Stretch-induced remodeling of engineered human cardiac microtissues derived from patients with hypoplastic left heart syndrome

Introduction: : Hypoplastic left heart syndrome (HLHS) is a congenital heart defect characterized by an under-developed and non-functional left ventricle. For HLHS patients to survive, surgical intervention is performed to redirect systemic blood flow through the right ventricle. However, even after surgery, HLHS patients are still at increased risk for heart failure. A major challenge limiting improved patient outcomes is that HLHS is a polygenic disease with no clearly defined genetic or environmental risk factors and thus the mechanisms driving heart failure are poorly understood. For these same reasons, it is exceptionally challenging to model HLHS and patient-specific outcomes in animal models. More recently, HLHS has been modeled in vitro with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) sourced from HLHS patients, which preserve the genome of the patient. However, these cells have largely been investigated in standard monolayer culture, without physiological or pathological biophysical factors that are known drivers of cardiac development and disease. We hypothesize that cyclic mechanical stretch of the ventricle plays a key role in HLHS pathophysiology. To test this hypothesis, our goal is to engineer a “Heart On A Chip” model of HLHS with patient-derived iPSC-CMs and measure cardiomyocyte structure and function in response to mechanical stretch. These data will enable us to correlate cardiomyocyte phenotypes with patient genetics, helping improve our understanding of disease mechanisms and genetic risk factors and guiding therapeutic strategies to improve patient outcomes in the future.

Materials and

Methods: : iPSCs were reprogrammed from mononuclear cells isolated from umbilical cord blood from eight antenatally diagnosed HLHS children at birth (6 males, 2 females; acquired from the Todd and Karen Wanek Family Program for HLHS at Mayo Clinic). Two HLHS samples carry a full chromosome 2 duplication (1 male, 1 female) (HLHS3 and HLHS4). Two control iPSC lines derived from peripheral blood mononuclear cells (1 male, 1 female) were acquired from Coriell. Ventricular-specific cardiomyocytes (CMs) (Figure 1A) were generated using a kit from STEMCELL Technologies, and cardiac troponin-T expression was quantified by flow cytometry (Figure 1B). To mimic native cardiac tissue architecture, we used microcontact printing to pattern PDMS-coated coverslips with 30 µm × 30 µm fibronectin lanes and transferred control and HLHS iPSC-CMs onto micropatterned substrates (Figure 1C). In addition to supporting maturation, this uniaxial orientation facilitates sarcomere immunostaining with α-actinin to quantify length and organization (Figure 2). After 20 days of culture, tissues were incubated with Fluo-4AM calcium dye and electrically paced at 1 Hz; calcium transients were acquired using a fluorescence microscope and high-speed camera (100 fps) to capture 3 seconds of calcium waves (Figure 3A). We imaged 4–5 coverslips from 2–3 independent differentiations. Using custom Python code, each lane was isolated (65–70/coverslip) and transients analyzed. Next, we similarly micropatterned stretchable silicone membranes (Figure 3B), seeded with iPSC-CMs, and subjected tissues to 1 Hz 20% cyclic strain for 48 hours, after which pacing and calcium imaging were repeated. All samples underwent one-way ANOVA with multiple comparisons.

Results, Conclusions, and Discussions:: We observed cardiomyocyte differentiation efficiencies of approximately 75% (Figure 1B), with HLHS samples yielding slightly higher percentages. Both control and HLHS iPSC-CMs formed aligned tissues on micropatterned fibronectin substrates (Figure 1C). Sarcomere length was comparable across all control and HLHS lines (Figure 2B). However, sarcomere organization was significantly reduced in HLHS1 and HLHS2 compared to Control1 (one-way ANOVA, p = 0.004 and p = 0.001, respectively) (Figure 2C).
We next examined calcium transients (Figure 3A). Notably, HLHS samples without the chromosome 2 duplication (HLHS2) exhibited shorter times to peak with significance between Control, whereas those with the duplication (HLHS3, HLHS4) showed longer times to peak with significance between Control2 (one-way ANOVA, p = 0.001, p = 0.001, and p = 0.007, respectively). Calcium decay kinetics remained largely unchanged (data not shown).
We then investigated the impact of cyclic stretch on calcium transients. In control tissues, cyclic stretch shortened the time to peak, suggesting stretch-induced maturation (Figure 3B). In contrast, HLHS tissues showed minimal response to stretch. These findings suggest that HLHS iPSC-CMs may have impaired mechanosensing or mechanoadaptation, potentially contributing to limited left ventricular development and poor adaptation to single-ventricle physiology. These results will be validated with additional replicates, and RNA sequencing will be used to explore underlying mechanisms.
In summary, we engineered a patient-specific HLHS model incorporating mechanical stretch to isolate the effects of genetic and biophysical cues on cardiomyocyte pathophysiology. Preliminary data suggest HLHS2 displays particularly impaired cardiac function, including disrupted sarcomere organization, altered calcium handling, and poor stretch responsiveness. We plan to expand our dataset to assess the impact of stretch on sarcomere structure, contractile force, and gene expression in HLHS versus control samples.
In the future, this approach may also enable evaluation of patient-specific disease progression, prediction of cardiac outcomes, and screening of therapeutic interventions aimed at improving long-term function in single-ventricle hearts.

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