The sinoatrial node (SAN), the heart's primary pacemaker, is essential for maintaining rhythmic activity. SAN dysfunctions (SND) and the induced bradycardia are often linked to genetic mutations, highlighting the need for human-relevant models. Differentiating human induced pluripotent stem cells into SAN-like cells (SAN-hiPSCs) provides an innovative platform to study SND mechanisms. In CPVT mouse models associated with RyR2 heterozygous single-point mutation, reduced L-type calcium current (ICaL) disrupts calcium homeostasis and pacemaker activity, leading to bradycardia. Here, we explore whether similar alterations occur in SAN-hiPSCs derived from a CPVT bradycardia-associated patient.

Our goal was to generate healthy control and patient-derived SAN-hiPSCs carrying the RyR2-H29D mutation and monitor their electrophysiological properties.

Control and CPVT patient-derived SAN-hiPSCs were differentiated using a 2D-sandwich-based method, modulating Wnt signaling. Maturity was enhanced by adding small molecules such as triiodothyronine, dexamethasone, and intracellular cyclic AMP (DTA cocktail). Patch-Clamp recordings were performed at day 40 post-differentiation.

Firstly, pacemaker profile has been electrophysiologically characterized in control SAN-hiPSCs, also testing adrenergic and cholinergic responses. CPVT SAN-hiPSCs showed a significant reduction in action potential (AP) frequency compared to control (Ctrl: 157.5±9.9 bpm; CPVT: 38.4±13.6 bpm). The pacemaker ‘funny’ current (If) was similar between groups (@−135mV Ctrl: −21,3±2,1 pA/pF; CPVT −21,8±2,6 pA/pF). In CPVT SAN-hiPSCs, a trend toward decreased ICaL was observed compared to control (@-10mV Ctrl: −37.6±9.5 pA/pF; CPVT: −25.9±4.2 pA/pF). G protein-gated inwardly rectifying potassium current (IKACh) was measured in control but remains under investigation in CPVT cells.

CPVT SAN-hiPSCs exhibit a bradycardic phenotype consistent with the clinical features. CPVT induced-bradycardia is not associated with alterations in the If current but it is most likely linked to a reduction in ICaL. Recent studies suggest that genetic ablation or pharmacological inhibition of IKACh can prevent SND-dependent bradycardia, highlighting its potential as a therapeutic target. Future research will focus on inhibiting IKACh in this model using a vector-based approach to restore SAN function in CPVT patients.