Engineering A Patient-specific Heart On A Chip Model Of Hypoplastic Left Heart Syndrome

Introduction:: Hypoplastic left heart syndrome (HLHS) is a congenital heart defect with variable degrees of underdevelopment of the left side of the heart. Surgical palliation of HLHS requires the right ventricle to perform as the systemic ventricle, under high strain and demand. This adversely impacts right ventricle cardiomyocytes increasing the risk for heart failure such that HLHS single ventricle transplant-free survival at 6 years is 60%. Some HLHS patients are at high risk for developing heart failure in response to the increased load and strain on a developmentally disadvantaged right ventricle. As HLHS is a polygenic disease with no single gene or environmental factor, existing animal models fail to recapitulate patient heterogeneity in post-operative survival and response to therapies.
Our objective is to create an in vitro “Heart On A Chip Model” of HLHS to correlate differences in cardiomyocyte function with patient genotype to identify single ventricular patients that are at higher risk for progression to heart failure. Our model can also be expanded to evaluate HLHS patient-specific functional responses to environmental factors common in HLHS, such as hypoxia and high strain, as well as predict responses to existing single ventricle therapies, such as digoxin.


Materials and Methods:: Clinical collaborators at the Mayo Clinic provided us with Induced Pluripotent Stem Cells derived from mononuclear cells (MCs) isolated from the umbilical cord blood from eight antenatally diagnosed HLHS children at birth (6 Males, 2 Females). Whole Exome Sequencing data for these patients were also provided. Three control cells were obtained from healthy peripheral blood MCs (2 Males, and 1 Female). We successfully created ventricular-specific cardiomyocytes from these cells using a commercially available kit from STEMCELL^TM Technologies. Additionally, using metabolic selection, we purified and matured the cardiomyocytes. After day 15, we used fluorescence-based calcium imaging to study the spontaneous calcium activity in 1 HLHS and 1 healthy line of cardiomyocytes. Briefly, cells were incubated using Fluo-4 calcium indicator, and image sequences of 3-4 regions were captured at 100fps. Transients were analyzed using an automated Python analysis adapted by our lab. In parallel, we are developing micropatterning techniques to align cardiomyocytes to mimic native tissue structure and organization. We will use these micropatterned tissues to evaluate sarcomere maturity and contraction.


Results, Conclusions, and Discussions:: We derived ventricular-specific human cardiomyocytes, as demonstrated by the expression of Myosin Light Chain 2 (Figure 1A). The onset of beating was delayed by an average of 6.85 ± 1.7 days in HLHS cardiomyocytes compared to controls, suggesting HLHS mutations reduce the efficiency of cardiac differentiation (Figure 1B). We also observed that calcium transients in HLHS- cardiomyocytes are altered, with reduced beats per minute (bpm) and increased rise and decay times compared to healthy controls (Figure 1C-D). Additionally, we have micropatterned substrates with fibronectin to facilitate cardiomyocyte alignment, as observed physiologically (Figure 1E). These aligned tissues will be used to measure contractile forces in ongoing experiments. Pathway analysis of whole exome sequencing in these 8 HLHS patients shows that they carry mutations associated with the developmental and metabolic processes (Figure 1F). Some of these genes include cardiac developmental genes, such as MYO5, JAG1, and NEB, as well as genes associated with heart failure, such as C1GALT1C1, MYZAP, and PKHD1L1. Future work will be aimed at optimizing the micropatterning of cardiomyocytes for muscular thin films or traction force microscopy to assess their systolic and diastolic stress. Collectively, we will create a patient-specific and functionally relevant model to elucidate key cardiac phenotypes in HLHS. Importantly, these phenotypes can be correlated with patient genotypes to elucidate patient-specific mechanisms that govern the progression to heart failure in single ventricle HLHS patients. Lastly, this model can be expanded to investigate the underlying molecular mechanisms of HLHS as well as be used as a platform to screen potential drug therapies and environmental risk factors associated with single ventricle failure.


Acknowledgements (Optional): : HLHS Mayo Consortium: patient iPSC cell lines and whole exome data. Ichida lab, Jasper Rubin-Sigler, Jonathan Cheng: iPSC training and healthy patient lines. Stemcell Technologies, James Hyun: Cardiomyocyte differentiation assistance. Boris Milkov: Python code
assistance. Seth Ruffins, Imaging Core USC: Imaging Core. McCain lab, Antonina Maxey, Natalie Khalil: Calcium transients technical assistance. Subramanyan lab, JongKyu Choi (past), Drayton Harvey, and Anna Olds


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