Subcellular mechanisms of delayed stretch-induced inotropy in rabbit myocardium

PDGF-AB AND FGF-2 TI’WGER DIFFERENT SIGNALUNO PAMAY M VASCULAR EEiY uu8cLE cELLs Delfull i3ihiilT$gs, Rene zlmm;nnmn*, Max-h-, +Kerckhoff-Klkrlk, Bad Nauitdm, Germany Proliferation of smooth muscle cells (SMCs) is essential for arteriogenesis and atherosclerosis. To determine key signalling pathways, we stimu-lated cultured SMCs with IO rig/ml platelet-derived growth factor-AB (PDGF) and IO rig/ml basic fibroblast growth factor (FGF-2) from 10 min to 12h. Proliferation was monitored by cell counting. Western blot using phospho-specific antibodies revealed an activated cytosolic Akt kinase (AKT; >lOx) by PDGF within 10 min declining over a period of 12h. The MAP kinases ERKl/2 were phosphotylated (5x) in a similar pattern. PDGF did not activate SAPWJNK and ~38. PKCG and PKCB phosphorylations increased significantly (>3x) from IO min remaining increased for 12h. A dramatic upregulation of the Egrl protein in the nuclear Fraction was detected. In contrast, FGF-2 did not activate AKT and PKCB, but led to significant phosphorylations of MEK1/2. The inhibitor UOl26 (10pM) completely abolished the MAPK activation and Egrl expression. Activation of PKC6, an upstream regulator of MAPK, was significantly downregulated (> 5x). In conclusion, our results point to PDGF specific signalling in SMCs. However, which of these pathways contribute to beneficial or pathological processessin arteries has to be elucidated. FUKCTIONAL EFFECTS AtiD SIGNAL TRAXDUCTIOK OF ICF-I IN HUMAN MYOCARDIUM Dlrk van Lewmski, Antomos Ciannakos, Swen Hulsmann”, Voss. Burken Pxske Abt Kardlologle u Pneumologlc, Abt fUr Neurophyslologle, August-Unwcrstt~ Gbttmgcn, Germany

Kersrm Georg-

Insulm-llke growth factor (IGF-I ) exerts a receptor medlatcd and (:a”dcpendenr posnwe motropx effect However, slgnal transducrmn mechamsms remam unclear Methods Isolated ventricular muscle strips from falling human hearts lsometnc contracnons (I Hz; 37°C) Recordings of Ca”-transients (Acquonn-method) or sarcoplasmattc reticulum Ca”-contenr (rapld coolmg contractures, RCC). lncubatlon with O.IpM IGF-I wth or wthout prcmcubatlon with wortmann~n (0 IpM; n=6). Ddttazem (~$vI, ny8). HOE642 (3lM. n=7). KB-R7943 (5pM; n=7). GFl09203X (I PM, n-4). and Cyclopwzonstiurc (CPA; 20pM) plus Ryanodm (IpM. n=7) Whole cell patch clamp L-type Ca”-current recordings in Isolated myocytes (n=S) Resulw IGF-I 0. IpM mcreases force and aequorm light signals to 146*9% and l5lrl2% of basal values, rcspect~vely (both p
EFFECTS OF ISCHAEMIA AND RE-OXYGWATION ON AKX’TC5LS AND NECRCXB FOLLOWING SIMULATED ISCHAEMlA-REOXYGENATION IN HUMAN ATRIAI. MYOCARDIUM. Hunaid A Vohra. Alan G Fowler & Manuel Galtines, University Clinical Sciences, Glenfield Hospital, Iaicesber, UK While essential for survival, reoxygenation subjects the tissue to an oxidative stress which myocyte cell death. We quantified apoptosis and in-vitro model of human atria1 myocardium prolonged periods of simulated ischaemia and (S/lR). The experiments (n-8 per group) were free-hand sections of right atria1 appendage

(reperfusion) may result in necrosis in an folIowing reoxy@nation performed on which were

subjected to periods of S/I (0,30,90 and l-in) folbwed by S/R (2, 8 and 24hrs). Cell damage was measured fdbwing release of myocyle spedfic creatine kinaae (CK-MB) assay. Cell apoptosis and necrosis were visual&d in tissue sections by means of fluorescent dyes using TUNJZL assay and Propidium Iodide, respectively. Quantification was by laser fluorescence confocal mlmscopy and NM-image software. Both CK-MB reelease and necrosis increased over the period of S/I in a dose-dependant manner. Apopto& peaked (32%) after 90min S/l with 2h S/R and increased (56%) after 90min S/I with 8hr S/R There was a deck in apotosis in tissue following 18Omin S/R. The smallest induction of apopta& occurred folbwing 24hrs S/ R In this model for ischaemia-reoxygenatkrn injury, apoptc& is the principal pathway to cell death folknving 30 to 90min simulakd ischaemia and 8hrs of reoxy~tion whewas mxmais is the principal mode of cell de& follom 24hrs reoy~nation.

SUBCELLULAR MECHANISMS OF DELAYED STRETCHIlYDUCED INOTROPY IN RABBIT MYOCARDfUM Dark v.Lewmstl, Burkhard Stumme. Claus Lum, ‘Lars S Maw, Burkert Pwske Abt. Kardtologle u. Pneumologle, Georg-August UmversltlIt Gbttmgen, Germany. “Dcpanment of Physiology, Loyola Unwerstty Chxago, USA ,Warnmal~an myocardlum IS characterized by a b1pha.w motroplc response to stretch wth an intoal rapId, followed by a delayed mcrease m force The tira phase is believed to result horn increased myofilament sens~tw~ty to Ca” In rat myocardwm. the second phase was attributed to a stretch-dependent paracrme release of endothelin- I. .Metbods: Rabbn right ventricular papillary muscles. electrical stlmulatlon. ~som-etnc confractions (I Hz; 37’C) Stretchmg to I_, for 30 min. again s-tretchmg to 98%1, Repetinon relaxmg to 88%1, of the same protocol ~11th the same muscle after blockmg the Na-lH’exchanger NHE-I (HOE 642 IOpM. n=l6), the Na-/Ca”-exchanger NCX (KB-R7943 SpM, n-g), stretch acwated channels, SAC (Gd” IOpM, n=lI) or the ET,-receptors (BQl23 0 IkM. n=9) Actlon potential durarton (APD) was rccordcd III 7 trabeculae at room temperature (floatmg elcctrodc method). Results: Stretchmg rabbn papillary muscles from 88% to 9&l, resulted m a btphastc response Durmg the mmal, rapld phase force increased by 144+9% (p-=0.05). Durmg phase 2, force mcreased further by 6 l+4% Neither HOE642, KB-R7943. Gd“ nor BQI 23 pretreatment affected the force mcrease in phase I. lnotroplc response m phase 2 was unaffected by pretreatment wth Gd” and BQI 23 In contrast, HOE642 and KB-R7943 sigmticantly blunted the inotropic response to stretch during phase 2 by 31*5% and 6li7%, rcspcttwely. (both p