Triadin Overexpression Stimulates Excitation-Contraction Coupling and Increases Predisposition to Cellular Arrhythmia in Cardiac Myocytes

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Abstract
Triadin 1 (TRD) is an integral membrane protein that associates with the ryanodine receptor (RyR2), calsequestrin (CASQ2) and junctin to form a macromolecular Ca signaling complex in the cardiac junctional sarcoplasmic reticulum (SR). To define the functional role of TRD, we examined the effects of adenoviral-mediated overexpression of the wild-type protein (TRDWT) or a TRD mutant lacking the putative CASQ2 interaction domain residues 200 to 224 (TRDDel.200–224) on intracellular Ca signaling in adult rat ventricular myocytes. Overexpression of TRDWT reduced the amplitude of ICa- induced Ca transients (at 0 mV) but voltage dependency of the Ca transients was markedly widened and flattened, such that even small ICa at low and high depolarizations triggered maximal Ca transients. The frequency of spontaneous Ca sparks was significantly increased in TRDWT myocytes, whereas the amplitude of individual sparks was reduced. Consistent with these changes in Ca release signals, SR Ca content was decreased in TRDWT myocytes. Periodic electrical stimulation of TRDWT myocytes resulted in irregular, spontaneous Ca transients and arrhythmic oscillations of the membrane potential. Expression of TRDDel.200–224 failed to produce any of the effects of the wild-type protein. The lipid bilayer technique was used to record the activity of single RyR2 channels using microsome samples obtained from control, TRDWT and TRDDel.200–224 myocytes. Elevation of TRDWT levels increased the open probability of RyR2 channels, whereas expression of the mutant protein did not affect RyR2 activity. We conclude that TRD enhances cardiac excitation-contraction coupling by directly stimulating the RyR2. Interaction of TRD with RyR2 may involve amino acids 200 to 224 in C-terminal domain of TRD.