Abstract
The cardiac ryanodine receptor (RyR2) is an intracellular ion channel that regulates Ca2+ release from the sarcoplasmic reticulum (SR) during excitation–contraction coupling in the heart. The glutathione transferases (GSTs) are a family of phase II detoxification enzymes with additional functions including the selective inhibition of RyR2, with therapeutic implications. The C-terminal half of GSTM2 (GSTM2C) is essential for RyR2 inhibition, and mutations F157A and Y160A within GSTM2C prevent the inhibitory action. Our objective in this investigation was to determine whether GSTM2C can enter cultured rat neonatal ventricular cardiomyocytes and influence contractility. We show that oregon green-tagged GSTM2C (at 1 μM) is internalized into the myocytes and it reduces spontaneous contraction frequency and myocyte shortening. Field stimulation of myocytes evoked contraction in the same percentage of myocytes treated either with media alone or media plus 15 μM GSTM2C. Myocyte shortening during contraction was significantly reduced by exposure to 15 μM GSTM2C, but not 5 and 10 μM GSTM2C and was unaffected by exposure to 15 μM of the mutants Y160A or F157A. The amplitude of the Ca2+ transient in the 15 μM GSTM2C - treated myocytes was significantly decreased, the rise time was significantly longer and the decay time was significantly shorter than in control myocytes. The Ca2+ transient was not altered by exposure to Y160A or F157A. The results are consistent with GSTM2C entering the myocytes and inhibiting RyR2, in a manner that indicates a possible therapeutic potential for treatment of arrhythmia in the neonatal heart.
Highlights
The glutathione transferases (EC 2.5.1.18) (GSTs) are a major family of phase II detoxification enzymes that conjugate the tripeptide glutathione (GSH) to a wide range of endogenous and exogenous toxins [1]
We find that the selective inhibition of RyR2 by GSTM2C is due to its interaction with amino acids within the divergent region 3 (D3 region) of cardiac ryanodine receptor [16]
The results presented in this manuscript show that the effects of GSTM2C on neonatal cardiac myocytes are consistent with the known actions of GSTM2C on RyR2 channel activity
Summary
The plasmids for the expression of recombinant GSTM2C and its mutants F157A and Y160A were constructed as described in our previous paper [12]. Recombinant GSTM2C was expressed in E. coli from a pHUE vector and purified by Ni-agarose affinity chromatography as previously described [11]. The poly His-ubiquitin tag used for doi:10.1371/journal.pone.0162415.g001 the affinity chromatography was subsequently cleaved by digestion with a catalytic fragment of the deubiquitylating enzyme mouse Usp 2 [23]. This strategy provides recombinant GSTM2C without any additional residues [12]. The numbering of residues mutated in the C-terminal domain is based on the whole GSTM2 protein
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