A Human Stem Cell Model of Fabry Disease Implicates LIMP-2 Accumulation in Cardiomyocyte Pathology.

Matthew J Birket,Matthew J Birket,Matthew J Birket,John P Leonard,John P Leonard,Miriam Lettieri,Gwenaelle Ret,Bing Wang,Cecile Orsini,Sandra Viale,Neil A Hanley,Neil A Hanley,Neil A Hanley,Pauline Cervello,Nicolas Redon,Antony D Adamson,Antony D Adamson,Miriam Lettieri,Sophie Raibaud,Vincent Mikol,Valerie Letang,Bruno Biton,Jean-Michel Itier,Jean-Claude Guillemot

doi:10.1016/j.stemcr.2019.07.004

Abstract

SummaryHere, we have used patient-derived induced pluripotent stem cell (iPSC) and gene-editing technology to study the cardiac-related molecular and functional consequences of mutations in GLA causing the lysosomal storage disorder Fabry disease (FD), for which heart dysfunction is a major cause of mortality. Our in vitro model recapitulated clinical data with FD cardiomyocytes accumulating GL-3 and displaying an increased excitability, with altered electrophysiology and calcium handling. Quantitative proteomics enabled the identification of >5,500 proteins in the cardiomyocyte proteome and secretome, and revealed accumulation of the lysosomal protein LIMP-2 and secretion of cathepsin F and HSPA2/HSP70-2 in FD. Genetic correction reversed these changes. Overexpression of LIMP-2 directly induced the secretion of cathepsin F and HSPA2/HSP70-2, implying causative relationship, and led to massive vacuole accumulation. In summary, our study has revealed potential new cardiac biomarkers for FD, and provides valuable mechanistic insight into the earliest pathological events in FD cardiomyocytes.

Highlights

Fabry disease (FD) is an inherited lysosomal storage disorder (LSD) caused by mutations in the GLA gene on the X chromosome, leading to the deficiency of a-galactosidase A (a-gal A)
Functional and Structural Characterization of Control and Fabry induced pluripotent stem cell (iPSC)-Derived CMs Reveals Increased Excitability in Fabry CMs iPSCs derived from two patients with FD carrying mutations in GLA (Fabry1, c.458G > A; and Fabry2, c.658C > T) that cause classic FD associated with
CMs were enriched using a suspension culture step (Nguyen et al, 2014), and purified by magnetic column/antibody-bead conjugates and returned to a monolayer; final purity was >95% by SIRPA labeling (Figure 1A; Video S1). a-Gal A deficiency was confirmed in Fabry CMs: by day 28 GL-3 levels were $5 times the control level and Fabry CMs were unable to clear exogenous GL-3 as control cells could (Figure 1B)

Summary

Introduction

Fabry disease (FD) is an inherited lysosomal storage disorder (LSD) caused by mutations in the GLA gene on the X chromosome, leading to the deficiency of a-galactosidase A (a-gal A). FD is characterized by the progressive intracellular accumulation of globotriaosylceramide (GL-3) throughout the body, in the vascular tree, nervous system, kidney, and heart (Nagueh, 2014). Most patients develop cardiac involvement, with common manifestations including left ventricular hypertrophy, arrhythmias and conduction disturbances (Linhart and Elliott, 2007). Extensive myocardial fibrosis and impaired left ventricular function can develop, and cardiac disease has become the main cause of mortality in FD (Mehta et al, 2009). In the heart, clearance of GL-3 from cardiomyocytes (CMs) has not been demonstrated, in contrast to the robust clearance from endothelial cells (Eng et al, 2001; Thurberg et al, 2009)

Results

Discussion

Conclusion