mTORC2 phosphorylation of Akt1: a possible mechanism for hydrogen sulfide-induced cardioprotection

Abstract

Hydrogen sulfide (H2S) is known to have cardiac protective effects through Akt activation. Akt acts as a 'central sensor' for myocyte survival or death; its activity is regulated by multiple kinases including PI3K, mTORC2, PDK1 and phosphatases including PTEN, PP2A and PHLPPL. Based on the previous finding that PI3K inhibitor LY294002 abolishes H2S-induced Akt phosphorylation and cardioprotection, it is accepted that PI3K is the mediator of H2S-induced Akt phosphorylation. However, LY294002 inhibits both PI3K and mTOR, and PI3K only recruits Akt to the membrane where Akt is phosphorylated by Akt kinases. We undertook a series of experiments to further evaluate the role of mTORC2, PDK1, PTEN, PP2A and PHLPPL in H2S-induced Akt phosphorylation and cardioprotection, which, we believe, has not been investigated before. Hearts from adult Sprague-Dawley rats were isolated and subjected to (i) normoxia, (ii) global ischemia and (iii) ischemia/reperfusion in the presence or absence of 50 µM of H2S donor NaHS. Cardiac mechanical function and lactate dehydrogenase (LDH) release were assessed. All hearts also were Western analyzed at the end of perfusion for Akt and a panel of appropriate Akt regulators and targets. Hearts pretreated with 50 µM NaHS had improved function at the end of reperfusion (Rate pressure product; 19±4×10(3) vs. 10±3×10(3) mmHg/min, p<0.05) and reduced cell injury (LDH release 19±10 vs. 170±87 mU/ml p<0.05) compared to untreated hearts. NaHS significantly increased phospho-Akt, phospho-mTOR, phospho-Bim and Bcl-2 in reperfused hearts (P<0.05). Furthermore using H9c2 cells we demonstrate that NaHS pretreatment reduces apoptosis following hypoxia/re-oxygenation. Importantly, PP242, a specific mTOR inhibitor, abolished both cardioprotection and protein phosphorylation in isolated heart and reduced apoptotic effects in H9c2 cells. Treating hearts with NaHS only during reperfusion produced less cardioprotection through a similar mechanism. These data suggest mTORC2 phosphorylation of Akt is a key mediator of H2S-induced cardioprotection in I/R.

Figure 1. Perfusion protocol and an illustration of LVDP.

(A) Protocol: There are total of 10 groups of hearts perfused under three different conditions: normoxia (N), 20 min global zero flow ischemia (I) and I followed by 60 min reperfusion (R). Each perfusion condition was further divided into control (C), 50 µM NaHS, a H₂S donor, pretreatment for 10 min (S) or post-treatment for 30 min (P–S) with or without 0.3 µM PP242, a specific mTORC2 inhibitor (-i). The 10 groups studied are NC, NS, IC, IS, RC, RS, RC-i, RS-i, P-RS and P-RS-i; n = 5 to 10 each group. (B) Illustrations of LVDP throughout perfusions under Normoxia, I/R control and I/R NaHS pretreatment (NC, RC, RS).

Figure 2. NaHS pretreatment improves cardiac functional recovery and reduces LDH release during reperfusion.

(A) LVDP, left ventricular developed pressure; (B) RPP, rate pressure product = LVDP×heart rate; (C) +dP/dt, the rate of increase of pressure over time; (D) −dP/dt, the rate of decrease of pressure over time; (E) LDH release. RC = Reperfusion Control, RS = Reperfusion NaHS pretreatment. *P<0.05, **P<0.01 vs. Reperfusion control (RC) by two-tail unpaired t test.

Figure 3. NaHS pretreatment regulates p-Akt, p-mTOR, p-Bim and Bcl-2 during normoxia, ischemia and reperfusion.

Expression levels are presented in the ratio of (A) p-Akt Ser473 relative to total Akt, (B) p-Akt Thr308 relative to total Akt, (C) p-mTOR relative to total mTOR, (D) p-Bim relative to total Bim, (E) Bcl-2 relative to GAPDH. GAPDH was used as a protein loading control. NC = Normoxia Control, NS = Normoxia NaHS treatment, IC = Ischemia Control, IS = Ischemia NaHS pretreatment, RC = Reperfusion Control, RS = Reperfusion NaHS pretreatment. *P<0.05 vs. RC by two-tail unpaired t test.

Figure 4. The effect of NaHS on the regulation of Akt kinase PDK1 and phosphotase PTEN, PP2A and PHLPPL.

Expression levels were normalized with GAPDH. Expression level is presented in (A) p-PTEN, (B) PDK1, (C) PP2A and (D) PHLPPL. No significant differences between Reperfusion Control (RC) and Reperfusion NaHS-pretreatment (RS) were observed.

Figure 5. PP242, a specific mTOR inhibitor, abolishes NaHS pretreatment-induced cardiac functional recovery and activation of Akt, Bim and Bcl-2.

(A) PRR recovery, (B) LDH release at the 30 and 60 min of reperfusion, (C) ratio of p-Akt Ser473 relative to total Akt, (D) ratio of p-Akt Thr308 relative to total Akt, (E) ratio of p-Bim relative to total Bim, and (F) ratio of Bcl-2 relative to GAPDH. RC = Reperfusion Control, RS = Reperfusion NaHS pretreatment, RC-i and RS-i = Reperfusion Control and Reperfusion NaHS pretreatment, respectively, with 0.3 µM PP242. *p<0.05, **p<0.01 vs. RC, ^††p<0.01 vs. RS by one-way ANOVA followed by Bonferroni post-hoc analysis.

Figure 6. PP242, a specific mTOR inhibitor, blocks NaHS pretreatment induced anti-apoptotic effect in H9c2 cells.

(A) and (B) Profiles of cell viability and apoptosis under normoxia and hypoxia conditions. (C) LDH release. RC = Reperfusion Control, RS = Reperfusion NaHS pretreatment, RC-i and RS-i = Reperfusion Control and Reperfusion NaHS pretreatment, respectively, with 0.3 µM PP242. **p<0.01 vs. RC, ^††p<0.01 vs. RS by two-way (A and B) and one-way (C) ANOVA followed by Bonferroni post-hoc analysis. N = 3, each with 3 technical triplicates.

Figure 7. PP242, a specific mTOR inhibitor, abolishes NaHS post-treatment- induced cardiac functional recovery and activation of Akt, Bim and Bcl-2.

(A) RPP recovery, (B) LDH release at the 30 and 60 min of reperfusion, (C) ratio of p-Akt Ser473 relative to total Akt, (D) ratio of p-Akt Thr308 relative to total Akt, (E) ratio of p-Bim relative to total Bim, and (F) ratio of Bcl-2 relative to GAPDH. RC = Reperfusion Control, P-RS = Reperfusion NaHS post-treatment, P-RS-i = Reperfusion NaHS post-treatment with 0.3 µM PP242. *p<0.05, **p<0.01 vs. RC, ^†p<0.05, ^††p<0.01 vs. P-RS by one-way ANOVA followed by Bonferroni post-hoc analysis.

Figure 8. A schematic diagram illustrates the mechanisms of H₂S-induced cardiac protection.

In addition to PI3K regulation, H₂S increases cell survival through mTORC2-mediated activation of Akt/Bim and Bcl-2 pro-survival cell signaling pathway. PI3K generates PIP3 from PIP2. PIP3 recruits Akt to the membrane and where Akt is phosphorylated by mTORC2. Solid lines indicate positive and dotted lines negative findings in H₂S-induced cardioprotection. PP242 inhibits mTORC2 induced Akt phosphorylation, Bcl-2 expression and Bim phosphorylation.