Unraveling common pathogenic pathway underlying the formation of hypoplastic left heart syndrome

Fonoudi H1,2, Bosman A1, Humphreys D1, Patrick R1, Blue G3, Hill A1, Ho J1, Winlaw D3 and Harvey R1,2

  1. Victor Chang Cardiac Research Institute, Sydney.
  2. St. Vincent׳s Clinical School, University of New South Wales, Sydney.
  3. 3. The Heart Centre for Children, The Children’s Hospital at Westmead, Sydney.

Hypoplastic left heart syndrome (HLHS) is a genetically complex disease, characterized by hypoplasia of the left side of the heart. Although being one of the most severe forms of congenital heart defects, our knowledge of the molecular underpinnings is very limited. Here, we have generated an in vitro model of HLHS using human induced pluripotent stem cells (hiPSCs) to uncover disease-causing factors. hiPSCs were generated from 10 HLHS patients and their parents (3 clones per individual; 87 hiPSC lines in total). To investigate differences during early cardiovascular development, hiPSCs were differentiated using both embryoid body and small moleculte cardiac-directed differentiation methods, and their cellular populations and gene expression were studied. Gene expression analysis of spontaneously differentiated cells showed lower expression of both cardiac and vascular smooth muscle markers in patients compared to controls. Flow cytometry analysis performed on hiPSC cultures after directed cardiac differentiation at 5-day intervals (day 0-30) showed cardiomyocyte differentiation in HLHS-hiPCSs was perturbed. Time-course RNA-seq of 5 HLHS families revealed down-regulation of cell cycle. This was confirmed using another 5 indipendent HLHS families. Cell phenotyping also indicated that beating cardiomyocytes derived from patients were immature and their calcium flux properties were significantly different. In summary, our findings suggest that the progression of cardiogenesis and vasculogenesis in HLHS-hiPSCs is perturbed, which may include problems in the cell cycle. Furthermore, the functionality of cardiomyocytes derived from HLHS-hiPSCs with respect to calcium flux properties was altered, suggestive of cardiomyocyte immaturity.