Intracellular two-phase Ca2+release and apoptosis controlled by TRP-ML1 channel activity in coronary arterial myocytes

Abstract

Activation of the death receptor Fas has been reported to produce a two-phase intracellular Ca2+release response in coronary arterial myocytes (CAMs), which consists of local Ca2+bursts via lysosomal transient potential receptor-mucolipin 1 (TRP-ML1) channels and consequent Ca2+release from the sarcoplasmic reticulum (SR). The present study was designed to explore the molecular mechanism by which lysosomal Ca2+bursts are coupled with SR Ca2+release in mouse CAMs and to determine the functional relevance of this lysosome-associated two-phase Ca2+release to apoptosis, a common action of Fas activation with Fas ligand (FasL). By confocal microscopy, we found that transfection of CAMs with TRP-ML1 small interfering (si)RNA substantially inhibited FasL (10 ng/ml)-induced lysosome Ca2+bursts and consequent SR Ca2+release. In contrast, transfection of CAMs with plasmids containing a full-length TRP-ML1 gene enhanced FasL-induced two-phase Ca2+release. We further demonstrated that FasL significantly increased the colocalization of the lysosomal marker Lamp1 with ryanodine receptor 3 and enhanced a dynamic trafficking of lysosomes to the SR. When CAMs were treated with TRP-ML1 siRNA, FasL-induced interactions between the lysosomes and SR were substantially blocked. Functionally, FasL-induced apoptosis and activation of calpain and calcineurin, the Ca2+sensitive proteins that mediate apoptosis, were significantly attenuated by silencing TRP-ML1 gene but enhanced by overexpression of TRP-ML1 gene. These results suggest that TRP-ML1 channel-mediated lysosomal Ca2+bursts upon FasL stimulation promote lysosome trafficking and interactions with the SR, leading to apoptosis of CAMs via a Ca2+-dependent mechanism.