Activation of Akt by SC79 protects myocardiocytes from oxygen and glucose deprivation (OGD)/re-oxygenation

SC79 is a novel Akt activator. The current study tested its potential effect against oxygen and glucose deprivation (OGD)/re-oxygenation-induced myocardial cell death. We showed that SC79 activated Akt and protected H9c2 myocardial cells and primary murine myocardiocytes from OGD/re-oxygenation. Reversely, Akt inhibitor MK-2206 or Akt1 shRNA knockdown almost completely abolished SC79-mediated myocardial cytoprotection. SC79 treatment in H9c2 cells inhibited OGD/re-oxygenation-induced programmed necrosis pathway, evidenced by mitochondrial depolarization and cyclophilin D-p53-ANT-1 (adenine nucleotide translocator 1) association. Further, SC79 activated Akt downstream NF-E2-related factor 2 (NRF2) signaling to suppress OGD/re-oxygenation-induced reactive oxygen species (ROS) production. Reversely, NRF2 shRNA knockdown in H9c2 cells largely attenuated SC79-induced ROS scavenging ability and cytoprotection against OGD/re-oxygenation. Together, we conclude that activation of Akt by SC79 protects myocardial cells from OGD/re-oxygenation.


INTRODUCTION
Ischemic heart diseases are major threat to human health, which contribute to significant human mortalities each year [1,2]. The incidence of these diseases, on the other hand, has been steadily rising [1,2]. Thus, understanding the associated pathological mechanisms and developing possible intervention strategies are vital to fight these diseases [1,2]. Our lab [3,4] and others have been applying oxygen glucose deprivation (OGD) in cultured myocardial cells to mimic ischemic cell damages [5][6][7]. Sustained OGD (> 1 hour) coupling with re-oxygenation is shown to disrupt mitochondrial function, causing reactive oxygen species (ROS) production and cell necrosis (but not apoptosis) [5][6][7].
Recent research efforts have developed a specific, potent and cell-permeable Akt small molecule activator, named SC79 [8]. SC79 inhibits Akt membrane translocation, yet simultaneously activates Akt in the cytosol [8]. Existing studies have reported the pro-survival potential of this compound in various experimental settings [8][9][10][11]. For example, Jo et al., showed that SC79 protects neurons from stroke in vivo and in vivo. Gong et al., recently demonstrated that SC79 activates Akt signaling and protects retinal pigment epithelium cells from UV radiation [11]. Similarly, this novel Akt activator could rescue osteoblasts from dexamethasone [10]. The potential effect and the underlying signaling mechanisms of SC79 against OGD/re-oxygenation-induced myocardial cell death were tested in the current study.
Next, the primary murine myocardiocytes were cultured (see Method). The above OGD (4 hours)/reoxygenation (24 hours) treatment again significantly inhibited cell survival ( Figure 1C), and provoked cell death ( Figure 1D). Similarly, pre-treatment with SC79 again dramatically decreased OGD/re-oxygenation-induced injuries to the primary murine myocardiocytes ( Figure 1C and 1D). Thus, SC79 efficiently protects myocardial cells from OGD/re-oxygenation. Notably, treatment with the SC79 by itself had no significant effect on survival and death of above myocardial cells ( Figure 1A-1D).

OGD/re-oxygenation-induced myocardial cell death is exacerbated with Akt inhibition, but is attenuated with forced-activation of Akt
Based on the results above, we would speculate that Akt inhibition may exacerbate OGD/re-oxygenationinduced myocardial cell death. MK-2206 and Akt1 shRNA were applied again to block Akt activation (p-Akt at Ser-473 and Thr-308) in H9c2 cells with OGD/re-oxygenation ( Figure 3A). Significantly, as shown in Figure 3B and 3C, MK-2206 and Akt1 shRNA largely intensified OGD/re-oxygenation-induced H9c2 cell viability reduction ( Figure 3B) and cell death ( Figure 3C). These results indicate that basal Akt activation is also important for the survival of H9c2 cells under OGD/re-oxygenation.
Remarkably, H9c2 cells expressing the ca-Akt1 were resistant to OGD/re-oxygenation ( Figure 3E and 3F). Therefore, similar to SC79 co-treatment, forced activation of Akt by expressing ca-Akt1 also inhibited OGD/reoxygenation damages in myocardial cells.
In the present study, SC79 treatment in myocardial cells also activated NRF2 signaling, as a key downstream of Akt, to block OGD/re-oxygenation-induced ROS production. Reversely, NRF2 shRNA knockdown significantly attenuated SC79-induced ROS scavenging and cytoprotective activities. Thus, activation of NRF2 signaling by SC79 is required for myocardial cytoprotection against OGD/re-oxygenation.

Primary culture of murine myocardiocytes
Primary culture of murine myocardiocytes was described previously [15]. Briefly, ventricles of C57BL6 mice (at day-1) were minced and digested in 0.5 mg/mL collagenase I (Sigma) for 40 min. The cell suspensions containing primary myocardiocytes were filtered. Cells were then cultured in M-199 medium supplemented with 10% FBS, and plated for 30 min to separate from nonmyocardiocytes. The myocardiocytes were then plated in M-199 supplemented with 10% FBS. A confluent monolayer with primary spontaneously beating cells was formed [15]. The protocol of culture of primary cells was approved by the Ethics Committee and IACUC of authors' institutions.

OGD/re-oxygenation
The detailed protocol of OGD/re-oxygenation was described previously [3]. Briefly, cells were cultured in a pre-warmed glucose-free balanced salt solution [3]. The solution was then bubbled with an anaerobic gas mix (95% N 2 , 5% CO 2 ). Cells were then incubated in the solution for 4 hours to produce oxygen and OGD and then reoxygenated for 3-24 hours.

LDH detection
Following the applied treatment, cell death was detected by lactate dehydrogenase (LDH) assay using a commercial available LDH kit (Takara, Tokyo, Japan). LDH release (×100 %) was calculated as follows: LDH in conditional medium/(LDH in conditional medium + LDH in cell lysates).

Western blots
Western blot assay was performed as described previously [3,40]. Band intensity was quantified and normalized to loading control.

Real-time PCR
RNA extraction and reverse transcription were performed as described previously [3] . Real-time PCR was performed on a Bio-Rad IQ5 multicolor detection system. After amplification, melt curve analysis was performed to analyze product melting temperature. GAPDH was tested as internal control, and 2 -ΔΔCT method [41] was applied to quantify mRNA expression. The primers for rat HO-1, GAPDH NQO-1 and GCLC were described previously [3].