ATP-sensitive K+ channels contribute to the protective effects of exogenous hydrogen sulfide against high glucose-induced injury in H9c2 cardiac cells

Abstract

Hyperglycemia, as well as diabetes mellitus, has been shown to impair ATP-sensitive K+ (KATP) channels in human vascular smooth muscle cells. Hydrogen sulfide (H2S) is also known to be an opener of KATP channels. We previously demonstrated the cardioprotective effects exerted by H2S against high-glucose (HG, 35 mM glucose)-induced injury in H9c2 cardiac cells. As such, we hypothesized that KATP channels play a role in the cardioprotective effects of H2S against HG-induced injury. In this study, to examine this hypothesis, H9c2 cardiac cells were treated with HG for 24 h to establish a model of HG-induced insults. Our findings revealed that treatment of the cells with HG markedly decreased the expression level of KATP channels. However, the decreased expression of KATP channels was reversed by the treatment of the cells with 400 µM sodium hydrogen sulfide (NaHS, a donor of H2S) for 30 min prior to exposure to HG. Additionally, the HG-induced cardiomyocyte injuries, including cytotoxicity, apoptosis, oxidative stress and mitochondrial damage, were ameliorated by treatment with NaHS or 100 µM diazoxide (a mitochondrial KATP channel opener) or 50 µM pinacidil (a non-selective KATP channel opener) for 30 min prior to exposure to HG, as indicated by an increase in cell viability, as well as a decrease in the number of apoptotic cells, the expression of cleaved caspase-3, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial membrane potential (MMP). Notably, treatment of the H9c2 cardiac cells with 100 µM 5-hydroxydecanoic acid (5-HD, a mitochondrial KATP channel blocker) or 1 mM glibenclamide (Gli, a non-selective KATP channel blocker) for 30 min prior to treatment with NaHS and exposure to HG significantly attenuated the above-mentioned cardioprotective effects exerted by NaHS. Notably, treatment of the cells with 500 µM N-acetyl‑L‑cysteine (NAC, a scavenger of ROS) for 60 min prior to exposure to HG markedly reduced the HG-induced inhibitory effect on the expression of KATP channels. Taken together, our results suggest that KATP channels play an important role in the cardioprotective effects of exogenous H2S against HG-induced injury. This study also provides novel data demonstraring that there is an antagonistic interaction between ROS and KATP channels in HG-exposed H9c2 cardiac cells.

The Opening of ATP-Sensitive K+ Channels Protects H9c2 Cardiac Cells Against the High Glucose-Induced Injury and Inflammation by Inhibiting the ROS-TLR4-Necroptosis Pathway

“…The cells were pre-treatment with 1 mM NAC for 60 min before exposure to HG for 24 h or co-treated with 30 µM TAK-242 or 100 µM Nec-1 and HG for 24 h. Similar to the anti-cytotoxicity effect of the opening of K ATP channels [7], the results of CCK-8 assay (Fig. 5) showed that pre-treatment of the cells with NAC or co-treatment of the cells with TAK-242 or Nec-1 dramatically blocked the HG-induced cytotoxicity, leading to an increase in cell viability, indicating the involvement of ROS-TLR4-necroptosis pathway activation in the HG-induced cytotoxicity.…”

“…Additionally, a previous study has indicated diabetes mellitus (DM) is associated with the dysfunction of cardiovascular K ATP channels [17]. More recently, we have demonstrated that high glucose (35 mM glucose, HG) significantly decreases the expression level of K ATP channels and that the opening of K ATP channels protects against the HG-induced injuries, including over-production of reactive oxygen species (ROS) in H9c2 cardiac cells [7]. However, what are the influences of these important channels on the HG-triggered intracellular signaling mechanisms is far less understood.…”

“…To date, multiple factors, such as ROS [7, 19-21], chronic inflammation [20, 22], cell death (including apoptosis and necroptosis) [7, 19-21], and activity of several signaling molecules, for example, toll-like receptors (TLRs) [22, 23], have been demonstrated to implicate in the hyperglycemia-induced cardiac injury. Importantly, the above factors can interact or crosstalk, consisting of the complicated intracellular signal mechanisms.…”

“…Accumulating evidence has shown that in the adaptive responses to pathophysiological stresses, such as ischemia [1-3] and hyperglycemia [4-7], ATP-sensitive K + (K ATP ) channels function as the high-fidelity metabolic sensors, which couple intracellular metabolic state to membrane excitability [8, 9] and play a homeostatic role ranging from blood glucose regulation to cardioprotection [3, 7, 10]. Cardiomyocytes contain K ATP channels in both the sarcolemma (surface membrane) [11] and mitochondria [12, 13].…”

Protective effects of exogenous and endogenous hydrogen sulfide in mast cell-mediated pruritus and cutaneous acute inflammation in mice

“…The activation of KATP channels by H2S has been shown as the mechanism underlying the inhibitory effects of this mediator in a variety of experimental approaches, including aspirin-induced leukocyte adherence in mesenteric venules [48] and high glucose-induced cardiac cells injury [51]. However, this not seem to be the cause beyond the protective effects of the spontaneous H2S donors against histamine-induced pruritus and skin inflammation, as pretreatment of mice with glibenclamide, a selective KATP channels blocker, did not antagonize the protective effects of H2S, even when used at doses that abolish the vasodilatation induced in the mouse dorsal skin by the KATP channel opener pinacidil.…”