ID: 136: N-TERMINAL REGION OF CARDIAC MYOSIN BINDING PROTEIN-C IS NECESSARY FOR CARDIAC FUNCTION

Rationale Cardiac myosin binding protein-C (cMyBP-C) is a trans-filament protein that has been shown to regulate cardiac function via its amino terminal (N′) region. In vitro studies have suggested the importance of the first 271 N′-residues of cMyBP-C (C0-C1f region) in slowing actin filament sliding over myosin to regulate cross-bridge cycling kinetics within the cardiac sarcomere. However, the role and necessity of the C0-C1f region of cMyBP-C in regulating contractile and cardiac function in vivo have not been elucidated. Hypothesis The N′-C0-C1f region of cMyBP-C is critical for proper cardiac function in vivo . Methods and Results Transgenic mice with approximately 95% expression of a mutant truncated cMyBP-C missing the N′-C0-C1f region (cMyBP-C 110 kDa ), compared to endogenous cMyBP-C, were generated and characterized at 3-months of age. cMyBP-C 110 kDa hearts had significantly elevated heart weight/body weight ratio, fibrosis, nuclear area and collagen content compared to hearts from non-transgenic (NTG) littermates. Electron microscopic analysis revealed normal sarcomere structure in cMyBP-C 110 kDa hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C 110 kDa hearts. Short axis M-mode echocardiography revealed a significant increase in left ventricular (LV) internal diameter during diastole in cMyBP-C 110 kDa hearts. Importantly, cMyBP-C 110 kDa hearts displayed a significant reduction in fractional shortening compared to hearts from NTG mice. We further observed a decrease in the thickness of the LV interventricular septum and free wall during systole in cMyBP-C 110 kDa hearts. Strain analysis using images acquired from ECG-Gated Kilohertz Visualization identified a significant deficit in global longitudinal strain in cMyBP-C 110 kDa hearts compared to NTG hearts. Consistent with cardiac hypertrophy, we observed a significant increase in the expression of the hypertrophic genes MYH7 and NPPA by real-time PCR analysis. As expected, the expression levels of the MYBPC3 gene were significantly elevated in cMyBP-C 110 kDa hearts compared to NTG hearts. Surprisingly, our Western blot analyses revealed no significant difference in total cMyBP-C levels between NTG and cMyBP-C 110 kDa heart homogenates. However, intriguingly, we observed a significant elevation in cMyBP-C phosphorylation at Ser-273, Ser-282, and Ser-302, sites important for cMyBP-C9s regulation of actomyosin interaction, in cMyBP-C 110 kDa heart homogenates compared to those from NTG mice. Conclusion The N′-C0-C1f region of cMyBP-C is essential for maintaining normal cardiac morphology and function in vivo and loss of this region promotes contractile dysfunction both at the molecular and tissue level.