Pasquie, J.-L. | bioRxiv Science

bioRxiv2026

Validation of differentiated sinoatrial-like hiPSCs as a model of native sinus node myocytes

BackgroundHuman induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) constitute an attractive system for basic research and pharmacologic screening of new molecules of clinical interest. Numerous protocols aiming at differentiating atrial- or ventricular-like cardiomyocytes (hiPSC-CMs) are available. Conversely, only a few are available for obtaining patient-derived sinoatrial node-like pacemaker myocytes (PM-hiPSC-CMs). Here we validate a new protocol to differentiate mature PM-hiPSC-CMs as a model of native sinoatrial node (SAN) myocytes. MethodsWe generated PM-hiPSC-CMs through a 2D matrix-sandwich method promoting epithelial-to-mesenchymal transition and small molecule-based temporal modulation of Wnt signaling pathway. In addition, we treated our cells with triiodothyronine, dexamethasone and intracellular cyclic AMP (DTA) to enhance expression of proteins involved in intracellular Ca2+ handling. ResultsProteomic analyses showed expression of key SAN proteins in DTA-treated PM-hiPSC-CMs. Importantly, expression of proteins related to Ca2+ handling was increased in DTA-treated PM-hiPSC-CMs compared to untreated ones. DTA-treated PM-hiPSC-CMs displayed action potentials, ionic currents and intracellular Ca2+ dynamics typical of native SAN. In addition, pacemaker activity responded to both {beta}-adrenergic and muscarinic stimulation. ConclusionsOur data indicate that the differentiation protocol effectively generates PM-hiPSC-CMs with typical native human SAN features. This protocol may serve as a potential approach to generate PM-hiPSC-CMs from patients with history of sinoatrial node disfunction (SND) carrying different mutations in ion channels underlying pacemaking. In addition, these in vitro models of SND could be used for testing long-term vector-based gene therapeutic strategies to handle bradycardia.

cell biology↗

bioRxiv2022

Impact of neurons on patient derived-cardiomyocytes using organ-on-a-chip and iPSC biotechnologies

In the heart, cardiac function is regulated by the autonomic nervous system (ANS) that extends through the myocardium and establish junctions at the sinus node and ventricular levels. Thus, an increase or decrease of neuronal activity acutely affects myocardial function and chronically affects its structure through remodeling processes. The neuro-cardiac junction (NCJ), which is the major structure of this system, is poorly understood and only few cell models allow us to study it. Here we present an innovant neuro-cardiac organ-on-chip model to study this structure to better understand the mechanisms involved in the establishment of NCJ. To create such a system, we used microfluidic devices composed of two separate cells compartment interconnected by asymmetric microchannels. Rat PC12 cells, were differentiated to recapitulate the characteristics of sympathetic neurons, and cultivated with cardiomyocytes derived from human induced pluripotent stem cells (hiPSC). We confirmed the presence of specialized structure between the two cell types that allow neuromodulation and observed that the neuronal stimulation impacts the excitation-contraction coupling properties including the intracellular calcium handling. Finally, we also co-cultivated human neurons (hiPSC-NRs) with human cardiomyocytes (hiPSC-CMs) both obtained from the same hiPSC line. Hence, we have developed a neuro-cardiac compartmentalized in vitro model system that allows to recapitulate structural and functional properties of neuro-cardiac junction and that can be used to better understand interaction between heart and brain in humans, as well as to evaluate the impact of drugs on a reconstructed human neuro-cardiac system.

physiology↗