1. Contractile dysfunction in human heart failure Jolanda van der Velden Ger Stienen Institute for Cardiovascular Research VU University Medical Center

2. Introduction Force measurements in permeabilized cardiomyocytes & contractile protein composition Establish methods Calcium sensitivity is increased in end stage failing hearts Effects of myosin light chain 2 phosphorylation Functional effects of PKC Atrial Fibrillation Diastolic versus systolic heart failure

3. Force measurements in cardiomyocytes

4. Isoform composition F D MHC - C-protein -  -actinin - Actin - -TnT tropomyosin - TnI - MLC-1 - MLC-2 - FDFD  -MHC  -MHC  -MHC  -MHC TnT TnI

5. 2D gel electrophoresis Isoelectric point Molecular weight MLC1 MLC2 TnT Desmin 16-3-2000 0.6 mgr sample patient 11 Actin Tm ATP synthase pH 4.55.5

6. Protein phosphorylation Ca 2+ myofibrils SR phospholamban SERCA 2 Ca 2+ Ryanodine receptor Ca 2+ sarcolemma cytoplasm PKC PKA MLCK CaM kinase

7. Calcium sensitivity of force development van der Velden et al Basic Res Cardiol (2002)

8. Effect of PKA on Force Relative force van der Velden et al Cardiovasc Res (2000)

9. Effects of MLC2-dephosphorylation pCa Relative force DonorFailing pCa van der Velden et al Cardiovasc Res (2004)

10. PKA after PKC van der Velden et al Cardiovasc Res (2005)

11. PKC after PKA

12. PKC and PKA

13. Systolic and diastolic dysfunction EF LVEDP norm. EF norm. LVEDP

14. Diastolic Heart Failure Myofilament dysfunction in diastolic heart failure? with normal cardiac function: Control (n=8) with pure diastolic dysfunction: DHF (n=12) Ventricular endomyocardial biopsies from patients: Borbély et al. Circulation (2005) *P<0.05

15. Force recordings Length pCa 9.0 4.5 9.0 5.8 Force

16. Calcium sensitivity of force development in DD patients Passive force (kN/m 2 ) Total force (kN/m 2 ) DD +PKA Borbély et al. Circulation (2005)

17. Patient groups 9Patients with sinus rhythm (SR) 11Patients with SR and atrial dilation (SR + AD) 16 Patients with persistent atrial fibrillation (AF) Methods: Atrial dimensions by Doppler Imaging. Protein composition by 1D- and 2D-gel electrophoresis. Force measurements in skinned cardiomyocytes ( F max ;pCa 50 ;K tr ).

18. Mean atrial dimensions Eiras et al JMCC (in press)

19. Myosin heavy chain composition  - desmin  - ALC-1  - ALC-2 - MHC  - troponin T 150 - 100 - 75 - 50 - 37 - 25 - 20 - kD  - actin SRAF SRAFSR+AD

20. 2D-gel electrophoresis Molecular weight IEF 4.55.5 ApoA1 SR

21. Alterations in protein composition

22. Protein (%)ANOVASRSR+ADAF  -MHC * 22.6  4.3#27.0  4.041.5  5.0 TnTD* 26.1  3.026.7  0.8#20.2  1.3 TnTP* 61.9  2.4#62.9  1.170.4  2.2 TnTPP 12.1  2.010.5  1.09.4  1.6 TnT/actin 21.0  1.720.4  2.518.6  2.4 ALC-1D 90.6  1.487.1  1.989.4  1.8 ALC-1P 9.4  1.412.9  1.910.6  1.8 ALC-1/actin 45.2  5.244.7  3.235.7  3.1 ALC-2D 71.3  4.368.2  2.668.2  2.8 ALC-2P 25.1  3.427.5  2.326.2  2.4 ALC-2PP 3.5  1.54.8  0.85.6  1.1 ALC-2/actin* 38.0  5.039.2  3.328.6  2.5 ALC-1/ALC-2 125  11117  9135  11 Desmin/actin* 23.0  3.1#22.0  2.7#39.3  2.9 ApoA-I* 13.1  2.416.3  2.710.1  1.6 ApoA-I/actin 19.4  2.814.8  2.69.9  2.3

23. Alterations in contractile function

24. Conclusions Atrial dilatation per se does not predispose nor cause the alterations in contractile protein composition and function in atrial fibrillation. Slowing of contraction might reduce ventricular filling.