The role of neurohumoral modulation in stem cell derived cardiac cells

Sevenster, Dane

The role of neurohumoral modulation in stem cell derived cardiac cells

Thesis / Dissertation

2026

Publisher

University of Cape Town

Department

Department of Human Biology

Faculty

Faculty of Health Sciences

Abstract

Background Cardiovascular diseases remain a leading cause of death in the world making up 33% of all global deaths. A commonality of these cardiac pathologies is the complex network of maladaptive remodelling signals that can arise and worsen disease outcomes, including sympathetic overdrive. The sympathetic nervous system provides neurohumoral signals to facilitate physiological remodelling factors within the heart, but the hyperactivity of these modulators can contribute to the development of cardiac failure. Sympathetic modulators such as norepinephrine and epinephrine induce a host of regulatory and remodelling effects on cardiac beating rate, contractility, and intracellular calcium handling via proteins such as sarcoendoplasmic reticulum calcium ATPase 2 (SERCA 2) and ryanodine receptor 2 (RYR 2). These modulators do not account for the full range of maladaptive cellular remodelling seen in the different stages of heart disease. However, neuropeptide Y (NPY) is an abundant modulator that is co-released with norepinephrine, but its pathophysiological contributions at the cellular level remain unclear. Specially, NPY is implicated in cardiovascular maladaptation to stress but with unknown effects on cardiac contraction and calcium handling processes. This project aims to assess the effects of NPY in modulating functional, electrical, and morphological remodelling changes in stem cell derived cardiac-like cells. Method Cardiac cell-like, beating embryoid bodies (EBs) were derived from pluripotent mouse embryonic stem cells through in vitro cardiac differentiation using the hanging drop method. EBs were split into NPY treated (100nM NPY) or control (saline) groups. After 24 hours, an EVOS M5000 light microscope with an external camera was used to capture video recordings of the pulsatile EBs. These recordings were used to extract beating characteristics and contractile profiles through using the open-source motion detection plugin for ImageJ, Myocyter v1.5. The software required adaptation and validation for the cellular contractile measurements, given the 3-dimensional and multicellular nature of the EB model. Calcium transients were investigated using the fluorescent Ca2+ indicator, Fluo-4 AM and fluorescent microscopy and were analysed using the LC-Pro v1 ImageJ plug-in. The detection of nuclei and the proteins SERCA 2 and RYR 2 were investigated using immunocytochemistry, confocal microscopy (Carl Zeiss LSM880) and ImageJ analysis. Results Spontaneously beating EBs were successfully generated from mouse embryonic stem cells and qualitatively, remained viable in the presence of NPY treatment. The parameters of EB beating characteristics obtained using the motion detection software Myocyter were qualitatively similar to those derived from a commercially available motion detection software PulseTM (free trial version provided by CuriBio), with the correlational analysis of the data points showing a positive correlation (r=0.858, p <0,0001). NPY treatment of beating EBs significantly decreased the beating rate by approximately 50% as well as impaired the rhythmic properties with an increase in the systole durations and an increase in the diastole durations (p<0.0001; n≥32 EBs). Furthermore, NPY decreased the amplitude of contraction by 42% as well as the upstroke- and downstroke rates of contraction by more than 60%. However, the measurements of calcium transients showed no significant differences in amplitudes. Furthermore, semi-quantitative analysis of the distribution of SERCA 2 relative to the nuclei was even throughout cells and showed little to no change in density or association, however RYR 2 showed uneven distribution and decreased signal intensity throughout cells. Mean fluorescence intensity measurements normalized to the number of cells showed no significant change for both SERCA 2 and RYR 2. NPY did not induce any significant change in the number of cells within EBs as detected by nuclei staining between the groups. Conclusion Results showed NPY induced changes in the beating and contractile functions that would translate into alterations at a clinically significant level. Although RYR 2 exhibited a trend of decreased signal intensity and uneven distribution, these observations cannot fully account for the contractile alterations seen, as the calcium transient amplitudes and mean fluorescence intensity measurements of SERCA 2 and RYR 2 showed no significant changes. Clarity on the downstream factors involved will require further investigations. The findings from this study highlight the significant impact of NPY on cardiac EBs derived from mouse embryonic stem cells. These results suggest that NPY may play a role in the maladaptive remodelling processes associated with other modulators of sympathetic overdrive and cardiac dysfunction. Targeting NPY signalling pathways could offer a novel therapeutic approach to mitigate sympathetic overdrive and its detrimental effects on the heart.

Keywords

cardiovascular diseases

neurohumoral modulation

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