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On the source of Ca2+ activating tonic component of cardiomyocyte contraction
HYSTERESIS IN THE CARDIAC FORCE-LENGTH RELATION VALIBATES THE EXISTENCE OF THE COOPERATIVITY MECHANISM. Carmit Levy, Amir Laadesberg. Dept of Biomedical Engineering, Technion-IIT, Hsifa, Israel. Cardiac muscle force length relation and cross-bridge (XB) recruitment are regulated by the cooperativity mechanism, whereby the affinity of troponin for calcium depends on the number of force-generating XBs. This hypothesis leads to the prediction that the generated force should lag sarcomere length (SL) changes at constant calcium concentration. We studied the force responses to slow (1Hz) and small (5Onm in amplitude) length oscillations, at the intact tetanized rat trabecula (from Sprague Dawley right ventricle, aged 2 months). Tetanus was elicited every 10 twitches using stimuli of 8H.z (pulse width of 40ms) in the presence of cyclopiazonic acid (3OpM) and calcium concentration of 6mM (K-H solution, 25’C). Sarcomere length (SL) was measured by laser’ difi%tion techniques (Dalsa Camera, frame rate of 75OOH2, Resolution of 3nm). Force was measured by silicon strain gauge (Resonance frequency of 2OOOHz). The stiffness was measured by additional high-frequency oscillation (50 or 200 Hz) with smaller amplitude (7mn). Both the stitiess and the force lagged the SL slow oscillations. The force response lagged the sarcomere length oscillations by 55.4ti8.8msec or 20+14 degree. Hysteresis was obtained between the force and SL. At the same SL the generated force was higher during SL shortening in accordance with the cooperativity mechanism whereby calcium affinity and the bound calcium are larger when reaching a SL from a longer SL. The study validates the existence of the cooperativity mechanism, which relates to the Frank-Starling law, and allows to quantify its rate constants.
FUNCTIONAL EFFECTS IN IIUMAN MYocARDwM
OF Na+/H+-EXCHANGE
Dirk v.Lewinski, Bu&bard Stumme, Claus Luem, Egbert Bisping, Btierl Pieske zk;k Cardiology and Pneumology, University of The Na’lIT-cxchanger m-1) is one of the most important pH-regulating proteins in the heart extruding H’ versus Na’ in a 1:1 stoicbiometric manner. However, little is known on functional effects especially in human myocardium. We investigated functional effects of NHE bbxkade in isolated human cardiac muscle. Metbodx Lwlated, electrically stimulate=3 (1 Hz) muscle strips from failing human bearts (n=lO), 37oC. Intluenee of HOE642, a selective NHE-I inhibitor w farce of eontraction and SR Ca”-content @CC). Influence of muscle stretch (l-) on NH&I activation. lotluencc of NHE-I blockade on atretcb dependent MAP-kinase activation (western Mot). Rerultr: Under basal exoerimental conditions. NHEl blockade resulted in a sli&, albeit consi& ‘negative inotropic effect, related to a deeline of the SR Ca”-content. Stretching muscle ships resulted in an initial, Eest increase in twitch force (Frank-Starling-mechanism) followed by a second, slower phase of force increase. This 2* phase could be largely reduced by HOE642. Stretch induced MAF’-kinase activation was prevented by HOE642 preiacubation, whitbout any effect of HOE 642 in umtretched mus&s. Conclurlons: NHYEI is activated in failin human myocardium and exerts positive inobupic effects throu& Ca”dependent pathways. These timctionai effects as well as stretchd~dent MAP-I&se activation can be partially prevented by NHE 1 inhibition.
ON THE SOURCE OF Cam ACTIVATING TONIC COMPONENT OF CARDIOYYOCYTE CONTRACTION. Bohden Lewmtoweki, Kmymtof Emanuel, Urezula Yackiiwicz. Department of Clinical Physiotogy, Medical Center of Postgraduate Education, Wanaw, Poland. Contractions of single, voltage clamped myocytes of guinea pig heart consist of a phasic component relaxing before the end of depolatization and a tonic component relaxing upon repolarization. The aim of this study was to find the source of Ca2* activating the phasic component. We found that neither component of contraction was inhibited by 5-10 mM Ni2’ which blocks Ca” currents and NaEa exchange. The tonic component was not selectively affected by 5.0 l.M KER7943, or intracellular dialysis with Na’ free solution which inhibit the reverse mode NalCa exchange. Thus the tonic component is activated neither by Ca” current nor by the revened Na/Ca exchange as currently proposed in literature. Both components of contraction were completely inhibited by blocking the SR Ca2+ release channels with 200 pM ryanodine in the cells in which Ca2+ current has been blocked with Ni2+. The phasic but not the tonic component was inhibited by nifedipine. We conclude that both components of contraction are activated by Ca” released from the SR, however, the site, kinetics and mechanism of release are quite different.
Induction of an sngiogenic phsnotyps In THP-1 monocytes by recombinant human rslsxln Marlyn Lewis, Usha Deshpande. Shu-Hul Uu, Mark Edkson, Qiang Zhang 8 Elaine N. Unamorl. Conneifes Corp., Palo Alto, CA. USA. We have previously shown in a ral model of acute myocardia) infarction that systemk administration of recombinant human relaxln (rhRLX), mutta in hweased ped-infarct expressfon of bask Rbrobkst growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A). As macmphages are an integral ampanent of the healing raepunse in the heart, we have examined the human monccytk THP1 cell line to detennine fhe potential effect of mRfx on the lnfllb’atlng macmphage. Mkroarray analysk indicated fhat *Rtx Induced Me exoressfon ~. ~~ of VEGF-A. ~~ bFGF. olatelet-derfved endothellal arowth factor (PD-ECGF), VEiF-B, and’ the transcription factor HIF-icz. A dose-related Increase in expreeslon of bFGF and the VEGFIe5 and VEGA,*, lsoforms al 24hr of rhRLX treatment was confirmed by quantitatfve RT-PCR (qAT-PCA). To examine the time course of this response, THP-1 cells were treated with 1 rig/ml rhRlx for 1,3.6,12 and 24 hr and examined for exuresskn of Mess fatiOm uslno uRTPCR. ELISA and Immunocytoch’emkby. Expressfon of boM Vi&F-A isoforms Increased slgnffkantly following rhRlx treatment. VEGA,,, expressfon peaked at 3hr wlm levels persistlng for 24hr. while VEGF,.5 expression levels increased gradually from 3hr to 24 hr. Bask FGF mRNA. but not acidic FGF, expression was induced rapidly, peaking at 3hr. with protein levels perslstlng through 24 hr. VEGF-B and PDECGF increased signffkantfy at the protein level by 24hr. tiRfx induced a rapid transient response in expression of HIFla mRNA. with protein levels remaining sfgniffcantly elevated through 24hr. Theae obaewaUons, along with the in vfvo effti of mRlx In fhe Infarcted heart, suggesf a potentfally Important role for relaxin on the angkgenk phenotype of lnfiltraflng macmphages klbwlng infarction.
A67
FUNCTIONAL EFFECTS IN IIUMAN MYocARDwM
OF Na+/H+-EXCHANGE
Dirk v.Lewinski, Bu&bard Stumme, Claus Luem, Egbert Bisping, Btierl Pieske zk;k Cardiology and Pneumology, University of The Na’lIT-cxchanger m-1) is one of the most important pH-regulating proteins in the heart extruding H’ versus Na’ in a 1:1 stoicbiometric manner. However, little is known on functional effects especially in human myocardium. We investigated functional effects of NHE bbxkade in isolated human cardiac muscle. Metbodx Lwlated, electrically stimulate=3 (1 Hz) muscle strips from failing human bearts (n=lO), 37oC. Intluenee of HOE642, a selective NHE-I inhibitor w farce of eontraction and SR Ca”-content @CC). Influence of muscle stretch (l-) on NH&I activation. lotluencc of NHE-I blockade on atretcb dependent MAP-kinase activation (western Mot). Rerultr: Under basal exoerimental conditions. NHEl blockade resulted in a sli&, albeit consi& ‘negative inotropic effect, related to a deeline of the SR Ca”-content. Stretching muscle ships resulted in an initial, Eest increase in twitch force (Frank-Starling-mechanism) followed by a second, slower phase of force increase. This 2* phase could be largely reduced by HOE642. Stretch induced MAF’-kinase activation was prevented by HOE642 preiacubation, whitbout any effect of HOE 642 in umtretched mus&s. Conclurlons: NHYEI is activated in failin human myocardium and exerts positive inobupic effects throu& Ca”dependent pathways. These timctionai effects as well as stretchd~dent MAP-I&se activation can be partially prevented by NHE 1 inhibition.
ON THE SOURCE OF Cam ACTIVATING TONIC COMPONENT OF CARDIOYYOCYTE CONTRACTION. Bohden Lewmtoweki, Kmymtof Emanuel, Urezula Yackiiwicz. Department of Clinical Physiotogy, Medical Center of Postgraduate Education, Wanaw, Poland. Contractions of single, voltage clamped myocytes of guinea pig heart consist of a phasic component relaxing before the end of depolatization and a tonic component relaxing upon repolarization. The aim of this study was to find the source of Ca2* activating the phasic component. We found that neither component of contraction was inhibited by 5-10 mM Ni2’ which blocks Ca” currents and NaEa exchange. The tonic component was not selectively affected by 5.0 l.M KER7943, or intracellular dialysis with Na’ free solution which inhibit the reverse mode NalCa exchange. Thus the tonic component is activated neither by Ca” current nor by the revened Na/Ca exchange as currently proposed in literature. Both components of contraction were completely inhibited by blocking the SR Ca2+ release channels with 200 pM ryanodine in the cells in which Ca2+ current has been blocked with Ni2+. The phasic but not the tonic component was inhibited by nifedipine. We conclude that both components of contraction are activated by Ca” released from the SR, however, the site, kinetics and mechanism of release are quite different.
Induction of an sngiogenic phsnotyps In THP-1 monocytes by recombinant human rslsxln Marlyn Lewis, Usha Deshpande. Shu-Hul Uu, Mark Edkson, Qiang Zhang 8 Elaine N. Unamorl. Conneifes Corp., Palo Alto, CA. USA. We have previously shown in a ral model of acute myocardia) infarction that systemk administration of recombinant human relaxln (rhRLX), mutta in hweased ped-infarct expressfon of bask Rbrobkst growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A). As macmphages are an integral ampanent of the healing raepunse in the heart, we have examined the human monccytk THP1 cell line to detennine fhe potential effect of mRfx on the lnfllb’atlng macmphage. Mkroarray analysk indicated fhat *Rtx Induced Me exoressfon ~. ~~ of VEGF-A. ~~ bFGF. olatelet-derfved endothellal arowth factor (PD-ECGF), VEiF-B, and’ the transcription factor HIF-icz. A dose-related Increase in expreeslon of bFGF and the VEGFIe5 and VEGA,*, lsoforms al 24hr of rhRLX treatment was confirmed by quantitatfve RT-PCR (qAT-PCA). To examine the time course of this response, THP-1 cells were treated with 1 rig/ml rhRlx for 1,3.6,12 and 24 hr and examined for exuresskn of Mess fatiOm uslno uRTPCR. ELISA and Immunocytoch’emkby. Expressfon of boM Vi&F-A isoforms Increased slgnffkantly following rhRlx treatment. VEGA,,, expressfon peaked at 3hr wlm levels persistlng for 24hr. while VEGF,.5 expression levels increased gradually from 3hr to 24 hr. Bask FGF mRNA. but not acidic FGF, expression was induced rapidly, peaking at 3hr. with protein levels perslstlng through 24 hr. VEGF-B and PDECGF increased signffkantfy at the protein level by 24hr. tiRfx induced a rapid transient response in expression of HIFla mRNA. with protein levels remaining sfgniffcantly elevated through 24hr. Theae obaewaUons, along with the in vfvo effti of mRlx In fhe Infarcted heart, suggesf a potentfally Important role for relaxin on the angkgenk phenotype of lnfiltraflng macmphages klbwlng infarction.
A67