Knockdown of nucleosome assembly protein 1-like 1 induces mesoderm formation and cardiomyogenesis via notch signaling in murine-induced pluripotent stem cells.

Low efficiency of cardiomyocyte differentiation from induced pluripotent stem cells (iPSCs) hinders the clinical application of iPSC technology for cardiac repair strategy. Recently, we screened out nucleosome assembly protein 1-like 1 (Nap1l1), which was downregulated during the differentiation of P19CL6 cells into cardiomyocytes. Here, we attempted to study the role of Nap1l1 in cardiomyogenesis of iPSC. Nap1l1 was downregulated during the differentiation of iPSC. Knockdown of Nap1l1 dramatically enhanced the differentiation of iPSC into functional cardiomyocytes while overexpression of Nap1l1 sharply lowered the differentiation. Moreover, although Nap1l1-knockdown had little effect on endoderm differentiation, the Nap1l1 modulation significantly accelerated mesoderm development. Re-expressing Nap1l1 in Nap1l1-knockdown-iPSC rescued the effects of Nap1l1. Inducibly overexpressing Nap1l1 at early stage of differentiation greatly inhibited mesoderm induction and cardiogenesis of iPSC. However, mesoderm stem cells (Flk-1-positive cells) originated from Nap1l1-knockdown- or -overexpression-iPSC showed no difference in further cardiomyocyte differentiation compared with that of control-iPSC. Further study revealed that Nap1l1-overexpression increased γ-secretase activity and the expression of Notch intracellular domain (NICD) and downstream genes during the differentiation of iPSC. γ-Secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester) greatly suppressed the production of NICD and abolished the inhibitory effects of Nap1l1-overexpression on mesoderm induction and cardiogenesis. These findings demonstrate that downregulation of Nap1l1 significantly enhances mesodermal induction and subsequent cardiogenesis of murine iPSC via inhibition of γ-secretase-regulated Notch signaling, which would facilitate the application of iPSC in heart diseases.

Urotensin II Protects Cardiomyocytes from Apoptosis Induced by Oxidative Stress through the CSE/H2S Pathway.

Plasma urotensin II (UII) has been observed to be raised in patients with acute myocardial infarction; suggesting a possible cardiac protective role for this peptide. However, the molecular mechanism is unclear. Here, we treated cultured cardiomyocytes with H2O2 to induce oxidative stress; observed the effect of UII on H2O2-induced apoptosis and explored potential mechanisms. UII pretreatment significantly reduced the number of apoptotic cardiomyocytes induced by H2O2; and it partly abolished the increase of pro-apoptotic protein Bax and the decrease of anti-apoptotic protein Bcl-2 in cardiomyocytes induced by H2O2. SiRNA targeted to the urotensin II receptor (UT) greatly inhibited these effects. Further analysis revealed that UII increased the production of hydrogen sulfide (H2S) and the level of cystathionine-γ-lyase (CSE) by activating the ERK signaling in H2O2-treated-cardiomyocytes. Si-CSE or ERK inhibitor not only greatly inhibited the increase in CSE level or the phosphorylation of ERK induced by UII but also reversed anti-apoptosis of UII in H2O2-treated-cadiomyocytes. In conclusion, UII rapidly promoted the phosphorylation of ERK and upregulated CSE level and H2S production, which in turn activated ERK signaling to protect cardiomyocytes from apoptosis under oxidative stress. These results suggest that increased plasma UII level may protect cardiomyocytes at the early-phase of acute myocardial infarction in patients.

Hydrogen Sulfide Regulates the [Ca2+]i Level in the Primary Medullary Neurons.

In the present study, we attempted to elucidate mechanisms for the regulation of intracellular calcium levels by H2S in primary rat medullary neurons. Our results showed that NaHS significantly increased the level of [Ca2+]i in rat medullary neurons in a concentration-dependent manner. L-Cysteine and SAM significantly raised the level of [Ca2+]i in the medullary neurons while HA and/or AOAA produced a reversal effect. In addition, L-cysteine and SAM significantly increased but HA and/or AOAA decreased the production of H2S in the cultured neurons. The [Ca2+]i elevation induced by H2S was significantly diminished by EGTA-Ca2+-free solutions, and this elevation was also reduced by nifedipine or nimodipine and mibefradil, suggesting the role of L-type and/or T-type Ca2+ channels. Moreover, the effect of H2S on [Ca2+]i level in neurons was significantly attenuated by BAPTA-AM and thapsigargin, suggesting the source of Ca2+. Therefore, we concluded that both exogenous and endogenous H2S elevates [Ca2+]i level in primarily cultured rat medullary neurons via both increasing calcium influx and mobilizing intracellular Ca2+ stores from ER.

Hydrogen Sulfide Targets the Cys320/Cys529 Motif in Kv4.2 to Inhibit the Ito Potassium Channels in Cardiomyocytes and Regularizes Fatal Arrhythmia in Myocardial Infarction.

AIMS: The mechanisms underlying numerous biological roles of hydrogen sulfide (H2S) remain largely unknown. We have previously reported an inhibitory role of H2S in the L-type calcium channels in cardiomyocytes. This prompts us to examine the mechanisms underlying the potential regulation of H2S on the ion channels. RESULTS: H2S showed a novel inhibitory effect on Ito potassium channels, and this effect was blocked by mutation at the Cys320 and/or Cys529 residues of the Kv4.2 subunit. H2S broke the disulfide bridge between a pair of oxidized cysteine residues; however, it did not modify single cysteine residues. H2S extended action potential duration in epicardial myocytes and regularized fatal arrhythmia in a rat model of myocardial infarction. H2S treatment significantly increased survival by ∼1.4-fold in the critical 2-h time window after myocardial infarction with a protection against ventricular premature beats and fatal arrhythmia. However, H2S did not change the function of other ion channels, including IK1 and INa. INNOVATION AND CONCLUSION: H2S targets the Cys320/Cys529 motif in Kv4.2 to regulate the Ito potassium channels. H2S also shows a potent regularizing effect against fatal arrhythmia in a rat model of myocardial infarction. The study provides the first piece of evidence for the role of H2S in regulating Ito potassium channels and also the specific motif in an ion channel labile for H2S regulation.

Hydrogen sulfide treatment promotes glucose uptake by increasing insulin receptor sensitivity and ameliorates kidney lesions in type 2 diabetes.

AIMS: To examine if hydrogen sulfide (H2S) can promote glucose uptake and provide amelioration in type 2 diabetes. RESULTS: Treatment with sodium hydrosulfide (NaHS, an H2S donor) increased glucose uptake in both myotubes and adipocytes. The H2S gas solution showed similar effects. The NaHS effects were blocked by an siRNA-mediated knockdown of the insulin receptor (IR). NaHS also increased phosphorylation of the IR, PI3K, and Akt. In Goto-Kakizaki (GK) diabetic rats, chronic NaHS treatment (30 µmol·kg(-1)·day(-1)) decreased fasting blood glucose, increased insulin sensitivity, and increased glucose tolerance with increased phosphorylation of PI3K and Akt in muscles. Similar insulin-sensitizing effects of NaHS treatment were also observed in Wistar rats. Moreover, glucose uptake was reduced in the cells with siRNA-mediated knockdown of the H2S-generating enzyme cystathionine γ-lyase in the presence or absence of exogenous H2S. Moreover, chronic NaHS treatment reduced oxygen species and the number of crescentic glomeruli in the kidney of GK rats. INNOVATION AND CONCLUSION: This study provides the first piece of evidence for the insulin-sensitizing effect of NaHS/H2S in the both in vitro and in vivo models of insulin resistance. REBOUND TRACK: This work was rejected during a standard peer review and rescued by the Rebound Peer Review (Antoxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Jin-Song Bian, Samuel Dudley, Hideo Kimura, and Xian Wang.

The hydrogen sulfide donor NaHS promotes angiogenesis in a rat model of hind limb ischemia.

It is not known whether H(2)S can promote angiogenesis with improvement of regional blood flow in ischemic organs. Sodium hydrosulfide (NaHS, a H(2)S donor) was administered once a day for 4 w following femoral artery ligation. Collateral vessel growth, capillary density, regional tissue blood flow, the expression of endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) and Akt were examined during or at the end of the treatment period. NaHS treatment significantly increased collateral vessel growth, capillary density, and regional tissue blood flow in ischemic hind limb muscles compared with the controls. These effects were associated with an increase in VEGF expression in the skeletal muscles and VEGFR2 phosphorylation in the neighboring vascular endothelial cells, suggesting a role of VEGF in mediating the NaHS effects in a cell-cell interaction pattern. Moreover, NaHS treatment also resulted in an increase in Akt phosphorylation in ischemic hind limb muscles. In conclusion, our observations with NaHS strongly suggest that H(2)S is a proangiogenic factor in chronic ischemia. The proangiogenic effect of NaHS may be mediated by interaction between the upregulated VEGF in the skeletal muscle cells and the VEGFR2 as well as its downstream signaling element Akt in the vascular endothelial cells.

Role of PKC in the novel synergistic action of urotensin II and angiotensin II and in urotensin II-induced vasoconstriction.

The intracellular signaling of human urotensin II (hU-II) and its interaction with other vasoconstrictors such as ANG II are poorly understood. In endothelium-denuded rat aorta, coadministration of hU-II (1 nM) and ANG II (2 nM) exerted a significant contractile effect that was associated with increased protein kinase C (PKC) activity and phosphorylation of PKC-alpha/betaII and myosin light chain, whereas either hU-II or ANG II administered alone at these concentrations had no statistically significant effect. This synergistic effect was abrogated by the PKC inhibitor chelerythrine (10 and 30 microM), the selective PKC-alpha/betaII inhibitor Gö-6976 (0.1 and 1 microM), the hU-II receptor ligand urantide (30 nM and 1 microM), or the ANG II antagonist losartan (1 microM). Moreover, in endothelium-intact rat aorta, the synergistic effect of hU-II and ANG II was not exerted any longer, and this synergistic effect was unmasked by pretreatment of the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. hU-II (10 nM) alone caused a long-lasting increase in phospho-PKC-theta, phospho-myosin light chain, and PKC activity, which was associated with long-lasting vasoconstriction. These changes were prevented by chelerythrine. Methoxyverapamil-thapsigargin treatment reduced the hU-II-induced vasoconstriction by approximately 50%. The methoxyverapamil-thapsigargin-resistant component of hU-II-induced vasoconstriction was dose-dependently inhibited by chelerythrine. In conclusion, hU-II induces a novel PKC-dependent synergistic action with ANG II in inducing vasoconstriction. PKC-alpha/betaII is probably the PKC isoform involved in this synergistic action. Nitric oxide produced in the endothelium probably masks this synergistic action. The long-lasting vasoconstriction induced by hU-II alone is PKC dependent and associated with PKC-theta phosphorylation.