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±4×103 vs. 10±3×103 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.
Highlights
Hydrogen sulfide (H2S) was first identified in 1996 [1] as an important endogenous regulator of a wide range of cell functions [2,3,4,5]
H2S potently protects against ischemia/ reperfusion (I/R) injury in myocytes, in isolated hearts and in intact animals [6,7,8,9,10]
In order to better define the mechanism of Akt activation during H2S cardioprotection, we investigated whether the PDK1 and Phosphatase and Tensin homolog (PTEN) kinases and/or the PP2A and PHLPPL phosphatases contribute to H2S cardioprotection
Summary
Hydrogen sulfide (H2S) was first identified in 1996 [1] as an important endogenous regulator of a wide range of cell functions [2,3,4,5]. In the cardiovascular system H2S produces three important effects. It induces the relaxation of isolated blood vessels [4] and serves as an in vivo regulator of blood pressure [2,5]. It has negative chronotropic and inotropic effects on heart muscle [3]. The activation of myocardial Akt is an important mediator of this ischemic cardioprotection [11,12,13,14]. All potential molecular mechanisms underpinning H2S-related cardioprotective Akt activation is not fully known
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