Contractile protein alterations leading to heart failure. Zoltán Papp. Acute and chronic pump function disorders. Contractile protein machinery
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1 Contractile alterations leading to heart failure Zoltán Papp University of Debrecen Institute of Cardiology Division of Clinical Physiology Debrecen, Hungary Acute and chronic pump function disorders - identification of alterations in the myofilament - the relationship between mechanical function and alterations Contractile machinery
2 Structure to function relation of contractile s transducer Motor A B Ca 2+ - contractures in permabilized cardiomyocytes Length 25 kn/m 2 active force sec passive force Determination of the Ca 2+ -sensitivity of the contractile system Maximal Ca 2+ -activated force (F o ) Forc ce (rel.) n Hill Ca 2+ -sensitivity ( 5 ) 2
3 Ca 2+ -sensitivity and contractile force contraction systole diastole [Ca 2+ ] Time Ca 2+ -sensitivity and contractile force contraction systole diastole Reduction in Ca 2+ -activated force [Ca 2+ ] Time Ca 2+ -sensitivity and contractile force contraction systole diastole Reduction in Ca 2+ -activated force Increase in Ca 2+ -sensitivity [Ca 2+ ] Time 3
4 The Frank-Starling-mechanism and the length-tension diagramm Ernest Henry Starling ce (%) Muscle forc heart Sarcomere length ( m) The Frank-Starling-mechanism and the length-tension diagramm skeletal muscle Ernest Henry Starling ce (%) Muscle forc heart Sarcomere length ( m). Is there a relationship between the length-dependent increase in Ca 2+ -sensitivity and the kinetics of the actin-myosin cycle in different mammalian species? 4
5 The length dependence of Ca 2+ -sensitivity is similar in different mammalian hearts Humán Human Mouse Egér Sertés Pig Normalized Normalizált fo erő rce SL: 2.3 m SL:.9 m : ~. Édes IF. et al., Am. J. Physiol. (in press) Determination of the actin-myosin turnover rate in isolated cardiomyocytes Length PF 25 kn/m passive 2 sec Determination of the actin-myosin turnover rate in isolated cardiomyocytes Length PF 25 kn/m passive 2 sec Length k tr 25 kn/m 2 sec 5
6 and actin-myosin turnover rate both increase with [Ca 2+ ] A : B P (relative unit) C (relative) 6 5 sec k tr (/sec) 6 5 The actin-myosin turnover rate is species specific, but does not depend on sarcomere length 9 Humán Human 9 Mouse Egér 9 Sertés Pig k tr (s - ) SL: 2.3 m SL:.9 m Édes IF. et al., Am. J. Physiol. (in press). Is there a relationship between the length-dependent increase in Ca 2+ -sensitivity and the kinetics of the actin-myosin cycle in different mammalian species? We did not see it! 6
7 Contractile alterations during short lasting ischaemia and reperfusion 2 min ischaemia Left ventricular pressure min mmhg Contractile alterations during short lasting ischaemia and reperfusion Left ventricular pressure 2 min ischaemia ischaemic contracture min mmhg ischaemic contractile failure 2. Can we explain the ischaemic contractile failure and the ischaemic contracture with opposing alterations in Ca 2+ -sensitivity? 7
8 Ischaemic contractile responses can be associated with the accumulation of metabolites tive force Relat mm MgATP Kontroll Control mm MgATP 4 mm MgADP ischaemia 3 2 Norma alized force ischaemia Model ischaemia: ph 6.2; [P i ]: 3 mm; [MgADP] +[MgATP] = 5 mm Stienen G. et al., J. Physiol, 58: , 999 Papp Z. & Édes I. LAM : Ebus J.P., et al., Pflügers Arch. 443:2-, 2 Papp Z. et al. J. Physiol. 543:77-89, Can we explain the ischaemic contractile failure and the ischaemic contracture with opposing alterations in Ca 2+ -sensitivity? Myocardial reperfusion injury involves several mechanisms 2 min ischaemia stunned myocardium Left ventricular pressure min mmhg 8
9 3. What are the effects of the potentially deleterious mechanisms of reperfusion injury on the contractile machinery? (proteases, SH-oxidation, peroxynitrite) What is the effect of Ca 2+ -dependent proteolysis on the contractile function? -calpain p prot + ein -calpain evokes structural alterations.. proteolysis * * * control calpain calpain + force µm control -calpain -calpain + force Papp Z. et al. Cardiovasc. Res. 45:98-993, 2 Papp Z. et al. Circulation 4:e57-e57, 2 9
10 Cytoskeletal alterations may contribute to the development of myocardial stunning. C 9-9+ Spectrin degrad.p.? Desmin C min (%) desmin 75 Desmin relative des time (min) Papp Z. et al. Cardiovasc. Res. 45:98-993, 2 Papp Z. et al. Circulation 4:e57-e57, 2 -calpain cleaves several s Incubation: 2 perc st -Ca 2+ 3mM Ca U -Calpain T: 3 C 2 kda - 6 kda - 97 kda - titin 3-33 kda -fodrin 284 kda?? -actinin 5 kda 6-8% gradient gel 66 kda - 42 kda - desmin 63 kda TnT 44 kda? TnI 3 kda Barta et al.,mol. Cell. Biochem. 25:83-88, 23 Barta J. et al., Mol. Cell. Biochem. 278:-8, 25 How does SH-group oxidation affect contractile function? SH HS Oxidation (DTDP) Reduction (DTT) S S
11 The effects of SH-oxidation are reversible Relative force. Control control DTDP DTT.5 red. ox. Normalized force. Control control DTDP DTT n= n=4 2 Reduced Free SH SH-groups (% of untreated) (%) ox. red. * Control control DTDP + DTT DTDP+DTT n=3 k tr (/s). ox. red. *.5. Control control DPDT DTDP DTT DTDP+DTT n=4 *:p<.5 Hertelendi et al., submitted S-thiolation of contractile s diminish Ca 2+ - activated force Free SH (% of maximum) force free SH DTDP (mm) (% of maximum) Hertelendi et al., submitted S-thiolation of actin and MLC- are probably involved in the contractile effects Free SH (% of maximum) force free SH DTDP (mm) (% of maximum) mm DTDP. mm DTDP.3 mm DTDP mm DTDP 3 mm DTDP 3 mm DTDP 3 mm DTDP 3 3 mm DTDP+DTT 3 kda 7 ACTIN MLC Hertelendi et al., submitted
12 What is the effect of peroxynitrite on the contractile function? OH + ONOO - nitration NO 2 OH Peroxynitrite decreases maximal Ca 2+ -activated force in a concentration dependent manner Relative force force Poly NT Mono NT Peroxynitrite peroxynitrite concentration ( M) 5 Nitrotyosine Nitrotyrosine level lev (% vel %) (% of maximum m) control 5 M peroxynitrite 2 m Borbély et al., Cardiovasc. Res. 67: , 25. Peroxynitrite decreased Ca 2+ -activated force without effects on the Ca 2+ - sensitivity and cross-bridge kinetics Relative force Control 5 M PN Normalized force Control 5 M PN k tr (/s) Control 5 M PN Borbély et al., Cardiovasc. Res. 67: , 25. 2
13 3. What are the effects of the potentially deleterious mechanisms of reperfusion injury on the contractile machinery? (proteases, SH-oxidation, peroxynitrite) Ca 2+ -sensitivity s effect on sarcomere and dk tr -calpain initially no effect titin, -fodrin, desmin structural damage TnT, TnI, -actinin SH-oxidation reduced MLC-, actin regulatory disorder (reversible) peroxynitrite no effect -actinin structural damage 4. What kind of myofibrillar alterations associate with chronic heart failure? P phosphorylation? Ca 2+ -sensitivity is increased in chronic heart failure. Relat ive force Donor Heart failure 5 = van der Velden et al. Cardiovasc Res 23;57:37-47 Papp, Z. et al. J. Muscle Res. Cell Motil. 24;25:
14 Phosphorylation deficit of contractile s is probable in chronic heart failure. Relat ive force Protein kinase A Donor 5 Heart failure = Donor PKA Heart failure 5 = van der Velden et al. Cardiovasc Res 23;57: What kind of myofibrillar alterations associate with chronic heart failure? - increased Ca 2+ -sensitivity - phosphorylation deficit (troponin I, titin) - arguable changes in expression Summary contraction systole diastole Reduction in Ca 2+ -activated force -inorganic phophate -acidosis -proteolysis -SH-oxidation -peroxynitrite Increase in Ca 2+ -sensitivity -sarcomere length -MgADP -phosphorylation deficit [Ca 2+ ] time 4
15 Coo-workers, partners Institute of Cardiology, Division of Clinical Physiology, Debrecen, Hungary István Édes Attila Tóth Judit Barta Szabolcs Szilágyi Annamária Bódi Attila Borbély Zita Hertelendi István Ferenc Édes Miklós Vaszily Zoltán Galajda Vrije Universiteit of Amsterdam, ICaRVU Ger J.M. Stienen Jolanda van der Velden Montpellier, INSERM U39/IFR3 Physiopathologie Guy Vassort Alain Lacampagne Olivier Cazorla Institut für Physiologische Chemie, Ruhr-Universität Bochum Kornelia Jaquet Thank you for your attention! 5
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