Abstract 15929: Heterozygous Expression cMyBP-C E258K Mutation Disrupts Contractile Function in Murine Engineered Cardiac Tissue Model
The cardiac myosin binding protein C (cMyBP-C) missense mutation E258K causes hypertrophic cardiomyopathy with highly variable disease severity amongst affected individuals. We developed a murine engineered cardiac tissue model (ECT) in which exclusive human cMyBP-CE258K expression resulted in decreased twitch force and maximally accelerated twitch kinetics. The human disease, however, is heterozygous implicating that coexpression of mutant and wild-type protein might impact disease severity. We tested the hypothesis that simultaneous expression of cMyBP-CE258K and wild-type cMyBP-C would impact contractile performance. We simultaneously transduced cMyBP-C null neonatal murine cardiomyocytes with virus containing either the human wild-type cMyBP-C (WT) or E258K mutation over a range of titers, and made ECTs by adding the cells to collagen and matrigel and casting into cylindrical constructs using a Flexcell system. After one week in culture the ECTs were mounted on a force transducer and force magnitude and kinetics were measured at 6 Hz electrical pacing at 37C. Protein was extracted and the ratio of WT to E258K expression quantified by immunoblotting using anti-HA and MYC antibodies.
ECTs with a 50:50 ratio of E258K:WT were compared to E258K alone, and WT (n=4 per group). Exclusive expression of E258K compared to WT had decreased force (0.20+/-0.02 vs. 0.74+/-0.04 mN, p<0.001), accelerated contraction time (0.05+/-0.002 vs. 0.04+/-0.005 s, p<0.05), and accelerated early relaxation time (0.04+/-0.002 vs. 0.03+/-0.001 s, p<0.05). The heterozygous E258K compared to WT had a greater decrease in force than E258K alone (0.03+/-0.001 s, p<0.001 vs. WT), and intermediate acceleration in contraction (0.05+/-0.002 s, p =0.17 vs. WT), and relaxation (0.03+/-0.001 s, p<0.05 vs. WT). In conclusion, the 50:50 expression of E258K:WT cMyBP-C results in accelerated contractile kinetics that are intermediate to the WT and homozygous E258K, while the twitch force amplitude is significantly depressed. These data indicate that heterozygous expression of mutant cMyBP-C modifies contractility and suggests a potential role for a dose-effect of mutant protein in modifying disease severity.
- © 2013 by American Heart Association, Inc.