Hypoplastic Left Heart Syndrome (HLHS) is a congenital defect marked by an underdeveloped left ventricle (LV) that is unable to provide adequate blood flow to the body. Currently, the standard of care for HLHS patients is surgical palliation or cardiac transplantation, both of which have serious complications. Fetal echocardiograms demonstrate that decreased LV filling during development results in a hypoplastic LV, thereby indicating a biomechanical mechanism for HLHS. Our rationale for this project is that understanding the role of biomechanical responsive pathways in the pathogenesis of HLHS will shift the current management paradigm of HLHS patients by enabling pharmacological treatment of this defect. Our overarching goals are 1) to discover novel molecular modulators that increase LV size in HLHS patients, and 2) to build and validate multi-scale computational models to evaluate the efficacy of these modulators at the cellular and whole-organ level. We hypothesize that increasing the activity of stretch-activated growth pathways will improve the growth of hypoplastic LVs in utero. This hypothesis is based on our data that stretch stimulates cardiomyocyte proliferation, growth, and ventricular growth. Furthermore, utilizing miRNA-Seq, we have identified a microRNA, miR-486, that is 1) stretch responsive in vitro/HLHS patients and 2) promotes cardiac growth in vivo. To test our hypothesis and to achieve our overarching goals, we will perform two specific aims: Aim 1. Predict and validate the effects of miR-486 treatment on mouse and human embryonic hearts developing HLHS. We postulate that miR-486 increases embryonic cardiac growth. Thus this aim will examine miR-486 role in LV growth, both in murine and human HLHS embryos. miR-486 effects on cell proliferation, size, and contractile function will also be studied in mouse and human iPS cardiomyocytes. These in vitro data will be incorporated into a novel 3D finite element (FE) model of embryonic cardiac growth. Finally, treatment of HLHS mouse embryos with miR-486 in utero will be used to validate FE modeling simulations. Aim 2. Prioritization of candidate miRNAs for in vivo testing based upon computational modeling of the miRNA?s potential to improve embryonic ventricular growth. We postulate that miRNA target predictions, mathematic molecular model of cardiomyocytes, and HLHS FE modeling can be coupled to test candidate stretch-responsive miRNAs for potential to increase LV size in HLHS hearts. The candidate miRNA that increases LV the most growth within simulations of mouse HLHS embryos will be tested in vivo. This miRNA will be tested in utero, to 1) examine the effects on LV growth and 2) validate the coupled model. The proposed studies using complementary in vitro (murine and human iPS cells), in vivo, and in silico methods will elucidate critical pathways by which biomechanical stress stimulates cardiac growth. Such a unique comprehensive approach is made possible by a multidisciplinary team that incorporates expertise in pediatric cardiology, cardiac biomechanics, molecular biology, and computational modeling.