(P017) WORSENED VIABILITY, METABOLISM AND RESPONSE TO HYPOXIC INJURY IN INDUCED PLURIPOTENT STEM CELL-CARDIOMYOCYTES DERIVED FROM PATIENTS WITH DILATED CARDIOMYOPATHY

Background: Heart failure can be caused by cardiomyopathy of diverse etiologies. Despite this, current guidelines dictate identical guideline-directed medical therapy for all patients with heart failure with reduced ejection fraction. Hypoxia-mediated injury is a prevalent form of acquired cellular damage in cardiomyocytes which leads to necrosis and triggering of apoptotic pathways. Patient-specific differences in response to hypoxic injury remain poorly understood. Recently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have gained traction to model heart disease in vitro. We show that iPSC-CMs derived from patients with dilated cardiomyopathy (DCM) exhibit altered beating rate, viability, metabolic activity, and calcium handling compared to healthy controls. We hypothesize DCM patient-derived iPSC-CMs have a greater response to hypoxic injury, measured by hypertrophy and mitochondrial dysfunction.

METHODS AND RESULTS: We generated iPSCs (n=4 healthy controls, n=8 DCM) from peripheral blood through transfection of reprogramming factors (Oct4, Sox2, Lin28, Klf4, L-Myc). Cell line quality was assessed via immunocytochemistry, RT-qPCR, trilineage differentiation and karyotyping. After iPSC-CM differentiation, we confirmed expression of cardiac markers and lack of pluripotency markers via immunocytochemistry. Hypoxic conditions were optimized by subjecting iPSC-CM lines to 0%, 0.4% or 1% oxygen for 6, 12 or 24 hours. Crystal violet and AlamarBlue assays were used to assess viability and metabolic activity. Having optimized the best injury conditions, we performed mitochondrial staining for imaging of iPSC-CMs with confocal microscopy.

Results show iPSC-CMs from patients with DCM had significantly lower expression of CXN43 (p < 0.01) and SERCA2a (p < 0.001) compared to healthy controls. Additionally, contractility rate (beats per minute) was significantly higher in DCM iPSC-CMs than in controls, over a period of 10 days. DCM iPSC-CMs also had significantly decreased viability and metabolic activity compared to controls, under normoxic (p < 0.01) and hypoxic conditions (p < 0.001). Preliminary data also shows differences in cell area (p < 0.01) and in mitochondrial localization in iPSC-CMs from patients with DCM.

Conclusion: We utilize a translational ‘Heart-in-a-Dish’ model to examine causes of clinical heart disease at the cellular level in patients with DCM. With in-depth characterization of iPSC-CM lines from patients with DCM, we demonstrate that hypoxic injury can recapitulate a diseased phenotype and be used to further explore underlying molecular mechanisms. By comparing iPSC-CMs from patients with DCM to normal controls, we confirm a diseased phenotype at baseline and worsened response to hypoxic injury. These findings pave the way for the identification of individualized molecular pathways underlying the pathogenesis of DCM and future personalized therapies.