Biology subjects

Sleiman, Y.

Publications and source records attributed to Sleiman, Y..

4 recordsLinked to original sources

bioRxiv2026

Cav1.3-C-terminus Restores Electrical and Contractile Functions in hiPSC-derived Cardiomyocytes Model of Heart Failure

Objective: Heart failure (HF) is a major global health concern characterized by impaired excitation-contraction coupling and extensive structural remodeling of the myocardium, often modeled using a potent vasoactive and neurohumoral mediator, endothelin-1 (ET-1). This study investigates whether AAV6-mediated delivery of the Cav1.3-C-terminus, acting as an auto-trans-regulator of gene transcription, can restore electrophysiological and contractile functions in an ET-1-induced HF model using hiPSC-CMs. Methods: hiPSC-CM monolayers were treated with 10 nM ET-1 for 48 hours to induce HF-like phenotype and then co-treated with an AAV6-Cav1.3-C-terminus. We subsequently evaluated gene expression via RT-qPCR, single-cell electrophysiological properties via patch-clamp, and contractile function using custom video analysis software. Results: ET-1 treatment induced a HF-associated remodeling phenotype, characterized by significant upregulation of hypertrophic (NPPA, NPPB, and ACTA1) and extracellular matrix-associated (COL1A1) genes, along with increased cell membrane capacitance. This structural remodeling was accompanied by marked electrophysiological alterations, including downregulation of KCNH2, CACNA1D, CACNA1C, and SCN5A, reduced potassium, calcium, and sodium current densities, and prolonged action potential duration. ET-1 impaired both the spontaneous beating rate and contraction amplitude of the monolayers. Co-treatment with the Cav1.3-C-terminus successfully restored CACNA1D and KCNH2 gene expression to baseline levels, accompanied by normalized L-type calcium and potassium current densities, normalized APD prolongation, and completely restored the chronotropic and inotropic functions to control levels. Conclusions: ET-1-treated hiPSC-CMs model effectively recapitulates the electrophysiological and contractile dysregulations of HF. Gene therapy by Cav1.3-C-terminus provides robust functional rescue myocardial contractility, highlighting its potential as a novel therapeutic strategy for HF.

cell biology↗

bioRxiv2026

Modeling Sympathetic Neuro-Cardiac Interactions in a hiPSC-Based Microphysiological System

The cardiac autonomic nervous system is a key driver of various cardiac disorders and arrhythmias. However, investigating neuronal regulation of the human heart has proven difficult due to immitted and reliable experimental models. Here, we present a novel microphysiological system utilizing a compartmentalized microfluidic device (MFD) to integrate co-cultured human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) and sympathetic neurons (hiPSC-SNs). MFD is composed of two wide-open chambers separated by microfluidic microchannels. hiPSC-SNs were characterized by confocal imaging and RT-qPCR for the expression of peripherin, tyrosine hydroxylase, and {beta}-tubulin III, as well as high levels of dopamine {beta}-hydroxylase and nicotinic acetylcholine receptors. Furthermore, patch-clamp techniques confirmed their functional maturity, showing spontaneous action potentials and positive responses to nicotine (1{micro}M). Co-culturing hiPSC-CMs and hiPSC-SNs within the MFD facilitated axonal projection into the cardiomyocyte chamber, establishing a physical connection between the two cell types. After 10 days of co-culture, functional integration was confirmed by a significant increase in the action potential frequency and beating rate of hiPSC-CMs, as recorded by patch-clamp and video motion tracking, respectively. Notably, nicotine application in the neuronal chamber accelerated these rates in hiPSC-CMs chamber, whereas the administration of the {beta}-blocker, propranolol (5{micro}M), effectively decreased the beating rates. Collectively, these data demonstrate the feasibility of differentiating hiPSCs into functional sympathetic neurons and establishing a robust neuro-cardiac interface. This microphysiological system represents a powerful platform for investigating disorders characterized by impaired neuro-cardiac interactions, offering a valuable tool for both disease modeling and pharmacological screening.

physiology↗

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↗

bioRxiv2025

Electrophysiological abnormalities associated with a CACNA1D variant are rescued by AAV6-Cav1.3-C-terminus gene therapy in patient-iPSC-CMs

Inherited arrhythmia syndromes are caused by genetic variants that alter cardiac ion channel function. We investigated a complex presentation in a pediatric patient with ventricular tachycardia and conduction abnormalities, harboring a de novo CACNA1D (c.3786G>T) variant, and two inherited variants, the SCN5A (c.2618C>G), and a DSP desmosome (c.1582C>G). The CACNA1D variant, which encodes Cav1.3 L-type calcium channel is the focus of this study, because the C-terminus fragment of Cav1.3 has recently been identified as a transcription auto-enhancer of its own gene and able to prevent arrhythmic events in a mouse model of ischemic heart failure. Leveraging this intrinsic property, we hypothesized that the Cav1.3-C-terminus could reverse the arrhythmic events associated with the CACNA1D variant. Patch-clamp and optical mapping experiments demonstrated a loss of Cav1.3 function, characterized by reduced L-type calcium current densities, and decrease of conduction velocity, leading to inducible re-entrant arrhythmias in human induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs). RNA sequencing confirmed this loss-of-function via the downregulation CACNA1D gene expression. Interestingly, Cav1.3-C-terminus treatment of hiPSC-CMs successfully normalized Cav1.3 gene expression, restored calcium currents, and conduction velocity, and prevented the susceptibility to arrhythmias. These findings highlight the electrophysiological consequences resulting from a de novo Cav1.3 variant and demonstrate an important transcriptional role of Cav1.3-C-terminus as a transcriptional regulator and as a promising therapeutic tool to restore normal electrical properties in patients with calcium channels loss of function.

physiology↗