cardiomyocytes. This facilitates myocardial relaxation and ventriculardiastolic distensibility, thus influencing the Frank–Starling mechanism.

Fish hearts appear particularly sensitive to the Frank–Starlingresponse, but the reason is unknown.

In the in vitro isolated working avascular heart of juvenile eels wehave shown that a basal endogenous NO release greatly affects theFrank–Starling response, making the heart more sensitive to preload(Imbrogno et al., J. Exp. Biol., 204: 17191727, 2001). Using the samebioassay, we now demonstrate that this effect involves a PI(3)kinase-mediated eNOS activation and, independently from the GC/cGMP/PKGpathway, it involves direct modulation of the SR CA2+ ATPase (SERCA)but not the ryanodine receptor. This suggests that in the eel heart NOmodulates Frank–Starling response through a beat-to-beat regulationof calcium reuptake, and thus ofmyocardial relaxation. However, otherNO-dependent/cGMP-independent mechanisms, including S-nitrosy-lation of effector proteins and ion channels, aswell as the production ofradical species, may play a role.

doi:10.1016/j.cbpa.2008.04.301

A7.16Hydrogen sulfide exerts protective effects against oxidative stressin cultured H9C2 myocytes

D. Mancardi, C. Penna, A. Merlino, F. Tullio, P. Pagliaro (Universitàdi Torino)

The endogenous hydrogen sulfide (H2S) is producedenzymatically invarious species. At low concentrations, this gasotransmitter limitsapoptosis and activates mitochondrial K+

ATP channels. This study wasdesigned to investigate the mechanisms of action of H2S donors inprotecting cultured myocytes against oxidative stress. An immortalizedmyoblasts line (H9c2) was used to this purpose: cells were kept inDMEM enriched with fetal bovine serum (10%) and penicillin/strepto-mycin (1%). The experimental groups have been divided as follows:Group 1) oxidative stresswas induced by incubationwith 400mMH2O2,Group 2) H9c2 were treated with different concentrations (0.1, 1, 10,100mMand1mM) ofH2S and after 24 h challengedwith400mMH2O2.After 2 h inH2O2mediumsampleswere collected and the concentrationof lactate dehydrogenase (LHD)wasmeasuredwith spectrophotometricassays. In Group 3 cells were lysed after treatment with H2S in theabsence of H2O2 challenge. A dose-dependent protective activity with amaximal reduction of LDH release at 10 mM H2S (50% reductioncompared to Group 1) was observed. At higher doses the protectiveeffect disappeared. Western blot showed a marked increase ofphosphorylated p-44/p/42 MAPK in cells treated with 10 mM H2S.Data suggest that pre-treatmentwith low doses of H2S exerts protectiveeffects on myoblasts against oxidative stress through a cascade thatincludes p44/p/42 MAPK activation.

doi:10.1016/j.cbpa.2008.04.302

A7.17Preconditioning-like effect of the platelet activating factor(PAF): Pre- and post-ischemic signaling pathway in isolatedrat hearts

C. Penna, B. Mognetti, F. Tullio, D. Gattullo, D. Mancardi, G. Alloatti, P.Pagliaro (Università di Torino)

We previously showed that endogenous or exogenous PAF canprecondition the rat heart. Here, we investigated the molecularmechanisms involved in PAF-induced cardioprotection againstischemia/reperfusion injury. In Group 1 control hearts, isolated rathearts underwent 30-minutes global ischemia and 2-hours ofreperfusion; Group 2) hearts were perfused for 19-min with PAF(2×10−11 M) before ischemia; Groups 3 and 4) before the ischemiahearts underwent co-infusion either with PAF and N-acetyl-L-cysteine or PAF and 5-hydroxydecanoate to scavenge ROS or toblock mitochondrial-ATP-sensitive K+ (mKATP) channels, respec-tively. Group 5) in PAF-pretreated hearts, Atractyloside, an opener ofmitochondrial permeability transition pore (mPTP), was infusedduring the initial 20 min of reperfusion. In sixteen additional heartsthe phosphorylation of kinases by PAF was determined both in pre-and post-ischemic period. PAF-pretreatment reduced infarct size(33±4% vs 64±4.6% of the area at risk of control hearts) andimproved post-ischemic left ventricular function. PAF beneficialeffects were abolished by N-acetyl-L-cysteine, 5-hydroxydecanoateor Atractyloside. PAF-pretreatment up-regulated phosphorylation/activation of protein kinases (PKCe and PKB/Akt) both in the pre-and post-ischemic period, and the phosphorylation/inactivation ofglycogen synthase kinase-3β (GSK-3β) at reperfusion. Thus, PAF-protective cascade requires both a pre-ischemic mKATP channelsand redox signaling activation and a post-ischemic PKB/Aktactivation, GSK-3β inhibition and closing of mPTP.

doi:10.1016/j.cbpa.2008.04.303

A7.18p53 is an important contributor of oxidative stress-mediateddoxorubicin-induced cardiac injury

J. Velez (University of Kentucky); R. Nithipongvanitch (University ofWisconsin, Mahidol University); T. Noel (University of Kentucky); C.Daosukho (University of Kentucky, Mahidol University); T. Oberley(University of Wisconsin); D. St. Clair (University of Kentucky)

Doxorubicin (Dox)-induced oxidative stress in cardiomyocytemitochondria has recently been linked to p53 translocation to thisorganelle. To further investigate the role of p53 in Dox-inducedoxidative stress and subsequent amplification of mitochondriamediated cardiac injury, we treated wild-type and p53 homozygousknockout (p53(−/−)) mice with 20 mg/kg Dox via intra-peritonealinjection. Cardiac left ventricular function was assessed by fractionalshortening and ejection fraction, using ultrasound technology. Nobasal differences were observed between genotypes, though cardiacfunction was significantly reduced in wild-type mice at three daysafter Dox-injection (pb0.05), but not in p53(−/−) mice. Ultrastruc-tural pathology revealed that levels of cardiac mitochondria injuryinduced byDox, were significantly higher in thewild-typemicewhencompared to the p53(−/−) mice (pb0.05). The levels of 4-hydroxy-2′-nonenal (4HNE)-adducted protein, a toxic lipid peroxidation product,were increased in both nuclei and mitochondria of wild-type(pb0.05) following Dox treatment. Similar 4-HNE induction by Doxwas observed in the nuclei of p53(−/−) mice (pb0.05), but not in themitochondria, supporting a role of p53 in the mitochondria. To testthe possibility that p53 may exert its cardiotoxicity by modulation ofredox signaling system, we determined the levels and activity ofantioxidant enzymes known to be present in mitochondria of cardiactissue, including manganese superoxide dismutase, catalase, glu-tathione peroxidase, and thioredoxin reductase-2. With the excep-tion of catalase, Dox did not cause significant changes in the levels of

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the enzymes examined in the p53(−/−) mice. Interestingly, Doxtreatment significantly decreased the activity of glutathione-S-transferase, a major enzyme involved in the removal of 4-HNE,in wild type mice (pb0.001), but did not cause any change inthe p53(−/−) mice. These results implicate the role of p53 in

Dox-induced oxidative stress and subsequent modulation ofredox signaling by 4-HNE.

doi:10.1016/j.cbpa.2008.04.304

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