Our findings demonstrate (i) that administration of NaHS induces cardioprotection via a cGMP/PKG/PLN pathway and (ii) contribution of nitric oxide to the H2S response is species-specific.

Hydrogen sulfide (H 2 S) is a signaling molecule with protective effects in the cardiovascular system. To harness the therapeutic potential of H 2 S, a number of donors have been developed. The present study compares the cardioprotective actions of representative H 2 S donors from different classes and studies their mechanisms of action in myocardial injury in vitro and in vivo. Exposure of cardiomyocytes to H 2 O 2 led to significant cytotoxicity, which was inhibited by sodium sulfide (Na 2 S), thiovaline (TV), GYY4137[morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate], and AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)-phenoxy)decyl) triphenylphospho-nium bromide]. Inhibition of nitric oxide (NO) synthesis prevented the cytoprotective effects of Na 2 S and TV, but not GYY4137 and AP39, against H 2 O 2 -induced cardiomyocyte injury. Mice subjected to left anterior descending coronary ligation were protected from ischemia-reperfusion injury by the H 2 S donors tested. Inhibition of nitric oxide synthase (NOS) in vivo blocked only the beneficial effect of Na 2 S. Moreover, Na 2 S, but not AP39, administration enhanced the phosphorylation of endothelial NOS and vasodilator-associated phosphoprotein. Both Na 2 S and AP39 reduced infarct size in mice lacking cyclophilin-D (CypD), a modulator of the mitochondrial permeability transition pore (PTP). Nevertheless, only AP39 displayed a direct effect on mitochondria by increasing the mitochondrial Ca 21 retention capacity, which is evidence of decreased propensity to undergo permeability transition. We conclude that although all the H 2 S donors we tested limited infarct size, the pathways involved were not conserved. Na 2 S had no direct effects on PTP opening, and its action was nitric oxide dependent. In contrast, the cardioprotection exhibited by AP39 could result from a direct inhibitory effect on PTP acting at a site different than CypD.

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Low dose NTG given prior to reperfusion reduces myocardial infarct size by preserving eNOS function, and the subsequent eNOS-dependent S-nitrosation of cyclophilin D, inhibiting cardiomyocyte necrosis. This novel pharmacological action of NTG warrants confirmation in clinical studies, although our data in human biopsies provide promising preliminary results.

Poly(ADP-ribose) polymerase-1 (PARP-1) activation is a hallmark of oxidative stress-induced cellular injury that can lead to energetic failure and necrotic cell death via depleting the cellular nicotinamide adenine dinucleotide (NAD + ) and ATP pools. Pharmacological PARP-1 inhibition or genetic PARP-1 deficiency exert protective effects in multiple models of cardiomyocyte injury. However, the connection between nuclear PARP-1 activation and depletion of the cytoplasmic and mitochondrial energy pools is poorly understood. By using cultured rat cardiomyocytes, here we report that ring finger protein 146 (RNF146), a cytoplasmic E3-ubiquitin ligase, acts as a direct interactor of PARP-1. Overexpression of RNF146 exerts protection against oxidant-induced cell death, whereas PARP-1-mediated cellular injury is augmented after RNF146 silencing. RNF146 translocates to the nucleus upon PARP-1 activation, triggering the exit of PARP-1 from the nucleus, followed by rapid degradation of both proteins. PARP-1 and RNF146 degradation occurs in the early phase of myocardial ischemia-reperfusion injury; it precedes the induction of heat shock protein expression. Taken together, PARP-1 release from the nucleus and its rapid degradation represent newly identified steps of the necrotic cell death program induced by oxidative stress. These steps are controlled by the ubiquitin-proteasome pathway protein RNF146. The current results shed new light on the mechanism of necrotic cell death. RNF146 may represent a distinct target for experimental therapeutic intervention of oxidant-mediated cardiac injury.

Background: Osteoarthritis (OA) is characterized by the formation and deposition of calcium-containing crystals in joint tissues, but the underlying mechanisms are poorly understood. The gasotransmitter hydrogen sulfide (H 2 S) has been implicated in mineralization but has never been studied in OA. Here, we investigated the role of the H 2 Sproducing enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) in cartilage calcification and OA development. Methods: 3-MST expression was analyzed in cartilage from patients with different OA degrees, and in cartilage stimulated with hydroxyapatite (HA) crystals. The modulation of 3-MST expression in vivo was studied in the meniscectomy (MNX) model of murine OA, by comparing sham-operated to MNX knee cartilage. The role of 3-MST was investigated by quantifying joint calcification and cartilage degradation in WT and 3-MST −/− meniscectomized knees. Chondrocyte mineralization in vitro was measured in WT and 3-MST −/− cells. Finally, the effect of oxidative stress on 3-MST expression and chondrocyte mineralization was investigated. Results: 3-MST expression in human cartilage negatively correlated with calcification and OA severity, and diminished upon HA stimulation. In accordance, cartilage from menisectomized OA knees revealed decreased 3-MST if compared to sham-operated healthy knees. Moreover, 3-MST −/− mice showed exacerbated joint calcification and OA severity if compared to WT mice. In vitro, genetic or pharmacologic inhibition of 3-MST in chondrocytes resulted in enhanced mineralization and IL-6 secretion. Finally, oxidative stress decreased 3-MST expression and increased chondrocyte mineralization, maybe via induction of pro-mineralizing genes. Conclusion: 3-MST-generated H 2 S protects against joint calcification and experimental OA. Enhancing H 2 S production in chondrocytes may represent a potential disease modifier to treat OA.

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Hydrogen sulfide (HS) exhibits beneficial effects in the cardiovascular system, many of which depend on nitric oxide (NO). Proline-rich tyrosine kinase 2 (PYK2), a redox-sensitive tyrosine kinase, directly phosphorylates and inhibits endothelial NO synthase (eNOS). We investigated the ability of HS to relieve PYK2-mediated eNOS inhibition and evaluated the importance of the HS/PYK2/eNOS axis on cardiomyocyte injury in vitro and in vivo. Exposure of H9c2 cardiomyocytes to HO or pharmacologic inhibition of HS production increased PYK2 (Y402) and eNOS (Y656) phosphorylation. These effects were blocked by treatment with NaS or by overexpression of cystathionine -lyase (CSE). In addition, PYK2 overexpression reduced eNOS activity in a HS-reversible manner. The viability of cardiomyocytes exposed to ΗΟ was reduced and declined further after the inhibition of HS production. PYK2 downregulation, l-cysteine supplementation, or CSE overexpression alleviated the effects of HO on H9c2 cardiomyocyte survival. Moreover, HS promoted PYK2 sulfhydration and inhibited its activity. In vivo, HS administration reduced reactive oxygen species levels, as well as PYK2 (Y402) and eNOS (Y656) phosphorylation. Pharmacologic blockade of PYK2 or inhibition of PYK2 activation by NaS reduced myocardial infarct size in mice. Coadministration of a PYK2 inhibitor and NaS did not result in additive effects on infarct size. We conclude that HS relieves the inhibitory effect of PYK2 on eNOS, allowing the latter to produce greater amounts of NO, thereby affording cardioprotection. Our results unravel the existence of a novel HS-NO interaction and identify PYK2 as a crucial target for the protective effects of HS under conditions of oxidative stress.