Cardiomyocyte contraction is mediated by electrically-evoked increases in intracellular calcium [Ca2+]i via the process of excitation-contraction coupling (ECC). During ECC, depolarization of the sarcolemma induces opening of voltage-gated L-type Ca2+ channels (Cav1.2), allowing Ca2+ entry into the cell. This Ca2+ influx in turn stimulates massive Ca2+ release via ryanodine receptor channels (RyR2) on the underlying sarcoplasmic reticulum. Cav1.2 and RyRs are colocalised in cellular microdomains termed dyads that are distributed throughout the volume of ventricular myocyte allowing homogenous Ca2+ release in response to the action potential. Cardiomyocyte Ca2+ handling is influenced by signals downstream of GPCR such as Endothelin (ET-1) and Angiotensin (Ang II) receptors, which promote inotropy and spontaneous Ca2+ release events (1). The molecular mechanisms underlying the modulation of dyadic Ca2+ signaling by ET-1 and Ang II are not resolved. Experiments were performed in ventricular myocytes isolated from 6 week old WKY rats. Rats were sacrificed in accordance with local ethical and welfare guidelines. Subcellular dyadic Ca2+ release following stimulation with ET-1 (100 nM) and Ang II (1 µM) was reported using a genetically encoded dyad-targeted GFP-based Ca2+ indicator (GCaMP6f-triadin), expressed via an adenovirus (2). GCaMP6f-triadin fluorescence was recorded at 23°C in field-stimulated myocytes by linescan imaging using a Nikon A1R confocal microscope. Images were processed using ImageJ and Matlab. Data are reported as the mean±SEM for n cells. Statistical analysis was by paired Student’s t-test or One-way ANOVA. GCaMP was targeted to the dyad and reported electrically-evoked and caffeine-induced increases in [Ca2+]i. ET-1 increased the rate of Ca2+ release (ΔF/ms) following electrical stimulation (ET-1: 0.30±0.02 vs 0.24±0.02 pre-treatment n=9, p<0.05) whereas it was not affected by Ang II (Ang II: 0.25±0.02 vs 0.27±0.02 pre-treatment n=8, p=0.59). Neither ET-1 nor Ang II affected the heterogeneity of Ca2+ release between dyads (ET-1: 34.72±2.654 vs 31.36±1.0 ms pre-treatment n=9, p=0.29, and Ang II: 36.08±1.28 vs 31.06±3.19 ms pre-treatment n=8, p=0.11, respectively). Ang II treatment significantly altered the mean latency (L) and mean deviation of the onset of Ca2+ release (Lmd) (L = 17.59±2.16 vs 21.94±1.41 ms, p=0.04; Lmd = 11.83±1.28 vs 16.00±0.82 ms for control vs Ang II, n=8, p=0.0008). Whereas ET-1 had no effect (L = 15.63±0.59 vs 17.27±1.24 ms, p=0.31; Lmd = 12.29±0.37 vs 12.79±0.69 ms, n=9, p=0.53, control vs ET-1). Our experiments show that dyads exhibit highly heterogeneous Ca2+ release that is further modified by GPCR stimulation. Our data also suggest that despite activating similar signaling cascades, Ang II and ET-1 elicit different effects on the local Ca2+ release.