Impaired cardiac cell phenotype in fabry disease using induced pluripotent stem cell-derived cardiomyocytes

Abstract Background Fabry disease (FD) is a systemic disorder of glycosphingolipid metabolism caused by deficient or absent lysosomal α-galactosidase A (GLA) activity, leading to the progressive accumulation of globotriaosylceramide (Gb3) in multiple organs, including the heart, kidneys, liver, and brain. Cardiac dysfunction is the leading cause of mortality in Fabry patients. Purpose This study aims to investigate the cardiac phenotype of Fabry patients by differentiating patient-derived cells into cardiomyocytes (CMs). Methods In this experimental study, human dermal fibroblasts from two Fabry patients were reprogrammed into human-induced pluripotent stem cells (iPSCs) and differentiated into CMs using the small molecules CHIR99021 and IWP2. Gene expression, protein expression, membrane potential, and endoplasmic reticulum (ER) calcium levels were evaluated in FD-derived CMs and control-derived CMs (C-derived CMs) at day 22 of differentiation. Results Generated FD-iPSCs expressed pluripotency markers OCT4, NANOG, TRA-1-60, and TRA-1-81. FD-related abnormalities, including reduced GLA gene and protein expression and Gb3 accumulation, were detected in FD-iPSCs and FD-derived CMs. The efficiency of cardiogenic differentiation was assessed by evaluating the gene and protein expression of cardiac-specific markers. qRT-PCR revealed downregulation of progenitor markers (ISL1, NKX2.5, GATA4, MEF2C), ion channels (KCNJ2), and calcium-handling machinery (TRDN, RYR2, SERCA2a, CACNA1C) in FD-derived CMs compared to C-derived CMs. However, the gene expression of contractile apparatus markers, including MYH7, MYL2, and TNNT2, was similar between FD-derived and C-derived CMs. Protein expression levels of LAMP2, CTNT, and α-actinin were also comparable between the two groups. Additionally, FD-derived CMs exhibited increased reactive oxygen species (ROS) production, which was normalized in C-derived CMs. ER calcium assessment using sequential 1 µM thapsigargin and 2 mM calcium challenges revealed that while ER calcium storage capacity remained unaffected in FD-derived CMs, extracellular calcium uptake was severely impaired. Conclusions These findings highlight impairments in the cardiac cell phenotype of Fabry patients, demonstrated through in vitro differentiation of patient-specific iPSCs into beating CMs.