In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants
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| Authors: | Shafaattalab, S; Li, AY; Lin, E; Stevens, CM; Dewar, LJ; Lynn, FC; Sanatani, S; Laksman, Z; Morin, RD; van Petegem, F; Hove-Madsen, L; Tieleman, DP; Davis, JP; Tibbits, GF |
| Year: | 2019 |
| Journal: | Proc. Natl. Acad. Sci. U. S. A. 116 Article Link (DOI) PubMed |
| Title: | In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants |
| Abstract: | Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1 R37C(+/-)) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in TNNI1 may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the TNNI1 R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C Tnl exhibited reduced Ca2+-binding sensitivity due to an increased Ca(2+)off-rate constant. Furthermore, we generated TNNI1 R37C(+/-)mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+ transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in TNNI1 R37C(+/-)at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI. |
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