Abstract 12435: Lack of Ca2+ Sensitization Induced by EMD 53998 in Engineered Heart Tissue Derived From Mybpc3-Knock-In and Mybpc3-Knock-Out Mice
Mutations in Mybpc3 encoding cardiac myosin-binding protein C cause hypertrophic cardiomyopathy (HCM), which likely involves increased Ca2+ sensitivity as a major pathomechanism. High phenotypic variability in HCM patients suggests a need for studying mechanisms and individual expression of the disease in vitro. We recently developed an automated 24-well fibrin-based engineered heart tissue (EHT) assay that provides a robust readout of force, frequency and rhythm and could therefore be a suitable in vitro test bed. Here we generated EHT from neonatal heart cells from Mybpc3-knock-out (KO) or Mybpc3-knock-in (KI) mice and compared to wild-type EHT (WT). Both homozygous KI and KO mice develop marked left ventricular hypertrophy and reduced fractional shortening over time. Spontaneous contractile activity of EHT was monitored repeatedly over up to 33 days and in a final measurement under electrical stimulation (5-6 Hz) and different Ca2+ concentrations. KO EHT (n=40) developed higher force and contraction velocity, and lower spontaneous frequency than WT (n=36) over time. Under electrical pacing, maximal force was reached at 0.6 mM Ca2+ in KO (n=15), compared to 1.0 mM Ca2+ in WT (n=11). The calcium sensitizer EMD 53998 (10 µM), added at 0.6 mM Ca2+, did not enhance force in KO, but increased it by ~50% in WT. Concomitantly, EMD 53998 decreased relaxation velocity by ~35% only in KO. KI EHT (n=44) also showed higher force over time than WT (n=12) and, in contrast to WT, did not increase force under EMD 53998. Moreover, 28% of KI EHT spontaneously developed a marked delay in relaxation (“cramps”). Taken together, KO and KI exhibited hypercontractility and unresponsiveness to the calcium sensitizer EMD 53998, KI additional intermittent relaxation deficits. The data recapitulate and extend former findings in HCM and suggest that the EHT platform is suitable to rapidly detect and study functional consequences of individual gene mutations, which will be particularly useful in combination with recent iPS technology.
- © 2011 by American Heart Association, Inc.