Tmbim5 loss causes muscle atrophy in zebrafish without exacerbating mcu or slc8b1 knockout phenotypes

Mitochondria generate ATP by establishing a proton gradient across the inner membrane, which also drives calcium uptake via the mitochondrial calcium uniporter (MCU), while sodium-dependent NCLX (encoded bySLC8B1) facilitates efflux. The absence of severe phenotypes inMCU-knockout models suggests redundant calcium transport mechanisms. We investigated the role of TMBIM5, a potential bidirectional mitochondrial calcium transporter, by generating zebrafish lacking Tmbim5, Slc8b1, or both.tmbim5-knockout fish exhibited growth impairment, muscle atrophy (particularly in slow-twitch fibers), increased brain cell death, and reduced mitochondrial membrane potential without altering steady-state mitochondrial calcium levels.slc8b1-deficient fish demonstrated significantly attenuated sodium-dependent calcium efflux and increased larval mortality but were otherwise normal. Surprisingly,tmbim5/mcuandtmbim5/slc8b1double knockouts were viable with normal Mendelian distribution.tmbim5/slc8b1double knockouts displayed impaired mitochondrial calcium uptake capacity, diminished calcium efflux, and severely disrupted cristae architecture not observed in single knockouts. These findings suggest that Tmbim5 functions predominantly in mitochondrial calcium efflux rather than influx and indicate that robust compensatory mechanisms maintain mitochondrial calcium homeostasis despite disruption of these transport pathways.