Background: Mutation of tmem161b in zebrafish causes skipped ventricular beats and a reduced heart rate. Electrophysiological analyses from isolated zebrafish and mouse cardiomyocytes suggest 1) this is associated with increased intracellular cytosolic Ca2+ and 2) evolutionary conserved. Yet, little is known about its function. In this study, we sought to uncover how Tmem161b functions at a molecular level.
Methods: Global knockout of Tmem161b in mice results in perinatal lethality. We therefore generated a conditional Tmem161b KO (cKO) mouse, creating a Tmem161b floxed allele and crossing it with the tamoxifen-inducible ubiquitous Cre line, Ubc-CreERT2. We deleted Tmem161b at 3 months of age and performed conscious ECGs, body composition analyses and lipidomics. In parallel, we performed pull down experiments to investigate what Tmem161b protein physically interacts with. Adult hearts of transgenic zebrafish expressing Tmem161b-Citrine fusion proteins were dissected, and pulldowns performed. Tmem161b-Citrine binding partners were then identified using LC-MS/MS. Identified protein interactors were then validated by immunofluorescence in mutant tmem161b zebrafish hearts and in vitro cell culture assays.
Results: At 5-weeks post tamoxifen cardiac electrical activity of cKO Tmem161b mice was affected. cKO Tmem161b mice showed decreased heart rate, increased QT interval and an increase in heart rate variability. Strikingly, cKO Tmem161b mice also rapidly lost body weight – predominantly fat mass. We therefore performed lipidomics on cKO Tmem161b hearts. Analysis of the lipidome in cKO Tmem161b hearts showed a reduction in triglyceride levels in addition to changes in lipid species levels. We next examined Tmem161b interacting proteins via pulldowns revealing 54 Tmem161b-Citrine binding partners. These included proteins such as Rtn1a/3, Apoba/b, Erlin1 that regulate or are localised in the endoplasmic reticulum (ER). Given the important role of the ER in Ca2+ handling and lipid synthesis, we next examined sarcoplasmic reticulum (a specialised ER) morphology in zebrafish tmem161b mutant cardiomyocytes. Mutant tmem161b cardiomyocytes displayed abnormal distribution of the sarcoplasmic reticulum. This was further validated in TMEM161B knockout cell lines which showed increased ER branching and tubulation.
Conclusion: Altogether, this study demonstrates that Tmem161b is involved in regulating ER morphology. Loss of Tmem161b leads to abnormal ER morphology and subsequently, dysregulated Ca2+ handling, lipid synthesis and ultimately, cardiac arrythmias.