Abstract:
The ryanodine receptor (RyR) is an ion channel that releases Ca2+ from the sarcoplasmic reticulum and is essential for excitation-contraction coupling and contraction in striated muscle. Previously we reported that the human muscle specific glutathione transferase (GSTM2-2) is an inhibitor of cardiac muscle RyR2 and a weak activator of skeletal muscle RyR1. Single channel experiments and Ca2+ release assays using the C-terminal half of GSTM2-2(GSTM2C) and the mutants, F157A and Y160A in the C terminal domain confirmed the importance of helix 6 in the C-terminal (non-enzymic) fold for inhibition of RyR2. The objective of this study was to determine the effect of GSTM2C on the cardiac myocyte function.
Primary cardiomyocytes were cultured from neonatal rats. Spontaneous or field- stimulated (1Hz with 3V, 2ms pulses) contraction was recorded in control and 15μM GSTM2C-treated cells using a JVC video camera KY/F550 attached to Nikon TE2000-U microscope. Images were analysed using Image Pro plus 6.2 software and percentage cell shortening measured.
Preliminary results showed that spontaneous contraction frequency fell from 42.5/min in the control group to 6.9 /min after GSTM2C treatment (P<0.001). The number of spontaneously beating cells fell from 6.6% in the control group to 1.9% after treatment (P<0.001). To determine whether the reduced contractions were due to the GSTM2 C terminus affecting action potentials or contraction, shortening was measured. Shortening in spontaneous contraction fell from 7.5±1.0% in control to 2.9±0.6% after GSTM2C treatment (P<0.001). Shortening after field stimulation also fell, from 7.3±0.8% before to 4.1±0.6% after, GSTM2C-treatment (P<0.01). These results are consistent with GSTM2 C reducing contraction in ventricular cardiomyocytes by reducing Ca2+ release through RyR2.
