1. 18 Sept 07 K 812 18 Sept 07 Cardiac Contractile Element 1

2. 18 Sept 07 Myosin Isoforms 2

3. 18 Sept 073

4. 4 Copyright ©2006 American Physiological Society Haddad, F. et al. Am J Physiol Heart Circ Physiol 290: H2351-H2361 2006 Coordinated regulation of cardiac MHC gene via antisense transcription

5. 18 Sept 075 Haddad, F. et al. Am J Physiol Heart Circ Physiol 290: H2351-H2361 2006 Myosin heavy chain (MHC) mRNA analyses by reverse transcription (RT)-polymerase chain reaction (PCR) using competitive PCR

6. 18 Sept 076 Haddad, F. et al. Am J Physiol Heart Circ Physiol 290: H2351-H2361 2006 Antisense beta-MHC RNA expression in NC and AbCon heart in 3 regions of the myocardium

7. Copyright ©2006 American Physiological Society Haddad, F. et al. Am J Physiol Heart Circ Physiol 290: H2351-H2361 2006 Fig. 2.  -MHC pre-mRNA expression in NC and AbCon heart

8. Copyright ©2006 American Physiological Society Haddad, F. et al. Am J Physiol Heart Circ Physiol 290: H2351-H2361 2006 Fig. 3.  -MHC mRNA expression in NC and AbCon heart

9. Copyright ©2006 American Physiological Society Haddad, F. et al. Am J Physiol Heart Circ Physiol 290: H2351-H2361 2006 Fig. 4. Antisense  -MHC RNA expression in NC and AbCon heart in 3 regions of the myocardium

10. Copyright ©2006 American Physiological Society Haddad, F. et al. Am J Physiol Heart Circ Physiol 290: H2351-H2361 2006 Fig. 5. Quantitative  -MHC RNA analyses in the apex region using real-time PCR and SYBRgreen fluorescence

11. Copyright ©2006 American Physiological Society Haddad, F. et al. Am J Physiol Heart Circ Physiol 290: H2351-H2361 2006; doi:10.1152/ajpheart.01111.2005 Fig. 6.  -pre-mRNA expression in NC and AbCon heart

12. Copyright ©2006 American Physiological Society Haddad, F. et al. Am J Physiol Heart Circ Physiol 290: H2351-H2361 2006; doi:10.1152/ajpheart.01111.2005 Fig. 7.  -MHC mRNA expression in NC and AbCon hearts

13. 18 Sept 0713 Titin

14. 18 Sept 0714

15. 18 Sept 0715 Granzier, H. L. et al. Circ Res 2004;94:284-295 Layout of titin in cardiac sarcomere

16. 18 Sept 0716 Granzier, H. L. et al. Circ Res 2004;94:284-295 Mechanism of passive and restoring force generation

17. 18 Sept 0717 Granzier, H. L. et al. Circ Res 2004;94:284-295 Exon-intron structure of human titin gene

18. 18 Sept 0718

19. Granzier, H. L. et al. Circ Res 2004;94:284-295 Passive tension/sarcomere length relations of cardiac myocytes that predominately express N2B titin and those that predominately express N2BA titin

20. Granzier, H. L. et al. Circ Res 2004;94:284-295 Contribution of titin and collagen to passive myocardial tension of mouse LV (MLV) and dog LV (DLV )

21. Titin summary Very large molecule which spans half a sarcomere from M- to Z-line Single gene which encodes for a protein of ~4.2 MDa Many isoforms exist which are the product of alternative splicing – each has different physical properties Cardiac muscle expresses two forms: a short one -N2B (~2.9 MDa) and a longer one N2BA (~3.3 MDa) Two cardiac forms have different physical properties – N2B is stiffer – and differential distributions N2B isoform can undergo PKA phosphorylation making it less stiff

22. Myosin binding protein C MyBP C

23. de Tombe, P. P. Circ Res 2006;98:1234-1236 Schematic structure of the cardiac sarcomere

24. Flashman, E. et al. Circ Res 2004;94:1279-1289 Rabbit psoas fibers labeled with goat antiserum to rabbit myosin binding protein C

25. Harris et al. Hypertrophic cardiomyopathy in cardiac myosin binding protein-C KO mice Circulation Research 90: 594, 2002

26. Figure 2. cMyBP-C mRNA transcript expression in knockout mice. A, RT-PCR of RNA using primers to exons 1 to 2 (a), 3 to 5 (b), and 25 to 34 (c) resulted in PCR products of expected size when cDNA from wild-type and heterozygous mice was amplified. cDNA from cMyBP-C-/- failed to yield product using primers to exons 3 to 5. Positive controls using cloned cMyBP-C cDNA and negative controls lacking reverse transcriptase (-RT) or DNA (H2O) are also shown. B, Northern blot using total RNA ( 3 µg/lane) was initially hybridized with a probe to exons 25 to 34 (3' probe). The blot was stripped and re-hybridized with a probe to exons 1 to 2 (5' probe).

27. Figure 3. cMyBP-C protein expression in knockout mice. Left, Representative SDS PAGE of rat cardiac (RC) and rat skeletal (RS) myofibrils, and total cell homogenates or myofibrils. Right, Western blots.

28. Figure 4. Cardiac hypertrophy in cMyBP-C knockout mice. A, Representative cross sections of hearts from adult (12 weeks) female littermate stained with Masson’s trichrome. Scale bar=1 mm. B, Heart (wet weight)-to-body weight ratios C, Relative expression of BNP, ANF, and  -skeletal actin RNA normalized to GAPDH D, Representative SDS-PAGE showing upregulation of ß-myosin heavy chain in littermate female cMyBP-C-/- mice (16 weeks).

30. Thin filament

31. 18 Sept 0731

32. 18 Sept 0732

33. 18 Sept 0733

35. 18 Sept 0735 161 amino acids 4 E-F hand Ca sites Sites III and IV –high affinity Ca/Mg sites Site I – dysfunctional Site II - low affinity Ca site – regulatory cardiac troponin C

36. 18 Sept 0736 EF hand Ca 2+ coordination E R V M F L C D D T V V M G G E D E S V D D I F C-terminus N-terminus Helix D Helix C Loop Ca 2+

37. 18 Sept 0737

38. 18 Sept 0738 When [Ca] = K d then [TnC] = [TnC · Ca]  K d = [Ca] at 50% [TnC · Ca] max

39. 18 Sept 0739 Non equilibrium states Rate of formation Rate of dissociation When rates of formation and dissociation are equal:

41. 18 Sept 0741

42. 18 Sept 0742 comparison of TnC Site I sequences X Y Z-Y–X -Z Mammals –fast skeletal DADGGGDISVKELGTV Mammals – cardiac LGAEDGCISTKELGKV Fish – cardiac QDAEDGCISTKELGKV

43. Bers – Table 8