The Role Of Of Ischemia And Diazoxide Preconditioning In Cardioprotection And Their Relation With PGC-1α

BackgroundMyocardium Ischemia and Reperfusion Injury (MIRI) has always been a hot topic issue. However, the mechanism is poorly understood. Mitochondrion is the most important cell organ, which is the center of supplying cell energy and paticipate in cell apoptosis and cell death. In heart, the most potent method for reducing ischemia/reperfusion injury is to precondition with brief ischemia, termed ischemic preconditioning (IPC). IPC is a biphasic event. The acute or early phase occurs immediately after the ischemic preconditioning stimulus and lasts for 1 to 3 hours while the delayed or late phase is seen 12 to 24 hours after the initial stimulus and lasts up to 72 hours. Unfortunately, the mechanism has been elusive. IPC may cause the heart produce endogenous substances and send them out. Then the incelluar signal transcriptional pathway was activated, followed by the result of protection against myocardium ischemia and reperfusion injury.Recent studys have identified the Peroxisome proliferator — activated receptor - γ coactivatorl a ( PGC -1α) as a regulator of mitochondrial function, such as energy homeostasis, thermal regulation, and glucose metabolism. Once PGC -1α is activated, it powerfully induces and coordinates gene expression that stimulates mitochondrial oxidative metabolism in brown fat, fiber - type switching in skeletal muscle, and multiple aspects of the fasted response in liver. The regulation of these metabolic and cell fate decisions by PGC - la is achieved through specific interaction with a variety of transcription factors, and muscle -spectific transcription factors. PGC - 1α therefore constitutes one of the first and clearest examples in which biological programs are chiefly regulated by a tran-scriptional coactivator in response to environmental stimuli. PGC -la's control of energy homeostasis suggests that it could be a target for antiobesity or diabetes drugs. PGC - la's role in IPC is not known yet.Recent data shows that mitochondria! ATP - sensitive K ( mito KATP) channels play a central role in protecting the heart from injury in ischemic preconditioning. IPC can be mimicked by pharmacologic preconditioning, in which stimulation of various protein kinase pathways or preposure to K channel openers that activate mitochondrial ATP - sensitive K ( mito KATP) channels results in a degree of cardioprotection. It has shown that diazoxide is a selective mito KATP channel opener. It is not known how opening of mito KATP channels can protect myocytes during ischemia and reperfusion.Therefore, this article describes the degree of myocardial injury at the sub-celluar level in hearts after IPC and DPC, through establishing Langendorff per-fusion system and with the method of S - P immunohistochemical study. To our knowledge, this is the first immunohistochemical study of PGC - 1 a in myocardium. We hypothesize that mocular regulation of mitochondrial energetics is integral to this cardioprotcetion program.Materials and MethodsAnimals: Eighteen healthy male Wistar rats weighing 250 ~ 300g.Surgical procedures: Animals were anesthetized with ether through rat tail vein for 5 min. The hearts were exposed through a left thoracotomy in the fourth intercostal space, arid the pericardium was opened. The hearts were quickly made an incision at the base of the heart and were put in ice - cold Krebs -Henseleit solution. The hearts were then attached to Langendorff perfusion system by the aortic root and passively perfused with Krebs - Henseleit bicarbonate buffer [ composed of(in mmol/1) 118.5 NaCl, 25.0 NaHCO3, 1.2 KH2P04, 4.8 KC1,1.2 MgSO4 ? 7H2O,1. 8 CaCl2 ? 7H2O,11 dextrose] at a constant pressure of 80 mmH20. The perfusate was bubbled with a 95% O2 -5% CO2 gas mixture, and the bubbling rate was adjusted to maintain physiological pH (7.4 -7. 5). Perfusate temperature was maintained at 38T1 by a circulatingwater jacket surrounding the buffer reservoirs. The heart was also maintained at 3SX. via a water jacketed housing in which it was suspended. The open top of the jacket was covered with a piece of parafilm to maintain the humidity and temperature.Protocols;Eighteen rats weighing 250 - 300g, were divided randomly into three groups;The control group (I/R group) had a 30 -min equilibration period and a 30 - min ischemia and a 60 - min reperfusion. The IPC group had a 10- min equilibration, then was elicited by two cycles of 5 min of ischemia interspersed with 5 min reperfusion prior to 30 min ischemia and a 60 - min reperfusion. The DPC group had a 10 - min equilibration and two cycles of 5 min of 100 |xM diazoxide perfusion followed by a 5 - min drug - free period before the 30 min ischemia and a 60 - min reperfusion. All hearts of three groups were packed with foil and stored in -70T! refrigerator.Immunohistochemical staining of PGC -la: Frozen sections were cut on a cryostat at 6|xm, air dried and fixed in a 50/50 mixture of acetone and metha-nol. They were then dried thoroughly and stored in foil at —20X1 prior to use. S— P method was used for immunohistochemical staining. The images were obtained from a digital camera and the staining intensity of PGC - 1 a was quantified using Meta Morph image analysis software.Observation the change of mitochondria ultrastructure: The blocks were immediately transfered into a 2. 5% glutaraldehyde solution buffered to a PH of 7. 05 with 0. 5M phosphate solution at 4T1. Each block was washed three times (each time for 30 min) , and was fixed with 1% osmium for 2 hours. Then each block was washed three times (each time for 30 min) with PBS solution. The blocks were dehydrated in graded series of ethanol and acetone ( 50 % ethanol 30 min—^70% ethanol 30 min—>-80% acetone—v90% acetone —?100% acetone 90 min) , Finally blocks were rountinely embedded in Epon for 48 hours at 60°C 70 nm ultrathin sections were made for observation. Then the sections were stained with uranyl acetate for 15 min and lead citrate for 20 min. Each section was observed under a JEM - 1200EX electron microscope. The mitochondria under each specimen were evaluated according to Flameng score.Statistical analysis;All analysis were performed with the SPSS 11.5. Datawere expressed as the means ± SD. One - way ANOVA and Student - Newman - Keuls post hoc test were used to compare the significance between the groups. P<0.05 was considered significant.Results1. The expression of PGC -la: The brown clour particle were the positive expression matter. The Intergrated OD Average of PGC - la in IPC or DPC group were significantly higher than the control groups (IPC group: P <0. 01;DPC group: P < 0. 05). There was no significant difference between IPC and DPC groups(P>0. 05).2. Ultrastructure change;The results of Mitochondrial score are illustrated in Table 1. In control group (I/R group) , mitochondrial score was added up to 1. 78 ±0. 14. But with the intervention of ischemic or diazoxide preconditioning before 30 - min ischemia, the mitochondrial score had significantly decreased than the control group( P <0. 01) . However, there was no significant difference between IPC and DPC groups (P > 0. 05 ) .As shown in Fig. 4, All the mitochondria are swollen. The mitochondria in control (I/R) group show severe damage;mitochondria have apparent swollen, the matrix shows clearing and condensation, and several mitochondria are destroyed with disruption of inner and/or outer membrane. In IPC and DPC groups, mitochondria are well preserved, with slight edema and the structure remaining intact.ConclusionIschemic or Diazoxide Preconditioning (IPC or DPC) can protect myocytes mitochondria from ischemia and reperfusion. The cardioprotection may be concerned with the activation and the high expression of PGC - la.