Human iPSC derived cardiac myocytes and sympathetic neurons in disease modelling

Background: Human induced pluripotent stem cells (hiPSCs) offer an unprecedented opportunity to generate a potentially unlimited source of cells to develop model systems that facilitate a mechanistic understanding of human disease. However, the predictive ability of hiPSC derived neurocardiac co-culture systems to recapitulate the human phenotype in diseased modelling is limited. Here, we optimized current methods for efficient and replicable induction of cardiac myocytes (hiPSC-CMs) and sympathetic neurons (hiPSC-SNs). The utility of healthy co-cultures was tested with pressor agents to develop a model of cardiac hypertrophy. Mono-cultures and co-cultures were also made from a patient with a catecholaminergic polymorphic ventricular tachycardia (CPVT) genotype (with isogenic pairing) to generate a model of triggered arrhythmia. Method: hiPSC-CMs and hiPSC-SNs were characterized by immunofluorescence, flow cytometry and calcium (Ca2+) imaging. Healthy hiPSC-CMs were incubated with angiotensin II (AngII) or endothelin-1 (ET-1) for 48 hours. Immunostaining, qPCR and ELISA were performed to measure cell size, pro-BNP expression and secretion in hypertrophic and control groups. cAMP was measured by Förster resonance energy transfer (FRET). Neurocardiac co-cultures were established by re-seeding spontaneously beating hiPSC-CMs (day 20) with mature hiPSC-SNs (day 40). Intracellular Ca2+ transients were measured in myocytes in response to isoprenaline, and in neurons in response to nicotinic stimulation. Results: Healthy hiPSC-SNs possessed neurite outgrowth, stained positive for PHOX2B, tyrosine hydroxylase and peripherin. Derived myocytes showed spontaneous beating, stained positive for cardiac troponin T and a-actinin. Cell surface area was significantly increased in older iPSC-CMs (day 40) (606.3±290.4μm² vs 3299.8±967.9μm², p<0.0001) compared to younger cells (day 20). Sarcomere length extended from 1.59μm to 1.88μm (p<0.0001). Healthy hiPSC-CMs exposed to AngII 0.1 μM (n=23), or ET-1 100nM (n=13) resulted in cell and nuclear enlargement, as well as enhanced proBNP gene expression and proBNP secretion. This overexpression was reversed by losartan in AngII treated cells. Cytoplasmic cAMP levels were increased by isoprenaline, which was higher in hypertrophic cells. CMs from the CPVT hiPSC line expressed a higher Ca2+ responsive to isoprenaline, caffeine and KCl stimulation when compared with healthy hiPSC-CMs. They also displayed spontaneous Ca2+ oscillations after isoprenaline. CPVT hiPSC-SNs had greater Ca2+ transients to nicotinic stimulation, indicating a diseased phenotype also resides in the neuron as well as the myocyte. Conclusion: We have recapitulated many features of the anatomy and (patho)physiology of SN and CM, where co-culture preparations behave in a manner that mimics key physiological responses seen in other mammalian systems. Whether our cell types have the full transcriptomic atlas of actual human cells remains to be established. CSC-COI Scholarship Programme, British Heart Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.