Characterisation of sympathetic neurons and cardiomyocytes derived from human pluripotent stem cells and their therapeutic mechanism

Heightened sympathetic drive is a powerful negative prognostic indicator for morbidity and mortality associated with arrhythmia and sudden cardiac death. In the heart, cardiac channelopathy, in particular catecholaminergic polymorphic ventricular tachycardia (CPVT), underpin a significant portion of lethal arrhythmia triggered by adrenergic stimulation. However, the molecular targets underlying dysautonomia remain unclear, and no therapeutic pathways have been validated against human tissue. 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. Chapter 1 introduces the autonomic innervation of the heart and how sympathetic dysfunction is related to cardiac arrhythmia in cardiac channelopathies. Intracellular calcium ([Ca2+]) is a critical modulator of neuronal function, which is subjected to cyclic nucleotides and membrane potential regulation. The development of hiPSCs is also reviewed, leading to the main of the aim of the thesis, which is concerned with testing the hypothesis that CPVT is also a disease of cardiac sympathetic neurons. Chapter 2 explains the methods used in the following results chapters. While primary human LQTS/CPVT cells are largely unavailable, hiPSCs have emerged as a tool to model inherited arrhythmias. In this chapter, methods for efficient and replicable induction of hiPSC-CMs and -SNs and phenotyping of these cells are provided. Chapter 3 depicts the physiological phenotypes of cardiac myocytes and sympathetic neurons derived from hiPSCs. They morphologically resemble primary cells, expressing tissue-specific markers. The electrophysiological properties of myocytes and the neurotransmission of sympathetic neurons are investigated. Chapter 4 demonstrates a model of cardiac hypertrophy using iPSC-CMs. Healthy iPSC-CMs develop anatomical alteration and beta hyper-responsiveness with angiotensin II. The results validate the utility of hiPSC in disease modelling when compared against a rat model. Chapter 5 Monocultures and co-cultures were made from hiPSC with a CPVT genotype (and isogenic pairing) to generate a model of triggered arrhythmia. Neuronal Ca2+ transients were enhanced in CPVT cells, which inspired further investigation into underlying dysregulated molecules, in particular cyclic nucleotide signalling (cAMP) and membrane excitability (M current). Chapter 6 provides a single-cell RNA sequencing dataset profiling transcriptome of hiPSC-differentiated cardiac myocytes and sympathetic neurons. Differential gene expression analysis discovered dysregulated genes in the CPVT sympathetic neurons. Chapter 7 is the concluding discussion that summarizes all the results, suggesting the applicability of hiPSC in disease modelling and the triggering role of sympathetic neurons in CPVT. It also highlights potential pathways for therapeutic intervention.