The Role Of Syntaxin-1A And Protective Drug Screening In Myocardial Ischemia-Reperfusion Injury

Background: Perioperative myocardial ischemia/reperfusion injury(IRI) which occurs to varying degrees in patients with cardiac surgery and in 1 to 4% of unselected non-cardiac surgical patients, is associated with increased morbidity and mortality. Methods and drugs that reduce perioperative myocardial IRI(such as ischemic preconditioning(IPC) and adenosine) also improve the prognosis of patients. Besides these efforts, the cardioprotective roles of volatile anesthetics such as isoflurane and sevoflurane are attracting increased interests. However, the mechanisms underlying volatile anaesthetics and IPC-induced cardioprotection remain elusive.Volatile anesthetics and IPC have been shown to work probably via the similar signaling pathways including PI3K/AKT/GSK3β, ERK1/2, JNK/STAT3, PKC, KATP channels and et al to alleviate cardiac IRI. These common traits encourage further search for more cardioprotective candidates that can be modulated by both volatile anesthetics and IPC. Recent studies indicated that syntaxin-1A, a membrane protein, which forms SNARE complex together with VAMP and SNAP25, might be one choice. The present study was designed to determine the protective role of syntaxin-1A against cardiac ischemia-reperfusion injury, and to discover new treatment strategies for cell non-autonomous death.Section 1: Syntaxin-1A Mediates Isoflurane Preconditioning but not Hypoxia Preconditioning-Induced Alleviation of Hypoxia-Reoxygenation Injury in Rat CardiomyocytesObjective: Syntaxin-1A is widely expressed in the brain, endocrine system, the heart as well as other organs. It functions primarily but not exclusively through the SNARE complex-mediated synaptic vesicle fusion. In particular, syntaxin-1A was found to be able to regulate myocardial IRI-related signaling pathways such as KATP channels and calcium channels[1-4]. Moreover, syntaxin-1A was up-regulated after ischemia[5]. These findings suggested a potential role of syntaxin-1A in cardiac IRI. More importantly, volatile anesthetics including halothane and isoflurane were found to bind syntaxin-1A. We hypothesized that(1) Cardiac Stx-1A levels were regulated by ischemia/reperfusion,hypoxia/reoxygenation, IPC and isoflurane pre-exposure.(2) Cardiomyocyte Stx-1A levels were related with IPC and isoflurane induced protection against cardiomyocyte HR injury.(3) Previously identified cardioprotective signalling pathways were involved in Stx-1A-mediated cardioprotection.Methods: Both in vivo myocardial IRI model and in vitro cardiomyocyte HR injury model were used in our study. Rats or neonatal ventricle cells were divided into five groups in each model: SHAM(control); Iso(isoflurane exposure only); IR(ischemia/reperfusion) or HR(hypoxia/reoxygenation); IPC(ischemia pre-conditioning) or HPC(hypoxia pre-conditioning) and Iso-PC(isoflurane pre-conditioning). To test hypothesis 1, Stx-1A protein levels were detected in vivo and in vitro. To test hypothesis 2, Stx-1A knockdown and overexpression in cardiomyocytes were achieved by adenoviral infection. In the experiments designed for hypothesis 3, the activities of PI3K/AKT/GSK3β, ERK1/2, STAT3 and PKC signaling were detected in vitro using Western blot.Results: Rat cardiomyocyte Stx-1A level was up-regulated by ischemia in vivo and hypoxia in vitro, and HPC/IPC as well as Iso-PC could further increase Stx-1A expression. Artificial regulation of Stx-1A levels had an influence on HR-induced cardiomyocyte apoptosis and cell unviability as well as the cardioprotective ability of Iso-PC but not HPC. Stx-1A knockdown reversed Iso-PC induced cardioprotection and increased AKT/GSK3β activities. Taken together, these findings suggest that Stx-1A was cardioprotective and isoflurane preconditioning might activate AKT/GSK3β signaling to increase Stx-1A expression and exert cardioprotection.Conclusion; Stx-1A is cardioprotective and a potential target of isoflurane induced cardioprotection. Stx-1A is possibly regulated by AKT/GSK3β signaling.Section 2: High Throughput Screening for Drugs against Secondary Hypoxia InjuryObjective: Two fundamentally distinct mechanisms contribute to cell death following hypoxia. Cell autonomous mechanisms are better defined and include the final effectors of cell death such as necrosis and apoptosis pathways. On the other hand, injured cells can release molecules that promote secondary injury of other cells. For example, potassium and glutamate are two molecules released from hypoxic neurons that can promote death of surrounding neurons by a nonautonomous mechanism. Nonautonomous mechanisms are thought to be particularly important in less severe hypoxic conditions in which some cells remain initially viable.Methods: Caenorhabditis elegans(C. elegans) mutant gc47:gc Is3 strain was used in our study. The gc47 is mapped based on its hypoxia resistant phenotype to the left arm of chromosome III. Sequencing of the rrt-1 gene in gc47 found a single mutation, a G to A transition at nucleotide 811, resulting in a missense reduction-of-function mutation in the cytoplasmic arginyl-t RNA synthetase gene rars-1 that was isolated in a mutagenesis screen for resistance to hypoxic organismal death. C. elegans gc47:gc Is3 were used to set up the protocol of high throughput screening for drugs against secondary hypoxia injury. The 2000 compounds in the SPECTRUM Collection were selected as drug library, including 1000 drug compounds, 580 pure natural products and 420 other bioactive components. The newly set up high throughput screening protocol was used to search for potential drugs against secondary hypoxia injury.Results: A protocol of high throughput screening using C. elegans gc47:gc Is3 strain was set up for drugs against secondary hypoxia injury. The hypoxia dose was 27 hours following 96 hours of recovery. After recovery, the morphology, movement, and size were accessed and the effect of one potential drug was determined by these indicators. With the high throughput screening protocol, 125 potential drugs were screened out of 2000 in the library. Two drugs were proved to be protective after another two rounds of repeat.Conclusions: The protocol of high throughput screening was effective in discovering new drugs against secondary screening. Meclocycline and phenazopyridine were screened out and proved to be protective from secondary hypoxia injury. Further experiments are need to study the underlying mechanism and test their effectiveness on mammalian models.