Impact of ANKRD1 mutations associated with hypertrophic cardiomyopathy on contraction parameters of engineered heart tissue

Abstract

Hypertrophic cardiomyopathy (HCM) is a myocardial disease associated with mutations in sarcomeric genes. Three mutations were found in ANKRD1, encoding ankyrin repeat domain 1 (ANKRD1), a transcriptional co-factor located in the sarcomere. In the present study, we investigated whether expression of HCM-associated ANKRD1 mutations affects contraction parameters after gene transfer in engineered heart tissues (EHTs). EHTs were generated from neonatal rat heart cells and were transduced with adeno-associated virus encoding GFP or myc-tagged wild-type (WT) or mutant (P52A, T123M, or I280V) ANKRD1. Contraction parameters were analyzed from day 8 to day 16 of culture, and evaluated in the absence or presence of the proteasome inhibitor epoxomicin for 24 h. Under standard conditions, only WT- and T123M-ANKRD1 were correctly incorporated in the sarcomere. T123M-ANKRD1-transduced EHTs exhibited higher force and velocities of contraction and relaxation than WT- P52A- and I280V-ANKRD1 were highly unstable, not incorporated into the sarcomere, and did not induce contractile alterations. After epoxomicin treatment, P52A and I280V were both stabilized and incorporated into the sarcomere. I280V-transduced EHTs showed prolonged relaxation. These data suggest different impacts of ANKRD1 mutations on cardiomyocyte function: gain-of-function for T123M mutation under all conditions and dominant-negative effect for the I280V mutation which may come into play only when the proteasome is impaired.

Rat engineered heart tissue: a novel tool in the safety pharmacology toolkit?

“…-channels [23]. Similarly, previous work by the authors of the present study indicated that mutations in the ankyrin repeat domain 1 (ANKRD1) gene associated with hypertrophic cardiomyopathy can affect contraction parameters of rat EHTs [2], and that EHTs from knock-in mice with a myosin-binding protein C binding show altered druginduced contractile parameters, but normal Ca 2? -transient properties [20].…”

Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice

“…The racks were then transferred to fresh 24-well plates containing culture medium (Dulbecco's modified Eagle's medium, 10% horse serum, 2% chick embryo extract, 1% penicillin/streptomycin, 10 mg ml À 1 insulin and 33 mg ml À 1 aprotinin) and maintained at 21% oxygen. On day 5 cytosine b-D-arabinofuranoside (25 mg ml À 1 ) was added to the culture medium for 48 h. AAV6 transduction (multiplicity of infection 1,000) was performed directly into the freshly isolated heart cells before adding reconstitution mix components and casting EHTs 43,44 . Contractility measurements were performed on day 19.…”

Cardiac Tissue Engineering

“…79 More recently, we used the model to overexpress potential disease-causing genetic variants of four and a half LIM domain protein 1, lamin A/C, or cardiac ankyrin repeat protein 1 in EHTs. [80][81][82] Functional differences induced by the different variants added to a better understanding of their role in disease. The group of Ralphe used ECTs from cardiac myosin-binding protein (cMyBP)-C knockout mice to characterize primary consequences of the HCM-related defect and reintroduced wild-type cMyBP-C by adenoviral overexpression to rescue the phenotype.…”