1. Homozygous mice that lack the proline-alanine rich region and C1 domain of cMyBP-C (p/a-C1 -/- mice, Figure 1) were developed by Witt et al.. 4 Figure 2A: Truncated cMyBP-C is expressed in p/a-C1 -/- mice. – Both full length cMyBP-C and cMyBP-C without the p/a-C1 region were detected on Coomassie stained gel and Western blot with antibodies directed against cMyBP-C. Figure 2B: Expression levels of cMyBP-C are comparable between p/a-C1 -/- and wild type (WT) mice. – Expression was normalized to -actinin expression. CONCLUSION: Deletion of the p/a region and C1 domain did not cause contractile dysfunction in mice either on a cellular level or in the whole heart. Discussion: Results in sedentary mice unexpectedly showed no cardiac dysfunction. – In contrast to in vitro studies suggesting a functional role for these regions. – Despite HCM associated mutations that are found in C1. Inter-species differences: – Human p/a and C1 had stronger functional effects in in vitro studies than mice. 3,5,6 – The function of cMyBP-C might be less critical in mammals with higher heart rates such as mice. Redundancy in domains of cMyBP-C: – Possibly other domains compensate for the absence of the p/a-C1 region. e.g. both C0 and C1 can bind to actin and myosin. 2 Possible (other) functions of the p/a region: – Provides flexibility to C0 domain (indirect function). – Mechanosensor (comparable to the PEVK region of titin). Force measurement in permeabilized cardiomyocytes. –Mice were treated with propranolol before being sacrificed. –Force measurements were done before and after incubation with protein kinase A (PKA). Figure 3A: cMyBP-C, cTnI and cTnT phosphorylation were lower in p/a-C1 -/- mice. Figure 3B: PKA treatment caused maximal phosphorylation of cMyBP-C and cTnI after 40 minutes. Figure 3C: Force development was comparable between p/a-C1 -/- and WT mice. – PKA decreased Ca 2+ -sensitivity of force. Echocardiograms were performed on mildly sedated mice (1-3% isoflurane). Figure 4: Results showed that cardiac morphometry and function comparable was between p/a-C1 -/- and WT mice. Isovolumetric relaxation time was shorter in p/a-C1 -/- mice (Table). –Morphometry: left ventricular peripheral wall thickness (LVPW), left ventricular internal diameter (LVID). –Systolic function: fractional shortening (FS), ejection fraction (EF), stroke volume (SV). –Diastolic function: blood flow over the mitral valve during early diastole (E) and atrial contraction (A), movement of the mitral valve annulus during early (E’) and late (A’) diastole, isovolumetric relaxation time (IVRT) and isovolumetric contraction time (IVCT). Normal cardiac contraction in mice lacking the proline-alanine rich region and C1 domain of cMyBP-C Sabine J. van Dijk 1, Christian C Witt 2, Samantha P. Harris 1 1 Department of Cellular and Molecular Medicine, University of Arizona, Tucson AZ, USA; 2 Department of Anaesthesiology and Operative Intensive Care, University Hospital Mannheim, Mannheim, Germany Background HYPOTHESIS: The p/a region and C1 domain (p/a-C1) of cardiac myosin binding protein C are essential to its function to regulate cross bridge cycling speed. – Without the p/a-C1 region, cross bridge cycling speed will be increased. – Activation and/or stabilization of cross bridges by the p/a-C1 region will aid in proper timing of systole. Myosin binding protein C (cMyBP-C) is essential for proper cardiac function. – Mutations in MYBPC3 are a frequent cause of hypertrophic cardiomyopathy (HCM). – Several cMyBP-C mouse models demonstrate cardiac dysfunction and remodeling. How cMyBP-C functions is elusive. – cMyBP-C interacts with multiple other sarcomeric proteins. – cMyBP-C is located in the C-zone, in the center 2/3 of the thick filament. – cMyBP-C inhibits cross bridge cycling. – The function of cMyBP-C is regulated by phosphorylation of the M-domain. The proline-alanine rich (p/a) region and C1: – The p/a region is little conserved among species and the % of proline and alanine in the p/a region is inversely correlated to heart rate in mammals. 1 – Multiple mutations linked to HCM found in C1. – C1 can bind both to myosin heads and actin. 2 – p/a and C1 regions are associated with both activating and inhibitory effects of cMyBP-C. 3 Figure 1. Schematic overview of the domains of cardiac myosin binding protein C (cMyBP ‑ C). The C0 and C1 domains are connected by a proline-alanine rich region (p/a). A second linker region, the M- domain, is located between C1 and C2 and contains phosphorylation sites (asterisks) that modulate the interaction of cMyBP ‑ C with other sarcomeric proteins. Regions with which cMyBP ‑ C binds to other sarcomeric proteins are indicated. The region of interest studied in this work, the p/a region and C1 domain are indicated by a green box. Methods and Results Normal force development and response to PKA in p/a-C1 -/- cardiomyocytes Normal cardiac morphology and function sjvandijk@email.arizona.edu p/a-C1 -/- mice express a shorter cMyBP-C at normal levels 2A)2B) Acknowledgements This work is supported by AHA 13POST14780089 to SJvD and NIH HL080367 to SPH. Conclusion and Discussion 3A) 3B) 3C) 4) 1) References 1.Shaffer et al. 2009, J Muscle Res Cell Motil 30:303-6. 2.Lu et al. 2011, J Mol Biol 431:908-13. 3.Razumova et al. 2006, J Biol Chem 281:35846-54. 4.Witt et al. 2001, J Biol Chem 276:5353-9. 5.Shaffer et al. 2010, J Biol Biotechnol 789798. 6.Herron et al. 2006, Circ Res 98:1290-8.