An Isogenic Human Myoblast Cell Model for Cystinosis Myopathy Reveals Alteration of Key Myogenic Regulatory Proteins.

<p><b>BACKGROUND</b></p><p>Cystinosis is a rare multisystem, autosomal recessive disease caused by dysfunction or loss of cystinosin (CTNS), which results in lysosomal cystine accumulation, primarily affecting the kidneys. Advances in renal transplantation, cysteamine treatment and improved medical care have increased life expectancy, revealing additional systemic phenotypes like myopathy later in life.

Muscle weakness is a major concern leading to life-threatening events in patients, and yet the aetiology of cystinosis myopathy remains to be elucidated.</p><p><b>METHODS</b></p><p>We generated human muscle cell-based models using CRISPR technology to explore the pathophysiology of cystinosis myopathy with the potential to develop new therapies. We used a 4-day differentiation protocol of myoblasts into myotubes to study the effect of CTNS loss in key regulators of myogenic differentiation using western blot analysis.

Afterwards, we used lentiviral (LV)-mediated CTNS WT cDNA addition in CTNS -/- cells to corroborate the CTNS-specific effect. As a next step, we performed multiomic analysis (proteomics, transcriptomics and metabolomics) to gain in-depth knowledge of affected mechanisms.</p><p><b>RESULTS</b></p><p>The polyclonal, isogenic human CTNS knock-out (KO; CTNS -/-) myoblasts exhibited unaltered growth characteristics and accumulated cystine.

Early-stage differentiation of myoblasts into myotubes showed a mild reduction in the fusion index of CTNS -/- myotubes. Upon examination of several key regulators of myogenic differentiation, we observed significantly decreased myosin heavy chain (MyHC) and ryanodine receptor (RyR) protein levels in CTNS -/- myotubes compared to WT cells.

Complementation with CTNS WT cDNA addition in CTNS -/- cells rescued the fusion index, cystine and altered protein levels to WT. In addition, proteomic analysis showed no differences at myoblast level upon the loss of CTNS, but following myotube differentiation, CTNS deletion led to an increase of five protein groups mainly involved in oxidative stress pathways, and a decrease of 18 protein groups biologically connected in myofibril assembly and muscle cell differentiation processes.

Importantly, LV-mediated CTNS addback reverted protein levels to WT levels. Moreover, metabolomics revealed a distinct clustering resulting from CTNS loss.</p><p><b>CONCLUSIONS</b></p><p>Muscle-specific complications are often overlooked in systemic cystinosis treatment.

We show that defective CTNS function impairs effective cystine mobilization from lysosomes, thereby affecting the protein levels of myogenic regulators. A deeper understanding of the molecular mechanisms underlying cystinosis myopathy holds promise for the development of targeted, personalized therapies to improve the quality of life for patients living with cystinosis.</p>