A Dynamic and Three-Dimensional Human Model of Hypoplastic Left Heart Syndrome to Probe Compromised Cardiomyocyte Proliferation

PROJECT SUMMARY/ABSTRACT
Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect in which the left side of the heart is
underdeveloped, with structural malformations to the ascending aorta, aortic/mitral valves, and a
characteristically small left ventricle. These anatomical abnormalities restrict proper blood circulation in infants
and can be lethal if left untreated. While palliative procedures have improved the prognosis of HLHS patients,
many still suffer downstream morbidities and a diminished quality of life. Unfortunately, the etiology of HLHS
remains elusive to scientists and clinicians. Genetic and environmental factors are thought to contribute to the
development of the disease; however, the exact cellular processes that modulate HLHS pathogenesis still need
to be elucidated. An inadequate understanding of the syndrome is exacerbated by limited experimental models
that can recapitulate and manipulate key features of HLHS. Restricted ethical access to human embryos, and
fundamental differences between animals and humans have failed to produce models that mimic clinical
phenotypes. Furthermore, standard 2D in-vitro cell culture spatially restricts tissue growth, which does not fully
recap three-dimensional development in nature. When compared to 2D culture, cardiomyocytes grown in 3D
have shown enhanced sarcomeric structure, contractility, mitochondrial respiration, cellular alignment, and
electrophysiological characteristics. Therefore, there exists a need to develop alternative strategies to study
abnormal cardiac development in-vitro. Tissue engineering techniques such as 3D bioprinting offer a unique
platform to deposit cells and biomaterials that mimic the extracellular matrix of tissues in an architecturally
controlled fashion. In addition to geometric control, the properties of these cell-laden hydrogels, such as stiffness,
can be controlled. Moreover, advances in iPSC technology offer an in-vitro biological system that retains genetic-
and disease-specific information from donors which provides a tool to probe genetically inheritable and
developmental diseases. Leveraging these technological advances, our proposed study aims to utilize a 3D-
bioprinted heart tube patterned into an endocardial layer (with iPSC-derived endothelial cells) and myocardial
layer (with iPSC-derived cardiomyocytes) to interrogate how dysregulated fluid-induced biomechanics and
microenvironmental stiffness impedes cardiac proliferation in-vitro. In this regard, we hypothesize aberrant
biomechanical forces induce stress-related endocardial-myocardial signaling that ultimately impedes
cardiomyocyte proliferation. We will test our hypothesis by (1) selectively varying endocardial stiffness within our
disease-specific 3D model and assessing intercellular signaling that dysregulates cardiomyocyte proliferation,
and (2) by applying varying degrees of flow-induced shear stress to the endocardial layer and studying
transcriptional shifts that occur in response to these perturbations at the single-cell level. Successful completion
of these aims will give novel etiological insights into how dysregulated biomechanical cues in HLHS impedes
cardiac growth and could shed light into alternative approaches to treat ventricular hypoplasia.

Grant Number: 5F32HL170581-02
NIH Institute/Center: NIH
Principal Investigator: Matthew Alonzo