Background: Acute myocardial infarction and chronic systolic heart failure result in heightened cardiac sympathetic activation¹, driving release of the co-transmitter neuropeptide-Y (NPY)², with circulating levels correlating with morbidity and mortality in both conditions³. The acute physiological effects of NPY on cardiac excitability are well studied⁴, but the effects of chronic NPY exposure, which may be important for subsequent ventricular remodelling, are poorly characterised. We therefore investigated this using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs).

Methods and Results: Immunohistochemistry and western blot analysis of hiPSC-CMs (SFC845, obtained from The James Martin Stem Cell Facility, Oxford) demonstrated NPY1R, NPY2R and NPY5R expression in these cells, receptors which were also found in ventricular biopsies from human patients undergoing valve surgery using qPCR (n=5). Incubation with NPY over 2 days significantly (Kruskal-Wallis, p<0.0001) increased cell area (following cytoskeletal immunofluorescent staining) at 1 nM (1669 [1242-1669] μm², n=98 cells), 10 nM (2163 [1531-2657] μm², n=94 cells) and 100 nM (1780 [1294-2399] μm², n=60 cells) compared to control (1126 [842.7-1603] μm², n=96 cells). NPY (10nM) also significantly increased ANP and BNP expression assessed by qPCR (unpaired t-test, p<0.05, n=6 wells) and reduced luciferase-based ATP cell viability (unpaired t-test, p<0.05, n=6 wells). NPY-mediated cell enlargement and cell viability reduction could be blocked with a NPY1R antagonist (BIBO 3304, 1 μM: 1414 [873.6-2387] μm², n=33 cells vs NPY+BIBO: 1167 [761.5-1659] μm², n=125 cells) and reversed with a NPY5R antagonist (CGP 71683A, 1 μM: 1488 [1051-2224] μm², n=49 cells vs NPY+CGP: 1222 [688.4-2158] μm², n=73 cells, MWU test, p<0.05), which did not occur in the presence of an NPY2R antagonist (BIIE 0246, 1 μM: 1047 [699-1686] μm², n=75 cells vs NPY+BIIE: 1517 [891.2-2287] μm², n=76 cells, p<0.05). The Forster resonance energy transfer-based sensor Epac-S^H187 was expressed in hiPSC-CMs to monitor the cyclic adenosine 3’,5’-monophosphate (cAMP) response to NPY. 1 and 10 nM NPY reduced intracellular cAMP levels, whilst higher doses of NPY (100 nM) slightly increased cAMP (n=65 cells). An NPY5R antagonist most effectively inhibited these cAMP changes (p<0.0001, n=40 cells).

Conclusions: In hiPSC-CMs, chronic NPY exposure promotes cellular hypertrophy and reduces cell viability via NPY1R and NPY5R signalling, associated with G_i-coupled pathways. NPY1R and NPY5R may represent potential drug targets to treat chronic heart failure.

1. Borovac, J. A., D'Amario, D., Bozic, J. & Glavas, D. Sympathetic nervous system activation and heart failure: Current state of evidence and the pathophysiology in the light of novel biomarkers. World journal of cardiology 12, 373-408 (2020).
2. Tan, C. M. J. et al. The Role of Neuropeptide Y in Cardiovascular Health and Disease. Frontiers in physiology 9, 1281 (2018).
3. Gibbs, T. et al. Neuropeptide‐Y Levels in ST‐Segment–Elevation Myocardial Infarction: Relationship With Coronary Microvascular Function, Heart Failure, and Mortality. Journal of the American Heart Association 11, e024850 (2022).
4. Kalla, M. et al. The cardiac sympathetic co-transmitter neuropeptide Y is pro-arrhythmic following ST-elevation myocardial infarction despite beta-blockade. Eur. Heart J. 41, 2168-2179 (2020).