Nadjet BELBACHIR, Stanford University - USA

Post-doctoral researcher, Cardiovascular institute of Stanford University - USA

Abstract

In the past 5 years alone, iPSC-CMs have been leveraged to achieve breakthroughs in understanding patient-specific pathogenic mechanisms of dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular dysplasia, Brugada’s Syndrome and numerous other disorders. The first project aimed to investigate the mechanism behind lethal LQT prolongation in two families carrying a mutation on the NAA10 gene that encodes for N-Alpha Acetylase 10 enzyme responsible for acetylation of over 40% of the whole proteasome including major ion channels likes Cav1.2 and Nav1.5. The affected subjects showed multiple defects (neurological, morphological and cardiac) and their life expectancy was estimated around 10 years, with the main cause of death remains cardiac arrhythmias. Our team obtained two cell lines carrying two different mutations on the NAA10 gene and exhibiting comparable phenotypes. We started by confirming the QT prolongation on the cellular level by performing action potential (AP) recordings and we observed an APD prolongation associated with arrhythmic events known to trigger Torsade de Pointes (TDP). We then performed patch clamp experiments in voltage-clamp configuration to evaluate ion currents potentially involved in the APD prolongation and we observed an increase of the L-type calcium current density (ICaL) with an increase of the calcium window current resulting from a positive shift of the inactivation and a negative shift of the activation kinetics. Mass spectrometry experiments have been conducted on CaV1.2, the ion channel responsible to generate ICaL, and several Acetylation sites have been identified to be differentially regulated by the mutated enzyme in the diseased lines. Intracellular calcium cycle showed an increase of the diastolic calcium level which is consistent with the observed phenotype. To further confirm the involvement of CaV1.2 in the affected patient’s phenotype, LTCC blockers have been administered acutely during multielectrode array (MEA) recordings and an appreciable decrease of the field potential duration has been observed along with a complete suppression of the arrhythmic events.
Despite such usefulness, the iPSC-CMs currently generated in culture suffer from a major limitation of cellular immaturity. The second project focuses on identifying a metabolic compounds combination to improve cardiac maturation in these cells. The iPSC-CMs resemble fetal cardiomyocytes in cellular morphology, metabolism, electrophysiology, contractility and response to calcium or β-adrenergic stimulation, unable to recapitulate the phenotypes of the adult. To date, various approaches to enhance maturation have been described, such as by 3D tissue engineering, electromechanical stimulation, alterations in culture substrate and hormone or steroid treatment. Nonetheless, no single method to date has been accepted as the ‘gold standard’ in achieving cellular maturity of iPSC-CMs in a high-throughput, scalable, and reproducible manner.

Biography

Dr Nadjet Belbachir is a postdoctoral fellow at the Cardiovascular institute of Stanford University under the supervision of Dr Joseph Wu. She earned her PhD at l’institut du Thorax at Nantes, France, in cardiac electrophysiology and arrhythmias in 2017. Her thesis, supervised by Dr Flavien Charpentier, focused on using cardiomyocytes derived from induced pluripotent stem cells to study the electrophysiological mechanisms leading to inherited cardiac arrhythmias, such as Brugada and long QT syndromes. She became extensively interested in deciphering the electrical mechanisms behind drug-induced and inherited cardiac defects. In the long-term future, her career goal as a scientist is to elucidate the interface of structural modulation of ion channel function in arrhythmic disorders using disease modeling, genetics and proteomics.