CRISPR/Cas9-mediated GLA gene editing in human iPSC-derived ventricular cardiomyocytes as a model of Fabry cardiomyopathy
J Branquinho, V Fogaca-Santos, P Nicolicht, P Varela-Calais, D Roman-Campos, J PesqueroAbstract
Background
Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the enzyme α-galactosidase A (α-Gal A). Enzyme deficiency results in multisystemic pathogenic alterations, including Fabry cardiomyopathy, the leading cause of death in affected patients, whose underlying mechanisms remain incompletely understood.
Purpose
To develop and characterize a genetically modified human left ventricular cardiomyocyte model derived from induced pluripotent stem cells (LV-iPSCs) in order to investigate the mechanisms underlying Fabry cardiomyopathy.
Methods
The CRISPR/Cas9 system was used to generate GLA variants via homology-directed repair with the GLA-N34D guide targeting exon 1. Edited and control cells were subjected to α-Gal A enzymatic activity assays, left ventricular-directed cardiomyocyte differentiation, and electrophysiological analyses.
Results
Gene editing generated two homozygous exon 1 variants: a missense mutation (p.N34D; p.Asn34Asp) and a 25-bp deletion (p.Leu32fs*). Both variants led to frameshift mutations with premature stop codons and nonfunctional proteins, while morphology, pluripotency, and cellular integrity were preserved, as confirmed by immunocytochemistry. Consistent with the FD phenotype, edited cells exhibited no detectable α-Gal A activity compared with controls (80.411 μmol/L/h; p < 0.0001). The left ventricular–directed differentiation protocol significantly shortened the time required to obtain more mature action potentials (APs) compared with the standard cardiomyocyte differentiation protocol (60-day reduction). LV-iPSC–derived cardiomyocytes exhibited increased capacitance, particularly in the presence of 5 µg/mL BMP4, indicating an expanded membrane area and enhanced structural maturation. Despite this, electrophysiological heterogeneity persisted, with predominantly sodium-driven APs and a subset of cells exhibiting calcium-mediated depolarization. These findings indicate that the left ventricular–specific differentiation protocol accelerates acquisition of a more mature ventricular phenotype while preserving subpopulations with electrophysiological features typical of earlier stages of cardiac development.
Conclusions
The generation of this novel in vitro model of human left ventricular–specific cardiomyocytes harboring GLA mutations provides a rapid disease-relevant platform for studying Fabry cardiomyopathy and broadens opportunities for drug screening and investigation of disease-related molecular mechanisms.