title: Glutathione transferase M2 variants inhibit ryanodine receptor function in adult mouse cardiomyocytes.
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.
authors.affiliation: John Curtin School of Medical Research, Australian National University, Australia.
authors.forename: Kaveenda
authors.initials: K
authors.lastname: Samarasinghe
authors.affiliation: John Curtin School of Medical Research, Australian National University, Australia.
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.affiliation: John Curtin School of Medical Research, Australian National University, Australia.
authors.forename: Padmaja
authors.initials: P
authors.lastname: Tummala
authors.affiliation: John Curtin School of Medical Research, Australian National University, Australia.
authors.forename: Jean
authors.initials: J
authors.lastname: Cappello
authors.affiliation: John Curtin School of Medical Research, Australian National University, Australia.
authors.forename: Suzy M
authors.initials: SM
authors.lastname: Pace
authors.affiliation: Cardiology Unit, The Canberra Hospital, Woden, ACT, Australia.
authors.forename: Leonard
authors.initials: L
authors.lastname: Arnolda
authors.affiliation: John Curtin School of Medical Research, Australian National University, Australia.
authors.forename: Marco G
authors.initials: MG
authors.lastname: Casarotto
authors.affiliation: John Curtin School of Medical Research, Australian National University, Australia.
authors.forename: Angela F
authors.initials: AF
authors.lastname: Dulhunty
authors.affiliation: John Curtin School of Medical Research, Australian National University, Australia. Electronic address: Philip.Board@anu.edu.au.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Board
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002478:Cells, Cultured; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics*; D008297:Male; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D002121:Calcium Channel Blockers; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; C000592748:ryanodine receptor 2. mouse; D002110:Caffeine; D005982:Glutathione Transferase; C412602:glutathione S-transferase Mu 2
keywords: Cardiac ryanodine receptor Ca(2+) channels; Excitation–contraction; Glutathione transferase M2; Mouse cardiomyocytes; coupling