The study, “A Human Stem Cell Model of Fabry Disease Implicates LIMP-2 Accumulation in Cardiomyocyte Pathology,” was published in Stem Cell Reports.
Fabry is a rare genetic disorder caused by mutations in the GLA gene. Located on the X chromosome, the gene provides instructions for the production of an enzyme called alpha-galactosidase A (alpha-GAL A).
These mutations typically affect the activity of alpha-GAL A, leading to the accumulation of a type of fat called globotriaosylceramide (GL-3) in different tissues and organs, including the heart, kidneys and nervous system, gradually compromising their normal function.
For this reason, most Fabry patients develop heart disease over the course of their lives, which may progress to heart failure, the most common cause of death among people living with the disorder.
“A major obstacle for advancing therapy for patients with [Fabry disease] is the knowledge gap between the direct molecular consequences of alpha-GAL A deficiency in CMs [cardiomyocytes, or heart cells] and the cascade of events driving disease in the heart; the inaccessibility of CMs from patients precludes adequate investigation of these events, especially at early stages,” the investigators wrote.
In a previous study, researchers describe the generation of induced pluripotent stem cells (iPSCs) from Fabry patients carrying nonsense mutations in the GLA gene. This gave them the possibility, for the first time, to study the impact of alpha-GAL A deficiency on heart cells derived from patients’ iPSCs grown in a lab dish.
(iPSCs are fully matured cells that are reprogrammed back to a stem cell state, where they are able to grow into any type of cell. A nonsense mutation is a mutation in which the alteration of a single nucleotide (the building blocks of DNA) makes proteins shorter.)
Investigators from Sanofi, in collaboration with researchers at the University of Manchester, further investigated the properties of heart cells derived from patients’ iPSCs. Their aim was to discover more clues about the molecular mechanisms involved in the development of heart disease linked to Fabry.
Functional and structural characterization experiments revealed that heart cells from Fabry patients had higher levels of GL-3, and showed a series of abnormalities in the way they responded to electrical stimuli and in how they regulated their calcium usage, compared to heart cells from healthy people serving as controls. Calcium is essential to coordinate the heart’s function by contributing to the electrical signals involved in heart muscle contraction.
When researchers analyzed the protein contents of heart cells grown in a lab dish, they found these cells produced more than 5,500 different proteins. This analysis also showed that compared to controls, heart cells from Fabry patients produced large amounts of lysosomal membrane protein 2 (LIMP-2) and heat shock-related 70 kDa protein 2 (HSPA2/HSP70-2).
(LIMP-2 is a protein normally found on the membrane of lysosomes — small structures within cells that accumulate, digest, and recycle materials — that regulates their transport within cells; HSPA2/HSP70-2 is a protein involved in cellular quality control, participating in the folding of other proteins and targeting abnormal proteins for degradation.)
Heart cells from Fabry patients released high amounts of cathepsin F, a protein that helps breakdown materials being transported inside lysosomes, as well as HSPA2/HSP70-2. As expected, when researchers corrected the genetic mutation associated with Fabry in heart cells derived from patients’ iPSCs, all these defects were reversed.
To confirm the validity of these proteins as Fabry biomarkers, researchers then forced healthy heart cells to produce high amounts of LIMP-2. They discovered this also triggered the release of large amounts of cathepsin F and HSPA2/HSP70-2, resulting in a massive accumulation of vacuoles (enclosed compartments filled with water and other substances) inside cells.
“In summary, our study has shown the power of the iPSC model to reveal early functional changes and the development of a distinctive biomarker expression profile in [Fabry disease] CMs. These biomarkers may be of utility in drug screening and in elucidating the earliest pathological events and cascades in [Fabry disease] cells. Quantification in patient plasma and urine samples will be an important next step toward validating their relevance in patients,” the researchers wrote.
“A better understanding of these mechanisms will no doubt accelerate the development of more effective and increasingly personalized therapies for patients,” they added.