Functional Characterization of Endogenously Expressed Human RYR1 Variants

Susan Treves; Thierry Girard; Francesco Zorzato

doi:10.3791/62196

Functional Characterization of Endogenously Expressed Human RYR1 Variants

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Medicine

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JoVE Journal Medicine

Functional Characterization of Endogenously Expressed Human RYR1 Variants

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07:59 min

June 9, 2021

DOI: 10.3791/62196-v

Susan Treves^1,2, Thierry Girard³, Francesco Zorzato^1,2

¹Department of Biomedicine,Basel University Hospital, ²Department of Life Sciences,University of Ferrara, ³Department of Anesthesia,Basel University Hospital

Overview

This protocol describes methods to study the functional effects of RYR1 mutations in patient-derived cells. By using Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy-derived satellite cells, researchers can gain insights into calcium homeostasis and potential therapeutic interventions.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Genetics

Background

RYR1 mutations are linked to various muscle disorders.
Understanding calcium homeostasis is crucial for diagnosing these conditions.
Patient-derived cells provide a more accurate biological response.
This study utilizes simple techniques that do not require sophisticated equipment.

Purpose of Study

To assess the functional impact of RYR1 mutations.
To evaluate the biological response of patient-derived cells.
To explore potential therapeutic options for RYR1-related disorders.

Methods Used

Isolation of EBV-transformed B lymphocytes from patients.
Incubation of cells with Fura-2 AM to monitor calcium levels.
Use of Krebs Ringer's solution for cell resuspension.
Testing of compounds to assess their therapeutic potential.

Main Results

Demonstrated functional changes in calcium homeostasis due to RYR1 mutations.
Validated the use of patient-derived cells for accurate biological assessments.
Provided a straightforward methodology for researchers.
Highlighted the potential for therapeutic testing using patient samples.

Conclusions

This protocol offers a reliable method for studying RYR1 mutations.
Using patient-derived cells enhances the relevance of findings.
Future research can build on these methods to explore new therapies.

Frequently Asked Questions

What are RYR1 mutations?

RYR1 mutations are genetic alterations in the ryanodine receptor 1 gene, associated with muscle disorders.

Why use patient-derived cells?

Patient-derived cells provide a more accurate reflection of the biological response in individuals with specific mutations.

What is the significance of calcium homeostasis?

Calcium homeostasis is critical for muscle function, and disruptions can lead to various muscle diseases.

How are the cells prepared for the experiment?

Cells are resuspended in Krebs Ringer's solution and incubated with Fura-2 AM to monitor calcium levels.

What equipment is needed for this protocol?

The protocol is simple and does not require sophisticated equipment, making it accessible for many researchers.

Can this method be used for therapeutic testing?

Yes, the methods can be adapted to test potential therapeutic compounds on patient-derived cells.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

This protocol is significant because it uses patient material. If the patient has the disease, using their cells should more accurately reflect the patient's biological response. This technique is simple to perform, straightforward, and does not require sophisticated equipment.

These methods can be used for diagnosis, for example, to prove that a RYR1 mutation functionally changes calcium homeostasis, as well as to test the potential therapeutic function of a compound. Depending on the knowledge and technical skills of the individual researcher, test experiments should be performed before actual patient samples are used. To monitor changes in the intracellular calcium concentration of EBV-transformed B lymphocytes, resuspend the immortalized B cells at a one times 10 to the seventh cells per milliliter concentration in Krebs Ringer's solution and Fura-2 AM to a final concentration of five micromolar for a 30-minute incubation at 37 degrees Celsius.

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RYR1 Variants Functional Characterization Calcium Homeostasis Patient Material Diagnosis Techniques B Lymphocytes Fura-2 AM Intracellular Calcium Concentration Spectrofluorometer Calcium Release Response Poly-L-lysine Fluorescent Microscope Software-controlled Camera Experimental Protocol