In Vitro Analyses of Suspected Arrhythmogenic Thin Filament Variants as a Cause of Sudden Cardiac Death in Infants
This week we profile a recent publication in PNAS from Sanam Shafaattalab (pictured) in the laboratory of Dr. Glen Tibbits at SFU.
Can you provide a brief overview of your lab’s current research focus?
The Tibbits lab (SFU / BC Children’s Hospital Research Institute) works on the mechanisms of inherited (and acquired) cardiomyopathies and arrhythmias and personalized treatments thereof. We use an array of techniques to address these questions, but in the last seven years we have focused on the use of human induced pluripotent stem cells differentiated into beating heart cells, or cardiomyocytes (hiPSC-CMs). These hiPSC-CMs are often derived from patients who are harboring genetic variants thought to be causal in these pathologies and which may result in sudden cardiac arrest (SCA). By making monolayers of hiPSC-CMs which form functional syncytia in Petri dishes, Sanam Shafaattalab studied the electrical and contractile behavior of these hiPSCs. Arrhythmias are complex phenomena which require the use of an integrated multicellular yet highly defined system to understand their mechanistic bases. We have invested significant energy and resources into developing sophisticated optical mapping (OM) technologies to monitor simultaneously both voltage (action potentials) and cytosolic Ca2+ transients in real time in multiple regions of interest across the monolayer of hiPSC-CMs to give critical insight into the underpinnings of these electrical disturbances.
What is the significance of the findings in this publication?
The significance of these findings are several fold. First, a medical examiner from another province requested if we could determine a cause of death related to SCA by using NGS sequencing of tissues from 191 infants and very young children who died suddenly but whose autopsies, exhaustive toxicological screens and other detailed post mortem analyses proved to be negative. We (Dr. Laura Dewar et al.) selected 72 genes that were potentially associated with SCA and on sequencing found a number of mutations associated with ion channelopathies that are well documented to be potentially associated with SCA. However, most surprising was that 10 infants (all of whom died within 24 months of age) harboured a variant in a contractile protein gene (troponin I) paralog that is only expressed in the human heart for the first 2 years of life. This variant TNNI1 R37C+/- is of unknown pathogenicity and not being a channelopathy precluded the use of conventional electrophysiological techniques. Second, we developed a unique pipeline of experimental techniques that included both in silico (phylogenetic analyses and molecular dynamic simulations) and in vitro (recombinant protein reconstitution of the human cardiac contractile unit and hiPSC-CMs that were genomically edited with CRISPR Cas9 to include the TNNI1 R37C+/- variant and its isogenic control and analyzed with OM) techniques to ascertain the pathogenicity of this genetic variant. The TNNI R37C+/− variant in this study was reliably and reproducibly disruptive to the normal physiology, in contrast to that in isogenic controls, across the battery of in silico and in vitro assays described. Third, this strengthens the evidence of pathogenicity and implicates this neonatal gene paralog encoding a sarcomeric contractile protein as having likely contributed to SCA through a proarrhythmic pathway and allows health care providers to inform the parents. Lastly, this platform has the potential to be applied broadly as part of a standardized and clinically relevant death investigation in cases of SCA where novel variants are identified and perhaps more important play a critical role in preventing these occurrences in the future. The detailed hiPSC-CM work was an important part of the PhD thesis of Dr. Sanam Shafaattalab and was critical to the final conclusion; however, other members of the lab: Alison Li and Drs. Eric Lin and Charlie Stevens also made significant contributions.
This work was funded by:
The work was generously funded by the Stem Cell Network of Canada and the Canadian Institutes of Health Research to Glen F. Tibbits.