Related Experiment Video
Updated: Apr 5, 2026
Functional Characterization of Endogenously Expressed Human RYR1 Variants
Published on: June 9, 2021
Glutathione transferase M2 variants inhibit ryanodine receptor function in adult mouse cardiomyocytes
Kaveenda Samarasinghe1, Dan Liu1, Padmaja Tummala1
1John Curtin School of Medical Research, Australian National University, Australia.
Abstract:
Release of Ca(2+) from the sarcoplasmic reticulum (SR) through the cardiac ryanodine receptor (RyR2) is an essential step in cardiac excitation-contraction coupling. Excess Ca(2+) release due to overactive RyR2 can cause arrhythmia that can lead to cardiac arrest. Fragments derived from the carboxy-terminal domain of human glutathione transferase M2 (GSTM2C) specifically inhibit RyR2 activity. Our aim was to further improve this inhibition by mutagenesis and to assess the therapeutic potential of GSTM2C based peptides to treat Ca(2+) release-based arrhythmia. We generated several mutant variants of the C-terminal fragment GSTM2C H5-8 and from those mutant proteins we identified two (RM13 and SM2) that exhibited significantly greater inhibition of cardiac SR Ca(2+) release and single RyR2 channel activity. Flow cytometry analysis showed that these two mutant proteins as well as GSTM2C H5-8 are taken up by isolated adult mouse cardiomyocytes without the aid of any additional compounds, Ca(2+) imaging and isolated cell contraction measurements revealed that GSTM2C H5-8, SM2 and RM13 reduce the SR Ca(2+) release rate and the fractional shortening of adult mouse cardiomyocytes, while importantly increasing the rate of Ca(2+) removal from the sarcoplasm. These observations indicate that peptides derived from GSTM2C inhibit RyR2 at a cellular level and thus they may provide the basis for a novel therapeutic agent to treat arrhythmia and heart attack.
Insights
New peptides derived from human glutathione transferase M2 (GSTM2C) fragments significantly inhibit cardiac sarcoplasmic reticulum Ca(2+) release. These GSTM2C-based peptides show therapeutic potential for treating Ca(2+) release-related heart arrhythmias.
Area of Science:
- Cardiovascular Biology
- Molecular Pharmacology
- Biochemistry
Background:
- Cardiac excitation-contraction coupling relies on calcium (Ca2+) release from the sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2).
- Dysfunctional RyR2 activity leading to excessive Ca2+ release is a primary cause of cardiac arrhythmias and potentially cardiac arrest.
- Naturally occurring carboxy-terminal fragments of human glutathione transferase M2 (GSTM2C) demonstrate inhibitory effects on RyR2.
Purpose of the Study:
- To enhance the RyR2 inhibitory properties of GSTM2C fragments through mutagenesis.
- To evaluate the therapeutic potential of modified GSTM2C peptides in treating Ca2+-mediated cardiac arrhythmias.
Main Methods:
- Site-directed mutagenesis was used to generate mutant variants of the GSTM2C H5-8 fragment.
- Inhibition of cardiac SR Ca2+ release and single RyR2 channel activity was assessed.
- Uptake of peptides by isolated adult mouse cardiomyocytes was confirmed using flow cytometry.
- Ca2+ imaging and isolated cell contraction measurements were performed to evaluate cellular effects.
Main Results:
- Two mutant variants, RM13 and SM2, exhibited significantly enhanced inhibition of cardiac SR Ca2+ release and RyR2 channel activity compared to GSTM2C H5-8.
- GSTM2C H5-8, RM13, and SM2 were effectively taken up by adult mouse cardiomyocytes.
- These peptides reduced SR Ca2+ release rates and fractional shortening in cardiomyocytes.
- Importantly, the peptides increased the rate of sarcoplasmic Ca2+ removal.
Conclusions:
- Mutagenesis of GSTM2C fragments yields peptides (RM13, SM2) with superior RyR2 inhibition.
- These GSTM2C-derived peptides are cell-permeable and modulate RyR2 activity at the cellular level.
- The findings suggest that GSTM2C-based peptides represent a promising novel therapeutic strategy for arrhythmias and heart attack.
