The cardioprotective effect of sevoflurane postconditioning (SPostC) is lost in diabetes that is associated with cardiac phosphatase and tensin homologue on chromosome 10 (PTEN) activation and phosphoinositide 3-kinase (PI3K)/Akt inactivation. T-LAK cell-originated protein kinase (TOPK), a mitogen-activated protein kinase- (MAPKK-) like serine/threonine kinase, has been shown to inactivate PTEN (phosphorylated status), which in turn activates the PI3K/Akt signaling (phosphorylated status). However, the functions of TOPK and molecular mechanism underlying SPostC cardioprotection in nondiabetes but not in diabetes remain unknown. We presumed that SPostC exerts cardioprotective effects by activating PTEN/PI3K/Akt through TOPK in nondiabetes and that impairment of TOPK/PTEN/Akt blocks diabetic heart sensitivity to SPostC. We found that in the nondiabetic C57BL/6 mice, SPostC significantly attenuated postischemic infarct size, oxidative stress, and myocardial apoptosis that was accompanied with enhanced p-TOPK, p-PTEN, and p-Akt. These beneficial effects of SPostC were abolished by either TOPK kinase inhibitor HI-TOPK-032 or PI3K/Akt inhibitor LY294002. Similarly, SPostC remarkably attenuated hypoxia/reoxygenation-induced cardiomyocyte damage and oxidative stress accompanied with increased p-TOPK, p-PTEN, and p-Akt in H9c2 cells exposed to normal glucose, which were canceled by either TOPK inhibition or Akt inhibition. However, either in streptozotocin-induced diabetic mice or in H9c2 cells exposed to high glucose, the cardioprotective effect of SPostC was canceled, accompanied by increased oxidative stress, decreased TOPK phosphorylation, and impaired PTEN/PI3K/Akt signaling. In addition, TOPK overexpression restored posthypoxic p-PTEN and p-Akt and decreased cell death and oxidative stress in H9c2 cells exposed to high glucose, which was blocked by PI3K/Akt inhibition. In summary, SPostC prevented myocardial ischemia/reperfusion injury possibly through TOPK-mediated PTEN/PI3K/Akt activation and impaired activation of this signaling pathway may be responsible for the loss of SPostC cardioprotection by SPostC in diabetes.

A small percentage of the 518 kinases in the human genome are known to play important roles in the pathophysiology of ischemia/reperfusion (I/R) injury in cardiomyocytes. However, there are many understudied kinases that may impact the damage to cardiomyocytes caused by I/R. Thus, there are likely unmet therapeutic opportunities in targeting kinases to reduce death caused by myocardial infarction. We utilized an unbiased mass spectrometry affinity chromatography method to investigate how simulated ischemia/reperfusion (sI/R) affected the activity of a large number of kinases in a cardiomyocyte cell line. Our data suggested that the activity of bone morphogenic protein 2‐inducible kinase (BMP2K) was elevated in cardiomyocytes exposed to simulated ischemia/reperfusion (sI/R) injury. BMP2K was cloned from a cell line that was induced to differentiate into an osteoblastic phenotype with BMP2. While no physiological substrates have been identified, BMP2K was observed to phosphorylate myelin basic protein and autophosphorylate in vitro. We determined that a reduction in the expression of BMP2K in cardiomyocytes using siRNA reduced apoptosis and overall cell death caused by sI/R injury. In addition, knocking down BMP2K reduced autophagic flux in cardiomyocytes under all the conditions that we tested. To identify potential BMP2K substrates and interacting proteins, we immunoprecipitated Flag‐BMP2K from HEK‐293 cells, performed SDS‐PAGE, and visualized proteins with Coomassie blue stain. We observed a 75 kD protein that was identified by mass spectrometry analysis as mortalin. Mortalin, which is member of the heat shock protein 70 (Hsp70) family, can function to suppress cell death during glucose deprivation. We validated our mass spectrometry results with co‐immunoprecipitation assays followed by immunoblotting with a mortalin antibody. Our future goals are to determine if mortalin is a direct substrate of BMP2K and to identify the functional significance of the BMP2K/mortalin interaction. In addition, we are currently investigating the mechanism by which BMP2K inhibition regulates both cell death and autophagy using both a cardiomyocyte cell line and primary adult rat ventricular cardiomyoctesSupport or Funding InformationThis research was supported by the American Heart Association Award: 15SDG25090279 (EMW) and by The Iowa Osteopathic Education and Research (IOER) Funds (EMW).