Digging into the pathophysiological complexity of CPVT5

Background Catecholaminergic Polymorphic Ventricular Tachycardia type 5 (CPVT5) is an atypical form of CPVT responsible for a rare, recessively inherited variant of catecholamine-mediated premature ventricular tachycardia caused by the absence of Triadin (TRDN), a small protein localized in the Junctional Sarcoplasmic Reticulum (jSR) and complexed to the Ryanodine Receptor 2 (RyR2). TRDN acts as a positive regulator of the RyR2, and helps anchor Calsequestrin 2 (CASQ2; the main intra-jSR Ca2+ buffer) to the RyR2. It has been demonstrated that the loss of TRDN leads to severe structural abnormalities in cardiomyocytes as well as alterations in the quantity and localization of key proteins involved in Ca2+ handling, such as CASQ2, Junctin (JNT) and Junctophilin2 (JPH2). Purpose Address the mechanism underpinning the reduced expression of our proteins of interest JPH2, CASQ2 and JNT, in TRDN-KO hearts. Methods We generated a TRDN-KO mouse model that recapitulates the phenotype of CPVT5 patients. The localization of our proteins of interest was studied relative to the RyR2 by immunostaining and confocal/super-resolution (STED) microscopy. Autophagic flux was studied by treatment of mice with chloroquine phosphate (CQ) before myocyte isolation. Involvement of the proteasome was assessed by culturing isolated ventricular myocytes in the presence of MG132 for 16 hours and subsequent western-blot. Results STED colocalization studies in TRDN-KO cells revealed that JPH2, CASQ2 or JNT are less colocalized with RyR2s vs. WT cells (p<0.0001, for each protein). No evidence of increased proteasomal involvement was found concerning the degradation of JPH2, CASQ2 or JNT in TRDN-KO hearts (p=0.61, p=0.24, p=0.24, respectively). Aggrephagy and lysosomal pathways were analyzed separately by measuring LC3 and LAMP1 granule size and abundance, with and without CQ treatment. Untreated TRDN-KO cells exhibited significantly larger and more numerous LC3 granules than their WT counterparts (p<0.0001), hinting an overall increase in aggrephagy in CPVT5 hearts. CQ treatment confirmed these findings, as aggresomal density further increased in TRDN-KO vs. WT cells. Furthermore, the colocalization of our proteins of interest with LC3 was greater in TRDN-KO vs. WT (p<0.0001, p=0.001, p<0.001), which persisted following CQ treatment (p=0.0008, p<0.0001, p=0.0002). Additionally supporting our hypothesis, LAMP1 immunostaining revealed larger and more abundant lysosomes in TRDN-KO vs. WT cells (p<0.0001). Conclusion In CPVT5 hearts, the absence of TRDN leads to mistargeting of JPH2, CASQ2, and JNT relative to the couplon. These stranded proteins in turn become degraded by aggrephagy and the lysosomal machinery, explaining their reduced expression.