research , who used a proteomic approach to measure changes in the amount of cardiomyocytes undergoing preconditioning.5 Relatively few of the proteins they identified changed at all. Of the 19 that did, about half were classified as mitochondrial proteins. The purpose of this editorial is not to review the …

Diazoxide and Ischaemic Preconditioning Act Differently on the Mitochondria: Is the Mitokatp Channel a Red Herring?

Diazoxide and Ischaemic Preconditioning Act Differently on the Mitochondria: Is the Mitokatp Channel a Red Herring?