Poster BB6 - 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 primarily affecting the left side of the heart, resulting in a non-functional left ventricle. For these patients to survive, surgical intervention redirects systemic blood flow through the right ventricle. However, after surgery, HLHS patients have an increased risk for heart failure such that transplant-free survival at 6 years is 60%. One likely contributing factor to heart failure is cardiomyocytes (CMs) of the right ventricle are under excessive strain due to univentricular circulation. However, the progression to heart failure in HLHS remains poorly characterized as HLHS is a polygenic disease with no single gene or environmental factor. Thus, existing animal models are limited in their ability to recapitulate patient-specific outcomes and disease progression. Furthermore, existing in vitro models for HLHS, that utilize induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in standard monolayer culture, have not assessed how patient genetic defects impact cardiomyocyte remodeling in response to pathological biophysical factors, such as high-strain.
Our goal is to engineer a “Heart On A Chip” model of HLHS to measure cardiomyocyte function in pathophysiological conditions and correlate phenotypes with patient genetics to determine disease mechanisms and identify patients who may be at higher risk for heart failure. We hypothesize that under pathological levels of strain, compared to healthy, HLHS iPSC-CMs exhibit altered contractile phenotypes, such as sarcomere organization and calcium handling. To test this our preliminary work has focused on developing assays to assess baseline HLHS patient-derived iPSC-CM phenotypes in the absence of strain.

Materials and

Methods: iPSCs were derived from mononuclear cells isolated from the umbilical cord blood from eight antenatally diagnosed HLHS children at birth (6 Males, 2 Females; a generous gift from Mayo Clinic). Whole Exome Sequencing data for these patients was also provided. Three control iPSC cell lines were derived from healthy peripheral blood MCs (1 Male, 2 Female). We successfully created ventricular-specific cardiomyocytes (CMs) from these iPSCs using a kit from STEMCELLTM Technologies and used metabolic selection to further purify and mature the iPSC-CMs. To mimic native cardiac tissue architecture, we used microcontact printing to pattern substrates with 30um x 30um lanes of fibronectin. We then optimized the dissociation and transfer of healthy and HLHS iPSC-CMs onto micropatterned substrates. In addition to benefitting maturation, this configuration is well-suited for immunostaining sarcomeres with α-actinin and quantifying sarcomere architecture because the cells are uniaxially oriented. After day 20, we used fluorescence-based calcium imaging to measure spontaneous and electrically paced calcium activity in HLHS and healthy iPSC-CMs. Briefly, cells were incubated with Fluo-4 calcium indicator, and images of regions with transients were captured at 100fps. We have also begun adapting these assays to assess adaptations in response to cyclic stretch in micropatterned HLHS and healthy iPSC-CMs using the Cytostretcher from CuriBio.

Results, Conclusions, and Discussions: Whole Exome sequencing from the HLHS patients showed no statistically significant enriched pathways, another indicator of the genetic heterogeneity. However, some of the genetic mutations in these patients had strong associations with pathways implicated in actin organization (RHO/GTPases) and ciliopathies (Figure 1A). We derived ventricular-specific iPSC-CMs as demonstrated by the expression of Myosin Light Chain 2 (Figure 1B). Using flow cytometry, we quantified cardiac troponin expression in our samples, with both Healthy and HLHS yielding similar CM differentiation efficiencies (Figure 1C). Additionally, micropatterned iPSC-CMs show alignment and orientation, similar to cardiomyocytes in the heart (Figure 1D). We evaluated sarcomere organization and length (Figure 1E) and our preliminary experiments (n=1) showed no difference in sarcomere organization. However, HLHS CMs did show longer sarcomere lengths when compared to Healthy (not shown). Using fluorescent live calcium imaging, we then evaluated calcium dynamics and parameters such as time to peak, amplitude, time to decay, and slope of decay (Figure 1F). We also electrically paced the tissues at native cardiac frequencies, such as 1Hz (Figure 1G). We observed that HLHS iPSC-CMs have lower spontaneous electrical activity compared to Healthy (Figure 1G). In most of our HLHS samples with pacing, the inherent calcium dynamics, such as time to peak and decay, were not adversely impacted. However, in one of our HLHS patients, we did observe a shorter time to peak (not shown). We are currently expanding this work to incorporate the effect of pathological cyclic strain, compared to physiological cyclic strain, in Healthy and HLHS cardiac microtissues. We plan to assess these phenotypes and correlate them to patient genotypes in hopes of identifying mechanisms that are associated with the progression to heart failure in HLHS patients. Lastly, this model can be used as a platform to screen potential drug therapies and environmental risk factors associated with single ventricle failure.

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