Nathalie Gaborit, Guillaume Lamirault, Patricia Lemarchand

Pursuing our work on deciphering the roles of IRX5 on cardiac function, we are now performing global transcriptomic and epigenetic studies on control and IRX5 mutated hiPSC-CMs, as well as on an isogenic line in which we deleted IRX5 gene using CRISPR technology. We are also investigating which other factors bind to IRX5 in a complex, to regulate gene expression, and already identified GATA4, TBX5 and IRX3. Altogether, this work should give further insight into the mechanisms by which IRX5 ensures proper development and function of human cardiomyocytes.

We then intend to pursue and go beyond the previous strategy to decipher the role of transcriptional regulatory elements in unknown mechanisms of genetically complex cardiac diseases. BrS is a major cause of SCD in young adults but what underlies its pathophysiology is still unclear. It has a complex mode of inheritance and screening of rare variants in coding loci is not sufficient to explain the disease risk. Interestingly, recent genetic studies conducted by team I are changing the paradigm of BrS inheritance, revealing a strong cumulative impact of 21 common haplotypes located in non-coding regions. The dominant postulate is that these non-coding haplotypes are transcriptional regulatory elements. Of further interest, 10 of the non-coding risk haplotypes are located near 8 cardiac transcription factors (TF) genes, including IRX3/5 and GATA4. Each of these have been implicated in the regulation of cardiac function, and for some, in the protection from rhythmic disorders. However, none have been functionally associated to BrS. Building from our previous research achievements, we will study the risk haplotypes located near IRX3/5 and GATA4, with the hypothesis that they are involved in BrS pathophysiology by altering their expression. Extending the concept to a haplotype located near a TF for which the role in human cardiomyocyte electrical function has not been yet revealed, we will also study the one near ZFPM2, known to interact with GATA4 in the heart.

Our goal is to functionally evaluate the relevance of these common non-coding haplotypes in the regulation of cardiac physiology and their impact on the disease risk. In this context, N Gaborit envisions a 2-year sabbatical in Mark Mercola Lab (Stanford University) to assemble all essential scientific expertise needed to embark on this challenging task. The acquired strong knowledge on an array of complementary hiPS-CMs approaches, which are at the forefront of stem cell research priorities, will then be transferred to the ITX:

  • CM maturation to get cells with an adult-like phenotype, more relevant to the study of cardiac arrhythmias.
  • 3D bioengineering to study CMs in an in-vivo-type environment.
  • Genome editing to generate appropriate isogenic lines and to attempt corrective strategies.
  • High throughput approaches to record, population-wide, the activity of CMs.
  • Automated arrhythmia classification by machine learning for high-throughput screens.

We will first study the impact of the deletion of the selected risk haplotypes on genome-wide gene expression, which will confirm their role as transcriptional regulatory elements and identify their targets in mature hiPSC-CMs. We will then investigate the impact of the haplotype deletions on CM function. However, efficient and reliable analysis of hiPSC-CMs functional phenotype is challenging. The new high-throughput functional phenotypic screening applications using mature hiPSC-CMs that have been developed by Mercola lab, will allow the parallel study of the multiple hiPSC lines. The main originality of this work is that it will test a detailed mechanistic hypothesis that should explain why the known BrS rare variants in coding loci are insufficient to explain the disease risk. Deciphering the role of common haplotypes in non-coding regions will have profound implications for understanding the pathophysiological mechanism of BrS, and beyond, on the regulation of physiological cardiac functions.