Fig. 3

Fig. 3 Tmem161b is required for mouse neonatal survival and wild-type Ca²⁺ oscillations in isolated cardiomyocytes. (A) Representative whole embryo images of heterozygous (Tmem161b^+/LacZ) and homozygous (Tmem161b^LacZ/LacZ) Tmem161b-LacZ (loss-of-function) mouse embryos at 17.5 dpc. Images show embryonic survival at 17.5 dpc and, with the exception of eye defects, are phenotypically wild type at a gross morphological level. (B) Graphical representation of neonatal survival at P0 showing that all Tmem161b^LacZ/LacZ embryos die at or soon after birth. (C) Dissected whole-mount hearts at 15.5 dpc stained for LACZ showing expression throughout the heart as well as in the region of the sinoatrial node (arrow). Homozygous Tmem161b^LacZ/LacZ hearts appear enlarged compared with heterozygous littermate hearts. (D) Graphical representation of heart weight, normalized to body weight, shows an increase in Tmem161b^LacZ/LacZ hearts compared with Tmem161b^+/+ and Tmem161b^+/LacZ (at 17.5 dpc). n = 9 wild types, 14 homozygotes, and 29 heterozygotes. **P < 0.001; *P < 0.05. (E) Quantification of cardiac cell number shows an increase in Tmem161b^LacZ/LacZ embryos (n = 10) compared with Tmem161b^+/+ (n = 8) and Tmem161b^+/LacZ (n = 13) (at 17.5 dpc). *P < 0.001. (F) Schematic of cardiomyocyte isolation to measure Ca²⁺ transients in mouse embryonic cardiomyocytes. (G) Representative traces of Ca²⁺ transients from isolated wild-type and Tmem161b^LacZ/LacZ cardiomyocytes. (H) Overview of parameters measured for cultured single cardiomyocytes stained with the Ca²⁺ indicator Fluo-4 AM and quantification of the rate of Ca²⁺ transients. Increased duration and variation (SD) between oscillations is observed in Tmem161b^LacZ/LacZ cardiomyocytes compared with wild-type cardiomyocytes. The mean peak duration is unchanged; however the frequency of oscillations (rate) is decreased in Tmem161b^LacZ/LacZ cardiomyocytes compared with wild type. n = 63 to 77 cells from three to four mice. *P < 0.05, ***P < 0.01; n.s., not significant.