title: Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice.
abstract: Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in engineered heart tissue (EHT) and yield novel mechanistic information on the function of cMyBP-C. EHTs were generated from cardiac cells of neonatal KI, heterozygous (HET) or wild-type controls (WT) and developed without apparent morphological differences. KI had 70% and HET 20% lower total cMyBP-C levels than WT, accompanied by elevated fetal gene expression. Under standard culture conditions and spontaneous beating, KI EHTs showed more frequent burst beating than WT and occasional tetanic contractions (14/96). Under electrical stimulation (6Hz, 37°C) KI EHTs exhibited shorter contraction and relaxation times and a twofold higher sensitivity to external [Ca(2+)]. Accordingly, the sensitivity to verapamil was 4-fold lower and the response to isoprenaline or the Ca(2+) sensitizer EMD 57033 2- to 4-fold smaller. The loss of EMD effect was verified in 6-week-old KI mice in vivo. HET EHTs were apparently normal under basal conditions, but showed similarly altered contractile responses to [Ca(2+)], verapamil, isoprenaline and EMD. In contrast, drug-induced changes in intracellular Ca(2+) transients (Fura-2) were essentially normal. In conclusion, the present findings in auxotonically contracting EHTs support the idea that cMyBP-C's normal role is to suppress force generation at low intracellular Ca(2+) and stabilize the power-stroke step of the cross bridge cycle. Pharmacological testing in EHT unmasked a disease phenotype in HET. The altered drug response may be clinically relevant.
authors.affiliation: Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Stöhr
authors.forename: Felix W
authors.initials: FW
authors.lastname: Friedrich
authors.forename: Frederik
authors.initials: F
authors.lastname: Flenner
authors.forename: Birgit
authors.initials: B
authors.lastname: Geertz
authors.forename: Alexandra
authors.initials: A
authors.lastname: Eder
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schaaf
authors.forename: Marc N
authors.initials: MN
authors.lastname: Hirt
authors.forename: June
authors.initials: J
authors.lastname: Uebeler
authors.forename: Saskia
authors.initials: S
authors.lastname: Schlossarek
authors.forename: Lucie
authors.initials: L
authors.lastname: Carrier
authors.forename: Arne
authors.initials: A
authors.lastname: Hansen
authors.forename: Thomas
authors.initials: T
authors.lastname: Eschenhagen
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics / Q000503:physiopathology; D002352:Carrier Proteins / Q000235:genetics*; D004195:Disease Models, Animal; D018390:Gene Targeting; D042541:Intracellular Space / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D009200:Myocardial Contraction / Q000187:drug effects* / Q000235:genetics*; D009206:Myocardium / Q000378:metabolism*; D023822:Tissue Engineering; D059467:Transcriptome; D014700:Verapamil / Q000494:pharmacology; D000099163:Myosin Binding Protein C
chemical_list: D000318:Adrenergic beta-Agonists; D002118:Calcium; D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D007545:Isoproterenol; D014700:Verapamil; D000099163:Myosin Binding Protein C
keywords: ATPase, Ca(2+) transporting, cardiac mRNA; Acta1; Alpha skeletal actin protein; Atp2a2; BPM; Beats per minute; Cardiac myosin-binding protein C; Cardiac myosin-binding protein C (cMyBP-C); Contraction time (spontaneous beating); Diastolic left ventricular internal diameter; Disease modeling; EC(50); EHT; EMD; EMD 57033; Engineered heart tissue; Engineered heart tissue (EHT); HCM; HET; Half maximal effective concentration; Half maximal inhibitory concentration; Heterozygous Mybpc3-targeted knock-in mice; Homozygous Mybpc3-targeted knock-in mice; Human cardiac myosin-binding protein C gene; Hypertrophic cardiomyopathy; IC(50); ISO; Induced pluripotent stem cells; Isoprenaline; KI; LVIDd; LVIDs; LVMBW; Left ventricular mass/body weight ratio; MYBPC3; Mouse cardiac myosin-binding protein C gene or mRNA; Mybpc3; Myh6; Myh7; Myofilament Ca(2+) sensitivity; Relaxation time (spontaneous beating); Slc8a1; Sodium calcium exchanger; Systolic left ventricular internal diameter; T1; T2; TTP; TTP50; TTR50; TTR90; Time to 100% of maximal twitch force; Time to 50% of maximal twitch force; Time to 50% of relaxation; Time to 90% of relaxation; cMyBP-C; iPSC; α-myosin heavy chain, α-MHC gene or mRNA; α-skAct; α-skeletal actin gene or mRNA; β-myosin heavy chain, β-MHC gene or mRNA